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Bioavailability of various vitamin E compounds in ruminants

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Bioavailability of various vitamin E compounds in ruminants
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Hidiroglou, Nicholas, 1961-
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English
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xi, 139 leaves : ill. ; 29 cm.

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Acetates ( jstor )
Animals ( jstor )
Cattle ( jstor )
Dosage ( jstor )
Liver ( jstor )
Plasmas ( jstor )
Rats ( jstor )
Sheep ( jstor )
Tocopherols ( jstor )
Vitamin E ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 124-138).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Nicholas Hidiroglou.

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BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS











BY

NICHOLAS HIDIROGLOU


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1989































This work is dedicated to my parents, Dr. and Mrs. Hidiroglou, for their continual support and encouragement throughout the doctoral program.















ACKNOWLEDGMENTS


The author wishes to express his sincere gratitude to Dr. L. R. McDowell, chairman of the supervisory committee, for his guidance, patience and friendship throughout the doctoral program. The author gratefully acknowledges the assistance and time provided by other members of the supervisory committee, including Drs. Douglas Bates, Joseph Conrad, Kermit Bachman and Rachel Shireman.

Deep appreciation is extended to Dr. Keith U. Ingold of the

National Research Council of Canada (NRCC) for providing laboratory facilities. The author is especially indebted to Dr. Graham W. Burton of the NRCC for his guidance, patience, and friendship. The technical assistance of Ann Webb, Dave Lindsay, Ewa Lusztyk of the NRCC is deeply appreciated. Many special thanks are extended to Dr. L. Arvanitis for his continual support and friendship throughout my graduate program.

Assistance of fellow graduate students, Oswaldo Balbuena, Pablo Cuesta, Edmundo Espinoza, Akhmad Probowo, Rodrigo Pastrana, Libardo Ochoa and Roger Merkel, during blood and tissue collections is appreciated. Special gratitude is expressed to Rodrigo Pastrana and Roger Merkel for continual support during tissue collections from animals.

Sincere appreciation is expressed to Mrs. Pat French for typing this dissertation.














TABLE OF CONTENTS


PAGE

DEDICATION.................................................... ii

ACKNOWLEDGMENTS................................................ iii

LIST OF TABLES ................................................. vi

LIST OF FIGURES ............................................... viii

ABSTRACT ....................................................... x

CHAPTER 1 INTRODUCTION...................................... 1

CHAPTER 2 REVIEW OF LITERATURE.............................. 3

Chemistry of Vitamin E............................ 3
Absorption and Storage of Vitamin E............... 12
The Metabolism of Vitamin E. ...................... 20
Vitamin E Deficiency.............................. 22
Biological Activity of Vitamin E.................. 28
Role of Vitamin E in the Respiratory Chain........ 41 Intervention of Vitamin E in Hematopoiesis........ 42 Biological Role of Vitamin E...................... 43
Role of a-Tocopherol as a Chain Breaking
Antioxidant...................................... 49

CHAPTER 3 PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP
FOLLOWING INTRAMUSCULAR ADMINISTRATION IN AN OIL
CARRIER........................................... 51

Introduction...................................... 51
Experimental...................................... 51
Analytical........................................ 52
Statistical Analysis.............................. 53
Results ........................................... 54
Discussion........................................ 59
Summary........................................... 61

CHAPTER 4 BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS
IN SHEEP .......................................... 63

Introduction...................................... 63
Materials and Methods............................. 63
Results ........................................... 67
Discussion........................................ 76
Summary ........................................... 77











PAGE


CHAPTER 5


LITERATURE CITED..............................................

BIOGRAPHICAL SKETCH...........................................


BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS...................

Introduction......................................
Materials and Method..............................
Results..........................................
Discussion........................................
Summary..........................................

PLASMA TOCOPHEROL IN RUMINANTS AFTER INGESTING FREE OR ACETYLATED TOCOPHEROL.....................

Introduction......................................
Materials and Methods.............................
Results..........................................
Discussion........................................
Summary..........................................

PLASMA AND TISSUES VITAMIN E CONCENTRATIONS IN SHEEP AFTER ADMINISTRATION OF A SINGLE INTRAPERITONEAL DOSE OF dl-a-TOCOPHEROL...........

Introduction......................................
Experimental Procedure............................
Results..........................................
Discussion........................................
Summary..........................................

GENERAL CONCLUSIONS...............................


CHAPTER 6


CHAPTER 7


CHAPTER 8















LIST OF TABLES


TABLE PAGE

2.1. NOMENCLATURE FOR TOCOPHEROLS........................... 7

2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS...... 10

2.3. RELATIVE ACTIVITY OF VARIOUS ALPHA-TOCOPHEROL STEREOISOMERS AS DETERMINED BY RESORPTION STERILITY
TEST IN FEMALE RATS.................................... 33

2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN THE IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER
AND WEISER (1968)...................................... 36

2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO
HEMOLYSIS TEST IN RATS; EXCERPT FROM BLISS AND
GYORGY (1969).......................................... 36

2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL............ 41

3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN SHEEP AFTER A SINGLE INTRAMUSCULAR ADMINISTRATION OF
VITAMIN E (40 mg/kg)................................... 56

3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (pg/g wet) OF SHEEP FOLLOWING A SINGLE INTRAMUSCULAR ADMINISTRATION
(40 mg/kg BODY WEIGHT) OF VARIOUS VITAMIN E
PREPARATIONS ........................................... 58

4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS IN TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E... 68

4.2. TISSUE a-TOCOPHEROL CONCENTRATIONS (pg/g FRESH TISSUE) IN THE FOUR DIETS...................................... 68

4.3. RETENTION TIME OF a-TOCOPHEROL FROM GAS-CHROMATOGRAPHY/ MASS SPECTROMETRY...................................... 71

4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS IN BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF
VITAMIN E .............................................. 74

4.5. AREA (AUC) UNDE THE PLASMA a-TOCOPHEROL CONCENTRATION CURVE (pg/ml b )...................................... 74










TABLE


5.1. COMPOSITION OF THE GRAIN PORTION OF THE DIET FED TO
COWS FOR 28 d.......................................... 81

5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (vg/ml) IN CATTLE
FED VARIOUS PREPARATIONS OF VITAMIN E.................. 85

5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (pg/g FRESH TISSUE)
IN CATTLE FED VARIOUS VITAMIN E PREPARATIONS........... 88

6.1. DL-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES (LEAST
SQUARE MEANS SE) IN SHEEP AFTER A SINGLE ORAL
ADMINISTRATION OF VITAMIN E (100 mg/kg BODY WEIGHT).... 100

6.2. TRIAL 2: D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES
(LEAST SQUARE MEANS SE) IN CATTLE AFTER A SINGLE
ORAL ADMINISTRATION OF VITAMIN E (50 mg/kg BODY
WEIGHT)................................................ 101

7.1. DIET OF SHEEP .......................................... 107

7.2. MEAN LEVELS OF a-TOCOPHEROL IN TISSUES (vg/g FRESH)
AND PLASMA (pg/ml) AT TIME OF SLAUGHTER................ 110

7.3. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG TISSUE
VALUES................................................. 112

7.4. MEANS OF THE RATIO OF TISSUE TO PLASMA LEVELS OF
a-TOCOPHEROL AT TIME OF SLAUGHTER...................... 114

7.5. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG RATIO OF
TISSUE TO PLASMA....................................... 115

7.6. PLASMA TOCOPHEROL (vg/ml) VALUES AND SUMMARIES FOR
THOSE ANIMALS SLAUGHTERED AT 28 DAYS................... 116


PAGE














LIST OF FIGURES


FIGURE PAGE 2.1 STRUCTURAL FORMULA OF a-TOCOPHEROL.................... 3

2.2 STRUCTURAL FORMULAS OF TOCOPHEROLS.................... 4

2.3 STRUCTURAL FORMULAS OF TOCOTRIENOLS................... 4

2.4 ASYMMETRIC CARBONS OF TOCOPHEROLS..................... 5

2.5 BIOSYNTHESIS OF THE RING STRUCTURE OF TOCOPHEROLS AND TOCOTRIENOLS.......................................... 9

2.6 STRUCTURE OF TOCOPHERYL QUINONE....................... 11

2.7 STRUCTURE OF a-TOCORED................................ 12

2.8 STRUCTURE OF a-TOCOPURPLE............................. 12

2.9 SCHEMATIC REPRESENTATION OF THE PEROXIDATION OF UNSATURATED FATTY ACIDS............................... 45

3.1 PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE I.M.
INJECTION OF D-a-TOCOPHEROL SUSPENDED IN STERILE
SESAME OIL ............................................ 55

4.1 a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 69

4.2 IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/ MASS SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD,
(B) a-TOCOPHEROL IN PANCREAS.......................... 72

4.3 MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A) AND a-TOCOPHEROL IN THE PANCREATIC TISSUE (B)
FOLLOWING HPLC COLLECTION............................. 73

4.4 a-TOCOPHEROL CONCENTRATIONS IN THE BLOOD PLASMA OF SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 75

5.1 a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS....... 87


viii










FIGURE


PAGE


5.2 MASS SPECTRAL SCANNING OF THE a-TOCOPHEROL STANDARD
(A) AND IN BLOOD PLASMA (B) FOLLOWING HPLC COLLECTION
AND TLC ............................................... 90

6.1 PLASMA TOCOPHEROL CONCENTRATION (pG/ML) IN SHEEP
FOLLOWING ADMINISTRATION OF A SINGLE MEGADOSE (100 MG/
KG BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL....................................... 96

6.2 PLASMA TOCOPHEROL CONCENTRATION (G/ML) IN CATTLE
FOLLOWING ADMINISTRATION OF A SINGLE DOSE (50 MG/KG
BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL....................................... 97














Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS IN RUMINANTS

By

NICHOLAS HIDIROGLOU

August 1989

Chairman: Dr. L. R. McDowell
Major Department: Animal Science

This research was designed to investigate the bioavailability of various tocopherol sources for ruminants. In experiment I, d-atocopherol and dl-a-tocopherol suspended in sesame oil were administered intramuscularly in sheep at a level of 40 mg/kg body weight (b.w.). Sheep administered d-a-tocopherol and killed after 360 hr resulted in a higher (P<.O1) bioavailability than for sheep injected with d-atocopherol or dl-a-tocopherol and sacrificed 240 hr after dosing. A more sustained increment in plasma a-tocopherol was observed with d-atocopherol than dl-a-tocopherol.

In experiment II, sheep were provided with 400 IU/d of either d-atocopherol, dl-a-tocopherol or their corresponding acetate forms for 28 d. Higher concentrations of a-tocopherol were observed in tissues of sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed d-a-tocopherol, the area under the plasma a-tocopherol time curve (AUC), was larger as compared to other forms of vitamin E supplementation.











Experiment Ill investigated the concentration of a-tocopherol in

plasma and tissues of beef cows following a daily oral administration of 1000 IU of four tocopherol sources for 28 d. The d-a-tocopherol and its acetate ester increased plasma tocopherol concentrations faster (P<.05) than the racemic products, with the greatest response occurring with d-atocopherol. Tissue analyses confirmed that in adrenal gland, kidney, liver and lung, a-tocopherol concentrations were higher (P<.05) following d-a-than dl-a-tocopherol supplementation.

In experiment IV, dl-a-tocopherol and its ester were administered to sheep or cattle (100 or 50 mg/kg b.w., respectively) in a single oral dose. Blood plasma a-tocopherol tolerance curve area was higher (P<.05) in the dl-a-tocopherol group than in its ester form, as well as quicker (P<.05) time (h) for maximum plasma c-tocopherol concentration. A greater (P<.05) plasma tolerance curve area was observed in the cattle following administration of dl-a-tocopherol than its acetylated form.

In experiment V plasma and tissue vitamin E concentrations were

determined following a 5 g dosage of dl-a-tocopherol intraperitoneally in sheep, which were slaughtered on d 3, 6, 10, 15 and 28 post dosing. There was a significant time effect for all tissues, while in most, the peak for a-tocopherol concentrations was observed at 3 d post dosing. Rate of uptake of vitamin E varied for different tissues, with liver, spleen and lung showing a pronounced uptake and muscle showing least.

Comparing the established biological potencies (IU/mg) of the

various tocopherol sources to the estimate values for ruminants are as follows, respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37).















CHAPTER 1
INTRODUCTION


Existence of an anti-sterility vitamin was brought to light in the early 1920s when evidence was obtained that female rats reared on a diet containing all the vitamins known at that time failed to reproduce, although they were apparently normal in other respects (Evans and Bishop, 1922; Mattill and Coulin, 1920; Sure, 1924). Although the rats would mate and conceive, pregnancy invariably was terminated by fetal death followed by resorption. The missing factor was characterized as a vitamin by Evans and Bishop (1922) and designated vitamin E. Evans et al. (1927), who found that the unsaponifiable fraction of wheat germ oil was a convenient raw material for chemical investigation, and isolated (1936) from wheatgerm oil two alcohols, a- and 6-tocopherol, both of which showed vitamin E activity.

The name tocopherol originated from the Greek tocos (childbirth), phero (to bear) and ol (alcohol). Two years later, its structure was elucidated (Fernholz, 1938), and shortly thereafter it was synthesized (Karrer et al., 1938). During the period 1930-1950, multiple varied deficiency disorders of animals were reported to be cured by vitamin E. Work by Schwarz and Folz (1957) led to the identification of selenium as the factor other than vitamin E that could also prevent the degeneration of liver in rats. Dose of selenium required for complete protection against liver degeneration was about one










five-hundredth that of vitamin E. Biological potencies of various sources of vitamin E have been established primarily through the rat fetal resorption assay (Mason and Harris, 1947). From this assay the following biological potencies were established: 1 mg of dl-atocopheryl acetate = 1.0 IU; 1 mg dl-a-tocopherol = 1.1 IU; 1 mg d-atocopheryl acetate = 1.36 IU; 1 mg d-a-tocopherol = 1.49 IU. Recently, considerable effort has been made to determine the biological activity of various vitamin E compounds in species other than rats, and specifically humans (Horwitt et al., 1984; Baker et al., 1986).

In ruminants, although much is known about clinical signs of

vitamin E deficiency (Rice and McMurray, 1982), very little attention has been given to the different potencies of various vitamin E compounds. This dissertation is particularly concerned with the biological activity of various vitamin E compounds in ruminants as well as the effect of mode of administration and its type of vehicle.











CHAPTER 2
REVIEW OF LITERATURE Chemistry of Vitamin E


Structure


The term "vitamin E" today applies to a group of related chemical compounds, the tocopherols and tocotrienols. The structural formula of a-tocopherol is indicated in Figure 2.1.









CH3
HO, CH CH CHCH 3 3
H3C O CH3
CH3


FIGURE 2.1.


STRUCTURAL FORMULA OF a-TOCOPHEROL


The following tocopherols (Fig. 2.2) and tocotrienols (Fig. 2.3) are known:











Chemical Name


RI R2 R3


a-Tocopherol 6-Tocopherol Y-Tocopherol 6-Tocopherol


CH3 CH 3 H3
H


CH3
H
CH3
H


CH3
CH3 CH3 CH3


5,7,8-Trimethyltocol 5,8-Dimethyltocol 7,8-Dimethyltocol 8-Methyltocol


(a)TococheroIs
R1
HO CHCH C3H

R CH
3
FIGURE 2.2. STRUCTURAL FORMULAS OF TOCOPHEROLS


Tocotrienols


Chemical Name


R1 R2 R3


a-Tocotrienol 6-Tocotrienol y-Tocotrienol 6-Tocotrienol


CH 3 CH3
H3
H


CH3
H
CH3
H


CH3 CH3 CH3 CH3


5,7,8-Trimethyltocotrienol 5,8-Dimethyltocotrienol 7,8-Dimethyltocotrienol 8-methyltocotrienol


STRUCTURAL FORMULAS OF TOCOTRIENOLS


Tocopherols


FIGURE 2.3.






5


Figures 2.2 and 2.3 show that vitamin E exists as eight plantderived compounds that have a common 6-chromanol ring structure but that differ in the side chain and number of methyl groups. The four tocols have a phytol side chain while the four trienols having trans double bonds at 3', 7' and 11' of the phytol side chain. The methyl substituents occur in the ring at positions 5,7,8 (a-), 5,8 (B-), 7,8

(y-), and 8 (6-) in both the tocol and trienol series.

The tocols contain three asymmetric carbons specifically at the 2 position (Fig. 2.4) in the ring and in the 4' and 8' position of the side chain, thus giving a total of eight possible optical isomers. The epimeric configuration at the 2 position is apparently dominant in determining biological activity.













CHS



HzC O0= F
CHz CHz CH3 H CHz CHz


FIGURE 2.4. ASYMMETRIC CARBONS OF TOCOPHEROLS










The tocotrienols possess only one center of asymmetry at C2, in addition to sites of geometrical isomerism at C3' and C Thus, a number of stereo-isomers of the tocopherols and tocotrienols can exist. Natural a-tocopherol was shown conclusively to have the 2R, 4'R, 8'R configuration (Table 2.1). Thus, RRR-a-tocopherol can be used to denote the natural tocopherol isomer. The epimer of natural a-tocopherol, i.e., (2S, 4'R, 8'R)-a-tocopherol can be named 2-epi-atocopherol. A mixture of RRR and SRR-a-tocopherol can be obtained synthetically and is named 2-ambo-a-tocopherol (ambo = Latin for both). The reduction product of natural a-tocopherol is a mixture of four diastereo-isomeric a-tocopherols and can be called 4' ambo, 8' ambo-a-tocopherol. Synthetic a-tocopherol from synthetic phytol or isophytol is a mixture of four racemates in equal proportions and should be named all-rac-a-tocopherol.

Bieri and Prival (1967) reported that a group of synthetic

tocopherol derivatives exhibited vitamin E activity. Among those reported were a-, 6-, and y-tocopheramines and N-methyl-B-, and Nmethyl-y-tocopheramines. The tocopheramine derivatives differ from the tocopherols in that an amino or an N-methyl amino group is substituted for the hydroxy group on the C-6 of the ring. Synthesis


Chemical

Karrer et al. (1938) were the first to synthesize a-tocopherol by condensation of trimethylhydroquinone with phytyl bromide. Phytyl bromide was soon replaced by the natural phytol (Karrer and Isler,










1938; Fieser et al., 1940) or isophytol (Karrer and Isler, 1941). All the tocopherols were subsequently synthesized and their structures elucidated.



TABLE 2.1. NOMENCLATURE FOR TOCOPHEROLS


Description Configuration Recommended Other designation


Natural 2R,4'R,8'R RRR a- [d]-a-tocopherol.
Steriochemically uniform
product (only RRR-a)

Semisynthetic 2R,4'R,8'R 1 ambo a dl-a-tocopherol.
Two stereoisomers in the
product (about 50% RRR-a
historical standard)

Synthetic Mixture of all All rac-a [dl]-a-tocopherol.
Eight stereoisomers in
the product (about 12.5%
RRR-a)

Synthetic 2S,4'R,8'R 2-epi-a [1]-a-tocopherol.
Steriochemically uniform
product (only SRR-a)




Two principal sources of vitamin E are in commercial use: d-atocopherol and esters (of acetate and succinate) of these compounds. The acetate esters are prepared chemically by reaction of the alcohol forms with acetic anhydride; they do not exist in nature. D-atocopherol is largely obtained from natural sources by molecular distillation. However, some of the d-a-tocopherol is prepared by further methylation of B-, y-, and 6-tocopherols, or by hydrogenation of a-tocotrienol.










Synthetic dl-a-tocopherol and its esters are prepared from

isophytol. This synthesis yields a mixture of eight isomers. Although semi-synthetic dl-a-tocopherol is racemic only at carbon 2 as prepared from natural phytol, samples of this compound may be available for research though it is not a commercial source of vitamin E. Biosynthesis

Although conclusive proof of the biosynthetic route is still

lacking, it is probable that the ring structure is formed from tyrosine by the pathway presented in Figure 2.5 (Whistance and Threlfall, 1967, 1968). Methylation of the ring structure by methyl transfer from methionine and formation of an initial isoprenoid side-chain from mevalonic acid completes the formation of a tocotrienol (Griffiths et al., 1968).


Properties of Vitamin E


Physical properties

Both d-a-tocopherol (RRR) and dl-a-tocopherol (all-rac) are

practically insoluble in water but are almost completely soluble in oils, fats, acetone, alcohol, chloroform, ether, benzene, and other fat solvents. All tocopherols are stable to heat and alkali in the absence of oxygen and are unaffected by acids up to 1000C; they are slowly oxidized by atmospheric oxygen, a process which is increased rapidly by heat and catalyzed by ferric or silver salts. On exposure to light, the tocopherols gradually darken. They are not precipitated by digitonin. The commercial forms of vitamin E are d-a-tocopheryl acetate and dl-a-tocopheryl acetate.





























Phe




OH
CH2COOH


O-glucose homogentisic acid glucoside O


CH2CHNH COOH


Tyr
OH


CH2COCOOH


p-hydroxyphentyl
OH pyruvate

H


homoarbutin
-gLcose


H
H


H3 deltatocotrenol Tocopherois









FIGURE 2.5. BIOSYNTHESIS OF THE RING STRUCTURE OF
TOCOPHEROLS AND TOCOTRIENOLS









The melting point of RRR-a-tocopherol is 2.5 to 3.50C; thus at room temperature, the compound is a viscous yellow oil which is soluble in aprotic solvents. Optical rotations of these tocopherols are very small and depend on the nature of the solvent. Table 2.2 gives the specific rotations of the spectra of natural tocopherols and tocotrienols in ethanol which show maxima in the range of 292-8 nm; infrared spectra show OH (2.8 to 3.0 pm) and CH (3.4 to 3.5 pm) stretching and a characteristic band at 8.6 pim.


TABLE 2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS 250
Compound Solvent 546.1


a-Tocopherol Ethanol +0.320 Benzene -3.00 6-Tocopherol Ethanol +2.90 Benzene +0.90 Y-Tocopherol Ethanol +3.20 Benzene -2.40 6-Tocopherol Ethanol +3.40 Benzene +1.10




Other properties include a molecular weight of 430.69; boiling point at 0.1 atm (used in molecular distillation) of 200 to 2200C; density 0.95 at 250C in reference to water at 40C, and a refractive index in sodium light spectrum at 200C of 1.5045.

8-Tocopherol is a viscous, pale yellow oil; absorption maxima of

17
297 nm; E = 86.4.
1 cm











1%
The absorption maximum for Y-tocopherol is 298 nm; E cm = 92.8; boiling point at 0.1 atm 210-2100C. Tocopheryl esters are more stable to oxygen but cannot function as anti-oxidants in this form. Metabolic Degradation Products of Tocopherol


The chemistry of the oxidation of tocopherols is highly complex and has been reviewed by Kasparek (1980). Complete chemical degradation of the tocopherol molecule occurs upon treatment with chromic acid and potassium permanganate, while milder oxidation by nitric acid, silver nitrate, ferric chloride, auric chloride, ceric sulphate and nitrogen dioxide leads to formation of a-tocopheryl quinone (Fig. 2.6). With nitric acid and ferric chloride, a-tocored (Fig. 2.7) and a-tocopurple (Fig. 2.8) are also formed.


FIGURE 2.6. STRUCTURE OF TOCOPHEROL QUINONE

























FIGURE 2.7. STRUCTURE OF a-TOCORED


FIGURE 2.8. Absorption


STRUCTURE OF o-TOCOPURPLE and Storage of Vitamin E


Mechanism of absorption is similar to that of other fat soluble vitamins (Weber, 1983). Its absorption is closely associated with that of fat, and is accelerated by the presence of bile.

Excess tocopherol is eliminated in the feces. Simon et al.

(1956) observed that rabbits receiving a single dose of 10 to 15 mg 5methyl-C14-d-a-tocopherol succinate eliminated 65% of the dose via the feces in 3 days and 80% in 6 days; 90% of the radioactivity was









identified as free a-tocopherol by isotope dilution. It was concluded that the vitamin was re-secreted into the intestinal tract from the blood or via the bile. In confirmation by Krishnamurthy and Bieri (1963), who administered the vitamin orally, a second increase in fecal tocopherol was noticed which they did not attribute to coprophagy since the rats had tail cups. Mellors and Mcbarnes (1966) demonstrated that no significant amount of either tocopherol or its metabolites was introduced into the lumen of the rat guts via the bile or through secretion from mucosal cells. By using a-tocopherol labelled with radioactive C14, Shantz (quoted by Harris and Ludwig, 1949) remarked that 80% of the vitamin E given in oil solution was excreted in the feces of rats.

Dju et al. (1950) observed that chickens receiving 1 g of atocopherol/day for a prolonged period eliminated 75% of the ingested quantity unchanged via the feces by 24 h. In contrast, chickens receiving a diet supplemented with 17.5 and 35 mg of a-tocopherol/kg only eliminated about 23% in the feces (Pudelkiewicz and Matterson, 1960).

Information on the absorption and excretion of tocopherols by farm animals is extremely sparse. In unpublished work with young calves, Blaxter and Brown (1952) indicated that about 25% of atocopherol added as the acetate to a diet of dried skimmed milk was excreted in the feces when the daily intake of the ester was from 25 to 100 mg. From such scanty data it is clearly impossible to draw the conclusion that species differ in their ability to absorb tocopherols, especially since great variation has been reported for a single










species. Also, with labile substances like the tocopherols, a simple estimation of absorption based upon amounts present in the food and feces may be inaccurate.

These and other metabolic studies have shown that tocopherols are incompletely absorbed. The amount absorbed seems to depend on the requirement of the organism (Klatskin and Molander, 1952). Absorption and elimination also seem to depend on the amount in the diet.

There is a linear relation between the logarithms of ingested and liver tocopherol in rats (Bolliger and Bolliger-Quaife, 1956). Griffiths (1960) noticed a linear relation between the logarithms of serum tocopherol and dietary concentration of vitamin E in chickens. The same author determined the ratio of pure a-, a-, Y-, and 6tocopherol in the livers of growing chicks as 100:41:19:0, but his findings are contradicted by Gray (1959), who found considerably lower tocopherol levels in rat plasma at high levels of tocopherol intake than Griffiths (1960) found in chicken serum.

Work of Dju et al. (1950) with hens included the observation that a-tocopherol was absorbed to a much greater extent than the y- and 6compounds. The serum tocopherol values of two hens which received weekly supplements of 1.6 g and 2.0 g a-tocopherol were 20.0 and 20.1 mg per 100 ml, but two hens that received weekly supplements of 0.8 and 1.0 g 6-tocopherol had serum tocopherol values of only 1.35 and

2.1 mg per 100 ml. Gamma-tocopherol appears to be only one-third to one-half as well absorbed as a-tocopherol in formula fed infants and supplemental vitamin E (1200 IU/day of all-rac-a-tocopherol: 800 mg atocopherol equivalents) in adults significantly reduced plasma Y-










tocopherol levels, which suggested that intestinal uptake or plasma transport is more efficient for a- than y-tocopherol (Jonsson et al., 1981). Tocopherol esters are hydrolyzed prior to absorption, and both bile and pancreatic juice are necessary for absorption to proceed (Gallo-Torres, 1970). These facts support the idea that free tocopherol is absorbed by diffusion from a mixed micelle of fatty acids, monoacyl glycerols, bile salts and acids, cholesterol and other fat-soluble vitamins. Maximal absorption occurs at the junction of the upper and middle thirds of the small intestine (Hollander et al., 1975). After crossing the brush border into intestinal mucosal cells, tocopherol is not re-esterified but is incorporated as the alcohol into chylomicrons in mammals and enters the plasma via the lymphatic system (Behrens et al., 1982).

Desai et al. (1965) showed, in confirmation of studies by Weber et al. (1962) with rats, that 1-a-tocopherol was absorbed as well as or better than the d-form of the vitamin. It appeared therefore that the differences in biopotency must be due to differences in retention whereby the d-epimer is retained much better than the 1-epimer in the blood and perhaps in other tissues of the body. The results indicated the existence of an active carrier of d-a-tocopherol in the blood and tissues which has a greater affinity for the d-epimer than for 1-atocopherol. Recently it has been reported that specific binding proteins exist for a-tocopherol in the cytosol and nuclei of rat liver tissue (Catignani et al., 1977; Guarnieri et al., 1980 Prasad et al., 1980) as well as in human erythrocytes (Kitabachi and Wimalasena, 1983) which are fairly specific for the natural stereoisomer. The









exact nature of the binding of a-tocopherol in the cell is an elusive problem which needs investigation. Experiments by Desai and Scott (1965) and Scott (1965), comparing the oral administration of d- and l-a-tocopheryl acetates in the presence of graded levels of dietary selenium, indicated that selenium is involved in some unknown way in the retention of d-a-tocopherol in plasma. It remains to be determined if the differences in plasma levels of d- and 1-epimers of a-tocopherol are the result of differences (1) in rate of excretion,

(2) in rate of destruction, (3) in the affinity of the epimers for specific carriers, or (4) in chemical activity influenced by structural configurations.

Gallo-Torres (1970) reported the obligatory role of bile for the intestinal absorption of vitamin E into the lymph of rats. Only negligible amounts of radioactivity could be detected in the thoracic duct lymph when both bile and pancreatic juice were absent from the duodenum.

The lipid-bile micelle structure is required to transport the fat-soluble vitamin E across the "unstirred water layer" which represents the aqueous phase in the intestinal lumen immediately adjacent to the brush border of the microvilli. Vitamin E is absorbed, together with free fatty acids, monoglycerides, and other fat-soluble vitamins, by penetrating the epithelial cell through the apical plasma membrane of the absorptive cells in the brush border. Studies on absorption and transport of a-tocopherol by Davies et al. (1971) showed that, for normal utilization of absorbed a-tocopherol, normal lipoprotein transport mechanisms are involved. Transfer of










vitamin E from the absorptive cell thus requires several stages. In mammals, it must first pass though the lateral or basal plasma membrane of the cell, then through the basal lamina before entering the fluid of the lamina propria. From this location the vitamin enters the capillaries of the lymph and is transported in the chylomicrons (Dobbins, 1975). Apparently only small amounts of tocopherol are transported from the intestine via the portal vein in mammals, whereas all of the tocopherol absorption in birds occurs via the portal vein directly to the liver (Machlin, 1984).

The absorption of orally fed vitamin E follows the pattern of

lipids in general and of fat soluble vitamins in particular (Desai et al., 1965; Wiss et al., 1962). The specific site of absorption is not well established. The small intestine is thought to be the major site of absorption for tocopherol even though some absorption takes place from the stomach of nonruminants and the rumen of ruminants (Blaxter and Brown, 1952; Roles, 1967). The presence of vitamin E in both the blood and lymph of animals suggests that absorbed vitamin E can be transferred by either the blood or lymphatic route (Roles, 1967).

Wiss et al. (1962) were also able to establish a mathematical relationship between the logarithms of tocopherol intake and plasma and liver concentration in chickens fed high doses of d-a-tocopheryl acetate (2,000 to 20,000 IU/kg of feed). Using [C14]-dl-a-tocopheryl acetate, they demonstrated that maximal liver concentration was reached only after several hours, and persisted longer than the synthetic antioxidant ethoxyquin (6-ethoxy-l1,2-dihydro-2,2,4trimethylquinoline), which attained a maximum within 30 min before









declining rapidly. Most of the tocopherol was bound to the structural components of the liver cells, primarily mitochondria and microsomes.

Vitamin E is transported in the blood via lipoproteins. A rapid exchange among lipoprotein classes occurs with vitamin E after it enters the circulation via the chylomicrons. Since plasma tocopherol level is correlated with the total plasma lipid content, low density lipoprotein (LDL), the most common lipoprotein in human plasma, carries most of the plasma vitamin E. There is also a rapid exchange between tocopherol in the erythrocyte membrane and lipoproteins such that approximately 20% of the plasma tocopherol concentration is carried by red blood cells. Delivery to other tissue cells appears to be in association with the receptor-mediated uptake of LDL (Traber and Kayden, 1984).

Type and composition of diet influence absorption of vitamin E from the gut. Pudelkiewicz and Matterson (1960) reported that only about one-third of the d-a-tocopherol in alfalfa is available to chicks. The poor utilization is attributed to a fat-soluble compound in alfalfa that acts antagonistically to a-tocopherol decreasing its availability. An antagonistic relationship also exists between absorption of vitamin E and unsaturated fatty acids (Bunyan et al., 1968).

Generally, in animals, the uptake of vitamin E from the small intestine is lowered when tocopherol is fed in an oily form. The uptake is facilitated by bile salts (Simon et al., 1956).

When tocopheryl acetate ester was fed, most fecal tocopherol

appeared as free tocopherol; however, a small portion appeared in the










esterified form (Krishnamurthy and Bieri, 1963) indicating that hydrolysis of the ester took place in the digestive tract. Rosenkrantz et al. (1951) observed that approximately 8% of atocopherol given to human subjects was recovered as tocopherol quinone, suggesting the oxidation of tocopherol in the human digestive system.

No tocopherol or tocopheryl quinone was excreted in rat urine although large doses of vitamin E were administered (Klatskin and Molander, 1952). McArthur and Watson (1939) failed to detect any tocopherol in cow urine. Simon et al. (1956) reported that about 18% of tocopherol intake appeared in the urine of the rabbit and man after intravenous administration of isotopic vitamin E. Urinary radio activity was not attributed to a-tocopherol or tocopheryl quinone but to two polar metabolites identified as an acid and its lactone which were subsequently named tocopheronic acid and tocopheronolactone (Green et al., 1967). Work by Chow et al. (1967) confirmed the findings of Simon and associates.

Krishnamurthy and Bieri (1963) reported that only 0.5% of total activity was in the urine when the rat was sacrificed 24 h after oral administration of C14 a-tocopherol. In a long-term experiment, the same authors found that the activity in the urine was very low. Furthermore, no significant amount of vitamin E was lost in the form of CO2.

Almost all studies on the pharmacological properties of vitamin E used doses between 200 and 800 mg/day. John Bieri, a leading American expert on vitamin E, had the following to say on dosage: "Nothing









indicates that plasma levels that have been raised to 1.5 to 2 times the normal levels are less effective than even higher levels. These plasma levels, in subjects with normal absorption, can be achieved within 2 to 3 weeks of doses of 3 x 100 or 200 IU/day" (Bieri et al., 1983). This statement is aimed at discouraging extremely high doses of vitamin E.

The conditions of physiological absorption discourage very high individual doses of vitamin E. Absorbing dietary vitamin E usually presents few problems, since the quantities involved are small and the required mechanisms of fat digestion, which play an important role also in the absorption of vitamin E, are triggered automatically. Large individual doses of vitamin E, however, are not fully absorbed, two-thirds of them usually being excreted unchanged. Rate of absorption varies between 20 and 60%, and is dose dependent. Absorption of tocopherols depends on various factors, e.g., 1) pancreatic enzymes, 2) bile acids, 3) pH level of the intestinal contents, 4) intestinal motility, 5) other food components, in particular the fatty acid composition (Simon-Schmoss et al., 1984).


Metabolism of Vitamin E


Although vitamin E is widely distributed among the tissues of

animals and much is known about the clinical signs and symptoms of its deficiency, very little is known about the metabolism of vitamin E. The relative ease with which the tocopherols and their metabolites can be oxidized in vitro has complicated efforts to elucidate their mechanism of action (Martius and Furer, 1963).









Investigations on the metabolites of a-tocopherol in animal

tissues were conducted by several workers with the anticipation that the nature of the metabolites would provide insight into the mechanism of action of vitamin E. Simon et al. (1956) isolated and characterized a metabolite of a-tocopherol from both rabbit and human urine. This oxidation product was given the common name tocopherol-5methyl-C14 succinate. The same authors found that urine contained mainly metabolized tocopherol, while intact tocopherol accounted for the majority of the radioactivity in the feces. Mervyn and Morton (1959) reported the presence of a-tocopheryl quinone in a nephritic human kidney deficient in vitamin E. Alaupovic et al. (1961) found no evidence of a-tocopheryl quinone, a-tocopheryl hydroquinone, atocopheroxide, tocopheronic acid or tocopheronolactone in pig and rat livers. Csallany et al. (1962) identified two new metabolites of atocopherol isolated from the liver of the rat after injection of [5methyl-14C] d-a-tocopherol. About 25% of the recovered activity was unchanged a-tocopherol, about 19% was a-tocopheryl quinone, and about 50% was dl-a-tocopherone, a dimer of oxidized -tocopherol. Csallany and Draper (1963, 1964) separated and identified the cis-and transisomers of dialpha-tocopherone. Draper et al. (1967) also identified a trimer of a-tocopherol in the mammalian liver. Krishnamurthy and Bieri (1963), and Plack and Bieri (1964) indicated that oral and intraperitoneal administration of 14C a-tocopherol to rats and chicks gave rise mostly to unchanged tocopherol in the tissues of the tested animals. Mellors and McBarnes (1966) confirmed the above findings. Following intravenous injection of 3H-a-tocopherol, Shiratori (1974)










observed large amounts of radioactivity in dermal tissue suggesting that skin may play a role for tocopherol secretion or excretion.


Vitamin E Deficiency


The absence of vitamin E in the diets of animals poses health problems and affects various functions. Effects of Deficiency on the Reproductive System


In 1920, Mattill and Conklin showed that a deficiency in vitamin E led to reproductive disorders and lesions of the genital apparatus in rats. The data were confirmed in 1922 by Evans and Bishop. As a result of some premature generalization of these observations, vitamin E came to be considered as an anti-sterility factor and its therapeutic use was widely advocated. Now, however, it is apparent that the role of this factor varies widely among species. In males

Rodents. In rats, vitamin E deficiency results in testicle

degeneration (Mason, 1954). The gland is reduced to half its normal size and becomes brownish and flaccid. Degenerative lesions affect the spermiduct and lead to the complete cessation of sperm production. Neither Sertoli cells nor the interstitial gland appear to be affected. Some changes in related glands (seminal vesicles and prostate) have been reported.

It appears that guinea pigs (Curto, 1966), hamsters and rabbits are also susceptible, although less markedly so. In rabbits, in particular, testicle lesions seem minor when compared to the









widespread effects of vitamin E deficiency on muscles (Hove and Harris, 1947).

Other species. Monkeys and roosters are also susceptible to vitamin E deficiency. Stallions, bulls, rams, billy goats, and perhaps male mice, are not (Baxter and Brown, 1952).


In females

Rodents. In rats, vitamin E deficiency does not appear to affect the development and functioning of the genital system (estrus, ovulation, fecundation and nesting), but it has a major impact on prenatal development, mainly during the days following nesting. The uterus and the ovaries become covered with brownish yellow pigment while the basic signs are early embryo mortality followed by resorption as a result of placental lesions which may sometimes hemorrhage. When there is a chronic deficiency, the rat may produce only one or two live young, while the others die (Martin and Moore, 1939). The survivors present serious muscular dystrophy lesions (Bonetti and Stirpe, 1963). This cause should be kept in mind when a reduction in fecundity in this species occurs.

Other species. Where there is vitamin E deficiency, fetoplacental hemorrhages and some degree of embryo mortality have occasionally been noted in sows and rats. Surviving piglets develop poorly and present muscular dystrophy. It may be concluded that sows are susceptible to vitamin E deficiency, though to less extent than rats (Lannek, 1962).

The effects on reproduction in cows, ewes, goats and mares are

nil, although these species are very susceptible to muscular dystrophy










lesions, especially in the very young (Gullickson, 1949). However, a high placental susceptibility to vitamin E deficiency as noted in rats and sows, apparently in mice and perhaps in rabbits, also appears to exist in cows according to Trinder et al. (1969). These authors noted a high frequency of placenta retentions (19%) in non-brucellic cattle in areas where muscular dystrophy in lambs was widespread. Intramuscular injections of vitamin E (680 IU) and potassium selenate (15 mg) one month before parturition markedly reduced the frequency of retention. Administrations of potassium selenate only were less effective, and earlier injections (7 weeks prepartum) ineffective.


A Variety of Disorders Due to Vitamin E Deficiency in Domestic Animals


Vitamin E deficiency causes various disorders in most animal species. An exhaustive description would be tedious and time consuming. Observations will be limited to three frequently affected species: chickens, lambs and calves. Chickens

Encephalomalacia. Clinical signs of nutrition-related

encephalomalacia are exclusively nerve-related (Dam, 1944). The first manifestation is ataxia. Next, there may be clonic contractions and especially trembling. The chicken keeps its head tucked under, as if rolled into a ball; then paralysis sets in and it falls down in lateral decubitus. Death is the usual outcome of this clinical form which entails 20 to 80% losses, depending on the intensity of the deficiency and the susceptibility of the subjects. Lesions are significant because they make it possible to distinguish this disorder









from another neural disease of viral origin in chickens, infectious encephalomyelitis.

Characteristics of nutrition-related encephalomalacia (Adamstone, 1947), are (1) discrete hepatic lesions; the liver is often pale, degenerated, speckled with sanguinous suffusions; (2) intestinal hemorrhagic lesions, occurring mainly around the duodenum and involving the muscularis; and (3) cerebral lesions, more specific, macroscopically detectable in some cases, but always visible upon histological examination.

Upon opening the cranium, the cerebellum is edematized, covered with hemorrhagic patches, and of very poor consistency. Microscopic examination confirms the edema and hemorrhages, and in addition makes it possible to detect changes in Purkinje cells, which appear retracted, with granulous cytoplasm and pycnic nuclei.

In viral encephalomyelitis, the nerve lesions are different from the encephalomalacia with perivascular lymphocytic infiltration. In addition, lymphocytic infiltration can be detected in the pancreas, the succenturiate ventricle and the muscularis of the gizzard.

The distinction between these two diseases is an important one, since in encephalomalacia the responsible agent is the feed, whereas in encephalomyelitis it is the breeder, since contamination of the chickens occurs in ovo.

Exudative diathesis Exudative diathesis appears in chickens at the same age as does encephalomalcia and is due to a vitamin E deficiency associated with a deficiency in sulfur amino acids (Bunyan et al., 1962). It is characterized by the appearance of subcutaneous








edema which deforms the head and neck and gives the feet a greenish tint, hence its other name of "green foot disease" (Dam and Glavind, 1939).

Lesions are also edematous and affect the entire subcutaneous connective tissue. This tissue has a gelatinous aspect and presents hemorrhagic spots, in particular on major muscle masses and in the periarticular area. In addition, whitish muscular dystrophy patches are found fairly regularly in the gizzard area.

Muscular dystrophy. Muscular dystrophy appears around the age of one month. It takes the form of locomotor troubles and weight loss and entails considerable mortality. Lesions affect striated muscle fibers which present zenkerism. They are closely comparable to those of myopathy-dyspnea in calves and muscular dystrophy in lambs. Muscular dystrophy in chickens appears to be due to a deficiency of both vitamin E and selenium.


Lambs

This deficiency is also associated with clinical manifestations appearing in areas where the soil is selenium-poor (Muth et al., 1961). Winter lambs born of ewes which are more or less underfed pay the heaviest consequence, particularly where ewes have two or three young, whose requirements cannot easily be met (Oldfield et al., 1963).

Clinical signs begin inconspicuously with listlessness; sick

lambs dislike moving about, and have trouble following the rest of the flock. However, the appetite remains normal. Gradually, the gait changes; steps become smaller, posteriors are stiff and splayed, and









are affected with trembling and very painful to pressure. Standing becomes increasingly difficult and then impossible, with animals dying in a few days from heart failure or malnutrition. Death is preceded by blindness in most cases. Lesions affect the large muscle masses and the most active muscles. They are found mainly on psoas, crural and also on intercostals and myocardium.

Affected areas are discolored, whitish, and have the appearance of fish flesh. Extent of discoloration is varied; sometimes an entire muscle group may be affected, sometimes only a few fibers--this gives the muscle a striated, combed effect. A histological examination of the lesions shows that the striated muscle fibers harbor zenkerism. Calves

Vitamin E deficiency is also accompanied by selenium deficiency, which is characterized by white muscle disease (Blaxter and Sharman, 1953). It usually manifests itself in calves which are fed on milk only and is associated with rapid-growing animals with high nutritional requirements. Clinical signs appear between 15 days and 3 months, either in stables or at the time of pasturing. In stables, clinical signs appear gradually. At first, the gait stiffens; animals are stooped and move slowly when going to suck. Stiffness increases and standing becomes impossible; polypnea develops.

The polypnea is the only respiratory manifestation of this syndrome. It is accompanied by heart disorders which are characterized by a pendulum rhythm and a reduction in heart sounds upon auscultation, the characteristic signs of myocarditis. Finally, calves remain in lateral decubitus, with swollen, painful muscles










affected by trembling, and die of either pulmonary edema or heart failure.

In pasture calves, the syndrome appears when they are released in the spring. They begin by running and jumping, but suddenly one or two may slow down and stand still, with a stiff, awkward stance. Then they lie down and are unable to get up. In this case also, heart and respiratory disorders lead to swift death (King and Maplesdon, 1960).

Vitamin E-selenium lesions are identical with those of lambs, both macro- and microscopically. From the foregoing it will appear that vitamin E deficiency appears to lead to two major types of syndromes: (1) degenerative muscular lesions, common to a number of species and generally related to selenium deficiency, and (2) vascular lesions (edema and hemorrhages), well known in poultry, affecting mainly nervous and subcutaneous connective tissues.


Biological Activity of Vitamin E


Biological activities of the various tocopherols are different. Extensive and very complex studies on several experimental animal species have been conducted to determine the biological activity of vitamin E components which have been described in the literature for many years.


Methods of Assay


Determination of biologically active vitamin E is complicated by many interrelated factors. Chemical analyses must contend with the fact that eight different tocopherols occur in nature, and synthetic










forms are supplemented in many foods and feeds as dl-a-tocopherol or as d- and dl-a-tocopheryl acetate or succinates. Bioassays may be confounded by the levels of antioxidants, prooxidants, selenium, and other factors which may alter the biological activity of the vitamin.

The biological activity of tocopherols (and of substances with vitamin E-like activity) can only be assessed with a bioassay technique which determines the ability of the tocopherol to reverse clinical signs of vitamin E deficiency. The manifestations of a deficiency differ markedly in different species. The signs of deficiency most commonly used in bioassay work are rat sterility (fetal resorption in the female and testicular atrophy in the male), rat erythrocyte hemolysis, and muscular dystrophy in a number of different species. A comprehensive review of bioassay methods for tocopherols has been reported by Bliss and Gyorgy (1967) and Ames (1971). Other tests, such as the liver storage, and elevation of plasma tocopherol, have also been used. These latter methods are not direct measures of biological activity in vivo, but they do reflect the relative absorption of the test compounds as well as their turnover in the liver or red blood cells. In general, the erythrocyte hemolysis and tissue storage tests correlate well with the in vivo procedures and may be correlated with alterations in red blood cell half-life observed in vitamin E-deficient humans. Therefore, although caution should be used in interpreting results, these methods are convenient and may be useful.










Resorption sterility test

The fetal resorption test in female rats is the classical means of conducting the bioassay of tocopherols (Mason and Harris, 1947; Ames et al., 1963). It measures the all-or-none response to the test substance by successfully mated vitamin E-deficient female rats in producing viable offspring.

The resorption sterility test in rats is frequently used in addition to the hemolysis test. This test system was used to determine the biological activity of the eight enantiomers of the synthetic all-rac-a-tocopheryl acetate (Weiser and Vechi, 1982).

The resorption sterility test is based on the fact that female rats supplied with a vitamin E-deficient diet are unable to carry their offspring to term. The fetuses die before the end of the gestation period and are subject to intra-uterine resorption. Weaned rats are given a vitamin E-free diet for 3 to 4 months at which time their sterility is assessed by test matings with fertile males; insemination is checked by vaginal smear for spermatozoa. Different levels of standard RRR-a-tocopherol and the unknown substances are added to olive oil (or sometimes to portions of diet) which is administered to pregnant females through the 5th to 9th days of pregnancy. If dosing is delayed beyond the 10th to 12th day of pregnancy, vitamin E is ineffective. The animals are killed on the 19th day, i.e., 2 days before parturition, and examined for living fetuses and for sites of implantation of fetuses that have been resorbed. Animals having less than four implantation sites are discarded, and those having one or more living fetuses are regarded as









positive, while rats with no live young are negative. The vitamin E activity of the test substance is determined by calculating the percentage of rats in each test group that showed a positive response and by plotting the calculated probits against dose (or log dose) to derive the 50% fertility dose. The ratio of the 50% fertility dose of the standard to the unknown substance is the relative vitamin E biopotency of the test substance. It will be clear from the foregoing that the fetal resorption test is tedious and time consuming and that meaningful results can be achieved only by carrying out a large number of tests on any given test substance, but it must still be regarded as the final reference point in assessing vitamin E biopotency of an unknown substance.

This test model is species-specific in its validity. The test results cannot be directly applied to man, but because this model can be standardized it can be used for comparisons of structure and activity relationships. As in any other specific animal model, its validity limits must be taken into account. The results obtained for relative activity of the free, easily oxidizable tocopherols vary greatly in this model from one team of investigators to another (different laboratories) since the free tocopherols are easily inactivated by other components in the diet of the experimental animals. Knowledge of this interference dates back to the 1940s, and consequently this model is used mainly to test and compare acetylated tocopherols.

The results tabulated in Table 2.3 show that it is the

configuration in the phytol side chain which decisively affects the









activity in the biological system. The 2R, 4'R, 8'R configuration, which is identical to the naturally occurring form, has the highest activity (100% relative biological activity). The configuration in the C-2 position is particularly important. It is noted that in all the disastereomer pairs the 2S form has less activity than the 2R form. The greatest difference in activity is noted in a comparison between the 2S, 4'R, 8'R-a-tocopheryl acetate and the 2R, 4'R,8'R-atocopheryl acetate, which is identical to the naturally occurring form. The relative activity is only 21 to 31% for the SRR form. The SSS isomer exhibits the surprisingly high relative activity of 60% compared to the RRR isomer.

Observations on the biological activity of stereoisomer mixtures vary. Ames (1979) showed on the basis of 19 tests that the activity of all-rac-a-tocopheryl acetate does not exceed the calculable activity. By contrast, Weiser and Vechi (1982) found in two experiments that stereoisomer mixtures had a synergistic effect in the rat resorption sterility test.


Erythrocyte hemolysis test

This test is based on the protection of erythrocytes by vitamin E against hemolysis induced by dialuric acid or hydrogen peroxide. Rats weighing about 100 g are fed a diet free of vitamin E for a depletion period of 3 to 4 weeks. A sample of blood is then taken from each rat and tested for hemolysis induction by addition of dialuric acid. Only animals showing a degree of hemolysis of 96 to 99% are retained in the test. In a preliminary trial with five animals per dose, dosages of vitamin E are determined which will reduce the hemolysis to a range of









TABLE 2.3. RELATIVE ACTIVITY OF VARIOUS a-TOCOPHEROL STEREOISOMERS
AS DETERMINED BY RESORPTION STERILITY TEST IN FEMALE RATS.


Standard with 100% biological activity: RRR-a-tocopherol acetate atocopherol acetate isomer


2R,4'R,8'R 2R,4'R,8'S 2R,4'S,8'S 2S,4'S,8'S 2R,4'S,8'R 2S,4'R,8'R 2S,4'R,8'S 2S,4'S,8'R all-rac-a (8 enantiomers) 2-ambo-a (2 enantiomers)


Relative activity in relation to the standard (in percent)


100

90 73 60 57 31 37 21 32 67-77

60.2

73










approximately 20 to 80% for both the standard and the test preparations. For the bioassay, two or three doses are selected from within this range for the standard and for each sample to be assayed. Each dose is dissolved in 0.2 ml olive oil and administered orally by stomach tube. After 40 to 44 hr the percent erythrocyte hemolysis is again measured. The use of dialuric acid as a hemolytic agent for erythrocytes derived from vitamin E-deficient animals was introduced by Rose and Gyorgy (1952), and later improved by Friedman et al. (1958).

As an alternative to dialuric acid, hydrogen peroxide may be used as the hemolytic agent (Gyorgy et al., 1952). It should be noted that there is no spontaneous hemolysis in vivo in vitamin E-deficient animals. However, the rat hemolysis test is not a direct measure in vivo of the biopotency of a vitamin E-active substance, since the test is carried out on blood derived from rats given a vitamin E-deficient diet. Furthermore, the results obtained need to be interpreted with great caution since the period of giving the deficient diet needs only be quite short before a positive test can be obtained, and the animal would not necessarily show overt signs of vitamin E deficiency such as sterility. The test is thus regarded by many as a measurement of the vitamin E content of the blood rather than as an assessment of the vitamin E status of the animal. With this difficulty in mind, however, the test provides a means of assessing the probable vitamin E activity of an unknown substance and has been found to agree well with the results obtained by fetal resorption (Harris and Ludwig, 1949) and chick liver storage (Pudelkiewicz et al., 1960).










Of all the test systems, the erythrocyte hemolysis test (EHT) is of special importance. This test can be done not only as a pure in vitro variant but also as an in vivo-in vitro form. The EHT provides a means of indirect measurement of the human vitamin E status and correlates well with the tocopherol concentrations in blood plasma. Individuals with decreased tocopherol concentration are characterized by an increased hydrogen peroxide-induced sensitivity of the erythrocytes to hemolysis in the blood serum.

The most commonly used hemolytic agent is hydrogen peroxide. Washed erythrocytes are incubated in a hydrogen peroxide solution (about 2%) over a period of 3 hr. Hemolysis liberates hemoglobin during incubation; correction for this hemoglobin is made by determining the amount of hemoglobin after incubation with distilled water which corresponds to 100% hemolysis (without addition). The hemolysis result obtained with hydrogen peroxide is given as a percentage.

The relative activity of d-a-tocopherol and d-y-tocopherol in the in vitro EHT is tabulated in Table 2.4 (Brubacher and Weiser, 1968). Erythrocytes from rats which were given a vitamin E-deficient diet were incubated in a test tube together with the various tocopherols and then hemolyzed with dialuric acid. The tocopherol esterified with acetic acid is inactive in this test. D-a-tocopherol has again been shown to be biologically superior to d-a-tocopherol.

The hemolysis test was originally conceived as an in vitro test but was later modified. This modified test differs from the abovementioned in vitro test in that the vitamin E active substance is











TABLE 2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN THE
IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER AND WEISER (1968).



Compound Relative activity (%)


d-a tocopherol 100 d-y tocopherol 30


administered orally to the experimental animals 2 days before blood samples are taken, after which the hemolysis test is undertaken. This test model shows that even the forms which are esterified with acetic acid exhibit vitamin E activity after they have been liberated by enzymes in the intestine (Table 2.5). Here again it is the d-a configuration, both in the free form and in the form esterified with




TABLE 2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO HEMOLYSIS TEST IN
RATS; EXCERPT FROM BLISS AND GYORGY (1969).


Compound


Relative activity (%)


3 d-a-tocopherol

4 D-a-tocopheryl acetate

5 all-rac-a-tocopheryl acetate

6 D-y-tocopherol

7 D-Y-tocopheryl acetate


132.8 147.4 100.0 (standard) 21.7 18.9









acetic acid, that possesses the highest biological activity. Although the test method is not precise enough to differentiate d-a-tocopherol from its acetate, the two forms with the natural configuration are nevertheless significantly superior to the synthetic all-rac-atocopheryl acetate.


Plasma and erythrocyte concentration test

Vitamin E acts as an antioxidant in biological membranes (Dam,

1957). Therapy is designed to achieve a high tocopherol concentration in these membranes.

In test rats which were reared on a vitamin E-deficient diet,

free d-a-tocopherol was compared with synthetic all-rac-a-tocopheryl acetate to determine their effect in producing changes in vitamin E concentration in plasma and in erythrocytes after intravenous or oral administration (Ogihara et al., 1985). The tocopherol concentration in the red blood cells was investigated as an indicator of the physiologically available tocopherol in biological membranes.

Although intravenous administration of all-rac-a-tocopheryl acetate produced a higher tocopherol level in plasma than RRR-atocopherol 6 h after infusion, the concentration in the erythrocytes at the same time increased by 100% after infusion of RRR-a-tocopherol compared to the response when all-rac-a-tocopheryl acetate was infused.

Differences between the natural, free vitamin E and the synthetic acetate ester were even more pronounced in these test animals after oral administration. Plasma concentration 6 h after oral administration of d-a-tocopherol was 40% higher than after all-rac-a-









tocopheryl acetate intake. Uptake in the erythrocytes 6 h after oral administration of d-a-tocopherol was 280% higher than after administration of the synthetic material. Direct comparison at 24 and 48 h after oral administration indicated that uptakes of the free d-atocopherol in the erythrocytes was 300 and 255% greater than uptake of the ester. These test results provided impressive support for the superiority of the natural vitamin E structure as previously shown in clinical studies.


Liver storage tests

Liver storage tests are based on the observation that amount of tocopherol stored in the liver of rats and chicks has a linear relationship to the level of vitamin E consumed.

Pudelkiewicz et al. (1960) used day-old White Plymouth Rock male chicks fed a vitamin E-deficient diet for 13 days for assay of liver storage of vitamin E. Smallest and largest chicks are discarded, with those remaining distributed by weight into groups of eight chicks each. The tocopherol standards and the unknown materials to be assayed are then mixed with the basal diet and the chicks in all lots are pair-fed to the lot eating the least amount of feed over a period of 14 days. Tocopherol content of pooled livers from each group was then determined by chemical assay using the Emmerie-Engel reaction after careful extraction, molecular distillation, and chromatographic separation of the tocopherols.











Muscular degeneration tests

Muscular dystrophy has been used as a means of measuring vitamin E biopotency in rabbits (Hove and Harris, 1947; Fitch and Diehl, 1965) and chicks (Scott and Desai, 1964; Desai and Scott, 1965).

In chickens, muscular degeneration is evaluated directly after 3 to 4 weeks on deficient or supplemented diets. The breast muscle is examined and scored from 0 to 4, depending on the severity of the lesion. Indirect measure of muscular degeneration, such as creatinuria and levels of plasma aspartate aminotransferase and lactic dehydrogenase, have also been used in ducks and rabbits. The time of onset of creatinuria or the extent of muscular dystrophy, assessed on a scale, are both used as biological measures, and the effect on these parameters of standard RRR-a-tocopherol or a test material is assessed as the basis of the assay. A method based on the reversal of muscular degeneration in the rat, using plasma pyruvate kinase levels as an index, appears to be quite reliable, rapid, and sensitive (Machlin et al., 1982).


D-a-Tocopherol Equivalents


A basis for establishing the biological activity of vitamin E

preparations is necessary to ensure uniformity of dosage. In 1978 the International Union of Nutritional Sciences (IUNS), therefore, recommended that d-a-tocopherol be used as the standard compound and that the biological activity be specified in activity equivalents (Fromming, 1984).










D-a-tocopheryl acetate can be easily obtained in chemically and isomerically pure form. The relative biological activities of the tocopherols have been determined in experimental animals and are now internationally accepted. They are calculated as follows:

1 d-a-tocopherol equivalent (mg)

= d-a-tocopherol x 1

= d-a-tocopheryl acetate x 0.91

= all-rac-a-tocopheryl acetate x 0.67

= d-y-tocopherol x 0.1.

These equivalence values have been disputed (Ames, 1979; Leth and Sandergaard, 1983), however, in 1983 they were confirmed by a WHO/FAO Expert Committee (WHO, 1983). Evidently the experts did not consider it necessary to change the relative biological activities of tocopherols based on animal experiments. More recent human studies suggest that the findings on which the conversion factors are based, i.e., experiments with rats given a vitamin E-deficient diet, should be regarded as species-specific for the experimental animals.

A comparison of the tocopherol concentrations in human tissues and cells emphasizes the physiological significance of free RRR-atocopherol relative to other tocopherols and derivatives. The vitamin E requirement in man is satisfied by the preferential utilization of d-a-tocopherol in the diet. That d-a-tocopherol possesses the relatively greatest biological activity has been confirmed by a multitude of relevant in vitro and in vivo tests and particularly by clinical studies. Accordingly, RRR-a-tocopherol is the actual vitamin E in man.












The United States Pharmacopoeia (USP) (1980) in Table 2.6 shows the recommended weight/unit relationships for six tocopherols:



TABLE 2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL.



1 mg all-rac-a-tocopherol = 1.1 units

1 mg all-rac-a-tocopheryl acetate = 1 unit

1 mg all-rac-a-tocopheryl succinate = 0.89 unit

1 mg RRR-a-tocopherol = 1.49 units

1 mg RRR-a-tocopheryl acetate = 1.36 units

1 mg RRR-a-tocopheryl succinate = 1.21 units






Role of Vitamin E in the Respiratory Chain


It has long been known that the hepatic necrosis that accompanies vitamin E deficiency is preceded by a reduction of cell respiration shown on cross sections of prenecrotic liver (Chernick et al., 1955). Vitamin E supplementation immediately reestablishes respiration, both in vivo and in vitro (Schwarz, 1955). Selenium is also effective, but only in vivo, which seems to indicate that a different mechanism is at work (Schwarz, 1972).

Cell respiration takes place at the level of the mitochondria,

through the respiratory chain, which consists of a series of hydrogentransport systems which intervene in an order determined by their oxidoreduction potentials. Energy released by this hydrogen transport













system (which corresponds to oxidation of the substrate) is partially stored in the form of adenosine triphosphate (ATP). Determination of the P/O ratio (number of atoms of inorganic P which disappear when one atom of oxygen is reduced), which is generally equal to 3, provides a measure of cellular respiratory activity.

One of the links in the respiratory chain is ubiquinone (or coenzyme Q) which intervenes between the flavin-nucleotide and cytochrome system. The structure of ubiquinone is similar to that of a-tocoquinone, a metabolite of a-tocopherol which, along with the corresponding hydroquinone can form a hydrogen transport system comparable to that of ubiquinone.

In the anemias observed when vitamin E deficiencies occur in

primates, an improvement is obtained upon administration of ubiquinone as with vitamin E, thereby indicating the similarity of action for the two substances.

It is likely that vitamin E intervenes in the respiratory chain, and thereby contributes to the metabolic equilibrium of cells (Schwarz, 1972).


Intervention of Vitamin E in Hematopoiesis


Anemia, which accompanies vitamin E deficiency in primates, and the decoloration of the muscles in muscular dystrophy in many species (resulting from rarefaction of the myoglobin) have led to research into the possible role of this vitamin in heme synthesis.

Heme synthesis results from a series of transformations starting with glycine and succinyl-CoA, leading to the formation of 6-











aminolevulinic acid (ALA), followed by the condensation of two molecules of ALA to form porphobilinogen. These two transformations are catalyzed by two enzymes, ALA-synthetase and ALA-dehydratase, respectively.

Nair et al. (1970) have shown that these two enzymes are under

the control of vitamin E. The fact that actinomycin D (which inhibits transcription from DNA to RNA) impeded the ability of vitamin E to reestablish heme formation in the mitochondria of deficient tissues suggest that vitamin E might intervene at this level by acting as a specific repressor of the transcription of the enzymes of ALA (Nair, 1972).

Since heme is not only a constituent of hemoglobin, but also of myoglobin, cytochromes and various oxidoreduction enzymes, it is possible to better understand certain aspects of vitamin E deficiency, namely the decoloration of the muscles in muscular dystrophy due to rarefaction of the myoglobin. Similarly, the decoloration which various researchers have observed in the mitochondria of deficient tissues reflects a rarefaction of the cytochromes, which contributes to a lowering of cell respiration by disruption of the respiratory chain at the level of the cytochromes.


Biological Role of Vitamin E

Antioxidant Activity

It has been known for some time that vitamin E is an excellent fat-soluble antioxidant (Dam, 1957; Tappel, 1962) although the physiological significance of this trait is only now becoming clear.












Because of its antioxidant effect, vitamin E is able to entrap free radicals before they can attack life-supporting cell structures. Free radicals are, by definition, chemical compounds which contain an odd number of electrons. Free radicals can be formed in biological systems through homolytic cleavage of covalently bonded organic compounds resulting in bond splitting, whereby each fragment retains one electron of the original bonding pair.

H

R C = C.

Bond splitting as such always gives rise to a pair of radicals. Radicals can also be formed from a capture of an electron by a molecule as illustrated below:

02 + e- 02*The formation of superoxide anion radical results in a net negative charge because of the extra electron in its posession. Capturement of electrons by molecular 02 is known to occur by its interaction with flavin oxidoreductase systems (Fridovich, 1976). For a better understanding of these phenomena, the effect of free radicals upon the breakdown of an essential fatty acid as described by Leibovitz and Siegel (1980) is presented in Fig. 2.9.

In a fatty acid, at a carbon atom adjoining a double bond, an agressive substance (aS) removes hydrogen (I), resulting in the formation of a fatty acid radical (II). This radical reacts with oxygen to form a peroxy fatty acid radical (III). This new radical in its turn removes hydrogen from another essential fatty acid, resulting in the formation of a hydroperoxy fatty acid (IV) and again in a fatty































I. R CH = CH CH2 R


aS.


Electron, Proton


R CH = CH CH- R


R CH = CH CH R R CH =CH -CH R


R CH = CH -


FIGURE 2.9.


0 0.


R CH = CH -


CH2 R


R CH = CH CH R


CH R
0 OH
0 OH


SCHEMATIC REPRESENTATION OF THE PEROXIDATION
OF UNSATURATED FATTY ACIDS


II. III.


1-










acid radical which can react further. Once this chain reaction has started, it continues until all unsaturated compounds have been broken up, self-quenching occurs, or the radical is entrapped by an antioxidant (Chow, 1979).

Autoxidation eventually results in some form of degradation of the material affected. For example, foodstuffs such as corn oil, butter and margarine become rancid. The process is generally represented by four elementary reactions:


Initiation: production of R' (1) Propagation: R. + 02 ----- R00 (2) RO. + RH ----- ROOH + R' (3) Termination: ROO. + RO0. ----- Molecular products (4) In this scheme, RH represents a lipid molecule and R. the carboncentered radical derived from it by removal of a hydrogen atom. (Propagation can also occur by addition of the peroxyl radical to a carbon-carbon double bond.) These reactions can be prevented by antioxidants (Lundberg, 1962).

ROO. + XH2 ----- ROOH + XH.

XH. + XH. ----- X + XXH2

XH = antioxidant


At the cellular level, autoxidation is clearly undesirable because it threatens the integrity, protective and organizational properties of the membranes which enclose cells and organelles, thus placing the survival of the cell itself in jeopardy.











In the last few years, much research has been done concerning free radicals and into how various substances act to inhibit them (Cheeseman et al., 1984).

Free radicals are involved in two types of biological phenomena. Some appear in normal cell reactions, others in abnormal reactions and in processes associated with the aging of organisms. All biological oxidations do not necessarily give rise to such radicals. The mechanisms by which they are produced are numerous as they result from many types of reactions. These reactions include the peroxidation of lipids, in the course of which, under certain conditions increase depending upon the rate of formation of free radicals. It has also been shown that some processes, whether enzymatic or not, produce reactions of the same type when some of the cell constituents released are exposed to the direct action of oxygen in a free environment or in the absence of inhibiting agents. Some researchers, such as Pryor (1970), do not rule out the possibility that radicals may escape an electron transport chain, then causing rapid and destructive oxidations.

It may seem paradoxical, as Pryor (1971) points out, that oxygen, which cells need to release energy from food, is so reactive that it manages to trigger, alongside the usual processes, some undesirable parallel reactions oriented toward actions detrimental to the phenomena essential to life. In the course of such oxidative reactions, it causes damage to the cells by producing unfavorable modifications in the structure of their membranes, in the membranes of the intracellular organelles and in the endoplasmic reticulum.










For its part, ozone, a layer of which shelters the planet from short-wavelength ultraviolet radiation and known to be harmful, also causes oxidations which are detrimental to biological cell systems.

Study of the chemical composition of the cell membranes shows that they are formed by an association of lipids and proteins performing important functions such as electronic phenomena, active transport, glycolysis, biosynthesis of lipids and proteins, etc. Lipids are the most important constituent not only of these cell membranes, but also those of the intra-cellular organelles including the the mitochondrias and microsomes. Many factors suggest that the membranes consist of a film of lipids, rich in triglycerides and phospholipids, between 40 and 65 A in thickness, covered on both sides by a layer of proteins each about 10 A thick (Lehninger, 1975). The peroxidation of the lipid constituents of the membranes, which contain a high proportion of mono and polyunsaturated fatty acids (C16:1' C18:1; C18:2; C18:3; and C20:4) disturb their metabolic functions (Tappel, 1972). The polyunsaturated fatty acid moieties of lipids are characterized by the presence of two or more carbon-carbon double bonds which are separated by a methylene-CH2-unit. Methylene units are very susceptible to attack by radicals as peroxyl and Roo* with an extremely rapid combination with oxygen to form peroxyl radicals (Tappel, 1972). Lipid peroxidation of polyunsaturated fatty acids particularly arachidonic acid results in the formation and accumulation of lipid peroxidation products known as lipofuscin pigments (Tappel, 1972). These pigments accumulate in tissues (myocardium, brain, liver, testicles, etc.) in response to vitamin E deficiency, oxidative stress and age. Results of chemical analyses











suggest that these lipofuscin pigments arise from membrane lipid and protein fragments formed in the course of their peroxidation phenomena (Porta and Hartroft, 1969).

Consequences of lipid peroxidation are alterations in structural (membrane permeability), functional components of the cell and their component enzymes particularly subcellular partices such as mitochondrias, microsomes, and lysosomes.


Role of a-Tocopherol as a Chain Breaking Antioxidant


A major biological function of a-tocopherol is to act as a chain breaking antioxidant, scavenging free radicals, capable of terminating chain reactions among unsaturated fatty acids (Zalkin and Tappel, 1960).

Chain-breaking antioxidants react directly with radicals. Many phenols (ArOH) of which vitamin E is an example, can stop a radical chain reaction because they are able to trap the chain-carrying peroxyl radicals. The free radical scavenger action of a-tocopherol depends on the ability of donation of the phenolic hydrogen atom to a fatty acyl free radical, which resolves the unpaired electron of the radical so that free radical attack on further molecules of unsaturated fatty acids is prevented (Burton, 1989) ArOH + ROO ROOH + ArO*.

The relatively stable phenoxyl radical produced is so unreactive to attack another polyunsaturated fatty acid moeity or 02 that instead it is inactivated by reaction with a second peroxy radical R00' + ArO" + inactive products or may in the presence of ascorbate undergo reduction back to the original phenol (Burton, 1989).







50


The latter mechanism is the basis of the so-called synergistic action of vitamins C and E. The antioxidant breaks the chain of oxidation and can dramatically reduce the length of the autoxidation chain and the extent of peroxidative damage. The better the antioxidant, the greater is the amount of material that is spared from peroxidative damage. Vitamin E appears to be a factor ensuring the integrity of cellular membranes protecting their constituent polyunsaturated fatty acids from peroxidation.














CHAPTER 3
PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP FOLLOWING
INTRAMUSCULAR ADMINISTRATION IN AN OIL CARRIER


Introduction


Vitamin E is a biological antioxidant that has been used

pharmacologically in young sheep and cattle for the prevention or treatment of conditions associated with nutritional muscular dystrophy (Jenkins et al., 1972). Assessments of the vitamin E status in the ruminant animal treated with various tocopherol preparations have relied on serum or plasma tocopherol concentrations (Jenkins et al., 1970). To our knowledge there is no information on the effects of various forms of vitamin E preparations on sheep tissue tocopherol levels. The purpose of the present investigation was to assess the bioavailability in sheep of two forms of vitamin E (d-a- and dl-atocopherols) administered intramuscularly using a serial blood plasma analysis of a-tocopherol concentration. In addition, the a-tocopherol levels were determined in a few selected tissues.


Experimental

Animals


Yearling crossbred wethers weighing 40-50 kg were used. All animals originated from a flock that was born and raised in total confinement (Heaney et al., 1980). The animals were fed ad libitum, a diet that consisted of providing grass (40%), hay (40%), and corn










silage (20%). The trial was carried out in three successive periods (2 months each), with each period utilizing one group of five sheep. Sheep were administered respective tocopherol treatments intramuscularly. The parenteral vitamin E preparations, suspended in sesame oil (200 mg/ml) were administered slowly at 40 mg/kg body weight into the muscle of the crural area of each leg. Treatments with respective slaughter times are as follows:

Group 1 dl-a-tocopherol at 240 h Group 2 d-a-tocopherol at 360 h Group 3 d-a-tocopherol at 240 h.

A group of six wethers originating from the same flock, was used as a control (Group 4).

Blood samples were taken from the jugular vein of treated sheep at specific time intervals after dosing ranging from zero to 360 h. The plasma was separated from the cellular blood components in a refrigerated centrifuge at 40C and stored at -200C until assayed for a-tocopherol. Tissue samples were taken from all sheep after slaughter and stored at -200C until analyzed for a-tocopherol content. Feed samples were taken during the experiment for vitamin E analysis.


Analytical


High pressure liquid chromatography (HPLC) was used for plasma atocopherol determination (McMurray and Blanchflower, 1979a). The HPLC system consisted of an M 6000 pump and W.K. septumless injector (Waters Associates Inc., Milford, Mass., USA) and Waters P Bondapak C12 (10 pm) column (3.9 x 300 mm) coupled to a Perkin-Elmer 650-105










fluorescense spectrophotometer set at 295 and 330 nm for excitation and emission, respectively. The column was eluted with methanol:water (97:3) at 3 min/ml. All solvents were HPLC or pesticide grade. Blood plasma (2 ml) samples were precipitated with ethanol (2 ml), and extracted with hexane (4 ml). Tissue samples were saponified and extracted with hexane (Thompson and Hidiroglou, 1983).

Identification and quantitation of the a-tocopherol was

accomplished by comparison of retention time and peak areas with d-atocopherol standard. Tocopherol determination in roughage was performed according to the techniques of McMurray and Blanchflower (1979b).


Statistical Analysis


The indexes of bioavailability of the various preparations of vitamin E were determined by analysis of variance. These indexes were: 1) the maximum a-tocopherol plasma (C max) concentration (peak of plasma a-tocopherol concentration curve), 2) the time of maximum (t max) a-tocopherol concentration, and 3) the area (AUC) under the atocopherol plasma concentration time curve (Balard, 1968; Koch-Weser, 1974). Differences in the vitamin E concentration between corresponding tissues in the four groups were determined by analysis of variance.











Results


Tocopherol Content of Roughages


Average tocopherol content of roughage at the time of consumption were 39, 69 and 34 (mg/kg DM) for maize-silage, grass-silage and hay, respectively. The only forms of vitamin E found in hay and grasssilage were a-tocopherol. In maize-silage a-tocopherol was, on the average about half the total tocopherol and was equal to the sum of a + a tocopherols (26%) and 6 tocopherol (24%).


Blood Plasma c-Tocopherol


After IM administration of d-a-tocopherol preparation, a lag period was noted in the sheep until vitamin E increased in the systemic circulation (Fig. 3.1; Group 2). There was no clear evidence of an elimination phase in the data. In all 3 groups there was a suggestion of a plateau near the peak. It would appear as though the system reached an equilibrium at or near peak levels.

In Group 2 (IM d-a-tocopherol injected sheep killed 360 hr after dosing) the most important index of bioavailability (Koch-Weser, 1979), the area under the plasma concentration-time curve (AUC) was the largest (P<0.01) compared to the other 2 groups (Table 3.1). This appeared to be due to a more sustained release into the plasma (t max much larger), resulting in a higher delivery of the vitamin. Maximum concentration (C max) was much higher (P




















































f~ 4 tocopheol


* 4


* *


0 0 0


27 66 14 143 181 29 256 296 334 373
TIME Owl


FIGURE 3.1.


MEAN PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE I.M. INJECTION OF D-a-TOCOPHEROL SUSPENDED IN STERILE SESAME OIL.


220, 211. 2 02193

I j3
1.
7175




I47 .1630.






120. 114.










TABLE 3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN SHEEP AFTER A
SINGLE INTRAMUSCULAR ADMINISTRATION OF VITAMIN E (40 mg/kg).


Vitamin E Cmax1 Tmal2 Co3 Cmax/Co Ct4 Ct/Co Cmax/Ct AUC5
Group preparations (vg/ml) (hr ) (pg/ml) (pg/ml) (ig/ml) (pg/ml) ( jg/ml/hr)


1 dl-a-tocopherol 1.76d 151b 1.12c 1.68d 1.47b 1.39d 1.23 286d 2 d-a-tocopherol 2.40c 254c 0.94d 2.68c 1.87a 2.06c 1.35 637c 3 d-a-tocopherol 1.84d 190a 0.71d 2.63c 1.30b 1.82c 1.54 277d SE 0.23 24.78 0.09 0.21 0.17 0.24 0.15 27

Sheep in Group 1 and 3 were slaughtered 240 d after dosing while sheep in Group
2 were killed 360 h after dosing.

1Maximum plasma concentration.

2Maximum time (hr).
3Initial plasma concentration.

4Terminal plasma concentration.

5Area under plasma concentration-time curve. abMeans in the same columns with different superscripts differ (P<0.05). cdMeans in the same columns with different superscripts differ (P










In Group 3 (IM d-a-tocopherol injected sheep killed 240 hr after dosing) there was also a more sustained delivery than in group 1 (t maximum 191>151). This resulted in a marginal increase in C max but no increase in AUC was noted. This may be due to the fact that initial values in Group 3 were very low compared to the other groups (Co=0.71). It was noted that relative to initial values the increase in vitamin E concentration in Group 3 sheep was much higher than for Group 1 and approached the relative increase for Group 2 (C max/Co=2.63).

The AUC measurement of Group 3 was comparable to that of group 1. This may have been due largely to the higher initial value in this group. The elimination rates of the three groups appeared to be very similar (no differences in Cmax/CT). Tissue Concentrations of Vitamin E


Sheep receiving d-a-tocopherol and slaughtered 360 hr after dosing had higher (P








TABLE 3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (pg/g wet)1 OF SHEEP FOLLOWING A SINGLE INTRAMUSCULAR ADMINISTRATION (40 mg/kg BODY WEIGHT) OF VARIOUS VITAMIN E PREPARATIONS.


Groups:
Vitamin E preparation:
Mode administration: dl-a-tocopherol d-a-tocopherol d-a-tocopherol Control Time after dosing (hr) 240 360 240


Tissues: Adrenal


Depot fat Muscle vastus medial Muscle:
brachiocephalicus Heart Kidney Lung Liver Pancreas Spleen


11.524.29ab 10.521.73ab 4.200.55b


2.740.41d

3.360.40ab 2.870.58ab 3.080.71e 4.270.37a 3.040.56b 2.720.56b


13.621.31ab 17.85il.69ab 7.750.60a 6.261.13c

5.331.03ab 6.100.93ab

7.521.10'

5.921.39a 6.941.00 a 6.08I.04a


16.82t2.52ab 4.921.01 b


5.770.84c

3.840.37ab

4.570.99ab

5.460.76d

4.180.48a 6.341.22a 5.65I.04a


12.841.68ab 3.880.94b


3.460.70d

5.761.59ab 3.440.91ab 2.910.23e 2.220.36b

4.320.50b 3.300.54b


ILeast square means SE. a,b
abMeans in the same rows with different superscripts differ (P









Discussion


Blood plasma vitamin E data showed that in sheep dosed with various vitamin E preparations, the movement of the active free atocopherol from the site of the IM injection to the blood stream was quite slow. In all sheep given the vitamin E preparation by IM oil depot injection, a delayed increase in plasma a-tocopherol levels occurred. D-a-tocopherol IM administration proved to be more potent than dl-a-tocopherol. The length of the experimental period had an effect on the bioavailabilities index. The IM dosed sheep with d-atocopherol and slaughtered 360 hr later had a bioavailability much higher than in sheep dosed similarly but killed 240 hr after dosing. During this 360 hr period a gradual but sustained response occurred.

Overman et al. (1954) reported that IM administration of vitamin E is ineffective in humans, regardless of the type of compound and the type of vehicle (oil or water) used. However, according to Caravaggi et al. (1968), IM injection of vitamin E is the best route for administration of this vitamin. These workers observed that in sheep, following IM injection of a commercial preparation of d-a-tocopherol acetate in aqueous suspension, the plasma a-tocopherol reached its peak 8 hr after its administration. The slow release of the IM injected vitamin E preparation in oil carrier could be due to myositis and inflammation of the lymphatic vessels (Bille et al., 1976), through which the vitamin E molecule is absorbed (Blomstrand and Forsgren, 1968; Machlin et al., 1979). It is also known (Voffey and Coutrice, 1956) that exercise influences the flow of fluid along the lymphatic vessels. When an animal is at rest, as in these











experiments, there is little or no lymphatic flow, and this would affect the release of the vitamin E preparations which probably was confined to connective tissue surrounding the muscle (Balard, 1968). Bille et al. (1976) showed that vitamin D injected by IM oil depot, into slaughter pigs, had a half-life of about 30-40 days at the injection site, and that oil granulomata had formed after dosing. Recently, it was reported (Dickson et al., 1986) that in sheep, following the IM injection of a vitamin E preparation (5 ml) suspended in arachis oil (400 mg dl-a-tocopherol acetate/ml) into quadriceps muscle, a considerable storage of vitamin E occurred in their lymph gland (iliac). These workers observed that in the injected sheep a continuous increase of vitamin E concentration occurred during 3 weeks (from 76 pg/g to 2687/pg/g lymph gland). On weeks 5 and 6 there were still high concentrations of vitamin E (1855 pg and 550 vg/g) in the lymph gland (iliac) of the killed sheep. The results of their experiment showed that the duration of the experimental period was relatively short and longer time was needed in order to establish accurately the various indexes of bioavailability. The planning of our experiment was based on work carried out on chicks (Marusich et al., 1967) and in dogs (Newmark et al., 1975) dosed IM with various forms of vitamin E. In their animals the plasma vitamin E showed its peak within 24 hr. The difference in initial d-a-tocopherol plasma values between the present 3 groups may have been due to different roughage batches used before the start of the trial. The results reported herein show that the time of slaughter had a major influence on tissue vitamin E levels. In sheep slaughtered 360 hr after IM










administration of d-a-tocopherol, tissue levels of a-tocopherol were higher than those of control. In some cases there were large individual variations in tissue a-tocopherol concentrations which made comparisons difficult between corresponding tissues in the various groups. It is apparent that of the tissues examined the depot fat is the preferred storage tissue for vitamin E. According to Machlin et al. (1979) adipose tissue tocopherol is not readily available upon metabolic demand. Tocopherol concentrations in the liver of control were low indicating low dietary intake. Rammel and Cunliffe (1983) using HPLC reported that levels of 2 mg of a-tocopherol per kg of fresh liver were observed in clinically normal sheep. According to these workers a-tocopherol hepatic levels less than 2 mg/kg become health problems. In deer, Meadows et al. (1982), also using HPLC, observed liver a-tocopherol concentrations of 5 pg/g wet tissue.

In conclusion, the results showed a delayed increase in plasma ctocopherol concentration in sheep administered IM two vitamin E preparations. A more sustained increment in plasma M-tocopherol was observed in sheep IM-injected with d-a-tocopherol than dl-atocopherol. Following d-a-tocopherol IM administration, higher tissue a-tocopherol levels occurred in sheep killed 360 hr after dosing than in those of control sheep.


Summary


The bioavailability of two vitamin E preparations (d-a-tocopherol and dl-a-tocopherol) suspended in sesame oil solution and administered intramuscularly was evaluated in sheep. In sheep administered d-a-









tocopherol and killed after 360 hr (Group 2), there was a higher (P<0.01) bioavailability as assessed by the area under the plasma concentration-time curve, than for sheep killed 240 hr after injection with d-a-tocopherol (Group 3) or dl-a-tocopherol (Group 1). In the 3 groups (5 sheep each) the highest blood plasma a-tocopherol increment occurred in the d-a-tocopherol injected sheep. For sheep injected with d-a-tocopherol and slaughtered at 360 hr (Group 2) the pancreatic, hepatic, lung, spleen and muscle tocopherol concentrations were higher (P<0.05) than in the control group. Also in group 2 there was a tendency for higher tissue tocopherol concentrations than in the other vitamin E treated sheep (Group 1 and 3).














CHAPTER 4
BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS IN SHEEP Introduction


The biological activity of the various tocopherol compounds has been established from experiments with laboratory animals. However, biopotency evaluation of different forms of vitamin E based on a specific animal model (i.e., rat antisterility test) may not be valid for other species. Biopotencies based on this test do not correlate with human data. Recently Baker et al. (1986) reported that human assay data confirmed the currently accepted biopotencies of 1.0 IU/mg and 1.36 IU/mg for dl-tocopheryl acetate and d-a-tocopheryl acetate, respectively. There are little data available for ruminants on the absorption and body distribution of vitamin E. In view of the unique digestive and physiological system in ruminants, the present investigation determined bioavailability of the various forms of vitamin E in sheep fed over a relatively long-term study (28 days).


Materials and Methods


Initially, twenty crossbred yearling wethers weighing 60 to 70 kg were used. All animals originated from a flock fed a commercial mix that consisted of 56.45% corn meal, 16.5% (44% CP) soybean meal, 25% cottonseed hulls, 1% trace mineral salt, dicalcium, 1% monocalcium phosphate, and .037% of vitamin A and D3. They were fed this diet throughout the experiment.











Sheep were placed in individual metabolism cages 10 days before administration of vitamin E as an adjustment period. They were randomly allotted to four dietary groups of five each that consisted of 400 International Units/day/sheep of either (1) dl-a-tocopherol,

(2) dl-a-tocopheryl acetate, (3) d-a-tocopherol (4) d-a-tocopheryl acetate, which was mixed with the commercial diet. Appropriate amounts of the various forms of tocopherols were dissolved in absolute ethanol prior to diet mixing. In the middle of the experiment, one sheep from group 3 and two sheep from group 4 stopped eating and were removed from the experiment.

Blood samples were taken at various intervals by jugular puncture with plasma separated from the cellular blood components in a refrigerated centrifuge and stored at -300C until assayed for vitamin E. All animals were slaughtered after 30 days. Samples from selected tissues (fat, muscles from the neck and leg, kidney, lung, heart, spleen, liver, pancreas) were collected and stored at -300C until analyzed for d-a-tocopherol.


Analytical Methods


Quantitation of d-a-tocopherol in sheep tissues, as well as

plasma samples, was performed by high-pressure liquid chromatography (HPLC) using a fluorescent detector, following tissue homogenization and heptane extraction. Identification and quantitation of atocopherol were obtained by comparison of retention time as well as peak areas with tocopherol standards. Standards were purchased from Eastman Kodak (Rochester, NY, USA).












Instrumentation


The chromatographic apparatus consisted of an M 6000 pump and a WK septumless injector (Waters Associates, Inc., Milford, Mass., USA). A Perkin-Elmer 650-150 fluorescence spectrophotometer, equipped with a microflow cell unit, was used for quantitation. Wavelength settings were 295 and 330 nm for excitation and emission, respectively. The column as a Bondapak C18 (3.9 mm x 30 cm) of 10 pm particle size purchased from Waters Associates (Milford, Mass., USA). The mobile phase was a solvent system consisting of methanol and water in a 97:3, with a flow rate of 3 ml/minute. All solvents used were HPLC grade.


Mass Spectrometry in Combination with Gas Chromatography


Further identification of a-tocopherol in various tissues was carried out by a combination of a Hewlett-Packard (HP) gas chromatograph 5790-mass spectrometer 5970A series with a data station (HP-9000). The mass spectrometer performs three basic functions. First, molecules are subjected to bombardment by a stream of highenergy electrons, converting some of the molecules to ions. The ions are accelerated in an electric field. Second, the accelerated ions are separated according to their mass-to-charge ratio in a magnetic field or electric field. Finally the ions with a particular mass-tocharge ratio are detected by a device which is able to count the number of ions which strike it. The detector's output is amplified and fed to a recorder (Roboz, 1968). Eluates from the HPLC were










purified for a-tocopherol by thin layer chromatography on silica gel using cyclohexane-diethylether (4:1 v/v) as the solvent.

Authentic standards of d-a-tocopherol, as well as tissue samples, were silylated before injection onto the GC/MS (Ingold et al., 1987).

The tocopherol trimethylsilyl ethers were injected (3 vl) into a 12 m x 2 mm i.d. ultra-i (OV 101 methyl silicone) capillary column maintained at 2800C with an injector temperature of 3500C and a helium carrier gas flow rate of 0.5 cc/min, which was connected to a series mass-selective detector. Mass spectra were obtained at an electron beam energy of 70 eV and an accelerating voltage of 1800 volts. Molecular ions ranging from 50 to 600 mass/charge were monitored continuously.


Statistical Methods


Indices of bioavailability of various preparations of vitamin E were performed by analysis of variance. These indices were: 1) the maximum a-tocopherol plasma concentration (peak of plasma a-tocopherol concentration curve), 2) the time of maximum (t max) a-tocopherol concentration, and 3) the area (AUC) under the a-tocopherol plasma concentration time curve. Analysis of variance was also used to estimate the difference in the vitamin E concentration between corresponding tissue in the four groups, as well as among tissues.










Results


Tissues


Diet and tissues were highly significant (P<0.0001) in their vitamin E concentrations, but diet x tissue interaction was not significant (P > 0.05) (Table 4.1). Higher a-tocopherol concentrations (P
1.16 SE) or dl-a-tocopherol (6.40 1.91 SE) supplemented sheep. In kidney, higher a-tocopherol concentrations were found in the d-atocopheryl acetate (7.20 0.27) than d-a-tocopherol (5.81 0.67 SE) or dl-a-tocopheryl acetate (4.78 1.84) or dl-a-tocopherol (4.11 0.63). However, analysis of variance did not show a difference (P >

0.05) among corresponding tissues among treatments. In all diets, pancreas contained the highest concentrations, followed by liver and spleen. Immediately after these tissues, heart, lung and kidney were










TABLE 4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS IN
TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.


Source DF MS F P


Model 35 142.76 16.10 0.0001 Error 117 8.86 Corrected total 152


Source DF SS F P


Diet 3 212.02 7.97 0.0001 Tissue 8 4318.39 60.86 0.0001 Diet x tissue 24 305.90 1.44 0.1049











TABLE 4.2. TISSUE a-TOCOPHEROL CONCENTRATIONS (pg/g FRESH TISSUE) IN THE FOUR DIETS.


Diet Mean N


d-a-tocopherol 10.43a 36 d-a-tocopheryl acetate 8.75b 27 dl-a-tocopheryl acetate 7.72b 45 dl-a-tocopherol 7.46b 45


In vertical row, numbers with the same letter (P>O.05).


are not different




































Diet 1 : dI-a-tocopherol


a I


I I I I I I I I D I Diet 3 : d-o-tocopherol Diet


Diet 2 : di-a-tocopherol acetate I I


I I


I I I I I I 1 1


4 : d-.a-tocopherol acetate






T I


0 10





,.1


F I I I I I I I I I FAT LEG NECK KION LAt HEAR SPL UV PAN


TISSUE TYPE


FIGURE 4.1.


a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS


S! I


I I I I I I I m FAT LEG NEOC KIDN LUN HEAR PL UV PAN


u ,


V


n


I
I










ranked lower (P<0.05). Lowest (P
Table 4.3 (and Figure 4.2) shows that the relative retention time (minutes) of the trimethylsilyl esters (TMSi) obtained from purified a-tocopherol tissues samples are very similar to that obtained from the TMSi a-tocopherol standard.

Mass spectra of the gas chromatographic peak corresponding to the c-tocopherol standard (TMSi) (Figure 4.3) and that corresponding to the peak with the same retention time obtained for the pancreas, for example, are identical, confirming the presence of a-tocopherol in these biological extracts.

The peak obtained at mass/charge 502 represents the molecular ion of a-tocopherol TMSi (430 a-tocopherol + 72 trimethylsilylether). The fragmentation pattern reveals peaks at mass/charge 277, resulting from the loss of the side chain (C16H33) and at mass/charge 237 due to the cleavage of the chroman structure accompanied by hydrogen rearrangement and the loss of a CH3-C~CH fragment (Nair and Luna, 1968).


Blood Plasma


Analysis of blood plasma a-tocopherol concentrations summarized in Table 4.4 show that diet and tissue source were significant (P<0.01) but not the interaction diet x day. Higher (P<0.01) etocopherol plasma concentrations were observed in sheep supplemented with d-a-tocopherol on days 9, 15, 17 and 25 (Figure 4.4) than in the other three groups. Also, in sheep fed d-a-tocopherol, the most











TABLE 4.3. RETENTION TIME OF a-TOCOPHEROL FROM GASCHROMATOGRAPHY/MASS SPECTROMETRY.


Relative retention time (minutes) Substance TMSia


Standard a-tocopherol 4.1 Pancreas 3.9 Liver 3.8 Heart 3.9 Spleen 3.9 Lung 3.9 aTMSi; Trimethylsilyl ether.





























R, a
(Cm)3 -so C CH,: R O' ICH CHz CH2 CMH CH3

R, R, R3 CH3


277
/
"ni


502


2.0E51.5E5


1.0E5


5.0E40



1.4E4

1.2E41000080006000 4000 2000

0


133 277


MASS/CHARGE


FIGURE 4.2.


IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/MASS SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD, (B) aTOCOPHEROL IN PANCREAS


237


193


207


kJl




























1.1E61.0E69.0E5 8.0E57.0E56.0E55.0E5 4.0E53.0E52.0E5 1.0E50



6.0E45.0E4


3.0E4 2.0E410000-


2.5 3.0


FIGURE 4.3.


3.5 4.0 TIME (min.)


MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A) AND a-TOCOPHEROL IN T-E PANCREATIC TISSUE (B) FOLLOWING HPLC COLLECTION


. .. I . . I I










TABLE 4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS IN
BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.

Source DF MS F P


Model 66 2.99 4.64 0.0001 Error 217 0.64 Corrected total 283


Source DF SS F P


Diet 3 44.79 23.09 0.0001 Tissue 16 225.13 11.13 0.0001 Diet x tissue 47 40.53 1.33 0.0883












TABLE 4.5. AREA (AUC) UNDER THE PLASMA a-TOCOPHEROL CONCENTRATION CURVE (pg/ml h- ).


Diet AUC (mean SE)


d-a-tocopherol 102.4 8.13a d-a-tocopheryl acetate 90.0 9.44b dl-a-tocopheryl acetate 75.0 7.31c dl-a-tocopherol 67.3 7.31d In vertical row, numbers with the same letter are not different (P>0.05).



































Diet 2 : di--tocopherol acetate


Diet 1 : di-a-tocopherol


iI


Diet 3 d~.~tOCOpherOI
I I *


Diet 3 : d-a-tocopherol

5



3-t~'


11 4


I 10 20 30 40
I I I "" 0 10 20 30 40

C


I
1


0


Diet 4 : d-a-tocopherol acetate 5


4




2-1





S 10 20 30 40 DAY


FIGURE 4.4.


a-TOCOPHEROL CONCENTRATIONS IN THE BLOOD PLASMA OF SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS










important indice of bioavailability, the area under the plasma atocopherol concentration time curve (AUC), was larger (P<0.05) as compared to dl-a-tocopheryl acetate or dl-a-tocopherol supplemented sheep (Table 4.5). No statistical difference (P>0.05) was observed in the maximum a-tocopherol concentration (pg/ml) between the four groups. They were 3.6 0.69 (SE) (dl-a-tocopherol) and 4.3 0.69 (dl-a-tocopheryl acetate), 5.11 0.77 (d-a-tocopherol) and 4.8 0.89 (d-a-tocopheryl acetate). Also, no difference on the time (days) of maximum concentration (P > 0.05) was observed. They were 18.2 3.24

(SE) (dl-a-tocopherol), 14.8 3.24 (dl-a-tocopheryl acetate), 11.5

4.30 (d-a-tocopherol) and 22.3 4.96 d-a-tocopheryl acetate. The elimination curve rates (concentration maximum/concentration terminal) in the four groups were very similar. They were 1.6 0.20 (SE) (dla-tocopheryl acetate), 1.8 0.22 (d-a-tocopherol) and 1.7 0.25 (da-tocopheryl acetate) and 1.4 0.20 (dl-a-tocopherol).


Discussion


Paucity of bioavailability data from various forms of tocopherol in sheep limits any comparison with the present data. Biological response of sheep to dietary intake of various forms of vitamin E was greater with the d-a-tocopherol than with its esterified form or the isomers. Indeed, the area plasma time curves, which provide a proper measure of relative availability for physiological function, and tissue a-tocopherol concentrations were highest in the d-a-tocopherol supplemented group. This indicates that in the ruminant body there may be a difference in the transfer rate between natural and unnatural










forms of vitamin E (Dunkley et al., 1967). As suggested in humans (Week et al., 1952), it could be that the physiological mechanism effecting hydrolysis in the ruminant duodenum of tocopherol esters is less effective than in rats. It is reasonable to assume that plasma tocopherol levels in this experiment reflect the relative efficacy of various forms of vitamin E on tissue storage. Indeed, as was reported for humans (Djuetal, 1958), the present data on -tocopherol concentrations in sheep tissue show a variable pattern which is largely a reflection of varied bioavailability of the different forms of vitamin E. It may be concluded that following ingestion of equivalent amounts (IU) of the various forms of tocopherol by sheep da-tocopherol was more potent than d-a-tocopheryl acetate or the 1isomeric forms of vitamin E.


Summary


Seventeen crossbred yearling wethers were randomly allotted to

four dietary groups that received 400 IU/day/sheep either of (1) dl-atocopherol (five sheep), (2) dl-a-tocopheryl acetate (five sheep), (3) d-a-tocopherol (four sheep) or (4) d-a-tocopheryl acetate (three sheep). Blood samples were taken at day 0 and then at frequent intervals for e-tocopherol determination. At the end of the 28-day experiment, animals were killed and various tissues sampled. Higher concentrations of a-tocopherol were observed in tissues of sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed d-atocopherol, the most important index of bioavailability, the area under the plasma a-tocopherol time curve (AUC) (Gibaldi and Perrier,







78


1975) was larger as compared to other forms of vitamin E supplementation.














CHAPTER 5
BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS
INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS


Introduction


Biopotencies of various tocopherol compounds are generally provided by the following relationship according to the National Formulary: 1 mg dl-a-tocopheryl acetate = 1.0 IU; 1 mg dl-atocopherol = 1.1 IU; 1 mg d-a-tocopheryl acetate = 1.36 IU; 1 mg d-atocopherol = 1.49 IU. These values have been based largely on small animal bioassay (rat anti-sterility assay). Horwitt (1980) and Horwitt et al. (1984) reported that in humans, on a mg basis, d-atocopheryl acetate was 2.16 times more potent than dl-a-tocopheryl acetate following a single oral dose. However, Baker et al. (1986) confirmed in humans following a continuous oral dosing that 1 mg of dl-a-tocopheryl acetate (all-racemic-a-tocopheryl acetate) has a biopotency of 1.0 IU whereas d-a-tocopheryl acetate (2R, 4'R, 8'R-atocopheryl acetate) has a biopotency of 1.36 IU (28 d). Horwitt (1980), Horwitt et al. (1984) and Baker et al. (1986) used the elevation of plasma a-tocopherol as a measure of bioavailability.

Although much is known about clinical signs of deficiency in calves and lambs (Rice and McMurray, 1982) very little attention is given to the different potencies of various vitamin E forms in cattle. The present authors (Hidiroglou and McDowell, 1987; Hidiroglou et al., 1988) have undertaken a series of studies on the utilization of









different chemical forms of vitamin E by sheep. That work was extended to cattle with the purpose of comparing plasma and tissue atocopherol concentrations following supplementation of various tocopherol compounds.


Materials and Method


Animals


Twenty-four Charolais-Hereford crossbred beef cows ranging from 2 to 10 years of age were culled 6 months after calving from the herd of the Nappan Experimental Farm, N.S., Canada. These unbred cows were stratified by age into four groups, and the six animals within each group were randomly assigned to four dietary tocopherol preparations. The treatments were d-a-tocopheryl acetate, d-a-tocopherol, dl-atocopherol and dl-a-tocopheryl acetate. Cows received daily 1,000 IU (a dosage above a physiological level) of their respective vitamin E preparation. This amounted to 735 mg of d-a-tocopheryl acetate, 671 mg of d-cra-tocopherol, 900 mg of dl-a-tocopherol and 1000 mg of dl-atocopheryl acetate, respectively. Each vitamin E preparation was diluted in alcohol to 3 ml and mixed with 25 g of dry molasses. All cows were individually fed 2 kg of average quality grass hay (8.7% CP, 35% ADF, 54% TDN, .51% Ca, .23% P, .02 ppm Se and 12 ppm vitamin E) and had ad libitum access to a barley-soybean mixture (Table 5.1) with no added a-tocopherol. The vitamin E-molasses mixture was prepared daily and was topdressed on the grain portion for each individual cow. The experiment lasted 28 days after which time the cows were sacrificed at commercial facilities.











TABLE 5.1. COMPOSITION OF THE GRAIN PORTION OF
THE DIET FED TO COWS FOR 28 da


Ingredient % as-fed basis


Barley 90 Soybean meal 7.5 Calcium carbonate 1.0 Dicalcium phosphate 1.0 Vitamin premixb .25 Trace mineral saltc .25


Analyzed for vitamin E and selenium
(8 ppm and .235 ppm, respectively). bProvided, per kilogram of diet:
5,000 IU vitamin A and 500 IU vitamin
D.
Provided, per kilogram of diet:
2.1 g NaC1, .22 mg I, .088 mg CO, 9 mg Fe, .725 mg Cu, 2.625 mg Mn and 8.75 mg Zn.











Blood Collection


Blood samples (15 ml) were obtained via jugular venipuncture on d 0, 1, 7, 14 and 28 of the feeding period and plasma was analyzed for a-tocopherol concentration. Samples were always collected in the morning before vitamin E treatments were provided. Blood samples were centrifuged at 1,000 x g for 15 min at 40C immediately after sampling, and plasma was stored at -200C until analyzed for a-tocopherol concentration.


Tissue Collection


On the last d of the experiment, cows were shipped to a local abattoir and slaughtered the following d. At that time, 10-50 g samples were collected from ten different tissues: heart, thyroid, liver, kidney, adrenal, pancreas, spleen, lung and neck muscle. These samples were frozen within 2 hours and stored at -200C until analyzed for a-tocopherol concentration using high pressure liquid chromatography.


Analytical Method

Quantification of a-tocopherol in tissues was performed by HPLC using a fluorescence detector as described by McMurray and Blanchflower (1979b) following tissue homogenization and heptane extraction by the method of Burton et al. (1985) and Ingold et al. (1987). Serum samples were prepared for HPLC analysis according to the method of McMurray and Blanchflower (1979b). Identification and quantification of a-tocopherol were by comparison of retention times










and peak areas with tocopherol standards. Selenium and a-tocopherol in the feed were determined, respectively, by the method of Hoffman et al. (1968) and McMurray and Blanchflower (1979a). Instrumentation


The chromatographic apparatus consisted of a M 6,000 pump and WK septumless injector. A Perkin-Elmer 650-150 fluorescence spectrophotometer equipped with a microflow cell unit was used for quantification. Wavelength settings were 295 and 330 nm for excitation and emission, respectively. The column was a P Bondapak C18 (3.9 mmx 30 cm) of 10 pm particle size. The mobile phase was a solvent system (HPLC grade) consisting of methanol and water (97:3) with a flow rate of 3 ml/min.


Mass Spectrometry in Combination with Gas Chromatography


Further confirmation of U-tocopherol in various tissues was

carried out by a combination Hewlett-Packard (HP) gas chromatograph 5790-mass spectrometer 5970A series with a data station (HP-9000). Eluate from the HPLC was purified for a-tocopherol by TLC on silica gel using cyclohexane-diethylether (4:1 v/v) as the solvent. Authentic standards of d-a-tocopherol as well as tissue samples were silylated after HPLC as described by Ingold et al. (1987) before injection onto the gas chromatograph/mass spectrometer. The tocopherol trimethylsilyl ethers were injected (3 pl) into an ultra-I (OV 101 methyl silicone) capillary column (12-m x 2-mm i.d.), maintained at 2800C with an injector temperature of 3500C and a helium










carrier gas flow rate of .5 cc/min, which was connected to a series mass selective detector. Mass spectra were obtained at an electron beam energy of 70 eV and an accelerating voltage of 1,800 volts. Molecular ions ranging from 50 to 600 mass/charge were monitored continuously.


Statistical Method


Analysis of variance (Steel and Torrie, 1980) was used to

estimate the difference in a-tocopherol concentration among similar time blood samplings as well as among corresponding tissues in the four treatment groups. An additional two way analysis was carried out to test groups and d for blood plasma and for groups and tissue types. Comparisons were made by using Duncan's multiple range test (Duncan, 1985). Statistical models used in the experiment were as follows. For the one-way analysis: Y = + gi + Ei, where gi is effect of .th th I group; Y. = p + d. +E.., where d. is effect of i d. For twoway analysis: Yijk = V + g. + t. + Ec.ijk, where g is effect of ith group and t. is effect of jth d.


Results


Plasma


Supplementation over a 1 to 7 d period of time with the various vitamin E preparations increased (P<.01) plasma a-tocopherol concentration above the baseline level (Table 5.2). Plasma atocopherol concentrations at 1 d were higher (P<.01) than the original baseline values in the d-a-tocopherol group but not for










TABLE 5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (pg/ml) IN CATTLE FED VARIOUS PREPARATIONS OF VITAMIN E.


Day Group
Item 0 1 7 14 28 SE mean


Treatment

d-a-tocopheryl acetate 1.49ag 2.46bfg 3.67bef 5.64bde 7.65abd .71 4.18a d-a-tocopherol 1.39af 3.82ae 5.25ae 8.41ad 9.22ad .58 5.61b dl-a-tocopherol 1.39ag 2.40bfg 3.14bef 3.98be 5.44bcd .44 3.27c dl-a-tocopheryl acetate 1.37af 2.65bef 3.10bde 4.02bde 4.62cd .52 3.16c SE .10 .25 .45 .82 .82 .27 Day mean 1.41d 2.83e 3.80f 5.51g 6.73h .31 abcMeans in the same column with different letters in their superscripts differ (P def'gMeans in the same row with different letters in their superscripts differ (P









the other groups (P>.05). To elevate plasma a-tocopherol concentration, d-a-tocopherol outranked all the other forms at all sampling dates except at 28 d, where its effect was not different (P>.05) from d-a-tocopheryl acetate (Table 5.2). D-a-tocopherol supplementation during the 28 d experimental period resulted in the highest blood plasma a-tocopherol concentration followed by d-atocopheryl acetate; the racemic mixtures ranked lower (Table 5.2) despite being dosed at equal units. Blood plasma a-tocopherol increased continuously from 0 to 28 d (Table 5.2). The difference in plasma a-tocopherol concentration (ig/ml) between d 28 and d 0 were as follows (Table 5.2). D-a-tocopheryl acetate = 6.16; d-a-tocopherol =

7.83; dl-a-tocopherol = 4.05; dl-a-tocopheryl acetate = 3.25. Consequently the relative bioavailabilities of the various tocopherols vs dl-a-tocopheryl acetate (1.0 IU/mg) could be calculated as follows:


(4.05 x 1.1 IU/mg)
dl-a-tocopherol = 1.37 IU/mg; d-a-tocopheryl acetate
3.25
(6.16 x 1.36 IU/mg) (7.83 x 1.1 x 1.36 IU/mg) = 2.58 IU/mg; d-a-tocopherol
3.25 3.25 = 3.60 IU/mg.


Tissues

There were differences among treatments (P<.01) and types of

tissue (P<.0001) in vitamin E tissue concentrations at slaughter 28 d after supplementation began Fig. 5.1 and Table 5.3). The adrenal gland and liver concentrations of a-tocopherol were greater (P<.01) for cattle fed d-a-tocopherol or d-a-tocopheryl acetate than when fed racemic forms of vitamin E. The a-tocopherol concentrations were





























40
DL-a-TOCOPHEROL 30


2010




40


DL-a-TOCOPHERYL ACETATE 30


2010
o
TYMSKD LUN SPL HEA V
THYMUSKIDLUN SPL HEA LIv "ADR


D- a-TOCOPHEROL

















D-a-TOCOPHERYL ACETATE









0


THYMUS KID LUN SPL HEA LIV ADR


TISSUE TYPE


FIGURE 5.1.


a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS











TABLE 5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (pg/g FRESH TISSUE) IN CATTLE FED VARIOUS VITAMIN E PREPARATIONS.


Dietary form
d-a-tocopheryl d-a-toco- dl-a-toco- dl-a-tocopheryl Tissue Tissue acetate pherol pherol acetate SE mean g/g fresh tissue

Adrenal gland 38.41a 38.07a 27.91b 28.75b 2.68 33.28d Heart 20.43a 18.71a 18.43a 16.12a 1.42 18.42 Kidney 12.07a 13.11a 8.81b 12.28a 1.00 11.56h Liver 24.20ab 27.00a 15.67c 19.88bc 1.79 21.68e Lung 15.73a 16.52a 10.64b 15.04a 1.08 14.48g Muscle 5.29a 5.79a 5.70a 5.79a .46 5.643 Spleen 17.95a 15.79ab 11.76b 13.88ab 1.38 14.84g Thyroid 3.40b 4.43ab 5.63a 4.35ab .46 4.45i SE 1.82 1.63 1.10 1.10 .80 Group mean 17.18a 17.42a 13.03b 14.53b .57 a,b,cMeans in the same row with different letters in their superscripts differ (P<0.05).
defghMeans in the same column with different letters in their superscripts differ (P











lower in the kidney (P<.05) and lung (P<.O1) of the cattle supplemented with dl-a-tocopherol than for cattle fed the other treatments. Higher concentrations (P<.05) of a-tocopherol were observed in the thyroid of cattle fed dl-a-tocopherol than in that of the cows receiving d-a-tocopheryl acetate, but the a-tocopherol concentrations were higher (P<.05) in the spleen of the group fed d-atocopheryl acetate than in that of the dl-a-tocopherol group. No difference (P>.05) was found for heart and muscle as a result of feeding the various forms of vitamin E.

Across all the treatments, the adrenal gland contained the

highest a-tocopherol concentration, followed generally by the liver or heart; the lowest concentrations were observed in the thyroid and muscle tissues (P<.05) (Table 5.3 and Figure 5.1).

Identity of a-tocopherol in tissues was confirmed by comparing the mass spectra of the a-tocopherol standard to those obtained by collecting the eluates from the HPLC and introducing it into the gas chromatography/mass spectrometry system following silylation. A good spectral match was obtained for the sampled compared with the atocopherol standard spectra. Mass spectra of the gas chromatographic peak corresponding to the a-tocopherol standard (TMSi; Fig. 5.2) and that corresponding to the peak with the same retention time obtained for the plasma, for example, are identical, confirming the presence of a-tocopherol in those biological extracts. The peak obtained at mass/charge 502 represents the molecular ion of a-tocopherol (TSMi: 430 for a-tocopherol + 72 for trimethylsilyl ether). The fragmentation pattern reveals peaks at mass/charge 277 resulting from




Full Text
27
are affected with trembling and very painful to pressure. Standing
becomes increasingly difficult and then impossible, with animals dying
in a few days from heart failure or malnutrition. Death is preceded
by blindness in most cases. Lesions affect the large muscle masses
and the most active muscles. They are found mainly on psoas, crural
and also on intercostals and myocardium.
Affected areas are discolored, whitish, and have the appearance
of fish flesh. Extent of discoloration is varied; sometimes an entire
muscle group may be affected, sometimes only a few fibersthis gives
the muscle a striated, combed effect. A histological examination of
the lesions shows that the striated muscle fibers harbor zenkerism.
Calves
Vitamin E deficiency is also accompanied by selenium deficiency,
which is characterized by white muscle disease (Blaxter and Sharman,
1953). It usually manifests itself in calves which are fed on milk
only and is associated with rapid-growing animals with high
nutritional requirements. Clinical signs appear between 15 days and 3
months, either in stables or at the time of pasturing. In stables,
clinical signs appear gradually. At first, the gait stiffens; animals
are stooped and move slowly when going to suck. Stiffness increases
and standing becomes impossible; polypnea develops.
The polypnea is the only respiratory manifestation of this
syndrome. It is accompanied by heart disorders which are
characterized by a pendulum rhythm and a reduction in heart sounds
upon auscultation, the characteristic signs of myocarditis. Finally,
calves remain in lateral decubitus, with swollen, painful muscles


20
indicates that plasma levels that have been raised to 1.5 to 2 times
the normal levels are less effective than even higher levels. These
plasma levels, in subjects with normal absorption, can be achieved
within 2 to 3 weeks of doses of 3 x 100 or 200 IU/day" (Bieri et al.,
1983). This statement is aimed at discouraging extremely high doses
of vitamin E.
The conditions of physiological absorption discourage very high
individual doses of vitamin E. Absorbing dietary vitamin E usually
presents few problems, since the quantities involved are small and the
required mechanisms of fat digestion, which play an important role
also in the absorption of vitamin E, are triggered automatically.
Large individual doses of vitamin E, however, are not fully absorbed,
two-thirds of them usually being excreted unchanged. Rate of
absorption varies between 20 and 60%, and is dose dependent.
Absorption of tocopherols depends on various factors, e.g., 1)
pancreatic enzymes, 2) bile acids, 3) pH level of the intestinal
contents, 4) intestinal motility, 5) other food components, in
particular the fatty acid composition (Simon-Schmoss et al., 1984).
Metabolism of Vitamin E
Although vitamin E is widely distributed among the tissues of
animals and much is known about the clinical signs and symptoms of its
deficiency, very little is known about the metabolism of vitamin E.
The relative ease with which the tocopherols and their metabolites can
be oxidized in vitro has complicated efforts to elucidate their
mechanism of action (Martius and Furer, 1963).


BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
BY
NICHOLAS HIDIROGLOU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989

This work is dedicated to my parents,
Dr. and Mrs. Hidiroglou, for their
continual support and encouragement
throughout the doctoral program.

ACKNOWLEDGMENTS
The author wishes to express his sincere gratitude to Dr. L. R.
McDowell, chairman of the supervisory committee, for his guidance,
patience and friendship throughout the doctoral program. The author
gratefully acknowledges the assistance and time provided by other
members of the supervisory committee, including Drs. Douglas Bates,
Joseph Conrad, Kermit Bachman and Rachel Shireman.
Deep appreciation is extended to Dr. Keith U. Ingold of the
National Research Council of Canada (NRCC) for providing laboratory
facilities. The author is especially indebted to Dr. Graham W. Burton
of the NRCC for his guidance, patience, and friendship. The technical
assistance of Ann Webb, Dave Lindsay, Ewa Lusztyk of the NRCC is
deeply appreciated. Many special thanks are extended to Dr. L.
Arvanitis for his continual support and friendship throughout my
graduate program.
Assistance of fellow graduate students, Oswaldo Balbuena, Pablo
Cuesta, Edmundo Espinoza, Akhmad Probowo, Rodrigo Pastrana, Libardo
Ochoa and Roger Merkel, during blood and tissue collections is
appreciated. Special gratitude is expressed to Rodrigo Pastrana and
Roger Merkel for continual support during tissue collections from
animals.
Sincere appreciation is expressed to Mrs. Pat French for typing
iii
this dissertation.

TABLE OF CONTENTS
PAGE
DEDICATION
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES viii
ABSTRACT x
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 REVIEW OF LITERATURE 3
Chemistry of Vitamin E 3
Absorption and Storage of Vitamin E 12
The Metabolism of Vitamin E 20
Vitamin E Deficiency 22
Biological Activity of Vitamin E 28
Role of Vitamin E in the Respiratory Chain 41
Intervention of Vitamin E in Hematopoiesis 42
Biological Role of Vitamin E 43
Role of a-Tocopherol as a Chain Breaking
Antioxidant 49
CHAPTER 3 PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP
FOLLOWING INTRAMUSCULAR ADMINISTRATION IN AN OIL
CARRIER 51
Introduction 51
Experimental 51
Analytical 52
Statistical Analysis 53
Results 54
Discussion 59
Summary 61
CHAPTER 4 BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS
IN SHEEP 63
Introduction 63
Materials and Methods 63
Results 67
Discussion 76
Summary 77
IV

PAGE
CHAPTER 5 BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN
E IN BEEF CATTLE AS INFLUENCED BY SUPPLEMENTATION
OF VARIOUS TOCOPHEROL COMPOUNDS 79
Introduction 79
Materials and Method 80
Results 84
Discussion 91
Summary 93
CHAPTER 6 PLASMA TOCOPHEROL IN RUMINANTS AFTER INGESTING
FREE OR ACETYLATED TOCOPHEROL 94
Introduction 94
Materials and Methods 94
Results 99
Discussion 102
Summary 105
CHAPTER 7 PLASMA AND TISSUES VITAMIN E CONCENTRATIONS IN
SHEEP AFTER ADMINISTRATION OF A SINGLE
INTRAPERITONEAL DOSE OF dl-ct-TOCOPHEROL 106
Introduction 106
Experimental Procedure 106
Results 109
Discussion 117
Summary 119
CHAPTER 8 GENERAL CONCLUSIONS 120
LITERATURE CITED 124
BIOGRAPHICAL SKETCH 139
IV

LIST OF TABLES
TABLE PAGE
2.1. NOMENCLATURE FOR TOCOPHEROLS 7
2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS 10
2.3. RELATIVE ACTIVITY OF VARIOUS ALPHA-TOCOPHEROL
STEREOISOMERS AS DETERMINED BY RESORPTION STERILITY
TEST IN FEMALE RATS 33
2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN
THE IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER
AND WEISER (1968) 36
2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR
ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO
HEMOLYSIS TEST IN RATS; EXCERPT FROM BLISS AND
GYORGY (1969) 36
2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL 41
3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN
SHEEP AFTER A SINGLE INTRAMUSCULAR ADMINISTRATION OF
VITAMIN E (40 mg/kg) 56
3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (yg/g wet) OF
SHEEP FOLLOWING A SINGLE INTRAMUSCULAR ADMINISTRATION
(40 mg/kg BODY WEIGHT) OF VARIOUS VITAMIN E
PREPARATIONS 58
4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS
IN TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E... 68
4.2. TISSUE a-TOCOPHEROL CONCENTRATIONS (yg/g FRESH TISSUE)
IN. THE FOUR DIETS 68
4.3. RETENTION TIME OF a-TOCOPHEROL FROM GAS-CHROMATOGRAPHY/
MASS SPECTROMETRY 71
4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS
IN BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF
VITAMIN E 74
4.5. AREA (AUC) UNDER THE PLASMA a-TOCOPHEROL CONCENTRATION
CURVE (yg/ml h_i) 74

TABLE PAGE
5.1. COMPOSITION OF THE GRAIN PORTION OF THE DIET FED TO
COWS FOR 28 d 81
5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (pg/ml) IN CATTLE
FED VARIOUS PREPARATIONS OF VITAMIN E 85
5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (Pg/g FRESH TISSUE)
IN CATTLE FED VARIOUS VITAMIN E PREPARATIONS 88
6.1. DL-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES (LEAST
SQUARE MEANS SE) IN SHEEP AFTER A SINGLE ORAL
ADMINISTRATION OF VITAMIN E (100 mg/kg BODY WEIGHT).... 100
6.2. TRIAL 2: D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES
(LEAST SQUARE MEANS SE) IN CATTLE AFTER A SINGLE
ORAL ADMINISTRATION OF VITAMIN E (50 mg/kg BODY
WEIGHT) 101
7.1. DIET OF SHEEP 107
7.2. MEAN LEVELS OF a-TOCOPHEROL IN TISSUES (pg/g FRESH)
AND PLASMA (pg/ml) AT TIME OF SLAUGHTER 110
7.3. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG TISSUE
VALUES 112
7.4. MEANS OF THE RATIO OF TISSUE TO PLASMA LEVELS OF
a-TOCOPHEROL AT TIME OF SLAUGHTER 114
7.5. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG RATIO OF
TISSUE TO PLASMA 115
7.6. PLASMA TOCOPHEROL (pg/ml) VALUES AND SUMMARIES FOR
THOSE ANIMALS SLAUGHTERED AT 28 DAYS 116
via

LIST OF FIGURES
FIGURE PAGE
2.1 STRUCTURAL FORMULA OF a-TOCOPHEROL 3
2.2 STRUCTURAL FORMULAS OF TOCOPHEROLS 4
2.3 STRUCTURAL FORMULAS OF TOCOTRIENOLS 4
2.4 ASYMMETRIC CARBONS OF TOCOPHEROLS 5
2.5 BIOSYNTHESIS OF THE RING STRUCTURE OF TOCOPHEROLS AND
TOCOTRIENOLS 9
2.6 STRUCTURE OF TOCOPHERYL QUINONE 11
2.7 STRUCTURE OF a-TOCORED 12
2.8 STRUCTURE OF a-TOCOPURPLE 12
2.9 SCHEMATIC REPRESENTATION OF THE PEROXIDATION OF
UNSATURATED FATTY ACIDS 45
3.1 PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE I.M.
INJECTION OF D-a-TOCOPHEROL SUSPENDED IN STERILE
SESAME OIL 55
4.1 a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 69
4.2 IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/
MASS SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD,
(B) a-TOCOPHEROL IN PANCREAS 72
4.3 MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A)
AND a-TOCOPHEROL IN THE PANCREATIC TISSUE (B)
FOLLOWING HPLC COLLECTION 73
4.4 a-TOCOPHEROL CONCENTRATIONS IN THE BLOOD PLASMA OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 75
5.1 a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE
SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS 87
viii

FIGURE PAGE
5.2 MASS SPECTRAL SCANNING OF THE a-TOCOPHEROL STANDARD
(A) AND IN BLOOD PLASMA (B) FOLLOWING HPLC COLLECTION
AND TLC 90
6.1 PLASMA TOCOPHEROL CONCENTRATION (pG/ML) IN SHEEP
FOLLOWING ADMINISTRATION OF A SINGLE MEGADOSE (100 MG/
KG BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL 96
6.2 PLASMA TOCOPHEROL CONCENTRATION (pG/ML) IN CATTLE
FOLLOWING ADMINISTRATION OF A SINGLE DOSE (50 MG/KG
BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL 97
IX

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
By
NICHOLAS HIDIROGLOU
August 1989
Chairman: Dr. L. R. McDowell
Major Department: Animal Science
This research was designed to investigate the bioavailability of
various tocopherol sources for ruminants. In experiment I, d-a-
tocopherol and dl-a-tocopherol suspended in sesame oil were administered
intramuscularly in sheep at a level of 40 mg/kg body weight (b.w.).
Sheep administered d-a-tocopherol and killed after 360 hr resulted in a
higher (P<.01) bioavailability than for sheep injected with d-a-
tocopherol or dl-a-tocopherol and sacrificed 240 hr after dosing. A
more sustained increment in plasma a-tocopherol was observed with d-a-
tocopherol than dl-a-tocopherol.
In experiment II, sheep were provided with 400 IU/d of either d-a-
tocopherol, dl-a-tocopherol or their corresponding acetate forms for 28
d. Higher concentrations of a-tocopherol were observed in tissues of
sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed
d-a-tocopherol, the area under the plasma a-tocopherol time curve (AUC),
was larger as compared to other forms of vitamin E supplementation.
x

Experiment III investigated the concentration of a-tocopherol in
plasma and tissues of beef cows following a daily oral administration of
1000 IU of four tocopherol sources for 28 d. The d-a-tocopherol and its
acetate ester increased plasma tocopherol concentrations faster (P<.05)
than the racemic products, with the greatest response occurring with d-a-
tocopherol. Tissue analyses confirmed that in adrenal gland, kidney,
liver and lung, a-tocopherol concentrations were higher (P<.05) following
d-a-than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered to
sheep or cattle (100 or 50 mg/kg b.w., respectively) in a single oral
dose. Blood plasma a-tocopherol tolerance curve area was higher (P<.05)
in the dl-a-tocopherol group than in its ester form, as well as quicker
(P<.05) time (h) for maximum plasma a-tocopherol concentration. A
greater (P<.05) plasma tolerance curve area was observed in the cattle
following administration of dl-a-tocopherol than its acetylated form.
In experiment V plasma and tissue vitamin E concentrations were
determined following a 5 g dosage of dl-a-tocopherol intraperitoneally in
sheep, which were slaughtered on d 3, 6, 10, 15 and 28 post dosing.
There was a significant time effect for all tissues, while in most, the
peak for a-tocopherol concentrations was observed at 3 d post dosing.
Rate of uptake of vitamin E varied for different tissues, with liver,
spleen and lung showing a pronounced uptake and muscle showing least.
Comparing the established biological potencies (IU/mg) of the
various tocopherol sources to the estimate values for ruminants are as
follows, respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37).
xi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
By
NICHOLAS HIDIROGLOU
August 1989
Chairman: Dr. L. R. McDowell
Major Department: Animal Science
This research was designed to investigate the bioavailability of
various tocopherol sources for ruminants. In experiment I, d-a-
tocopherol and dl-a-tocopherol suspended in sesame oil were administered
intramuscularly in sheep at a level of 40 mg/kg body weight (b.w.).
Sheep administered d-a-tocopherol and killed after 360 hr resulted in a
higher (P<.01) bioavailability than for sheep injected with d-a-
tocopherol or dl-a-tocopherol and sacrificed 240 hr after dosing. A
more sustained increment in plasma a-tocopherol was observed with d-a-
tocopherol than dl-a-tocopherol.
In experiment II, sheep were provided with 400 IU/d of either d-a-
tocopherol, dl-a-tocopherol or their corresponding acetate forms for 28
d. Higher concentrations of a-tocopherol were observed in tissues of
sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed
d-a-tocopherol, the area under the plasma a-tocopherol time curve (AUC),
was larger as compared to other forms of vitamin E supplementation.

Experiment III investigated the concentration of a-tocopherol in
plasma and tissues of beef cows following a daily oral administration of
1000 IU of four tocopherol sources for 28 d. The d-a-tocopherol and its
acetate ester increased plasma tocopherol concentrations faster (PC.05)
than the racemic products, with the greatest response occurring with d-a-
tocopherol. Tissue analyses confirmed that in adrenal gland, kidney,
liver and lung, ct-tocopherol concentrations were higher (PC.05) following
d-a-than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered to
sheep or cattle (100 or 50 mg/kg b.w., respectively) in a single oral
dose. Blood plasma a-tocopherol tolerance curve area was higher (PC.05)
in the dl-a-tocopherol group than in its ester form, as well as quicker
(PC.05) time (h) for maximum plasma a-tocopherol concentration. A
greater (PC.05) plasma tolerance curve area was observed in the cattle
following administration of dl-a-tocopherol than its acetylated form.
In experiment V plasma and tissue vitamin E concentrations were
determined following a 5 g dosage of dl-a-tocopherol intraperitoneally in
sheep, which were slaughtered on d 3, 6, 10, 15 and 28 post dosing.
There was a significant time effect for all tissues, while in most, the
peak for a-tocopherol concentrations was observed at 3 d post dosing.
Rate of uptake of vitamin E varied for different tissues, with liver,
spleen and lung showing a pronounced uptake and muscle showing least.
Comparing the established biological potencies (IU/mg) of the
various tocopherol sources to the estimate values for ruminants are as
follows, respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37).

CHAPTER 1
INTRODUCTION
Existence of an anti-sterility vitamin was brought to light in
the early 1920s when evidence was obtained that female rats reared on
a diet containing all the vitamins known at that time failed to
reproduce, although they were apparently normal in other respects
(Evans and Bishop, 1922; Mattill and Coulin, 1920; Sure, 1924).
Although the rats would mate and conceive, pregnancy invariably was
terminated by fetal death followed by resorption. The missing factor
was characterized as a vitamin by Evans and Bishop (1922) and
designated vitamin E. Evans et al. (1927), who found that the
unsaponifiable fraction of wheat germ oil was a convenient raw
material for chemical investigation, and isolated (1936) from wheat-
germ oil two alcohols, a- and 6-tocopherol, both of which showed
vitamin E activity.
The name tocopherol originated from the Greek tocos (childbirth),
phero (to bear) and ol (alcohol). Two years later, its structure was
elucidated (Fernholz, 1938), and shortly thereafter it was synthesized
(Karrer et al., 1938). During the period 1930-1950, multiple varied
deficiency disorders of animals were reported to be cured by vitamin
E. Work by Schwarz and Folz (1957) led to the identification of
selenium as the factor other than vitamin E that could also prevent
the degeneration of liver in rats. Dose of selenium required for
complete protection against liver degeneration was about one
1

2
five-hundredth that of vitamin E. Biological potencies of various
sources of vitamin E have been established primarily through the rat
fetal resorption assay (Mason and Harris, 1947). From this assay the
following biological potencies were established: 1 mg of dl-a-
tocopheryl acetate =1.0 IU; 1 mg dl-a-tocopherol = 1.1 IU; 1 mg d-a-
tocopheryl acetate = 1.36 IU; 1 mg d-a-tocopherol = 1.49 IU.
Recently, considerable effort has been made to determine the
biological activity of various vitamin E compounds in species other
than rats, and specifically humans (Horwitt et al., 1984; Baker et
al., 1986).
In ruminants, although much is known about clinical signs of
vitamin E deficiency (Rice and McMurray, 1982), very little attention
has been given to the different potencies of various vitamin E
compounds. This dissertation is particularly concerned with the
biological activity of various vitamin E compounds in ruminants as
well as the effect of mode of administration and its type of vehicle.

CHAPTER 2
REVIEW OF LITERATURE
Chemistry of Vitamin E
Structure
The term "vitamin E" today applies to a group of related chemical
compounds, the tocopherols and tocotrienols. The structural formula
of a-tocopherol is indicated in Figure 2.1.
FIGURE 2.1. STRUCTURAL FORMULA OF a-TOCOPHEROL
The following tocopherols (Fig. 2.2) and tocotrienols (Fig. 2.3)
are known:
3

4
Tocopherols Chemical Name
R1 R2 R3
a-Tocopherol
CH
CH-
CH-
5,7,8-Trimethyltocol
3-Tocopherol
CH-
H 3
CHo
5,8-Dimethyltocol
Y-Tocopherol
H 3
CH-
CH-
7,8-Dimethyltocol
3-Tocopherol
H
H 3
CH3
8-Methyltocol
(a)Tocopherois
FIGURE
2.2.
STRUCTURAL
FORMULAS OF TOCOPHEROLS
Tocotrienols
Chemical Name
R1
R2
R3
a-Tocotrienol
CH-
CHo
CH-
5,7,8-Trimethyltocotrienol
3-Tocotrienol
CHo
H 3
CH-
5,8-Dimethyltocotrienol
Y-Tocotrienol
H 3
CH
CH-
7,8-Dimethyltocotrienol
6-Tocotrienol
H
H 3
CHo
8-methyltocotrienol
FIGURE 2.3. STRUCTURAL FORMULAS OF TOCOTRIENOLS

5
Figures 2.2 and 2.3 show that vitamin E exists as eight plant-
derived compounds that have a common 6-chromanol ring structure but
that differ in the side chain and number of methyl groups. The four
tocols have a phytol side chain while the four trienols having trans
double bonds at 3', 7' and 11' of the phytol side chain. The methyl
substituents occur in the ring at positions 5,7,8 (a-), 5,8 (B-), 7,8
(y), and 8 (6-) in both the tocol and trienol series.
The tocols contain three asymmetric carbons specifically at the 2
position (Fig. 2.4) in the ring and in the 4' and 8' position of the
side chain, thus giving a total of eight possible optical isomers.
The epimeric configuration at the 2 position is apparently dominant in
determining biological activity.
FIGURE 2.4. ASYMMETRIC CARBONS OF TOCOPHEROLS

6
The tocotrienols possess only one center of asymmetry at C2, in
addition to sites of geometrical isomerism at C^' and Cy'. Thus, a
number of stereo-isomers of the tocopherols and tocotrienols can
exist. Natural -tocopherol was shown conclusively to have the 2R,
4'R, 8'R configuration (Table 2.1). Thus, RRR-a-tocopherol can be
used to denote the natural tocopherol isomer. The epimer of natural
a-tocopherol, i.e., (2S, 4'R, 8'R)-a-tocopherol can be named 2-epi-a-
tocopherol. A mixture of RRR and SRR-a-tocopherol can be obtained
synthetically and is named 2-ambo-a-tocopherol (ambo = Latin for
both). The reduction product of natural -tocopherol is a mixture of
four diastereo-isomeric a-tocopherols and can be called 4' ambo, 8'
ambo-a-tocopherol. Synthetic a-tocopherol from synthetic phytol or
isophytol is a mixture of four racemates in equal proportions and
should be named all-rac-a-tocopherol.
Bieri and Prival (1967) reported that a group of synthetic
tocopherol derivatives exhibited vitamin E activity. Among those
reported were a-, 8-, and y-tocopheramines and N-methyl-8-, and N-
methyl-y-tocopheramines. The tocopheramine derivatives differ from
the tocopherols in that an amino or an N-methyl amino group is
substituted for the hydroxy group on the C-6 of the ring.
Synthesis
Chemical
Karrer et al. (1938) were the first to synthesize a-tocopherol by
condensation of trimethylhydroquinone with phytyl bromide. Phytyl
bromide was soon replaced by the natural phytol (Karrer and Isler,

7
1938; Fieser et al., 1940) or isophytol (Karrer and Isler, 1941). All
the tocopherols were subsequently synthesized and their structures
elucidated.
TABLE 2.1. NOMENCLATURE FOR TOCOPHEROLS
Description
Configuration
Recommended
Other designation
Natural
2R,4'R,8'R
RRR a-
[d]-a-tocopherol.
Steriochemically uniform
product (only RRR-)
Semisynthetic
2R,4'R,8'R
1 ambo a
dl-a-tocopherol.
Two stereoisomers in the
product (about 50% RRR-a
historical standard)
Synthetic
Mixture of all
All rac-a
[dl]-a-tocopherol.
Eight stereoisomers in
the product (about 12.5%
RRR-a)
Synthetic
2S,4'R,8R
2-epi-a
[ 1 ]-ot-tocopherol.
Steriochemically uniform
product (only SRR-a)
Two principal sources of vitamin E are in commercial use: d-a-
tocopherol and esters (of acetate and succinate) of these compounds.
The acetate esters are prepared chemically by reaction of the alcohol
forms with acetic anhydride; they do not exist in nature. D-a-
tocopherol is largely obtained from natural sources by molecular
distillation. However, some of the d-a-tocopherol is prepared by
further methylation of B-, y-, and 6-tocopherols, or by hydrogenation
of a-tocotrienol.

8
Synthetic dl-a-tocopherol and its esters are prepared from
isophytol. This synthesis yields a mixture of eight isomers. Although
semi-synthetic dl-a-tocopherol is racemic only at carbon 2 as prepared
from natural phytol, samples of this compound may be available for
research though it is not a commercial source of vitamin E.
Biosynthesis
Although conclusive proof of the biosynthetic route is still
lacking, it is probable that the ring structure is formed from tyrosine
by the pathway presented in Figure 2.5 (Whistance and Threlfall, 1967,
1968). Methylation of the ring structure by methyl transfer from
methionine and formation of an initial isoprenoid side-chain from
mevalonic acid completes the formation of a tocotrienol (Griffiths et
al., 1968).
Properties of Vitamin E
Physical properties
Both d-a-tocopherol (RRR) and dl-a-tocopherol (all-rac) are
practically insoluble in water but are almost completely soluble in
oils, fats, acetone, alcohol, chloroform, ether, benzene, and other fat
solvents. All tocopherols are stable to heat and alkali in the absence
of oxygen and are unaffected by acids up to 100C; they are slowly
oxidized by atmospheric oxygen, a process which is increased rapidly by
heat and catalyzed by ferric or silver salts. On exposure to light,
the tocopherols gradually darken. They are not precipitated by
digitonin. The commercial forms of vitamin E are d-a-tocopheryl
acetate and dl-a-tocopheryl acetate.

9
CH2CHNH2COOH
Phe
OH
ch2cooh
O-glucose
homogentisic
acid glucoside
OH
1
Tyr
CH2COCOOH
p-hydroxyphenyl
Oh pyuvate
OH
homoarbulin
O-glucose
FIGURE 2.5. BIOSYNTHESIS OF THE RING STRUCTURE OF
TOCOPHEROLS AND TOCOTRIENOLS

10
The melting point of RRR-a-tocopherol is 2.5 to 3.5C; thus at
room temperature, the compound is a viscous yellow oil which is
soluble in aprotic solvents. Optical rotations of these tocopherols
are very small and depend on the nature of the solvent. Table 2.2
gives the specific rotations of the spectra of natural tocopherols and
tocotrienols in ethanol which show maxima in the range of 292-8 nm;
infrared spectra show OH (2.8 to 3.0 pm) and CH (3.4 to 3.5 pm)
stretching and a characteristic band at 8.6 pm.
TABLE 2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS
Compound
Solvent
25
a
546.1
a-Tocopherol
Ethanol
+0.32
Benzene
-3.0
6-Tocopherol
Ethanol
+2.9
Benzene
+0.9
Y-Tocopherol
Ethanol
+3.2
Benzene
-2.4
6-Tocopherol
Ethanol
+3.4
Benzene
+1.1
Other properties include a molecular weight of 430.69; boiling
point at 0.1 atm (used in molecular distillation) of 200 to 220C;
density 0.95 at 25C in reference to water at 4C, and a refractive
index in sodium light spectrum at 20C of 1.5045.
8-Tocopherol is a viscous, pale yellow oil; absorption maxima of
297 nm; E.1% = 86.4.
1 cm

11
boiling point at 0.1 atm 210-210C. Tocopheryl esters are more stable
to oxygen but cannot function as anti-oxidants in this form.
Metabolic Degradation Products of Tocopherol
The chemistry of the oxidation of tocopherols is highly complex
and has been reviewed by Kasparek (1980). Complete chemical
degradation of the tocopherol molecule occurs upon treatment with
chromic acid and potassium permanganate, while milder oxidation by
nitric acid, silver nitrate, ferric chloride, auric chloride, ceric
sulphate and nitrogen dioxide leads to formation of a-tocopheryl
quinone (Fig. 2.6). With nitric acid and ferric chloride, a-tocored
(Fig. 2.7) and ot-tocopurple (Fig. 2.8) are also formed.
3
FIGURE 2.6. STRUCTURE OF TOCOPHEROL QUINONE

12
FIGURE 2.7. STRUCTURE OF a-TOCORED
FIGURE 2.8. STRUCTURE OF a-TOCOPURPLE
Absorption and Storage of Vitamin E
Mechanism of absorption is similar to that of other fat soluble
vitamins (Weber, 1983). Its absorption is closely associated with
that of fat, and is accelerated by the presence of bile.
Excess tocopherol is eliminated in the feces. Simon et al.
(1956) observed that rabbits receiving a single dose of 10 to 15 mg 5-
methyl-C^-d-a-tocopherol succinate eliminated 65% of the dose via the
feces in 3 days and 80% in 6 days; 90% of the radioactivity was

13
identified as free a-tocopherol by isotope dilution. It was concluded
that the vitamin was re-secreted into the intestinal tract from the
blood or via the bile. In confirmation by Krishnamurthy and Bieri
(1963), who administered the vitamin orally, a second increase in
fecal tocopherol was noticed which they did not attribute to
coprophagy since the rats had tail cups. Mellors and Mcbarnes (1966)
demonstrated that no significant amount of either tocopherol or its
metabolites was introduced into the lumen of the rat guts via the bile
or through secretion from mucosal cells. By using a-tocopherol
labelled with radioactive C^, Shantz (quoted by Harris and Ludwig,
1949) remarked that 80% of the vitamin E given in oil solution was
excreted in the feces of rats.
Dju et al. (1950) observed that chickens receiving 1 g of a-
tocopherol/day for a prolonged period eliminated 75% of the ingested
quantity unchanged via the feces by 24 h. In contrast, chickens
receiving a diet supplemented with 17.5 and 35 mg of a-tocopherol/kg
only eliminated about 23% in the feces (Pudelkiewicz and Matterson,
1960).
Information on the absorption and excretion of tocopherols by
farm animals is extremely sparse. In unpublished work with young
calves, Blaxter and Brown (1952) indicated that about 25% of a-
tocopherol added as the acetate to a diet of dried skimmed milk was
excreted in the feces when the daily intake of the ester was from 25
to 100 mg. From such scanty data it is clearly impossible to draw the
conclusion that species differ in their ability to absorb tocopherols,
especially since great variation has been reported for a single

14
species. Also, with labile substances like the tocopherols, a simple
estimation of absorption based upon amounts present in the food and
feces may be inaccurate.
These and other metabolic studies have shown that tocopherols are
incompletely absorbed. The amount absorbed seems to depend on the
requirement of the organism (Klatskin and Molander, 1952). Absorption
and elimination also seem to depend on the amount in the diet.
There is a linear relation between the logarithms of ingested and
liver tocopherol in rats (Bolliger and Bolliger-Quaife, 1956).
Griffiths (1960) noticed a linear relation between the logarithms of
serum tocopherol and dietary concentration of vitamin E in chickens.
The same author determined the ratio of pure a-, B-, Y-, and <5-
tocopherol in the livers of growing chicks as 100:41:19:0, but his
findings are contradicted by Gray (1959), who found considerably lower
tocopherol levels in rat plasma at high levels of tocopherol intake
than Griffiths (1960) found in chicken serum.
Work of Dju et al. (1950) with hens included the observation that
a-tocopherol was absorbed to a much greater extent than the y- and 6-
compounds. The serum tocopherol values of two hens which received
weekly supplements of 1.6 g and 2.0 g a-tocopherol were 20.0 and 20.1
mg per 100 ml, but two hens that received weekly supplements of 0.8
and 1.0 g 6-tocopherol had serum tocopherol values of only 1.35 and
2.1 mg per 100 ml. Gamma-tocopherol appears to be only one-third to
one-half as well absorbed as a-tocopherol in formula fed infants and
supplemental vitamin E (1200 IU/day of all-rac-a-tocopherol: 800 mg a-
tocopherol equivalents) in adults significantly reduced plasma Y-

15
tocopherol levels, which suggested that intestinal uptake or plasma
transport is more efficient for a- than y-tocopherol (Jonsson et al.,
1981). Tocopherol esters are hydrolyzed prior to absorption, and both
bile and pancreatic juice are necessary for absorption to proceed
(Gallo-Torres, 1970). These facts support the idea that free
tocopherol is absorbed by diffusion from a mixed micelle of fatty
acids, monoacyl glycerols, bile salts and acids, cholesterol and other
fat-soluble vitamins. Maximal absorption occurs at the junction of
the upper and middle thirds of the small intestine (Hollander et al.,
1975). After crossing the brush border into intestinal mucosal cells,
tocopherol is not re-esterified but is incorporated as the alcohol
into chylomicrons in mammals and enters the plasma via the lymphatic
system (Behrens et al., 1982).
Desai et al. (1965) showed, in confirmation of studies by Weber
et al. (1962) with rats, that 1-a-tocopherol was absorbed as well as
or better than the d-form of the vitamin. It appeared therefore that
the differences in biopotency must be due to differences in retention
whereby the d-epimer is retained much better than the 1-epimer in the
blood and perhaps in other tissues of the body. The results indicated
the existence of an active carrier of d-a-tocopherol in the blood and
tissues which has a greater affinity for the d-epimer than for 1-a-
tocopherol. Recently it has been reported that specific binding
proteins exist for a-tocopherol in the cytosol and nuclei of rat liver
tissue (Catignani et al., 1977; Guarnieri et al., 1980 Prasad et al.,
1980) as well as in human erythrocytes (Kitabachi and Wimalasena,
1983) which are fairly specific for the natural stereoisomer. The

16
exact nature of the binding of a-tocopherol in the cell is an elusive
problem which needs investigation. Experiments by Desai and Scott
(1965) and Scott (1965), comparing the oral administration of d- and
1-a-tocopheryl acetates in the presence of graded levels of dietary
selenium, indicated that selenium is involved in some unknown way in
the retention of d-a-tocopherol in plasma. It remains to be
determined if the differences in plasma levels of d- and 1-epimers of
a-tocopherol are the result of differences (1) in rate of excretion,
(2) in rate of destruction, (3) in the affinity of the epimers for
specific carriers, or (4) in chemical activity influenced by
structural configurations.
Gallo-Torres (1970) reported the obligatory role of bile for the
intestinal absorption of vitamin E into the lymph of rats. Only
negligible amounts of radioactivity could be detected in the thoracic
duct lymph when both bile and pancreatic juice were absent from the
duodenum.
The lipid-bile micelle structure is required to transport the
fat-soluble vitamin E across the "unstirred water layer" which
represents the aqueous phase in the intestinal lumen immediately
adjacent to the brush border of the microvilli. Vitamin E is
absorbed, together with free fatty acids, monoglycerides, and other
fat-soluble vitamins, by penetrating the epithelial cell through the
apical plasma membrane of the absorptive cells in the brush border.
Studies on absorption and transport of a-tocopherol by Davies et al.
(1971) showed that, for normal utilization of absorbed a-tocopherol,
normal lipoprotein transport mechanisms are involved. Transfer of

17
vitamin E from the absorptive cell thus requires several stages. In
mammals, it must first pass though the lateral or basal plasma
membrane of the cell, then through the basal lamina before entering
the fluid of the lamina propria. From this location the vitamin
enters the capillaries of the lymph and is transported in the
chylomicrons (Dobbins, 1975). Apparently only small amounts of
tocopherol are transported from the intestine via the portal vein in
mammals, whereas all of the tocopherol absorption in birds occurs via
the portal vein directly to the liver (Machlin, 1984).
The absorption of orally fed vitamin E follows the pattern of
lipids in general and of fat soluble vitamins in particular (Desai et
al., 1965; Wiss et al., 1962). The specific site of absorption is not
well established. The small intestine is thought to be the major site
of absorption for tocopherol even though some absorption takes place
from the stomach of nonruminants and the rumen of ruminants (Blaxter
and Brown, 1952; Roles, 1967). The presence of vitamin E in both the
blood and lymph of animals suggests that absorbed vitamin E can be
transferred by either the blood or lymphatic route (Roles, 1967).
Wiss et al. (1962) were also able to establish a mathematical
relationship between the logarithms of tocopherol intake and plasma
and liver concentration in chickens fed high doses of d-ot-tocopheryl
acetate (2,000 to 20,000 IU/kg of feed). Using [C^]-dl-a-tocopheryl
acetate, they demonstrated that maximal liver concentration was
reached only after several hours, and persisted longer than the
synthetic antioxidant ethoxyquin (6-ethoxy-l,2-dihydro-2,2,4-
trimethylquinoline), which attained a maximum within 30 min before

18
declining rapidly. Most of the tocopherol was bound to the structural
components of the liver cells, primarily mitochondria and microsomes.
Vitamin E is transported in the blood via lipoproteins. A rapid
exchange among lipoprotein classes occurs with vitamin E after it
enters the circulation via the chylomicrons. Since plasma tocopherol
level is correlated with the total plasma lipid content, low density
lipoprotein (LDL), the most common lipoprotein in human plasma,
carries most of the plasma vitamin E. There is also a rapid exchange
between tocopherol in the erythrocyte membrane and lipoproteins such
that approximately 20% of the plasma tocopherol concentration is
carried by red blood cells. Delivery to other tissue cells appears to
be in association with the receptor-mediated uptake of LDL (Traber and
Kayden, 1984).
Type and composition of diet influence absorption of vitamin E
from the gut. Pudelkiewicz and Matterson (1960) reported that only
about one-third of the d-ci-tocopherol in alfalfa is available to
chicks. The poor utilization is attributed to a fat-soluble compound
in alfalfa that acts antagonistically to a-tocopherol decreasing its
availability. An antagonistic relationship also exists between
absorption of vitamin E and unsaturated fatty acids (Bunyan et al.,
1968).
Generally, in animals, the uptake of vitamin E from the small
intestine is lowered when tocopherol is fed in an oily form. The
uptake is facilitated by bile salts (Simon et al., 1956).
When tocopheryl acetate ester was fed, most fecal tocopherol
appeared as free tocopherol; however, a small portion appeared in the

19
esterified form (Krishnamurthy and Bieri, 1963) indicating that
hydrolysis of the ester took place in the digestive tract.
Rosenkrantz et al. (1951) observed that approximately 8% of ex-
tocopherol given to human subjects was recovered as tocopherol
quinone, suggesting the oxidation of tocopherol in the human digestive
system.
No tocopherol or tocopheryl quinone was excreted in rat urine
although large doses of vitamin E were administered (Klatskin and
Molander, 1952). McArthur and Watson (1939) failed to detect any
tocopherol in cow urine. Simon et al. (1956) reported that about 18%
of tocopherol intake appeared in the urine of the rabbit and man after
intravenous administration of isotopic vitamin E. Urinary radio
activity was not attributed to a-tocopherol or tocopheryl quinone but
to two polar metabolites identified as an acid and its lactone which
were subsequently named tocopheronic acid and tocopheronolactone
(Green et al., 1967). Work by Chow et al. (1967) confirmed the
findings of Simon and associates.
Krishnamurthy and Bieri (1963) reported that only 0.5% of total
activity was in the urine when the rat was sacrificed 24 h after oral
14
administration of C a-tocopherol. In a long-term experiment, the
same authors found that the activity in the urine was very low.
Furthermore, no significant amount of vitamin E was lost in the form
of CO2.
Almost all studies on the pharmacological properties of vitamin E
used doses between 200 and 800 mg/day. John Bieri, a leading American
expert on vitamin E, had the following to say on dosage: "Nothing

20
indicates that plasma levels that have been raised to 1.5 to 2 times
the normal levels are less effective than even higher levels. These
plasma levels, in subjects with normal absorption, can be achieved
within 2 to 3 weeks of doses of 3 x 100 or 200 IU/day" (Bieri et al.,
1983). This statement is aimed at discouraging extremely high doses
of vitamin E.
The conditions of physiological absorption discourage very high
individual doses of vitamin E. Absorbing dietary vitamin E usually
presents few problems, since the quantities involved are small and the
required mechanisms of fat digestion, which play an important role
also in the absorption of vitamin E, are triggered automatically.
Large individual doses of vitamin E, however, are not fully absorbed,
two-thirds of them usually being excreted unchanged. Rate of
absorption varies between 20 and 60%, and is dose dependent.
Absorption of tocopherols depends on various factors, e.g., 1)
pancreatic enzymes, 2) bile acids, 3) pH level of the intestinal
contents, 4) intestinal motility, 5) other food components, in
particular the fatty acid composition (Simon-Schmoss et al., 1984).
Metabolism of Vitamin E
Although vitamin E is widely distributed among the tissues of
animals and much is known about the clinical signs and symptoms of its
deficiency, very little is known about the metabolism of vitamin E.
The relative ease with which the tocopherols and their metabolites can
be oxidized in vitro has complicated efforts to elucidate their
mechanism of action (Martius and Furer, 1963).

21
Investigations on the metabolites of a-tocopherol in animal
tissues were conducted by several workers with the anticipation that
the nature of the metabolites would provide insight into the mechanism
of action of vitamin E. Simon et al. (1956) isolated and
characterized a metabolite of a-tocopherol from both rabbit and human
urine. This oxidation product was given the common name tocopherol-5-
methyl-C^ succinate. The same authors found that urine contained
mainly metabolized tocopherol, while intact tocopherol accounted for
the majority of the radioactivity in the feces. Mervyn and Morton
(1959) reported the presence of a-tocopheryl quinone in a nephritic
human kidney deficient in vitamin E. Alaupovic et al. (1961) found no
evidence of a-tocopheryl quinone, a-tocopheryl hydroquinone, a-
tocopheroxide, tocopheronic acid or tocopheronolactone in pig and rat
livers. Csallany et al. (1962) identified two new metabolites of a-
tocopherol isolated from the liver of the rat after injection of [5-
methyl-^C] d-a-tocopherol. About 25% of the recovered activity was
unchanged a-tocopherol, about 19% was a-tocopheryl quinone, and about
50% was dl-a-tocopherone, a dimer of oxidized -tocopherol. Csallany
and Draper (1963, 1964) separated and identified the cis-and trans-
isomers of dialpha-tocopherone. Draper et al. (1967) also identified
a trimer of a-tocopherol in the mammalian liver. Krishnamurthy and
Bieri (1963), and Plack and Bieri (1964) indicated that oral and
intraperitoneal administration of a-tocopherol to rats and chicks
gave rise mostly to unchanged tocopherol in the tissues of the tested
animals. Mellors and McBarnes (1966) confirmed the above findings.
Following intravenous injection of H-a-tocopherol, Shiratori (1974)

22
observed large amounts of radioactivity in dermal tissue suggesting
that skin may play a role for tocopherol secretion or excretion.
Vitamin E Deficiency
The absence of vitamin E in the diets of animals poses health
problems and affects various functions.
Effects of Deficiency on the Reproductive System
In 1920, Mattill and Conklin showed that a deficiency in vitamin
E led to reproductive disorders and lesions of the genital apparatus
in rats. The data were confirmed in 1922 by Evans and Bishop. As a
result of some premature generalization of these observations, vitamin
E came to be considered as an anti-sterility factor and its
therapeutic use was widely advocated. Now, however, it is apparent
that the role of this factor varies widely among species.
In males
Rodents. In rats, vitamin E deficiency results in testicle
degeneration (Mason, 1954). The gland is reduced to half its normal
size and becomes brownish and flaccid. Degenerative lesions affect
the spermiduct and lead to the complete cessation of sperm production.
Neither Sertoli cells nor the interstitial gland appear to be
affected. Some changes in related glands (seminal vesicles and
prostate) have been reported.
It appears that guinea pigs (Curto, 1966), hamsters and rabbits
are also susceptible, although less markedly so. In rabbits, in
particular, testicle lesions seem minor when compared to the

23
widespread effects of vitamin E deficiency on muscles (Hove and
Harris, 1947).
Other species. Monkeys and roosters are also susceptible to
vitamin E deficiency. Stallions, bulls, rams, billy goats, and
perhaps male mice, are not (Baxter and Brown, 1952).
In females
Rodents. In rats, vitamin E deficiency does not appear to affect
the development and functioning of the genital system (estrus,
ovulation, fecundation and nesting), but it has a major impact on
prenatal development, mainly during the days following nesting. The
uterus and the ovaries become covered with brownish yellow pigment
while the basic signs are early embryo mortality followed by
resorption as a result of placental lesions which may sometimes
hemorrhage. When there is a chronic deficiency, the rat may produce
only one or two live young, while the others die (Martin and Moore,
1939). The survivors present serious muscular dystrophy lesions
(Bonetti and Stirpe, 1963). This cause should be kept in mind when a
reduction in fecundity in this species occurs.
Other species. Where there is vitamin E deficiency, feto
placental hemorrhages and some degree of embryo mortality have
occasionally been noted in sows and rats. Surviving piglets develop
poorly and present muscular dystrophy. It may be concluded that sows
are susceptible to vitamin E deficiency, though to less extent than
rats (Lannek, 1962).
The effects on reproduction in cows, ewes, goats and mares are
nil, although these species are very susceptible to muscular dystrophy

24
lesions, especially in the very young (Gullickson, 1949). However, a
high placental susceptibility to vitamin E deficiency as noted in rats
and sows, apparently in mice and perhaps in rabbits, also appears to
exist in cows according to Trinder et al. (1969). These authors noted
a high frequency of placenta retentions (19%) in non-brucellic cattle
in areas where muscular dystrophy in lambs was widespread. Intra
muscular injections of vitamin E (680 IU) and potassium selenate (15
mg) one month before parturition markedly reduced the frequency of
retention. Administrations of potassium selenate only were less
effective, and earlier injections (7 weeks prepartum) ineffective.
A Variety of Disorders Due to Vitamin E Deficiency in Domestic Animals
Vitamin E deficiency causes various disorders in most animal
species. An exhaustive description would be tedious and time
consuming. Observations will be limited to three frequently affected
species: chickens, lambs and calves.
Chickens
Encephalomalacia. Clinical signs of nutrition-related
encephalomalacia are exclusively nerve-related (Dam, 1944). The first
manifestation is ataxia. Next, there may be clonic contractions and
especially trembling. The chicken keeps its head tucked under, as if
rolled into a ball; then paralysis sets in and it falls down in
lateral decubitus. Death is the usual outcome of this clinical form
which entails 20 to 80% losses, depending on the intensity of the
deficiency and the susceptibility of the subjects. Lesions are
significant because they make it possible to distinguish this disorder

25
from another neural disease of viral origin in chickens, infectious
encephalomyelitis.
Characteristics of nutrition-related encephalomalacia (Adamstone,
1947), are (1) discrete hepatic lesions; the liver is often pale,
degenerated, speckled with sanguinous suffusions; (2) intestinal
hemorrhagic lesions, occurring mainly around the duodenum and
involving the muscularis; and (3) cerebral lesions, more specific,
macroscopically detectable in some cases, but always visible upon
histological examination.
Upon opening the cranium, the cerebellum is edematized, covered
with hemorrhagic patches, and of very poor consistency. Microscopic
examination confirms the edema and hemorrhages, and in addition makes
it possible to detect changes in Purkinje cells, which appear
retracted, with granulous cytoplasm and pycnic nuclei.
In viral encephalomyelitis, the nerve lesions are different from
the encephalomalacia with perivascular lymphocytic infiltration. In
addition, lymphocytic infiltration can be detected in the pancreas,
the succenturiate ventricle and the muscularis of the gizzard.
The distinction between these two diseases is an important one,
since in encephalomalacia the responsible agent is the feed, whereas
in encephalomyelitis it is the breeder, since contamination of the
chickens occurs jt_ ovo.
Exudative diathesis Exudative diathesis appears in chickens at
the same age as does encephalomalcia and is due to a vitamin E
deficiency associated with a deficiency in sulfur amino acids (Bunyan
et al., 1962). It is characterized by the appearance of subcutaneous

26
edema which deforms the head and neck and gives the feet a greenish
tint, hence its other name of "green foot disease" (Dam and Glavind,
1939).
Lesions are also edematous and affect the entire subcutaneous
connective tissue. This tissue has a gelatinous aspect and presents
hemorrhagic spots, in particular on major muscle masses and in the
periarticular area. In addition, whitish muscular dystrophy patches
are found fairly regularly in the gizzard area.
Muscular dystrophy. Muscular dystrophy appears around the age of
one month. It takes the form of locomotor troubles and weight loss
and entails considerable mortality. Lesions affect striated muscle
fibers which present zenkerism. They are closely comparable to those
of myopathy-dyspnea in calves and muscular dystrophy in lambs.
Muscular dystrophy in chickens appears to be due to a deficiency of
both vitamin E and selenium.
Lambs
This deficiency is also associated with clinical manifestations
appearing in areas where the soil is selenium-poor (Muth et al.,
1961). Winter lambs born of ewes which are more or less underfed pay
the heaviest consequence, particularly where ewes have two or three
young, whose requirements cannot easily be met (Oldfield et al.,
1963).
Clinical signs begin inconspicuously with listlessness; sick
lambs dislike moving about, and have trouble following the rest of the
flock. However, the appetite remains normal. Gradually, the gait
changes; steps become smaller, posteriors are stiff and splayed, and

27
are affected with trembling and very painful to pressure. Standing
becomes increasingly difficult and then impossible, with animals dying
in a few days from heart failure or malnutrition. Death is preceded
by blindness in most cases. Lesions affect the large muscle masses
and the most active muscles. They are found mainly on psoas, crural
and also on intercostals and myocardium.
Affected areas are discolored, whitish, and have the appearance
of fish flesh. Extent of discoloration is varied; sometimes an entire
muscle group may be affected, sometimes only a few fibersthis gives
the muscle a striated, combed effect. A histological examination of
the lesions shows that the striated muscle fibers harbor zenkerism.
Calves
Vitamin E deficiency is also accompanied by selenium deficiency,
which is characterized by white muscle disease (Blaxter and Sharman,
1953). It usually manifests itself in calves which are fed on milk
only and is associated with rapid-growing animals with high
nutritional requirements. Clinical signs appear between 15 days and 3
months, either in stables or at the time of pasturing. In stables,
clinical signs appear gradually. At first, the gait stiffens; animals
are stooped and move slowly when going to suck. Stiffness increases
and standing becomes impossible; polypnea develops.
The polypnea is the only respiratory manifestation of this
syndrome. It is accompanied by heart disorders which are
characterized by a pendulum rhythm and a reduction in heart sounds
upon auscultation, the characteristic signs of myocarditis. Finally,
calves remain in lateral decubitus, with swollen, painful muscles

28
affected by trembling, and die of either pulmonary edema or heart
failure.
In pasture calves, the syndrome appears when they are released in
the spring. They begin by running and jumping, but suddenly one or
two may slow down and stand still, with a stiff, awkward stance. Then
they lie down and are unable to get up. In this case also, heart and
respiratory disorders lead to swift death (King and Maplesdon, 1960).
Vitamin E-selenium lesions are identical with those of lambs,
both macro- and microscopically. From the foregoing it will appear
that vitamin E deficiency appears to lead to two major types of
syndromes: (1) degenerative muscular lesions, common to a number of
species and generally related to selenium deficiency, and (2) vascular
lesions (edema and hemorrhages), well known in poultry, affecting
mainly nervous and subcutaneous connective tissues.
Biological Activity of Vitamin E
Biological activities of the various tocopherols are different.
Extensive and very complex studies on several experimental animal
species have been conducted to determine the biological activity of
vitamin E components which have been described in the literature for
many years.
Methods of Assay
Determination of biologically active vitamin E is complicated by
many interrelated factors. Chemical analyses must contend with the
fact that eight different tocopherols occur in nature, and synthetic

29
forms are supplemented in many foods and feeds as dl-a-tocopherol or
as d- and dl-a-tocopheryl acetate or succinates. Bioassays may be
confounded by the levels of antioxidants, prooxidants, selenium, and
other factors which may alter the biological activity of the vitamin.
The biological activity of tocopherols (and of substances with
vitamin E-like activity) can only be assessed with a bioassay
technique which determines the ability of the tocopherol to reverse
clinical signs of vitamin E deficiency. The manifestations of a
deficiency differ markedly in different species. The signs of
deficiency most commonly used in bioassay work are rat sterility
(fetal resorption in the female and testicular atrophy in the male),
rat erythrocyte hemolysis, and muscular dystrophy in a number of
different species. A comprehensive review of bioassay methods for
tocopherols has been reported by Bliss and Gyorgy (1967) and Ames
(1971). Other tests, such as the liver storage, and elevation of
plasma tocopherol, have also been used. These latter methods are not
direct measures of biological activity iji vivo, but they do reflect
the relative absorption of the test compounds as well as their
turnover in the liver or red blood cells. In general, the erythrocyte
hemolysis and tissue storage tests correlate well with the i£ vivo
procedures and may be correlated with alterations in red blood cell
half-life observed in vitamin E-deficient humans. Therefore, although
caution should be used in interpreting results, these methods are
convenient and may be useful.

30
Resorption sterility test
The fetal resorption test in female rats is the classical means
of conducting the bioassay of tocopherols (Mason and Harris, 1947;
Ames et al., 1963). It measures the all-or-none response to the test
substance by successfully mated vitamin E-deficient female rats in
producing viable offspring.
The resorption sterility test in rats is frequently used in
addition to the hemolysis test. This test system was used to
determine the biological activity of the eight enantiomers of the
synthetic all-rac-a-tocopheryl acetate (Weiser and Vechi, 1982).
The resorption sterility test is based on the fact that female
rats supplied with a vitamin E-deficient diet are unable to carry
their offspring to term. The fetuses die before the end of the
gestation period and are subject to intra-uterine resorption. Weaned
rats are given a vitamin E-free diet for 3 to 4 months at which time
their sterility is assessed by test matings with fertile males;
insemination is checked by vaginal smear for spermatozoa. Different
levels of standard RRR-a-tocopherol and the unknown substances are
added to olive oil (or sometimes to portions of diet) which is
administered to pregnant females through the 5th to 9th days of
pregnancy. If dosing is delayed beyond the 10th to 12th day of
pregnancy, vitamin E is ineffective. The animals are killed on the
19th day, i.e., 2 days before parturition, and examined for living
fetuses and for sites of implantation of fetuses that have been
resorbed. Animals having less than four implantation sites are
discarded, and those having one or more living fetuses are regarded as

31
positive, while rats with no live young are negative. The vitamin E
activity of the test substance is determined by calculating the
percentage of rats in each test group that showed a positive response
and by plotting the calculated probits against dose (or log dose) to
derive the 50% fertility dose. The ratio of the 50% fertility dose of
the standard to the unknown substance is the relative vitamin E
biopotency of the test substance. It will be clear from the foregoing
that the fetal resorption test is tedious and time consuming and that
meaningful results can be achieved only by carrying out a large number
of tests on any given test substance, but it must still be regarded as
the final reference point in assessing vitamin E biopotency of an
unknown substance.
This test model is species-specific in its validity. The test
results cannot be directly applied to man, but because this model can
be standardized it can be used for comparisons of structure and
activity relationships. As in any other specific animal model, its
validity limits must be taken into account. The results obtained for
relative activity of the free, easily oxidizable tocopherols vary
greatly in this model from one team of investigators to another
(different laboratories) since the free tocopherols are easily
inactivated by other components in the diet of the experimental
animals. Knowledge of this interference dates back to the 1940s, and
consequently this model is used mainly to test and compare acetylated
tocopherols.
The results tabulated in Table 2.3 show that it is the
configuration in the phytol side chain which decisively affects the

32
activity in the biological system. The 2R, 4'R, 8'R configuration,
which is identical to the naturally occurring form, has the highest
activity (100% relative biological activity). The configuration in
the C-2 position is particularly important. It is noted that in all
the disastereomer pairs the 2S form has less activity than the 2R
form. The greatest difference in activity is noted in a comparison
between the 2S, 4'R, 8'R-a-tocopheryl acetate and the 2R, 4'R,8'R-a-
tocopheryl acetate, which is identical to the naturally occurring
form. The relative activity is only 21 to 31% for the SRR form. The
SSS isomer exhibits the surprisingly high relative activity of 60%
compared to the RRR isomer.
Observations on the biological activity of stereoisomer mixtures
vary. Ames (1979) showed on the basis of 19 tests that the activity
of all-rac-a-tocopheryl acetate does not exceed the calculable
activity. By contrast, Weiser and Vechi (1982) found in two
experiments that stereoisomer mixtures had a synergistic effect in the
rat resorption sterility test.
Erythrocyte hemolysis test
This test is based on the protection of erythrocytes by vitamin E
against hemolysis induced by dialuric acid or hydrogen peroxide. Rats
weighing about 100 g are fed a diet free of vitamin E for a depletion
period of 3 to 4 weeks. A sample of blood is then taken from each rat
and tested for hemolysis induction by addition of dialuric acid. Only
animals showing a degree of hemolysis of 96 to 99% are retained in the
test. In a preliminary trial with five animals per dose, dosages of
vitamin E are determined which will reduce the hemolysis to a range of

33
TABLE 2.3. RELATIVE ACTIVITY OF VARIOUS o-TOCOPHEROL STEREOISOMERS
AS DETERMINED BY RESORPTION STERILITY TEST IN FEMALE RATS.
Standard with 100% biological activity:
RRR-a-tocopherol acetate a- Relative activity in relation
tocopherol acetate isomer to the standard (in percent)
2R,4'R,8'R
100
2R,4R,8'S
90
2R,4'S,8'S
73
2S,4'S,8'S
60
2R,4'S,8'R
57
2S,4'R,8'R
31
2S,4R,8'S
37
2S,4'S,8'R
21
all-rac-a
32
(8 enantiomers)
67-77
2-ambo-a
60.2
(2 enantiomers)
73

34
approximately 20 to 80% for both the standard and the test
preparations. For the bioassay, two or three doses are selected from
within this range for the standard and for each sample to be assayed.
Each dose is dissolved in 0.2 ml olive oil and administered orally by
stomach tube. After 40 to 44 hr the percent erythrocyte hemolysis is
again measured. The use of dialuric acid as a hemolytic agent for
erythrocytes derived from vitamin E-deficient animals was introduced
by Rose and Gyorgy (1952), and later improved by Friedman et al.
(1958).
As an alternative to dialuric acid, hydrogen peroxide may be used
as the hemolytic agent (Gyorgy et al., 1952). It should be noted that
there is no spontaneous hemolysis iri vivo in vitamin E-deficient
animals. However, the rat hemolysis test is not a direct measure in
vivo of the biopotency of a vitamin E-active substance, since the test
is carried out on blood derived from rats given a vitamin E-deficient
diet. Furthermore, the results obtained need to be interpreted with
great caution since the period of giving the deficient diet needs only
be quite short before a positive test can be obtained, and the animal
would not necessarily show overt signs of vitamin E deficiency such as
sterility. The test is thus regarded by many as a measurement of the
vitamin E content of the blood rather than as an assessment of the
vitamin E status of the animal. With this difficulty in mind,
however, the test provides a means of assessing the probable vitamin E
activity of an unknown substance and has been found to agree well with
the results obtained by fetal resorption (Harris and Ludwig, 1949) and
chick liver storage (Pudelkiewicz et al., 1960).

35
Of all the test systems, the erythrocyte hemolysis test (EHT) is
of special importance. This test can be done not only as a pure in
vitro variant but also as an iin vivo-in vitro form. The EHT provides
a means of indirect measurement of the human vitamin E status and
correlates well with the tocopherol concentrations in blood plasma.
Individuals with decreased tocopherol concentration are characterized
by an increased hydrogen peroxide-induced sensitivity of the
erythrocytes to hemolysis in the blood serum.
The most commonly used hemolytic agent is hydrogen peroxide.
Washed erythrocytes are incubated in a hydrogen peroxide solution
(about 2%) over a period of 3 hr. Hemolysis liberates hemoglobin
during incubation; correction for this hemoglobin is made by
determining the amount of hemoglobin after incubation with distilled
water which corresponds to 100% hemolysis (without addition). The
hemolysis result obtained with hydrogen peroxide is given as a
percentage.
The relative activity of d-a-tocopherol and d-y-tocopherol in the
in vitro EHT is tabulated in Table 2.4 (Brubacher and Weiser, 1968).
Erythrocytes from rats which were given a vitamin E-deficient diet
were incubated in a test tube together with the various tocopherols
and then hemolyzed with dialuric acid. The tocopherol esterified with
acetic acid is inactive in this test. D-ct-tocopherol has again been
shown to be biologically superior to d-a-tocopherol.
The hemolysis test was originally conceived as an i^ vitro test
but was later modified. This modified test differs from the above-
mentioned in^ vitro test in that the vitamin E active substance is

36
TABLE 2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN THE
IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER AND WEISER (1968).
Compound
Relative activity (%)
d-a tocopherol
100
d-y tocopherol
30
administered orally to the experimental animals 2 days before blood
samples are taken, after which the hemolysis test is undertaken. This
test model shows that even the forms which are esterified with acetic
acid exhibit vitamin E activity after they have been liberated by
enzymes in the intestine (Table 2.5). Here again it is the d-a
configuration, both in the free form and in the form esterified with
TABLE 2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR
ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO HEMOLYSIS TEST IN
RATS; EXCERPT FROM BLISS AND GYORGY (1969).
Compound
Relative activity (%)
3 d-a-tocopherol 132.8
4 D-a-tocopheryl acetate 147.4
5 all-rac-a-tocopheryl acetate 100.0 (standard)
6 D-y-tocopherol 21.7
7 D-y-tocopheryl acetate
18.9

37
acetic acid, that possesses the highest biological activity. Although
the test method is not precise enough to differentiate d-a-tocopherol
from its acetate, the two forms with the natural configuration are
nevertheless significantly superior to the synthetic all-rac-a-
tocopheryl acetate.
Plasma and erythrocyte concentration test
Vitamin E acts as an antioxidant in biological membranes (Dam,
1957). Therapy is designed to achieve a high tocopherol concentration
in these membranes.
In test rats which were reared on a vitamin E-deficient diet,
free d-a-tocopherol was compared with synthetic all-rac-a-tocopheryl
acetate to determine their effect in producing changes in vitamin E
concentration in plasma and in erythrocytes after intravenous or oral
administration (Ogihara et al., 1985). The tocopherol concentration
in the red blood cells was investigated as an indicator of the
physiologically available tocopherol in biological membranes.
Although intravenous administration of all-rac-a-tocopheryl
acetate produced a higher tocopherol level in plasma than RRR-a-
tocopherol 6 h after infusion, the concentration in the erythrocytes
at the same time increased by 100% after infusion of RRR-a-tocopherol
compared to the response when all-rac-a-tocopheryl acetate was
infused.
Differences between the natural, free vitamin E and the synthetic
acetate ester were even more pronounced in these test animals after
oral administration. Plasma concentration 6 h after oral
administration of d-a-tocopherol was 40% higher than after all-rac-a-

38
tocopheryl acetate intake. Uptake in the erythrocytes 6 h after oral
administration of d-a-tocopherol was 280% higher than after
administration of the synthetic material. Direct comparison at 24 and
48 h after oral administration indicated that uptakes of the free d-a-
tocopherol in the erythrocytes was 300 and 255% greater than uptake of
the ester. These test results provided impressive support for the
superiority of the natural vitamin E structure as previously shown in
clinical studies.
Liver storage tests
Liver storage tests are based on the observation that amount of
tocopherol stored in the liver of rats and chicks has a linear
relationship to the level of vitamin E consumed.
Pudelkiewicz et al. (1960) used day-old White Plymouth Rock male
chicks fed a vitamin E-deficient diet for 13 days for assay of liver
storage of vitamin E. Smallest and largest chicks are discarded, with
those remaining distributed by weight into groups of eight chicks
each. The tocopherol standards and the unknown materials to be
assayed are then mixed with the basal diet and the chicks in all lots
are pair-fed to the lot eating the least amount of feed over a period
of 14 days. Tocopherol content of pooled livers from each group was
then determined by chemical assay using the Emmerie-Engel reaction
after careful extraction, molecular distillation, and chromatographic
separation of the tocopherols.

39
Muscular degeneration tests
Muscular dystrophy has been used as a means of measuring vitamin
E biopotency in rabbits (Hove and Harris, 1947; Fitch and Diehl, 1965)
and chicks (Scott and Desai, 1964; Desai and Scott, 1965).
In chickens, muscular degeneration is evaluated directly after 3
to 4 weeks on deficient or supplemented diets. The breast muscle is
examined and scored from 0 to 4, depending on the severity of the
lesion. Indirect measure of muscular degeneration, such as
creatinuria and levels of plasma aspartate aminotransferase and lactic
dehydrogenase, have also been used in ducks and rabbits. The time of
onset of creatinuria or the extent of muscular dystrophy, assessed on
a scale, are both used as biological measures, and the effect on these
parameters of standard RRR-a-tocopherol or a test material is assessed
as the basis of the assay. A method based on the reversal of muscular
degeneration in the rat, using plasma pyruvate kinase levels as an
index, appears to be quite reliable, rapid, and sensitive (Machlin et
al., 1982).
D-a-Tocopherol Equivalents
A basis for establishing the biological activity of vitamin E
preparations is necessary to ensure uniformity of dosage. In 1978 the
International Union of Nutritional Sciences (IUNS), therefore,
recommended that d-a-tocopherol be used as the standard compound and
that the biological activity be specified in activity equivalents
(Fromming, 1984).

40
D-a-tocopheryl acetate can be easily obtained in chemically and
isomerically pure form. The relative biological activities of the
tocopherols have been determined in experimental animals and are now
internationally accepted. They are calculated as follows:
1 d-a-tocopherol equivalent (mg)
= d-a-tocopherol x 1
= d-a-tocopheryl acetate x 0.91
= all-rac-a-tocopheryl acetate x 0.67
= d-y-tocopherol x 0.1.
These equivalence values have been disputed (Ames, 1979; Leth and
Sandergaard, 1983), however, in 1983 they were confirmed by a WH0/FA0
Expert Committee (WHO, 1983). Evidently the experts did not consider
it necessary to change the relative biological activities of
tocopherols based on animal experiments. More recent human studies
suggest that the findings on which the conversion factors are based,
i.e., experiments with rats given a vitamin E-deficient diet, should
be regarded as species-specific for the experimental animals.
A comparison of the tocopherol concentrations in human tissues
and cells emphasizes the physiological significance of free RRR-a-
tocopherol relative to other tocopherols and derivatives. The vitamin
E requirement in man is satisfied by the preferential utilization of
d-a-tocopherol in the diet. That d-a-tocopherol possesses the
relatively greatest biological activity has been confirmed by a
multitude of relevant i^n vitro and Tn vivo tests and particularly by
clinical studies. Accordingly, RRR-a-tocopherol is the actual vitamin
E in man.

41
The United States Pharmacopoeia (USP) (1980) in Table 2.6 shows
the recommended weight/unit relationships for six tocopherols:
TABLE 2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL.
1 mg all-rac-o-tocopherol = 1.1 units
1 mg all-rac-a-tocopheryl acetate = 1 unit
1 mg all-rac-a-tocopheryl succinate = 0.89 unit
1 mg RRR-a-tocopherol = 1.49 units
1 mg RRR-a-tocopheryl acetate = 1.36 units
1 mg RRR-a-tocopheryl succinate = 1.21 units
Role of Vitamin E in the Respiratory Chain
It has long been known that the hepatic necrosis that accompanies
vitamin E deficiency is preceded by a reduction of cell respiration
shown on cross sections of prenecrotic liver (Chernick et al., 1955).
Vitamin E supplementation immediately reestablishes respiration, both
in vivo and in vitro (Schwarz, 1955). Selenium is also effective, but
only in^ vivo, which seems to indicate that a different mechanism is at
work (Schwarz, 1972).
Cell respiration takes place at the level of the mitochondria,
through the respiratory chain, which consists of a series of hydrogen-
transport systems which intervene in an order determined by their
oxidoreduction potentials. Energy released by this hydrogen transport

42
system (which corresponds to oxidation of the substrate) is partially
stored in the form of adenosine triphosphate (ATP). Determination of
the P/0 ratio (number of atoms of inorganic P which disappear when one
atom of oxygen is reduced), which is generally equal to 3, provides a
measure of cellular respiratory activity.
One of the links in the respiratory chain is ubiquinone (or
coenzyme Q) which intervenes between the flavin-nucleotide and
cytochrome system. The structure of ubiquinone is similar to that of
a-tocoquinone, a metabolite of a-tocopherol which, along with the
corresponding hydroquinone can form a hydrogen transport system
comparable to that of ubiquinone.
In the anemias observed when vitamin E deficiencies occur in
primates, an improvement is obtained upon administration of ubiquinone
as with vitamin E, thereby indicating the similarity of action for the
two substances.
It is likely that vitamin E intervenes in the respiratory chain,
and thereby contributes to the metabolic equilibrium of cells
(Schwarz, 1972).
Intervention of Vitamin E in Hematopoiesis
Anemia, which accompanies vitamin E deficiency in primates, and
the decoloration of the muscles in muscular dystrophy in many species
(resulting from rarefaction of the myoglobin) have led to research
into the possible role of this vitamin in heme synthesis.
Heme synthesis results from a series of transformations starting
with glycine and succinyl-CoA, leading to the formation of 6 -

43
aminolevulinic acid (ALA), followed by the condensation of two
molecules of ALA to form porphobilinogen. These two transformations
are catalyzed by two enzymes, ALA-synthetase and ALA-dehydratase,
respectively.
Nair et al. (1970) have shown that these two enzymes are under
the control of vitamin E. The fact that actinomycin D (which inhibits
transcription from DNA to RNA) impeded the ability of vitamin E to
reestablish heme formation in the mitochondria of deficient tissues
suggest that vitamin E might intervene at this level by acting as a
specific repressor of the transcription of the enzymes of ALA (Nair,
1972).
Since heme is not only a constituent of hemoglobin, but also of
myoglobin, cytochromes and various oxidoreduction enzymes, it is
possible to better understand certain aspects of vitamin E deficiency,
namely the decoloration of the muscles in muscular dystrophy due to
rarefaction of the myoglobin. Similarly, the decoloration which
various researchers have observed in the mitochondria of deficient
tissues reflects a rarefaction of the cytochromes, which contributes
to a lowering of cell respiration by disruption of the respiratory
chain at the level of the cytochromes.
Biological Role of Vitamin E
Antioxidant Activity
It has been known for some time that vitamin E is an excellent
fat-soluble antioxidant (Dam, 1957; Tappel, 1962) although the
physiological significance of this trait is only now becoming clear.

44
Because of its antioxidant effect, vitamin E is able to entrap free
radicals before they can attack life-supporting cell structures. Free
radicals are, by definition, chemical compounds which contain an odd
number of electrons. Free radicals can be formed in biological
systems through homolytic cleavage of covalently bonded organic
compounds resulting in bond splitting, whereby each fragment retains
one electron of the original bonding pair.
H
R C = C.
Bond splitting as such always gives rise to a pair of radicals.
Radicals can also be formed from a capture of an electron by a
molecule as illustrated below:
O2 + e 0^* .
The formation of superoxide anion radical results in a net negative
charge because of the extra electron in its posession. Capturement of
electrons by molecular is known to occur by its interaction with
flavin oxidoreductase systems (Fridovich, 1976). For a better
understanding of these phenomena, the effect of free radicals upon the
breakdown of an essential fatty acid as described by Leibovitz and
Siegel (1980) is presented in Fig. 2.9.
In a fatty acid, at a carbon atom adjoining a double bond, an
agressive substance (aS) removes hydrogen (I), resulting in the
formation of a fatty acid radical (II). This radical reacts with
oxygen to form a peroxy fatty acid radical (III). This new radical in
its turn removes hydrogen from another essential fatty acid, resulting
in the formation of a hydroperoxy fatty acid (IV) and again in a fatty

45
I.
II.
III.
R CH = CH CH2 R
Electron,
Proton
R CH = CH CH R ^
R CH = CH CH R
0-0-
R CH = CH CH2 R
R CH = CH CH R -
IV. R CH = CH CH R
I
0 OH
FIGURE 2.9. SCHEMATIC REPRESENTATION OF THE PEROXIDATION
OF UNSATURATED FATTY ACIDS

46
acid radical which can react further. Once this chain reaction has
started, it continues until all unsaturated compounds have been broken
up, self-quenching occurs, or the radical is entrapped by an
antioxidant (Chow, 1979).
Autoxidation eventually results in some form of degradation of
the material affected. For example, foodstuffs such as corn oil,
butter and margarine become rancid. The process is generally
represented by four elementary reactions:
Initiation: production of R* (1)
Propagation: R. + 0^ R00' (2)
ROO. + RH R00H + R' (3)
Termination: ROO. + ROO. Molecular products (4)
In this scheme, RH represents a lipid molecule and R. the carbon-
centered radical derived from it by removal of a hydrogen atom.
(Propagation can also occur by addition of the peroxyl radical to a
carbon-carbon double bond.) These reactions can be prevented by
antioxidants (Lundberg, 1962).
R00. + XH2 R00H + XH.
XH. + XH. X + XXH2
XH = antioxidant
At the cellular level, autoxidation is clearly undesirable because it
threatens the integrity, protective and organizational properties of
the membranes which enclose cells and organelles, thus placing the
survival of the cell itself in jeopardy.

47
In the last few years, much research has been done concerning
free radicals and into how various substances act to inhibit them
(Cheeseman et al., 1984).
Free radicals are involved in two types of biological phenomena.
Some appear in normal cell reactions, others in abnormal reactions and
in processes associated with the aging of organisms. All biological
oxidations do not necessarily give rise to such radicals. The
mechanisms by which they are produced are numerous as they result from
many types of reactions. These reactions include the peroxidation of
lipids, in the course of which, under certain conditions increase
depending upon the rate of formation of free radicals. It has also
been shown that some processes, whether enzymatic or not, produce
reactions of the same type when some of the cell constituents released
are exposed to the direct action of oxygen in a free environment or in
the absence of inhibiting agents. Some researchers, such as Pryor
(1970), do not rule out the possibility that radicals may escape an
electron transport chain, then causing rapid and destructive
oxidations.
It may seem paradoxical, as Pryor (1971) points out, that oxygen,
which cells need to release energy from food, is so reactive that it
manages to trigger, alongside the usual processes, some undesirable
parallel reactions oriented toward actions detrimental to the
phenomena essential to life. In the course of such oxidative
reactions, it causes damage to the cells by producing unfavorable
modifications in the structure of their membranes, in the membranes of
the intracellular organelles and in the endoplasmic reticulum.

48
For its part, ozone, a layer of which shelters the planet from
short-wavelength ultraviolet radiation and known to be harmful, also
causes oxidations which are detrimental to biological cell systems.
Study of the chemical composition of the cell membranes shows
that they are formed by an association of lipids and proteins
performing important functions such as electronic phenomena, active
transport, glycolysis, biosynthesis of lipids and proteins, etc.
Lipids are the most important constituent not only of these cell
membranes, but also those of the intra-cellular organelles including
the the mitochondrias and microsomes. Many factors suggest that the
membranes consist of a film of lipids, rich in triglycerides and
phospholipids, between 40 and 65 A in thickness, covered on both sides
by a layer of proteins each about 10 A thick (Lehninger, 1975). The
peroxidation of the lipid constituents of the membranes, which contain
a high proportion of mono and polyunsaturated fatty acids (C16:1 *
C18:1; C18:2; C18:3; and C20:4) disturb their metabolic functions
(Tappel, 1972). The polyunsaturated fatty acid moieties of lipids are
characterized by the presence of two or more carbon-carbon double
bonds which are separated by a methylene-CH2-unit. Methylene units
are very susceptible to attack by radicals as peroxyl and Roo' with an
extremely rapid combination with oxygen to form peroxyl radicals
(Tappel, 1972). Lipid peroxidation of polyunsaturated fatty acids
particularly arachidonic acid results in the formation and
accumulation of lipid peroxidation products known as lipofuscin
pigments (Tappel, 1972). These pigments accumulate in tissues
(myocardium, brain, liver, testicles, etc.) in response to vitamin E
deficiency, oxidative stress and age. Results of chemical analyses

49
suggest that these lipofuscin pigments arise from membrane lipid and
protein fragments formed in the course of their peroxidation phenomena
(Porta and Hartroft, 1969).
Consequences of lipid peroxidation are alterations in structural
(membrane permeability), functional components of the cell and their
component enzymes particularly subcellular partices such as
mitochondrias, microsomes, and lysosomes.
Role of a-Tocopherol as a Chain Breaking Antioxidant
A major biological function of a-tocopherol is to act as a chain
breaking antioxidant, scavenging free radicals, capable of terminating
chain reactions among unsaturated fatty acids (Zalkin and Tappel,
1960).
Chain-breaking antioxidants react directly with radicals. Many
phenols (ArOH) of which vitamin E is an example, can stop a radical
chain reaction because they are able to trap the chain-carrying
peroxyl radicals. The free radical scavenger action of a-tocopherol
depends on the ability of donation of the phenolic hydrogen atom to a
fatty acyl free radical, which resolves the unpaired electron of the
radical so that free radical attack on further molecules of
unsaturated fatty acids is prevented (Burton, 1989)
ArOH + ROO -* R00H + ArO.
The relatively stable phenoxyl radical produced is so unreactive to
attack another polyunsaturated fatty acid moeity or 0£ that instead it
is inactivated by reaction with a second peroxy radical R00 +
ArO" -* inactive products or may in the presence of ascorbate
undergo reduction back to the original phenol (Burton, 1989).

50
The latter mechanism is the basis of the so-called synergistic
action of vitamins C and E. The antioxidant breaks the chain of
oxidation and can dramatically reduce the length of the autoxidation
chain and the extent of peroxidative damage. The better the
antioxidant, the greater is the amount of material that is spared from
peroxidative damage. Vitamin E appears to be a factor ensuring the
integrity of cellular membranes protecting their constituent
polyunsaturated fatty acids from peroxidation.

CHAPTER 3
PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP FOLLOWING
INTRAMUSCULAR ADMINISTRATION IN AN OIL CARRIER
Introduction
Vitamin E is a biological antioxidant that has been used
pharmacologically in young sheep and cattle for the prevention or
treatment of conditions associated with nutritional muscular dystrophy
(Jenkins et al., 1972). Assessments of the vitamin E status in the
ruminant animal treated with various tocopherol preparations have
relied on serum or plasma tocopherol concentrations (Jenkins et al.,
1970). To our knowledge there is no information on the effects of
various forms of vitamin E preparations on sheep tissue tocopherol
levels. The purpose of the present investigation was to assess the
bioavailability in sheep of two forms of vitamin E (d-a- and dl-a-
tocopherols) administered intramuscularly using a serial blood plasma
analysis of a-tocopherol concentration. In addition, the a-tocopherol
levels were determined in a few selected tissues.
Experimental
Animals
Yearling crossbred wethers weighing 40-50 kg were used. All
animals originated from a flock that was born and raised in total
confinement (Heaney et al., 1980). The animals were fed ad libitum, a
diet that consisted of providing grass (40%), hay (40%), and corn
51

52
silage (20%). The trial was carried out in three successive periods
(2 months each), with each period utilizing one group of five sheep.
Sheep were administered respective tocopherol treatments
intramuscularly. The parenteral vitamin E preparations, suspended in
sesame oil (200 mg/ml) were administered slowly at 40 mg/kg body
weight into the muscle of the crural area of each leg. Treatments
with respective slaughter times are as follows:
Group 1 dl-a-tocopherol at 240 h
Group 2 d-a-tocopherol at 360 h
Group 3 d-a-tocopherol at 240 h.
A group of six wethers originating from the same flock, was used as a
control (Group 4).
Blood samples were taken from the jugular vein of treated sheep
at specific time intervals after dosing ranging from zero to 360 h.
The plasma was separated from the cellular blood components in a
refrigerated centrifuge at 4C and stored at -20C until assayed for
a-tocopherol. Tissue samples were taken from all sheep after
slaughter and stored at -20C until analyzed for a-tocopherol content.
Feed samples were taken during the experiment for vitamin E analysis.
Analytical
High pressure liquid chromatography (HPLC) was used for plasma a-
tocopherol determination (McMurray and Blanchflower, 1979a). The HPLC
system consisted of an M 6000 pump and W.K. septumless injector
(Waters Associates Inc., Milford, Mass., USA) and Waters y Bondapak
C^2 (10 ym) column (3.9 x 300 mm) coupled to a Perkin-Elmer 650-105

53
fluorescense spectrophotometer set at 295 and 330 nm for excitation
and emission, respectively. The column was eluted with methanol:water
(97:3) at 3 min/ml. All solvents were HPLC or pesticide grade. Blood
plasma (2 ml) samples were precipitated with ethanol (2 ml), and
extracted with hexane (4 ml). Tissue samples were saponified and
extracted with hexane (Thompson and Hidiroglou, 1983).
Identification and quantitation of the a-tocopherol was
accomplished by comparison of retention time and peak areas with d-a-
tocopherol standard. Tocopherol determination in roughage was
performed according to the techniques of McMurray and Blanchflower
(1979b).
Statistical Analysis
The indexes of bioavailability of the various preparations of
vitamin E were determined by analysis of variance. These indexes
were: 1) the maximum a-tocopherol plasma (C max) concentration (peak
of plasma a-tocopherol concentration curve), 2) the time of maximum (t
max) a-tocopherol concentration, and 3) the area (AUC) under the a-
tocopherol plasma concentration time curve (Balard, 1968; Koch-Weser,
1974). Differences in the vitamin E concentration between
corresponding tissues in the four groups were determined by analysis
of variance.

54
Results
Tocopherol Content of Roughages
Average tocopherol content of roughage at the time of consumption
were 39, 69 and 34 (mg/kg DM) for maize-silage, grass-silage and hay,
respectively. The only forms of vitamin E found in hay and grass-
silage were a-tocopherol. In maize-silage a-tocopherol was, on the
average about half the total tocopherol and was equal to the sum of a
+ 3 tocopherols (26%) and 6 tocopherol (24%).
Blood Plasma a-Tocopherol
After IM administration of d-a-tocopherol preparation, a lag
period was noted in the sheep until vitamin E increased in the
systemic circulation (Fig. 3.1; Group 2). There was no clear evidence
of an elimination phase in the data. In all 3 groups there was a
suggestion of a plateau near the peak. It would appear as though the
system reached an equilibrium at or near peak levels.
In Group 2 (IM d-a-tocopherol injected sheep killed 360 hr after
dosing) the most important index of bioavailability (Koch-Weser,
1979), the area under the plasma concentration-time curve (AUC) was
the largest (P<0.01) compared to the other 2 groups (Table 3.1). This
appeared to be due to a more sustained release into the plasma (t max
much larger), resulting in a higher delivery of the vitamin. Maximum
concentration (C max) was much higher (P<0.01) in group 2 than in the
others.

tOCOptofOl pf/ml plxmi
55
FIGURE 3.1. MEAN PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE
I.M. INJECTION OF D-ct-TOCOPHEROL SUSPENDED IN STERILE
SESAME OIL.

56
TABLE 3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN SHEEP AFTER A
SINGLE INTRAMUSCULAR ADMINISTRATION OF VITAMIN E (40 mg/kg).
Vitamin E
Group preparations
Cmax1
(pg/ml)
Tmax3
(hr1)
Co3
(pg/ml)
Cmax/Co
(Pg/ml)
Ct4
(Pg/ml)
Ct/Co Cmax/Ct
(pg/ml) (
AUC5
pg/ml/hr)
1
dl-a-tocopherol
1.76d
151b
1.12c
1.68d
1.47b
1.39d 1.23
286d
2
d-a-tocopherol
2.40c
254c
0.94d
2.68c
1.87a
2.06c
1.35
637c
3
d-a-tocopherol
1.84d
190a
0.71d
2.63c
1.30b
1.82c
1.54
277d
SE
0.23
24.78
0.09
0.21
0.17
0.24
0.15
27
Sheep in Group 1 and 3 were slaughtered 240 d after dosing while sheep in Group
2 were killed 360 h after dosing.
^Maximum plasma concentration.
2
Maximum time (hr).
3
Initial plasma concentration.
Terminal plasma concentration.
3Area under plasma concentration-time curve.
a A
c,d
Means in the same columns with different superscripts differ (P<0.05),
Means in the same columns with different superscripts differ (P<0.01),

57
In Group 3 (IM d-a-tocopherol injected sheep killed 240 hr after
dosing) there was also a more sustained delivery than in group 1 (t
maximum 191>151). This resulted in a marginal increase in C max but
no increase in AUC was noted. This may be due to the fact that
initial values in Group 3 were very low compared to the other groups
(Cq=0.71). It was noted that relative to initial values the increase
in vitamin E concentration in Group 3 sheep was much higher than for
Group 1 and approached the relative increase for Group 2 (C
max/Co=2.63).
The AUC measurement of Group 3 was comparable to that of group 1.
This may have been due largely to the higher initial value in this
group. The elimination rates of the three groups appeared to be very
similar (no differences in Cmax/C^,).
Tissue Concentrations of Vitamin E
Sheep receiving d-a-tocopherol and slaughtered 360 hr after
dosing had higher (P<0.05) pancreatic, hepatic, lung, spleen and
muscle a-tocopherol levels than the control group (Table 3.2). There
also was higher a-tocopherol concentration (P<0.05) in the pancreas in
sheep of Group 2 than Group 3. In the lung, brachiocephalicus muscle,
pancreas and spleen of sheep slaughtered 240 hr after dosing, higher
a-tocopherol concentrations were found in the d-a-tocopherol than dl-
a-tocopherol administered animals. In these tissues a-tocopherol
concentrations in Group 3 had a tendency to be higher than those of
Group 1 sheep, a-tocopherol concentrations in all groups were higher
(P<0.01) in the adipose and adrenal than in other tissues analyzed.

58
TABLE 3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (p g/g wet)1 OF SHEEP FOLLOWING
A SINGLE INTRAMUSCULAR ADMINISTRATION (40 mg/kg BODY WEIGHT)
OF VARIOUS VITAMIN E PREPARATIONS.
Groups:
Vitamin E preparation:
Mode administration:
Time after dosing (hr)
dl-a-tocopherol
240
d-a-tocopherol
360
d-a-tocopherol
240
Control
Tissues:
Adrenal
11.524.29ab
13.621.31ab
12.491.99ab
17.6416.36ab
Depot fat
10.521.73ab
17.851.69ab
16.822.52ab
12.84H ,68ab
Muscle vastus medial
4.200.55b
7.750.60a
4.921.01b
3.88i0.94b
Muscle:
brachiocephalicus
2.740.41d
6.261.13c
5.770.84c
3.46i0.70d
Heart
3.360.40ab
5.331.03ab
3.840.37ab
5.76H .59ab
Kidney
2.870.58ab
6.100.93ab
4.570.99ab
3.44i0.91ab
Lung
3.080.71e
7.521.10c
5.460.76d
2.91l0.23e
Liver
4.270.37a
5.921.39a
4.180.48a
2.22i0.36b
Pancreas
3.040.56b
6.941.00a
6.341.22a
4.32i0.50b
Spleen
2.720.56b
6.081.04a
5.6511.04a
3.30i0.54b
Least square means SE.
3 b
Means in the same rows with different superscripts differ (P<0.05).
c d G
Means in the same rows with different superscripts differ (P<0.01).

59
Discussion
Blood plasma vitamin E data showed that in sheep dosed with
various vitamin E preparations, the movement of the active free ex-
tocopherol from the site of the IM injection to the blood stream was
quite slow. In all sheep given the vitamin E preparation by IM oil
depot injection, a delayed increase in plasma a-tocopherol levels
occurred. D-a-tocopherol IM administration proved to be more potent
than dl-a-tocopherol. The length of the experimental period had an
effect on the bioavailabilities index. The IM dosed sheep with d-a-
tocopherol and slaughtered 360 hr later had a bioavailability much
higher than in sheep dosed similarly but killed 240 hr after dosing.
During this 360 hr period a gradual but sustained response occurred.
Overman et al. (1954) reported that IM administration of vitamin
E is ineffective in humans, regardless of the type of compound and the
type of vehicle (oil or water) used. However, according to Caravaggi
et al. (1968), IM injection of vitamin E is the best route for
administration of this vitamin. These workers observed that in sheep,
following IM injection of a commercial preparation of d-a-tocopherol
acetate in aqueous suspension, the plasma a-tocopherol reached its
peak 8 hr after its administration. The slow release of the IM
injected vitamin E preparation in oil carrier could be due to myositis
and inflammation of the lymphatic vessels (Bille et al., 1976),
through which the vitamin E molecule is absorbed (Blomstrand and
Forsgren, 1968; Machlin et al., 1979). It is also known (Voffey and
Coutrice, 1956) that exercise influences the flow of fluid along the
lymphatic vessels. When an animal is at rest, as in these

60
experiments, there is little or no lymphatic flow, and this would
affect the release of the vitamin E preparations which probably was
confined to connective tissue surrounding the muscle (Balard, 1968).
Bille et al. (1976) showed that vitamin D injected by IM oil depot,
into slaughter pigs, had a half-life of about 30-40 days at the
injection site, and that oil granulomata had formed after dosing.
Recently, it was reported (Dickson et al., 1986) that in sheep,
following the IM injection of a vitamin E preparation (5 ml) suspended
in arachis oil (400 mg dl-ct-tocopherol acetate/ml) into quadriceps
muscle, a considerable storage of vitamin E occurred in their lymph
gland (iliac). These workers observed that in the injected sheep a
continuous increase of vitamin E concentration occurred during 3 weeks
(from 76 ug/g to 2687/yg/g lymph gland). On weeks 5 and 6 there were
still high concentrations of vitamin E (1855 pg and 550 pg/g) in the
lymph gland (iliac) of the killed sheep. The results of their
experiment showed that the duration of the experimental period was
relatively short and longer time was needed in order to establish
accurately the various indexes of bioavailability. The planning of
our experiment was based on work carried out on chicks (Marusich et
al., 1967) and in dogs (Newmark et al., 1975) dosed IM with various
forms of vitamin E. In their animals the plasma vitamin E showed its
peak within 24 hr. The difference in initial d-ot-tocopherol plasma
values between the present 3 groups may have been due to different
roughage batches used before the start of the trial. The results
reported herein show that the time of slaughter had a major influence
on tissue vitamin E levels. In sheep slaughtered 360 hr after IM

61
administration of d-a-tocopherol, tissue levels of <*-tocopherol were
higher than those of control. In some cases there were large
individual variations in tissue a-tocopherol concentrations which made
comparisons difficult between corresponding tissues in the various
groups. It is apparent that of the tissues examined the depot fat is
the preferred storage tissue for vitamin E. According to Machlin et
al. (1979) adipose tissue tocopherol is not readily available upon
metabolic demand. Tocopherol concentrations in the liver of control
were low indicating low dietary intake. Rammel and Cunliffe (1983)
using HPLC reported that levels of 2 mg of a-tocopherol per kg of
fresh liver were observed in clinically normal sheep. According to
these workers a-tocopherol hepatic levels less than 2 mg/kg become
health problems. In deer, Meadows et al. (1982), also using HPLC,
observed liver a-tocopherol concentrations of 5 yg/g wet tissue.
In conclusion, the results showed a delayed increase in plasma o-
tocopherol concentration in sheep administered IM two vitamin E
preparations. A more sustained increment in plasma a-tocopherol was
observed in sheep IM-injected with d-a-tocopherol than dl-a-
tocopherol. Following d-a-tocopherol IM administration, higher tissue
a-tocopherol levels occurred in sheep killed 360 hr after dosing than
in those of control sheep.
Summary
The bioavailability of two vitamin E preparations (d-a-tocopherol
and dl-a-tocopherol) suspended in sesame oil solution and administered
intramuscularly was evaluated in sheep. In sheep administered d-a-

62
tocopherol and killed after 360 hr (Group 2), there was a higher
(P<0.01) bioavailability as assessed by the area under the plasma
concentration-time curve, than for sheep killed 240 hr after injection
with d-a-tocopherol (Group 3) or dl-a-tocopherol (Group 1). In the 3
groups (5 sheep each) the highest blood plasma a-tocopherol increment
occurred in the d-a-tocopherol injected sheep. For sheep injected
with d-a-tocopherol and slaughtered at 360 hr (Group 2) the
pancreatic, hepatic, lung, spleen and muscle tocopherol concentrations
were higher (P<0.05) than in the control group. Also in group 2 there
was a tendency for higher tissue tocopherol concentrations than in the
other vitamin E treated sheep (Group 1 and 3).

CHAPTER 4
BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS IN SHEEP
Introduction
The biological activity of the various tocopherol compounds has
been established from experiments with laboratory animals. However,
biopotency evaluation of different forms of vitamin E based on a
specific animal model (i.e., rat antisterility test) may not be valid
for other species. Biopotencies based on this test do not correlate
with human data. Recently Baker et al. (1986) reported that human
assay data confirmed the currently accepted biopotencies of 1.0 IU/mg
and 1.36 IU/mg for dl-tocopheryl acetate and d-a-tocopheryl acetate,
respectively. There are little data available for ruminants on the
absorption and body distribution of vitamin E. In view of the unique
digestive and physiological system in ruminants, the present
investigation determined bioavailability of the various forms of
vitamin E in sheep fed over a relatively long-term study (28 days).
Materials and Methods
Initially, twenty crossbred yearling wethers weighing 60 to 70 kg
were used. All animals originated from a flock fed a commercial mix
that consisted of 56.45% corn meal, 16.5% (44% CP) soybean meal, 25%
cottonseed hulls, 1% trace mineral salt, dicalcium, 1% monocalcium
phosphate, and .037% of vitamin A and D^. They were fed this diet
throughout the experiment.
63

64
Sheep were placed in individual metabolism cages 10 days before
administration of vitamin E as an adjustment period. They were
randomly allotted to four dietary groups of five each that consisted
of 400 International Units/day/sheep of either (1) dl-a-tocopherol,
(2) dl-a-tocopheryl acetate, (3) d-a-tocopherol (4) d-a-tocopheryl
acetate, which was mixed with the commercial diet. Appropriate
amounts of the various forms of tocopherols were dissolved in absolute
ethanol prior to diet mixing. In the middle of the experiment, one
sheep from group 3 and two sheep from group 4 stopped eating and were
removed from the experiment.
Blood samples were taken at various intervals by jugular puncture
with plasma separated from the cellular blood components in a
refrigerated centrifuge and stored at -30C until assayed for vitamin
E. All animals were slaughtered after 30 days. Samples from selected
tissues (fat, muscles from the neck and leg, kidney, lung, heart,
spleen, liver, pancreas) were collected and stored at -30C until
analyzed for d-a-tocopherol.
Analytical Methods
Quantitation of d-a-tocopherol in sheep tissues, as well as
plasma samples, was performed by high-pressure liquid chromatography
(HPLC) using a fluorescent detector, following tissue homogenization
and heptane extraction. Identification and quantitation of a-
tocopherol were obtained by comparison of retention time as well as
peak areas with tocopherol standards. Standards were purchased from
Eastman Kodak (Rochester, NY, USA).

65
Instrumentation
The chromatographic apparatus consisted of an M 6000 pump and a
WK septumless injector (Waters Associates, Inc., Milford, Mass., USA).
A Perkin-Elmer 650-150 fluorescence spectrophotometer, equipped with a
microflow cell unit, was used for quantitation. Wavelength settings
were 295 and 330 nm for excitation and emission, respectively. The
column as a Bondapak C18 (3.9 mm x 30 cm) of 10 pm particle size
purchased from Waters Associates (Milford, Mass., USA). The mobile
phase was a solvent system consisting of methanol and water in a 97:3,
with a flow rate of 3 ml/minute. All solvents used were HPLC grade.
Mass Spectrometry in Combination with Gas Chromatography
Further identification of a-tocopherol in various tissues was
carried out by a combination of a Hewlett-Packard (HP) gas
chromatograph 5790-mass spectrometer 5970A series with a data station
(HP-9000). The mass spectrometer performs three basic functions.
First, molecules are subjected to bombardment by a stream of high-
energy electrons, converting some of the molecules to ions. The ions
are accelerated in an electric field. Second, the accelerated ions
are separated according to their mass-to-charge ratio in a magnetic
field or electric field. Finally the ions with a particular mass-to-
charge ratio are detected by a device which is able to count the
number of ions which strike it. The detector's output is amplified
and fed to a recorder (Roboz, 1968). Eluates from the HPLC were

66
purified for a-tocopherol by thin layer chromatography on silica gel
using cyclohexane-diethylether (4:1 v/v) as the solvent.
Authentic standards of d-a-tocopherol, as well as tissue samples,
were silylated before injection onto the GC/MS (Ingold et al.f 1987).
The tocopherol trimethylsilyl ethers were injected (3 yl) into a
12 m x 2 mm i.d. ultra-1 (OV 101 methyl silicone) capillary column
maintained at 280C with an injector temperature of 350C and a helium
carrier gas flow rate of 0.5 cc/min, which was connected to a series
mass-selective detector. Mass spectra were obtained at an electron
beam energy of 70 eV and an accelerating voltage of 1800 volts.
Molecular ions ranging from 50 to 600 mass/charge were monitored
continuously.
Statistical Methods
Indices of bioavailability of various preparations of vitamin E
were performed by analysis of variance. These indices were: 1) the
maximum a-tocopherol plasma concentration (peak of plasma a-tocopherol
concentration curve), 2) the time of maximum (t max) a-tocopherol
concentration, and 3) the area (AUC) under the a-tocopherol plasma
concentration time curve. Analysis of variance was also used to
estimate the difference in the vitamin E concentration between
corresponding tissue in the four groups, as well as among tissues.

67
Results
Tissues
Diet and tissues were highly significant (P<0.0001) in their
vitamin E concentrations, but diet x tissue interaction was not
significant (P > 0.05) (Table 4.1). Higher a-tocopherol
concentrations (P<0.01) were observed in the tissues of sheep fed the
d-a-tocopherol than other vitamin E compounds (Table 4.2). Analysis
of variance for each individual tissue of the four diets showed
differences (P<0.01) only for heart, kidney and lung (Figure 4.1).
Higher concentrations (pg/g fresh tissue) of a-tocopherol were
observed in the sheep's heart fed d-a-tocopherol (14.74 1.64 SE)
than in the d-a-tocopheryl acetate (9.65 1.64 SE), dl-a-tocopheryl
acetate (8.47 2.84 SE) or dl-a-tocopherol (6.20 2.50 SE)
supplemented sheep. Also, in d-a-tocopherol fed sheep, higher
concentrations were observed in lung (12.13 2.58 SE) than in d-a-
tocopheryl acetate (8.99 1.51 SE) or dl-a-tocopheryl acetate (6.14
1.16 SE) or dl-a-tocopherol (6.40 1.91 SE) supplemented sheep. In
kidney, higher a-tocopherol concentrations were found in the d-a-
tocopheryl acetate (7.20 0.27) than d-a-tocopherol (5.81 0.67 SE)
or dl-a-tocopheryl acetate (4.78 1.84) or dl-a-tocopherol (4.11
0.63). However, analysis of variance did not show a difference (P >
0.05) among corresponding tissues among treatments. In all diets,
pancreas contained the highest concentrations, followed by liver and
spleen. Immediately after these tissues, heart, lung and kidney were

68
TABLE 4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS IN
TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.
Source
DF
MS
F
P
Model
35
142.76
16.10
0.0001
Error
117
8.86
Corrected total
152
Source
DF
SS
F
P
Diet
3
212.02
7.97
0.0001
Tissue
8
4318.39
60.86
0.0001
Diet x tissue
24
305.90
1.44
0.1049
TABLE 4.2. TISSUE a-TOCOPHEROL
THE
CONCENTRATIONS (pg/g FRESH TISSUE) IN
FOUR DIETS.
Diet
Mean
N
d-a-tocopherol
10.43a
36
d-a-tocopheryl acetate
8.75b
27
dl-a-tocopheryl acetate
7.72b
45
dl-a-tocopherol
7.46b
45
In vertical row, numbers with the same letter are not different
(P>0.05).

TOCOPHEROL Wg FRESH TISSUE)
69
Diet I : dl-otocopherol
Diet 2 : dl-otocopherol acetate
c
Diet 4 : d-o-tocopherol acetate
TISSUE TYPE
FIGURE 4.1. a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS

70
ranked lower (P<0.05). Lowest (P<0.05) concentrations of a-tocopherol
were observed in the muscle and adipose tissue (Figure 4.1).
Table 4.3 (and Figure 4.2) shows that the relative retention time
(minutes) of the trimethylsilyl esters (TMSi) obtained from purified
a-tocopherol tissues samples are very similar to that obtained from
the TMSi a_tocopherol standard.
Mass spectra of the gas chromatographic peak corresponding to the
a-tocopherol standard (TMSi) (Figure 4.3) and that corresponding to
the peak with the same retention time obtained for the pancreas, for
example, are identical, confirming the presence of a-tocopherol in
these biological extracts.
The peak obtained at mass/charge 502 represents the molecular ion
of a-tocopherol TMSi (430 a-tocopherol + 72 trimethylsilylether). The
fragmentation pattern reveals peaks at mass/charge 277, resulting from
the loss of the side chain (C^H-^) and at mass/charge 237 due to the
cleavage of the chroman structure accompanied by hydrogen
rearrangement and the loss of a CH3-C=CH fragment (Nair and Luna,
1968).
Blood Plasma
Analysis of blood plasma a-tocopherol concentrations summarized
in Table 4.4 show that diet and tissue source were significant
(P<0.01) but not the interaction diet x day. Higher (P<0.01) a-
tocopherol plasma concentrations were observed in sheep supplemented
with d-a-tocopherol on days 9, 15, 17 and 25 (Figure 4.4) than in the
other three groups. Also, in sheep fed d-a-tocopherol, the most

71
TABLE 4.3. RETENTION TIME OF a-TOCOPHEROL FROM GAS-
CHROMATOGRAPHY/MASS SPECTROMETRY.
Relative retention time (minutes)
Substance
TMSia
Standard oi-tocopherol
4.1
Pancreas
3.9
Liver
3.8
Heart
3.9
Spleen
3.9
Lung
3.9
aTMSi; Trimethylsilyl ether.

ABUNDANCE
72
MASS/CHARGE
FIGURE 4.2. IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/MASS
SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD, (B) a-
TOCOPHEROL IN PANCREAS

ABUNDANCE
73
FIGURE 4.3. MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A) AND
a-TOCOPHEROL IN THE PANCREATIC TISSUE (B) FOLLOWING
HPLC COLLECTION

74
TABLE 4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS IN
BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.
Source
DF
MS
F
P
Model
66
2.99
4.64
0.0001
Error
217
0.64
Corrected total
283
Source
DF
SS
F
P
Diet
3
44.79
23.09
0.0001
Tissue
16
225.13
11.13
0.0001
Diet x tissue
47
40.53
1.33
0.0883
TABLE 4.5. AREA (AUC)
UNDER THE PLASMA a-TOCOPHEROL CONCENTRATION
CURVE (pg/ml h_1).
Diet
AUC (mean SE)
d-a-tocopherol
102.4 8.13a
d-a-tocopheryl acetate
90.0 9.44b
dl-ot-tocopheryl acetate
75.0 7.31c
dl-a-tocopherol
67.3 7.31d
In vertical row, numbers with the same letter are not different
(P>0.05).

TOCOPHEROL Wml PLASMA)
75
Diet 1 : dlatocopherol
Diet 2 : dla-tocopherol acetate
Diet 3 : da-tocopherol
DAY
FIGURE 4.4.
SEES SS5SS 0F SHEEP

76
important indice of bioavailability, the area under the plasma a-
tocopherol concentration time curve (AUC), was larger (P<0.05) as
compared to dl-a-tocopheryl acetate or dl-a-tocopherol supplemented
sheep (Table 4.5). No statistical difference (P>0.05) was observed in
the maximum o-tocopherol concentration (pg/ml) between the four
groups. They were 3.6 0.69 (SE) (dl-a-tocopherol) and 4.3 0.69
(dl-a-tocopheryl acetate), 5.11 0.77 (d-a-tocopherol) and 4.8 0.89
(d-a-tocopheryl acetate). Also, no difference on the time (days) of
maximum concentration (P > 0.05) was observed. They were 18.2 3.24
(SE) (dl-a-tocopherol), 14.8 3.24 (dl-a-tocopheryl acetate), 11.5
4.30 (d-a-tocopherol) and 22.3 4.96 d-ar-tocopheryl acetate. The
elimination curve rates (concentration maximum/concentration terminal)
in the four groups were very similar. They were 1.6 0.20 (SE) (dl-
a-tocopheryl acetate), 1.8 0.22 (d-a-tocopherol) and 1.7 0.25 (d-
a-tocopheryl acetate) and 1.4 0.20 (dl-a-tocopherol).
Discussion
Paucity of bioavailability data from various forms of tocopherol
in sheep limits any comparison with the present data. Biological
response of sheep to dietary intake of various forms of vitamin E was
greater with the d-a-tocopherol than with its esterified form or the
isomers. Indeed, the area plasma time curves, which provide a proper
measure of relative availability for physiological function, and
tissue a-tocopherol concentrations were highest in the d-a-tocopherol
supplemented group. This indicates that in the ruminant body there
may be a difference in the transfer rate between natural and unnatural

77
forms of vitamin E (Dunkley et al., 1967). As suggested in humans
(Week et al., 1952), it could be that the physiological mechanism
effecting hydrolysis in the ruminant duodenum of tocopherol esters is
less effective than in rats. It is reasonable to assume that plasma
tocopherol levels in this experiment reflect the relative efficacy of
various forms of vitamin E on tissue storage. Indeed, as was reported
for humans (Djuetal, 1958), the present data on -tocopherol
concentrations in sheep tissue show a variable pattern which is
largely a reflection of varied bioavailability of the different forms
of vitamin E. It may be concluded that following ingestion of
equivalent amounts (IU) of the various forms of tocopherol by sheep d-
a-tocopherol was more potent than d-ot-tocopheryl acetate or the 1-
isomeric forms of vitamin E.
Summary
Seventeen crossbred yearling wethers were randomly allotted to
four dietary groups that received 400 IU/day/sheep either of (1) dl-a-
tocopherol (five sheep), (2) dl-a-tocopheryl acetate (five sheep), (3)
d-a-tocopherol (four sheep) or (4) d-a-tocopheryl acetate (three
sheep). Blood samples were taken at day 0 and then at frequent
intervals for ct-tocopherol determination. At the end of the 28-day
experiment, animals were killed and various tissues sampled. Higher
concentrations of ct-tocopherol were observed in tissues of sheep fed
d-a-tocopherol than the other tocopherol forms. In sheep fed d-a-
tocopherol, the most important index of bioavailability, the area
under the plasma ot-tocopherol time curve (AUC) (Gibaldi and Perrier,

78
1975) was larger as compared to other forms of vitamin E
supplementation.

CHAPTER 5
BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS
INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS
Introduction
Biopotencies of various tocopherol compounds are generally
provided by the following relationship according to the National
Formulary: 1 mg dl-a-tocopheryl acetate = 1.0 IU; 1 mg dl-a-
tocopherol = 1.1 IU; 1 mg d-a-tocopheryl acetate = 1.36 IU; 1 mg d-a-
tocopherol = 1.49 IU. These values have been based largely on small
animal bioassay (rat anti-sterility assay). Horwitt (1980) and
Horwitt et al. (1984) reported that in humans, on a mg basis, d-a-
tocopheryl acetate was 2.16 times more potent than dl-a-tocopheryl
acetate following a single oral dose. However, Baker et al. (1986)
confirmed in humans following a continuous oral dosing that 1 mg of
dl-a-tocopheryl acetate (all-racemic-a-tocopheryl acetate) has a
biopotency of 1.0 IU whereas d-a-tocopheryl acetate (2R, 4'R, 8'R-a-
tocopheryl acetate) has a biopotency of 1.36 IU (28 d). Horwitt
(1980), Horwitt et al. (1984) and Baker et al. (1986) used the
elevation of plasma a-tocopherol as a measure of bioavailability.
Although much is known about clinical signs of deficiency in
calves and lambs (Rice and McMurray, 1982) very little attention is
given to the different potencies of various vitamin E forms in cattle.
The present authors (Hidiroglou and McDowell, 1987; Hidiroglou et al.,
1988) have undertaken a series of studies on the utilization of
79

80
different chemical forms of vitamin E by sheep. That work was
extended to cattle with the purpose of comparing plasma and tissue a-
tocopherol concentrations following supplementation of various
tocopherol compounds.
Materials and Method
Animals
Twenty-four Charolais-Hereford crossbred beef cows ranging from 2
to 10 years of age were culled 6 months after calving from the herd of
the Nappan Experimental Farm, N.S., Canada. These unbred cows were
stratified by age into four groups, and the six animals within each
group were randomly assigned to four dietary tocopherol preparations.
The treatments were d-o-tocopheryl acetate, d-a-tocopherol, dl-a-
tocopherol and dl-a-tocopheryl acetate. Cows received daily 1,000 IU
(a dosage above a physiological level) of their respective vitamin E
preparation. This amounted to 735 mg of d-a-tocopheryl acetate, 671
mg of d-a-tocopherol, 900 mg of dl-a-tocopherol and 1000 mg of dl-a-
tocopheryl acetate, respectively. Each vitamin E preparation was
diluted in alcohol to 3 ml and mixed with 25 g of dry molasses. All
cows were individually fed 2 kg of average quality grass hay (8.7% CP,
35% ADF, 54% TDN, .51% Ca, .23% P, .02 ppm Se and 12 ppm vitamin E)
and had ad libitum access to a barley-soybean mixture (Table 5.1) with
no added a-tocopherol. The vitamin E-molasses mixture was prepared
daily and was topdressed on the grain portion for each individual cow.
The experiment lasted 28 days after which time the cows were
sacrificed at commercial facilities.

81
TABLE 5.1. COMPOSITION OF THE GRAIN PORTION OF
THE DIET FED TO COWS FOR 28 da
Ingredient
% as-fed basis
Barley
90
Soybean meal
7.5
Calcium carbonate
1.0
Dicalcium phosphate
1.0
Vitamin premix^
.25
Trace mineral saltc
.25
g
Analyzed for vitamin E and selenium
(8 ppm and .235 ppm, respectively).
^Provided, per kilogram of diet:
5,000 IU vitamin A and 500 IU vitamin
D.
c
Provided, per kilogram of diet:
2.1 g NaCl, .22 mg I, .088 mg CO,
9 mg Fe, .725 mg Cu, 2.625 mg Mn
and 8.75 mg Zn.

82
Blood Collection
Blood samples (15 ml) were obtained via jugular venipuncture on d
0, 1, 7, 14 and 28 of the feeding period and plasma was analyzed for
a-tocopherol concentration. Samples were always collected in the
morning before vitamin E treatments were provided. Blood samples were
centrifuged at 1,000 x g for 15 min at 4C immediately after sampling,
and plasma was stored at -20C until analyzed for a-tocopherol
concentration.
Tissue Collection
On the last d of the experiment, cows were shipped to a local
abattoir and slaughtered the following d. At that time, 10-50 g
samples were collected from ten different tissues: heart, thyroid,
liver, kidney, adrenal, pancreas, spleen, lung and neck muscle. These
samples were frozen within 2 hours and stored at -20C until analyzed
for a-tocopherol concentration using high pressure liquid
chromatography.
Analytical Method
Quantification of a-tocopherol in tissues was performed by HPLC
using a fluorescence detector as described by McMurray and
Blanchflower (1979b) following tissue homogenization and heptane
extraction by the method of Burton et al. (1985) and Ingold et al.
(1987). Serum samples were prepared for HPLC analysis according to
the method of McMurray and Blanchflower (1979b). Identification and
quantification of a-tocopherol were by comparison of retention times

83
and peak areas with tocopherol standards. Selenium and a-tocopherol
in the feed were determined, respectively, by the method of Hoffman et
al. (1968) and McMurray and Blanchflower (1979a).
Instrumentation
The chromatographic apparatus consisted of a M 6,000 pump and WK
septumless injector. A Perkin-Elmer 650-150 fluorescence
spectrophotometer equipped with a microflow cell unit was used for
quantification. Wavelength settings were 295 and 330 nm for
excitation and emission, respectively. The column was a P Bondapak
C18 (3.9 mmx 30 cm) of 10 pm particle size. The mobile phase was a
solvent system (HPLC grade) consisting of methanol and water (97:3)
with a flow rate of 3 ml/min.
Mass Spectrometry in Combination with Gas Chromatography
Further confirmation of -tocopherol in various tissues was
carried out by a combination Hewlett-Packard (HP) gas chromatograph
5790-mass spectrometer 5970A series with a data station (HP-9000).
Eluate from the HPLC was purified for a-tocopherol by TLC on silica
gel using cyclohexane-diethylether (4:1 v/v) as the solvent.
Authentic standards of d-a-tocopherol as well as tissue samples were
silylated after HPLC as described by Ingold et al. (1987) before
injection onto the gas chromatograph/mass spectrometer. The
tocopherol trimethylsilyl ethers were injected (3 pi) into an ultra-1
(0V 101 methyl silicone) capillary column (12-m x 2-mm i.d.),
maintained at 280C with an injector temperature of 350C and a helium

84
carrier gas flow rate of .5 cc/min, which was connected to a series
mass selective detector. Mass spectra were obtained at an electron
beam energy of 70 eV and an accelerating voltage of 1,800 volts.
Molecular ions ranging from 50 to 600 mass/charge were monitored
continuously.
Statistical Method
Analysis of variance (Steel and Torrie, 1980) was used to
estimate the difference in a-tocopherol concentration among similar
time blood samplings as well as among corresponding tissues in the
four treatment groups. An additional two way analysis was carried out
to test groups and d for blood plasma and for groups and tissue types.
Comparisons were made by using Duncan's multiple range test (Duncan,
1985). Statistical models used in the experiment were as follows.
For the one-way analysis: Y_ = p + g^ + e_, where g^ is effect of
th th
i group; Y = p + d^ +£ij> where d^ is effect of i d. For two-
way analysis: Y. .. = p + g. + t. + e. where g. is effect of i^
ljk 6i j ijk 6i
group and t^ is effect of d.
Results
Plasma
Supplementation over a 1 to 7 d period of time with the various
vitamin E preparations increased (P<.01) plasma a-tocopherol
concentration above the baseline level (Table 5.2). Plasma a-
tocopherol concentrations at 1 d were higher (PC.01) than the original
baseline values in the d-a-tocopherol group but not for

85
TABLE 5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (pg/ml) IN CATTLE FED VARIOUS
PREPARATIONS OF VITAMIN E.
Item
Day
SE
Group
mean
0
1
7
14
28
Treatment
d-a-tocopheryl acetate
1.49ag
2.46bfg
3.67bef
5.64bde
7.65abd
.71
4.18a
d-a-tocopherol
1.39af
3.82ae
5.25ae
8.41ad
9.22ad
.58
5.61b
dl-a-tocopherol
1.39ag
2.40bfg
3.14bef
3.98be
5.44bcd
.44
3.27c
dl-a-tocopheryl acetate
1.37af
2.65bef
3.10bde
4.02bde
4.62cd
.52
3.16c
SE
.10
.25
.45
.82
.82
.27
Day mean
1.41d
2.83e
3.80f
5.518
6.73h
.31
3 b c
Means in the same column with different letters in their superscripts
differ (PCO.Ol).
d G f 2
Means in the same row with different letters in their superscripts differ
(P<0.01).

86
the other groups (P>.05). To elevate plasma a-tocopherol
concentration, d-a-tocopherol outranked all the other forms at all
sampling dates except at 28 d, where its effect was not different
(P>.05) from d-a-tocopheryl acetate (Table 5.2). D-a-tocopherol
supplementation during the 28 d experimental period resulted in the
highest blood plasma a-tocopherol concentration followed by d-a-
tocopheryl acetate; the racemic mixtures ranked lower (Table 5.2)
despite being dosed at equal units. Blood plasma a-tocopherol
increased continuously from 0 to 28 d (Table 5.2). The difference in
plasma a-tocopherol concentration (yg/ml) between d 28 and d 0 were as
follows (Table 5.2). D-a-tocopheryl acetate = 6.16; d-a-tocopherol =
7.83; dl-a-tocopherol = 4.05; dl-a-tocopheryl acetate = 3.25.
Consequently the relative bioavailabilities of the various tocopherols
vs dl-a-tocopheryl acetate (1.0 IU/mg) could be calculated as follows:
(4.05 x 1.1 IU/mg)
dl-a-tocopherol = 1.37 IU/mg; d-a-tocopheryl acetate
3.25
(6.16 x 1.36 IU/mg) (7.83 x 1.1 x 1.36 IU/mg)
= 2.58 IU/mg; d-a-tocopherol
3.25 3.25
=3.60 IU/mg.
Tissues
There were differences among treatments (P<.01) and types of
tissue (PC.OOOl) in vitamin E tissue concentrations at slaughter 28 d
after supplementation began Fig. 5.1 and Table 5.3). The adrenal
gland and liver concentrations of a-tocopherol were greater (P<.01)
for cattle fed d-a-tocopherol or d-a-tocopheryl acetate than when fed
racemic forms of vitamin E. The a-tocopherol concentrations were

-TOCOPHEROL, pg/g FRESH TISSUE
87
40n
n
DL-a-TOCOPHEROL
D-a-TOCOPHEROL
30-
.
D
I
20-
5
-
5 S 5
s S 5
s 2
D
10-

D D

c D
0-
1 1 IT I 1 1 1 1 1 1 1
40-]
DL-a-TOCOPHERYL ACETATE 5
+-i
,
D-a-TOCOPHERYL ACETATE
30-
T
-
20-
x 5
5 S
-
5 5
5
5 *
10-
-


D 0
0-1
THY MUS KID LUN SPL HEA LIV ADR THY MUS KID LUN SPL HEA LIV ADR
TISSUE TYPE
FIGURE 5.1. a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE
SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS

88
TABLE 5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (yg/g FRESH TISSUE) IN CATTLE FED
VARIOUS VITAMIN E PREPARATIONS.
Dietary
form
Tissue
mean
d-
Tissue
a-tocopheryl
acetate
d-a-toco-
pherol
dl-a-toco-
pherol
dl-a-tocopheryl
acetate SE
pg/g fresh tissue
Adrenal gland
38.41a
38.07a
27,91b
28.75b
2.68
33.28d
Heart
20.43a
18.71a
18.43a
16.12a
1.42
18.42f
Kidney
12.07a
13.11a
8.81b
12.28a
1.00
11.56h
Liver
24.20ab
27.00a
15.67c
19.88bc
1.79
21.68e
Lung
15.73a
16.52a
10.64b
15.04a
1.08
14.48
Muscle
5.29a
5.79a
5.70a
5.79a
.46
5.64j
Spleen
17.95a
15.79ab
11.76b
13.88ab
1.38
14.848
Thyroid
3.40b
4.43ab
5.63a
4.35ab
.46
4.45J
SE
1.82
1.63
1.10
1.10
.80
Group mean
17.18a
17.42a
13,03b
14.53b
.57
Means in the same row with different letters in their superscripts differ
(P<0.05).
d,e,f,g,h
Means in the same column with different letters in their superscripts
differ (PC0.0001).

89
lower in the kidney (PC.05) and lung (PC.01) of the cattle
supplemented with dl-a-tocopherol than for cattle fed the other
treatments. Higher concentrations (PC.05) of a-tocopherol were
observed in the thyroid of cattle fed dl-a-tocopherol than in that of
the cows receiving d-a-tocopheryl acetate, but the a-tocopherol
concentrations were higher (PC.05) in the spleen of the group fed d-a-
tocopheryl acetate than in that of the dl-a-tocopherol group. No
difference (P>.05) was found for heart and muscle as a result of
feeding the various forms of vitamin E.
Across all the treatments, the adrenal gland contained the
highest a-tocopherol concentration, followed generally by the liver or
heart; the lowest concentrations were observed in the thyroid and
muscle tissues (PC.05) (Table 5.3 and Figure 5.1).
Identity of a-tocopherol in tissues was confirmed by comparing
the mass spectra of the a-tocopherol standard to those obtained by
collecting the eluates from the HPLC and introducing it into the gas
chromatography/mass spectrometry system following silylation. A good
spectral match was obtained for the sampled compared with the a-
tocopherol standard spectra. Mass spectra of the gas chromatographic
peak corresponding to the a-tocopherol standard (TMSi; Fig. 5.2) and
that corresponding to the peak with the same retention time obtained
for the plasma, for example, are identical, confirming the presence of
-tocopherol in those biological extracts. The peak obtained at
mass/charge 502 represents the molecular ion of a-tocopherol (TSMi:
430 for a-tocopherol + 72 for trimethylsilyl ether). The
fragmentation pattern reveals peaks at mass/charge 277 resulting from

ABUNDANCE
90
MASS/CHARGE
FIGURE 5.2. MASS SPECTRAL SCANNING OF THE a-TOCOPHEROL STANDARD
(A) AND IN BLOOD PLASMA (B) FOLLOWING HPLC COLLECTION
AND TLC.

91
the loss to the cleavage of the chroman structure accompanied by
hydrogen rearrangement and the loss of a CH^-C=CH fragment.
Discussion
The data show that, in cattle, a greater elevation of plasma
levels of -tocopherol occurred over pre-treatment levels after
ingestion of the naturally occurring d-a-tocopherol than its ester
forms or the 1-epimer of -tocopherol. These results agree with those
of Baker et al. (1980) who reported in humans that plasma -tocopherol
was considerably higher when the free tocopherol was orally
administered rather than its acetate ester. According to Machlin and
Gabriel (1982), the higher blood plasma -tocopherol attained
following supplementation with -tocopherol compared to -tocopheryl
acetate suggests that hydrolysis of the acetate may be a limiting
factor when high levels of the vitamin are administered. Harris and
Ludwig (1949) reported lower blood plasma a-tocopherol levels in
humans dosed with the nonesterified rather than esterified forms.
This is related to a diet-mediated oxidative destruction of the free
form in the gut prior to its absorption. This does not seem to be the
case with ruminants. Indeed, the stability of d-a-tocopherol in
ruminal liquor is high (Astrup et al., 1974b). Vitamin E is not
metabolized or oxidized to any great extent in the gastrointestinal
tract of cattle (Astrup et al., 1974a).
Plasma pre-treatment -tocopherol concentrations were low.
Levels less than 1.5 yg/ml in blood plasma were considered by several
workers as potentially hazardous for cattle (McMurray and Rice, 1982;

92
Peterson and Hakkarainen, 1986). Following vitamin E administration,
the levels increased progressively for all treatments during the 4 wk
experimental period to the range (4 to 9 yg/ml) reported acceptable
for beef cattle (Bayfield and Mylrea, 1969).
In humans and monkeys, Machlin and Gabriel (1982) reported that
it is advisable to treat subjects with high levels of a-tocopherol for
at least 1 to 3 wk to attain maximal plasma a-tocopherol levels.
In order to evaluate the effectiveness of the various vitamin E
preparations, most studies (Horwitt et al., 1984; Baker et al., 1986)
have relied on serum a-tocopherol concentrations and have paid little
attention to tocopherol concentrations in tissue. In the present
experiment, tissue analysis showed higher concentrations following d-
a-tocopherol or d-a-tocopheryl acetate supplementation than with the
racemic forms. These results suggest strongly that d-a-tocopherol or
its acetate form is more available to bovine tissue than the 1-epimer.
Cattle, like the rat, appear to discriminate well between 2R, 4'R,
8'R, and 2S, 4'R, 8'R a-tocopherol (Ingold et al., 1987).
Dju et al. (1958) reported that, in the human body, there is a
wide variation in the distribution of a-tocopherol in the various
tissues. In the present experiment, highest concentrations were
detected in the adrenal gland and liver. The liver is the major
storage organ for a-tocopherol (Machlin and Gabriel, 1982) and (for a
short time at least) helps maintain plasma tocopherol levels when the
intake of vitamin E becomes inadequate. As indicated earlier, in rats
(Behrens and Madere, 1986), the adrenal gland accumulated more
tocopherol per gram of tissue than other tissues, regardless of the

93
source of vitamin E supplementation. In general, the apparent
biopotency of various vitamin E sources for cattle are comparable to
results reported for humans by Horwitt et al. (1984).
Summary
Twenty-four crossbred beef cows were used to investigate the
concentration of (-tocopherol) in plasma and tissues following oral
administration of four tocopherol sources. Animals were assigned to
the following treatments: dl-a-tocopherol, d-a-tocopherol, dl--
tocopheryl acetate and d-a-tocopheryl acetate. Animals received a
daily oral dose of 1,000 IU of the respective tocopherol treatment for
28 d and then were slaughtered. Blood samples were collected on d 0,
1, 7, 14 and 28 for tocopherol concentration assays, and samples from
ten different tissues were collected from slaughtered cows.
Identification of -tocopherol in tissues was confirmed by HPLC
retention times and by comparison of mass spectra with that of ot-
tocopherol standards. The d-ot-tocopherol and its acetate ester
increased plasma tocopherol concentration faster than the racemic
products with the greatest response occurring with d-a-tocopherol.
Across all treatments, the highest -tocopherol concentrations were
noted in the adrenal gland and liver with the lowest found in muscle
and thyroid tissue. Tissue analyses confirmed that in adrenal gland,
kidney, liver and lung, -tocopherol concentrations were higher
following d- than dl-a-tocopherol supplementation.

CHAPTER 6
PLASMA TOCOPHEROL IN RUMINANTS AFTER INGESTING FREE OR ACETYLATED
TOCOPHEROL
Introduction
Administration of a massive dose of vitamin E is one of the usual
methods in the prophylaxes of vitamin E deficiencies (Ogihara et al.,
1985). Overman et al. (1954) reported in humans that a single massive
oral dose of vitamin E is effective in increasing the free tocopherol
plasma level to a significant degree after six hours. This temporary
rise with ensuing drop has been designated as a tolerance curve. The
tolerance curve mirrors the intestinal absorption of vitamin E.
However, on this subject, the data for ruminant animals are sparse.
Indeed, in ruminant species with their unique digestive system and
physiologic differences in metabolic mechanisms, there are only few data
on the bioavailability of the various forms of vitamin E. For this
reason, the objective of the experiment was to gain information on blood
plasma response to ot-tocopherol in sheep or cattle provided with
different chemical forms of tocopherols but with identical weights of
these compounds, namely dl-cx-tocopherol and dl-a-tocopheryl acetate.
Materials and Methods
Trial 1
Ten yearling crossbred wethers weighing 40 to 50 kg were used. All
animals originated from a flock fed a commercial diet that
94

95
consisted of corn meal, 56.45%; soybean meal (44% CP), 16.5%;
cottonseed hulls, 25%; trace mineral salt, 1%; monocalcium phosphate,
1%; and .037% of vitamin A and D^. They were continued on this diet
throughout the experiment. Sheep were placed in individual metabolism
cages 10 days before administrations of vitamin E as an adjustment
period. They were then randomly assigned to two groups of five sheep
each. Each animal received a single intraruminal dose of either (1)
dl-o-tocopherol (i mg = 1.10 IU) or (2) dl-a-tocopheryl acetate (1 mg
=1.0 IU) at a rate of 100 mg/kg body weight.
Trial 2
Four crossbred dairy heifers (averaging 250 kg) were used in a
crossover design with two 20 d periods. They were given ad libitum
access to hay and water during the study. In the first period, two
heifers were administered orally with dl-a-tocopherol and two with dl-
a-tocopheryl acetate. The single dose given for each component was 50
mg/kg body weight. The end of the first period was followed by a
three-wk washing period. Then the treatment was reversed and the
procedures were repeated in period 2.
Blood Samples
Blood samples were withdrawn from the jugular vein in heparinized
vacutainers and immediately were centrifuged. They were collected
before vitamin E administrations and at precise intervals (Figures 6.1
and 6.2). Plasma was separated, frozen and stored at -20C until
analyzed for a-tocopherol.

-TOCOPHEROL fig/ml PLASMA cr-TOCOPHEROL pg/ml PLASMA
96
FIGURE 6.1. PLASMA TOCOPHEROL CONCENTRATION (uG/ML) IN SHEEP
FOLLOWING ADMINISTRATION OF A SINGLE MEGADOSE (100 MG/KG
BODY WEIGHT) OF (A) DL-ot-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL

97
HOUR
. PLASMA TOCOPHEROL CONCENTRATION (vG/ML) IN CATTLE
FOLLOWING ADMINISTRATION OF A SINGLE DOSE (50 MG/KG
BODY WEIGHT) OF (A) DL-ct-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL
FIGURE 6.2

98
Analytical Method
Plasma samples were prepared for high pressure liquid
chromatography (HPLC) according to the method of McMurray and
Blanchflower (1979). The chromatographic apparatus consisted of a model
6000 pump and WK septumless injector (Waters Associates, Milford,
Mass.). A Perkin-Elmer 650-150 fluorescence spectrophotometer, equipped
with a microflow cell unit, was used for quantification. Wavelength
settings were 295 and 330 nm for excitation and emission, respectively.
The column was a y Bondapak C18 (3.9 mm x 30 cm) of 10 ym particle size
purchased from Waters Associates (Milford, Mass.) The mobile phase, a
solvent system (HPLC grade), consisted of methanol and water in a 97:3
ratio with a flow rate of 3 ml/minute.
Statistical Analysis
The indices of bioavailability were calculated for each individual
sheep or cattle. These indices were: (1) the maximum a-tocopherol
plasma (C max) concentration (peak of plasma a-tocopherol curves); (2)
the time of maximum (t max) a-tocopherol concentration; and (3) the area
(AUC) under the a-tocopherol plasma concentration time curve (Gibaldi
and Perrier, 1975). Statistical analysis was carried out by analysis of
variance (sheep) or covariance (cattle) by the general model procedure
(SAS, 1980). The following model was used for the sheep,
Yij = y+ t. + Ei j;
where y = mean;
ti = ith type i = 1,2
E^j = random error;

99
while for cattle the model used was,
Yij = y + CQ + tA + ij
where C = value at time 0.
Results
Trial 1
Following the oral administration of the two vitamin E
preparations to sheep, there was an increase in the plasma a-
tocopherol level (Figure 6.1). The peak of this increase was reached
faster (P<.05) in the dl-a-tocopherol than in its ester form (Table
6.1). The shorter T-maximum observed in the dl-a-tocopherol dosed
group may have contributed to a faster onset of biological action.
The data show that C plasma a-tocopherol concentrations had a
maxima r
tendency (P>0.05) to be higher in the dl-a-tocopherol than in the
ester-dosed group. A direct comparison of the plasma tolerance curve
area (AUC) between the two groups (Table 6.1), showed that the AUC was
higher (P<.05) in the dl-a-tocopherol than in the sheep administered
with dl-a-tocopheryl acetate (Table 6.1).
Maximum plasma vitamin E concentration, as well the tolerance
curve, are dependent chiefly on factors affecting the absorption of
the vitamin from the intestine. Our results showed that the
effectiveness of the absorption indicated by the tolerance curve area
was higher in the sheep dosed with free alcohol form than with the
acetylated group. The higher peak observed in the plasma tolerance
curve in the dl-a-tocopherol group than in its acetate form probably
reflects its higher potency. At the end of the two-wk experimental

100
TABLE 6.1. DL-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES (LEAST SQUARE
MEANS SE) IN SHEEP AFTER A SINGLE ORAL ADMINISTRATION
OF VITAMIN E (100 mg/kg BODY WEIGHT).
Cmax1
Tmax3
(hr1)
Co3
CmaxCo Ct^ Ct/Co
Cmax/Ct
AUC5
(pg/ml)
(pg/ml)
(pg/ml) (pg/ml) (pg/ml)
(pg/ml)
(pg/ml/hr)
dl-a-tocopherol
3.58.22
26.514.04
.421.06
8.9911.13 .731.07 1.801.25
4.951.54
332.18120.27
dl-a-tocopheryl acetate
3.05.20
40.413.61
.411.05
7.6711.01 .591.06 1.501.22
5.361.48
269.63118.13
Significance (P)
<.05
<.05
Maximum plasma concentration.
I
Maximum time (hr).
Initial plasma concentration.
Terminal plasma concentration.
Area under plasma concentration time curve.
Significance tested following analysis of variance.

101
TABLE 6.2. TRIAL 2: D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES
(LEAST SQUARE MEANS SE) IN CATTLE AFTER A SINGLE ORAL
ADMINISTRATION OF VITAMIN E (50 mg/kg BODY WEIGHT).
Vitamin E
Preparations
Cmax^
(pg/ml)
Tmax3
(hr-1)
Cmax/Co
(pg/ml)
CT3
(pg/ml)
CT
(pg/ml)
Cmax/CT
(pg/ml)
AUC4
(u/ml/hr-1)
dl-a-tocopherol
10.84
35.38
3.88
3.71
1.36
3.04
1995
dl-a-tocopheryl
acetate
9.23
48.61
3.43
3.21
1.23
2.84
1589
SE
0.61
14.89
0.37
0.40
0.43
.39
88
Significance (P)
<.05
Maximum plasma concentration.
I
Maximum time (hr).
Terminal plasma concentration.
Area under plasma concentration time curve.

102
period, the terminal plasma tocopherol levels in both groups were
higher (P<.05) than the original values.
Trial 2
A few hours after oral dosing in both groups of cattle there was
an increase in plasma a-tocopherol levels (Figure 6.2). The free form
was absorbed with higher effectiveness than its ester form. This is
shown by its greater plasma tolerance curve area and its tendency to
higher maxima concentration and speedier t maxima appearance. There
was significantly less utilization of the ester form versus the
alcohol form. In the present experiment, the system of rotation used
in administering the two vitamin E doses minimized the individual
variations.
Discussion
In the present experiment, the plasma persistence curve was
mainly used to provide a criterion of availability of vitamin E for
physiological function following oral dosage to ruminants with two
isomeric forms.
The present results show that in the ruminal environment, despite
a strong reducing system, the dl-ot-tocopherol was preserved
sufficiently to induce a superior persistence curve area response to
that of identical weight of the esterified form. It is possible that
the degree of hydrolysis could be an important factor in the shape of
the tolerance curve. As the stability of free tocopherol is known to
be inferior to that of tocopherol esters, it may be hypothesized that

103
the relative less favorable response to tocopherol ester is due to
factors influencing the hydrolysis of these esters.
It seems probable that in cattle, like other mammals, dl-a-
tocopheryl acetate following hydrolysis is split in the intestinal
mucosa and is absorbed as free tocopherol from the gut and passed to
the systemic circulation via the lymphatic system. According to
Tikriti (1969), in cattle the acetylated form of vitamin E is
hydrolyzed in the digestive system, beginning in the rumen, and
appears to be utilized somewhat less efficiently than the free form.
One cannot rule out, as it was suggested by Gallo-Torres et al.
(1971), the possibility of a rate-limiting hydrolytic reaction
occurring in the lumen of the intestine prior to entrance of the
vitamin E into the intestinal wall of the ruminant animal.
Our results showed that T-maxima is much longer in ruminants than
in humans following oral loading with vitamin E. Indeed, Hashim and
Schuttinger (1966) observed that in normal human subjects plasma
tocopherol levels began to rise between 2 and 4 h and peaked at 7.5 h
following oral administration of vitamin E. They also observed that
at 24 h the plasma tocopherol level had declined and that at 48 h
post-absorptive levels were reached. Marusich et al. (1967) reported
that in chickens, following the administration of a single oral dose
(50 IU of dl-<*-tocopheryl acetate), the peak plasma values obtained at
6 h were followed by a steady decline. However, in rats, Marusich et
al. (1968) observed that plasma tocopherol levels peaked at 24 h,
following administration of a single oral dose of dl-a-tocopheryl
acetate.

104
The fact that the biological response to dl-a-tocopherol was
greater than with its acetylated form agrees with the results reported
in humans (Baker et al., 1980). These workers observed that following
administration of a single oral dose (800 mg) of dl-a-tocopheryl
acetate, plasma d-a-tocopherol peaked (50% from the initial value) 8 h
after ingestion. The same dose of free tocopherol induced a greater
rise (60% from the original value) in 5 h.
It seems that dl-a-tocopherol serves more pharmacological action
when ingested in amounts many times the generally recognized
nutritional requirements than its esterified form. The results
demonstrated that T max was faster following oral dosing of the free
than acetylated form. This suggests that the hydrolysis rate of
esterified tocopherol to the physiologically active free tocopherol is
quite slow.
In this study, the area (AUC) under the a-tocopherol plasma
concentration time curve undoubtedly was influenced by the amount of
vitamin E activity again. Indeed, the higher vitamin E potency of dl-
a-tocopherol (1.1 IU/mg) compared to 1.0 IU/mg, for the acetate form
probably contributed to the tendency of a higher plasma vitamin E peak
as well as the greater AUC.
In the present experiment, initial plasma tocopherol
concentrations (2 to 3 yg/ml) from hay-fed cows were in the range of
values observed by Pehrson and Hakkarainen (1986) and Hakkarainen et
al. (1987). The initial value of a-tocopherol in the plasma of sheep
fed a commercial diet was very low. In grain-fed sheep, Caravagi
(1969) reported plasma concentrations of .89 .16 SD yg/ml. Storer

105
(1974) observed higher plasma tocopherol concentrations (1.28 .07
pg/ml) for sheep fed dry feed. It appears that in the ruminant
animal, following a single oral dose of dl-a-tocopherol or its
acetylated form, the terminal values were higher in sheep and cattle
than the initial values. This may be related to a slow saturation of
tissues with both forms of tocopherol. However, no difference was
observed in the elimination rates (C max/CT). In conclusion, the data
in ruminants confirm the higher biological potency of the dl-a-
tocopherol vs. dl-a-tocopheryl acetate.
Summary
Two trials were carried out in order to evaluate the
bioavailability of dl-a-tocopherol and dl-a-tocopheryl acetate
administered to sheep or cattle in a single oral dose. In the first
trial, two groups of five sheep were used. They received 100 mg/kg
body weight of either dl-a-tocopherol or its acetylated form. The
blood plasma tolerance curve area and the maximum plasma concentration
were higher in the dl-a-tocopherol group than in its ester form. The
time (h) for maximum plasma a-tocopherol concentration was quicker
(P<0.05) in the dl-a-tocopherol group than in its ester form. In a
second trial, four heifers received the two forms (50 mg/kg body
weight) in rotation after an appropriate washing period between the
two dosings. Again, a greater plasma tolerance curve area was
observed in the cattle following administration of dl-a-tocopherol
than its acetylated form.

CHAPTER 7
PLASMA AND TISSUE VITAMIN E CONCENTRATIONS IN SHEEP AFTER
ADMINISTRATION OF A SINGLE INTRAPERITONEAL DOSE OF dl-a-TOCOPHEROL
Introduction
There have been remarkably few systematic investigations on the
disposition of pharmacologic doses of vitamin E in sheep. Massive
doses in sheep can be given orally, intramuscularly (IM) or
intravenously (IV) (Hidiroglou and Karpinski, 1987). Bioavailability
of vitamin E after intramuscular (IM) administration has recently been
reported (Hidiroglou and McDowell, 1987).
A potential alternative to IV or IM dosing could be the delivery
of vitamin E into the peritoneal space. Since no studies have been
published on the disposition kinetics of vitamin E in sheep plasma and
tissues following administration of a single intraperitoneal (IP) dose
of vitamin E, such a study using dl-a-tocopherol was undertaken.
Experimental Procedure
Animals
Twenty-five clinically normal, one-year-old, crossbred wethers
were used in the study. The sheep weighed from 30 to 35 kg at the
beginning of the experiment. A standard diet (Table 7.1) and water
were available ad libitum. Five sheep were selected at random and
slaughtered at d 0 as the control with no treatment. Thereafter, 4
106

107
TABLE 7.1. DIET OF SHEEP3
Ingredient
(%)
Corn meal
56.45
Soybean meal
16.50
Cottonseed hulls
25.00
Trace mineral salt*3
1.00
Dicalcium, monocalcium phosphate
1.00
Vitamins A, D^c
0.037
2
Analyzed for vitamin E and Se (8 ppm and .235 ppm, respectively).
^Provided, per kilogram of diet: 2.1 g NaCl, .22 mg I, .088 mg Co,
9 mg Fe, .725 mg Cu, 2.625 mg Mn and 8.75 mg Zn.
Q
Provided, per kilogram of diet: 5,000 IU vitamin A and 500 IU
vitamin D.

108
sheep were selected at random and killed at each of days 3, 6, 10, 15
and 28 after dosing.
Five g of dl-a-tocopherol were given intraperitoneally (IP) to
each treated sheep as a single injection in emulsion (Tween 80) with a
final volume of 75 ml. The IP dose was administered using the
technique of Hurter (1987). Blood samples were withdrawn by jugular
puncture at designated times prior to (0) and after vitamin E
administration (3, 6, 10, 15 and 28 d) and collected in heparinized
tubes. Plasma was obtained upon centrifugation at 500 x g for 15 min.
The animals were killed by exsanguination. Portions of kidney, liver,
adrenal, pancreas, skeletal muscles, heart, spleen and lung were
removed. All tissues were rinsed in water and their surfaces dried
with filter-paper. During removal of all tissues, care was taken to
dissect away superfluous adipose tissue. All tissues and plasma were
stored at -20C until analyzed for a-tocopherol concentration.
Analytical Methods
Quantitation of d-a-tocopherol in blood plasma and tissues was
performed by high pressure liquid chromatography (HPLC) using a
fluorescent detector (McMurray and Blanchflower, 1979). The HPLC
consisted of an M6000 pump and WK septumless injector. A Perkin-Elmer
650-150 fluorescence spectrophotometer equipped with a microflow-cell
unit was used for quantification. Wavelength settings were 295 and
330 nm for excitation and emission, respectively. The column was a y
Bondapak (3.9 mm x 30 cm) of 10 ym particle size. Elution was
performed with methanol:water (97:3) solvent with a flow rate of 3

109
ml/min. All solvents used for the HPLC mobile phase or for the
extraction were HPLC or pesticide grade. Tissue samples for vitamin
E determination were prepared according to the method of Burton et al.
(1985).
Statistics
One-way analysis of variance (ANOVA) was performed on the
logarithms of the tissue values and the tissue to plasma ratios.
Logarithms were taken to stabilize the variances which increased with
increasing concentrations. To further study the effect of time, five
contrasts were examined (SAS Institute, 1985). Untransformed means
have been presented with approximate standard errors based on the
analysis of the logarithms. Residuals were examined and an outlier
was identified using Tietjen and Moore's statistic (Tietjen and Moore,
1972). The changes which occurred when this outlier was removed from
the analysis are noted.
Plasma values were also analyzed by analysis of variance. This
analysis was a one-way ANOVA of time within the animal. Here also, a
transformation to logarithms was necessary and the time effect was
examined by several contrasts.
Results
Mean levels of dl-a-tocopherol in tissues and plasma at the time
of slaughter are given in Table 7.2. The accompanying analysis of
variance of the logarithms of these values at slaughter is given in

no
TABLE 7.2. MEAN LEVELS OF ct-TOCOPHEROL IN TISSUES (pg/g FRESH) AND PLASMA
(yg/ml) AT TIME OF SLAUGHTER.
0
3
Time (da
6
y)
10
15
28
SEMa
Kidney
0.96
28.07
(33.09)b
8.25
6.80
5.46
3.96
. 21M
(.19M)
Liver
0.97
212.61
(270.01)
69.26
37.20
34.42
15.71
. 26M
(.17M)
Adrenal
1.70
42.25
(47.67)
24.59
35.51
20.21
33.86
.21M
(.20M)
Pancreas
11.36
28.50
(31.59)
23.19
24.40
20.74
28.60
. 11M
(.12M)
Neck
1.47
7.65
(9.24)
4.54
4.57
5.24
4.49
. 25M
(.23M)
Leg
1.56
7.55
(9.38)
4.59
4.52
6.63
3.96
.25M
(.19M)
Heart
2.90
23.55
(27.82)
16.84
16.40
14.73
11.03
. 18M
(.16M)
Spleen
2.73
125.20
(160.60)
35.12
27.93
25.13
9.74
. 35M
(27M)
Lung
2.09
164.85
(216.34)
128.75
102.66
33.22
12.19
.43M
(.25M)
Plasma
0.56
5.43
(5.97)
3.13
2.23
2.22
1.96
. 14M
(.14M)
Q
The standard error of the mean is based on the analysis of variance on the
transformed scale and is approximate. It varies with the mean, M, and has been
expressed in terms of it.
bThe values in parentheses are the results when one outlier was removed.

Ill
Table 7.3. There was a significant time effect for all the tissues.
In most tissues, the tocopherol concentrations recorded at 3 d after
dosing were the largest, but the various tissues responded differently
to vitamin E injection. Examination of the tissue data 3 d postdosing
revealed a striking accumulation of injected vitamin E in the liver.
The lungs had the next highest concentration at that date. The spleen
invariably contained a considerable amount of a-tocopherol 3 d post
initiation. ot-Tocopherol concentrations in the remaining tissues,
also 3 d after dosing, showed a considerable increase but to a smaller
degree. The contrasts over time give some indication of when a
plateau had been reached, but it must be kept in mind that
significance may fail to be achieved because of large variances or
insufficient data. For example, for the kidney the mean of days 15 to
28 is not different (P>.05) from the mean at day 10. This indicates
there is no further significant change from the level reached by day
6, suggesting a plateau has been reached. However, as the means
continue to decrease up to and including day 28, it would seem
inappropriate to draw this conclusion. Similarly, the significance
tests suggest the neck, leg, heart, adrenal and pancreas have reached
a plateau by 3 d. The liver and lung do not appear to have reached a
plateau by 15 d, however. The contrasts suggest the spleen has
reached a plateau by day 10, although the mean decreases on d 28.
There were particularly low values recorded for one animal at d
3. Tietjen and Moore (1972) statistics indicated this value was an
outlier (P<.05) for liver, spleen and lung and it was an extreme

112
TABLE 7.3. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG TISSUE VALUES.
Tissue Overall
time effect
DOF 5
3-28a
versus 0
1
Source of variation
6-28 10-28 15-28
versus 3 versus 6 versus 10
1 1 1
28
versus 15
1
Residual
19
Kidney
5.036**
17.612**
6.424**
0.687*
0.286b
0.171
0.152
Liver
14.382**
59.183**
8.517**
2.350**
0.566(*)
1.294*(**) 0.235
Neck
1.413**
6.709**
0.312(*)
0.019
0.004
0.019
0.212
Leg
1.527**
6.907**
0.187(*)
0.009
0.028
0.503
0.210
Heart
2.466**
11.353**
0.484(**)
0.162
0.238
0.091
0.117
Spleen
7.057**
24.121**
7.422**
1.368(*)
1.276(*)
1.097
0.413
Lung
11.984**
43.916**
2.144(**)
3.898*(**)
7.572**
2.388(**
) 0.574
Adrenal
4.522**
20.818**
0.647(*)
0.086
0.398
0.658(*)
0.149c
Pancreas
0.468**
2.063**
0.083
0.003
0.000
0.169
0.063c
aMean of
treatments
3, 6, 10,
15 and 28
(3-28) are
compared
to control
group 0.
Significance levels in parentheses note changes which occurred when one outlier
was removed.
c
Residual is based on 17 degrees of freedom because of missing observations.
* (**)Indicates a significant effect at 5% (1%) level.

113
observation for several other variables. The effect it has on the
results is noted in the tables. The main effect of its omission was a
decrease in the variances and an increase in the values at d 3. The
result is, the contrast tests shift the plateau to d 6 for the neck,
leg, heart and adrenal which seems quite reasonable studying the
means. As well, the increase at d 28 becomes significant (P<.05) for
the adrenal. At 28 d, the skeletal muscles and kidney had the lowest
values among the various tissues, while the adrenal ranked first
followed by the pancreas.
The means for the ratio of the tissue to plasma (t/p) levels are
given in Table 7.4 and the associated analysis of variance in Table
7.5. The observations at d 3 did not fail the Tietjen and Moore test
(P>.05) for any variable. However, its effect has been noted in the
tables. Here there is no effect (P>.05) over time for the neck and
heart. While the overall test is not significant (P>.05), the
contrast of d 3 to later times indicates d 3 values were depressed for
these two tissues. The leg data show a similar pattern although the
overall time effect was significant in this case. After a significant
increase, the kidney data appear to achieve a plateau by d 6, the
spleen by d 10. Liver and lung data also show a significant increase
and than a decrease (P<.01). Although the tests indicate a plateau is
reached, the means suggest that a decrease may continue to d 28. The
adrenal and pancreas data show a significant time effect, but without
a plateau.
In Table 7.6, summary statistics are given for the four animals
whose plasma values were recorded throughout the experiment. These

114
TABLE 7.4. MEANS OF THE RATIO OF TISSUE TO PLASMA LEVELS OF cx-TOCOPHEROL AT
TIME OF SLAUGHTER.
0
3
Time (day)
6 10
15
28
SEMa
Kidney
1.82
5.18
(5.78)
2.65
3.08
2.56
2.04
.20M
(.20M)b
Liver
1.97
36.84
(45.60)
21.80
17.02
16.57
8.26
.25M
(.21M)
Adrenal
3.72
7.95
(8.34)
7.84
16.15
9.89
17.00
. 24M
(.25M)
Pancreas
18.36
5.29
(5.37)
7.73
10.94
9.74
14.62
. 12M
(.13M)
Neck
2.74
1.37
(1.58)
1.42
2.09
2.35
2.32
. 21M
(.21M)
Leg
2.81
1.36
(1.63)
1.48
2.08
3.09
2.03
.21M
(.18M)
Heart
6.09
4.24
(4.73)
5.43
7.40
6.35
5.60
. 17M
(.17M)
Spleen
5.13
21.50
(27.01)
11.16
12.53
11.16
4.96
. 30M
(.24M)
Lung
4.54
29.29
(38.15)
42.54
47.52
15.82
6.07
.43M
(.31M)
3
The standard error of the mean is based on the analysis of variance on the
transformed scale and is approximate. It varies with the mean, M, and has been
expressed in terms of it.
^The values in parentheses are the results when one outlier was removed.

115
TABLE 7.5. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG RATIO OF TISSUE TO
PLASMA.
Tissue Overall
time effect
DOF 5
3-28a
versus 0
1
Source of variation
6-28 10-28 15-28
versus 3 versus 6 versus 10
1 1 1
28
versus 15
1
Residual
19
Kidney
0.432*
0.765*
1.128*(**)b
0.021
0.181
0.064
0.138
Liver
4.657**
19.108**
1.090**
0.719(*)
0.414
0.955(*)
0.222
Neck
0.415
0.535
0.836*(NS)
0.676
0.029
0.001
0.165
Leg
0.510*(NS)
0.481
1.081*(N S)
0.611(*)
0.076
0.301
0.153
Heart
0.170
0.002
0.604*(NS)
0.080
0.144
0.020
0.109
Spleen
1.231*(**)
2.527*(**
) 1.567*(**)
0.235
1.042(*)
0.787
0.310
Lung
4.298**
10.924**
0.000
1.663(*)
6.985**
1.919(**
) 0.577
Adrenal
1.189**
3.323**
0.447
0.958*
0.273
0.943*
0.202a
Pancreas
0.739**
1.520**
1.416**
0.368*
0.012
0.326*
0.058a
g
Residual is based on 17 degrees of freedom because of missing observations.
^Significance levels in parentheses note changes which occurred when one outlier
was removed.
* (**)Indicates a significant effect at 5% (1%) level. NS indicates non
significance at 5% level.

116
TABLE 7.6. PLASMA TOCOPHEROL (yg/ml) VALUES AND SUMMARIES FOR THOSE ANIMALS
SLAUGHTERED AT 28 DAYS.
Days
1
Animal
2 3
4
Mean
SEM
Day 0
0.58
0.47
0.78
0.48
0.58
0.072
Day 1
6.41
6.09
7.27
3.59
5.84
0.790
Day 2
2.89
3.67
6.64
6.56
4.94
0.972
Day 3
1.95
2.81
4.53
8.13
4.36
1.368
Day 6
1.64
2.34
3.59
2.66
2.56
0.405
Day 10
1.56
1.72
1.87
1.44
1.65
0.094
Day 15
1.44
1.15
a
1.64
1.41
0.142
Day 28
1.88
1.64
2.03
2.27
1.96
0.132
Area^
51.43
52.56
74.77
71.96
62.68
6.199
Maximum
concentration
plasma (yg/ml)
6.41
6.09
7.27
8.13
6.98
0.459
Time to maximum
concentration (d)
1.00
1.00
1.00
3.00
1.50
0.500
c
Elimination
rate
3.41
3.71
3.58
3.58
3.57
0.062
aNo measurement was
taken at
this point
in time.
Total area under the plasma concentration curve.
c
Elimination rate is defined as the maximum/terminal plasma level.

117
values were typical of those recorded for all the animals up to the
time of their slaughter.
Discussion
Liver and spleen were characterized by a high accumulation of
vitamin E. Similar results were observed in the liver and spleen of
rats following IP dosing with a large amount of vitamin E (Peake and
Bieri, 1971). This route of administration did not cause any
alteration in the metabolism of vitamin E. Indeed, Black and Bieri
(1964) demonstrated that following IP dosing of rats with ^C-d-a-
tocopherol the radioactivity recovered in the livers was an unchanged
-tocopherol. There is an increase in the intensity of exchange by
the tissues, metabolism and elimination from the body when this
vitamin is given to sheep IP.
The important uptake of vitamin E by the sheep liver supports the
concept that the liver is a target for vitamin E action. Vitamin E is
delivered to the liver via the portal vein and returned to the rest of
the body by the hepatic vein. According to Kayden and Traber (1987),
there must be mechanisms in the liver for the regulation of tocopherol
incorporation into lipoproteins, its storage in the liver and for its
excretion. When a massive dose of vitamin E was given to sheep IP,
there was a peak 3 d post dosing in the vitamin E concentration in all
tissues and then a decline. The greatest rate of loss occurred in the
hepatic tissue, where the loss was proportional to its concentration
in vitamin E. Bieri (1972) reported that all tissues (except depot

118
fat) contain a labile pool of a-tocopherol, which mobilizes rapidly,
and a fixed component which is retained for long periods.
The high concentrations of vitamin E in the liver, spleen and
lung, 3 d after initiation of the IP administration to sheep are in
agreement with the results of Gallo-Torres (1971) in rats where these
organs contained high levels of vitamin E following parenteral
administration of this vitamin. In the lung very high concentrations
of vitamin E could be attributed to this organ's abundant blood
supply. The high tissue/plasma (t/p) ratio observed in the adrenal
and pancreas of sheep provided with IP vitamin E characterizes the
intensity of metabolism in these organs. Tikriti et al. (1968)
observed in dairy cows that, following parenteral administration of
radiotocopherol, pancreas and adrenal were relatively high in vitamin
E activity. The heart t/p indicated an intermediate vitamin E
activity while in the muscle the t/p was low. This might indicate
that the muscle has a different mechanism of action concerning
handling of vitamin E than other organs.
Gallo-Torres (1982) noted that information on the fate of vitamin
E after parenteral administration is scarce. The present work
provided data on the fate of a massive single IP dose of vitamin E in
the body of sheep, indicating a differential handling by the various
organs. Judging from the results of vitamin E distribution, it
appears that the IP method is a convenient route of vitamin E
administration to sheep.

119
Summary
Blood plasma and tissue vitamin E were determined in twenty-five
crossbred sheep following intraperitoneal injection of dl-a-
tocopherol. Five sheep were used as controls (no treatment and killed
at d 0). The remaining 20 sheep were administered intraperitoneally
with 5 g of dl-a-tocopherol. From these twenty vitamin E dosed sheep,
four were slaughtered at each of d 3, 6, 10, 15 and 28 after dosing.
There was a significant time effect for all on a-tocopherol
concentrations for all tissues. In most tissues, the peak for ot-
tocopherol concentrations was observed at 3 d post dosing. There was
a varied rate of uptake for different tissues examined. Three d post
IP dosing, a large uptake of vitamin E by the liver was noticed, and
this supports the concept of the hepatic tissues as a target organ for
vitamin E action. Also at d 3, a pronounced uptake was observed in
the spleen and lung. Vitamin E concentrations in the remaining
tissues at d 3 post dosing showed also a considerable increase, but to
a lesser degree than those in liver, spleen and lung. A decline of
vitamin E concentration in all tissues occurred after d 3. This
study, through the use of contrasts over time (d), provided some
indication of when a plateau in vitamin E concentrations had been
reached for each individual tissue following an IP dosing.

CHAPTER 8
GENERAL CONCLUSIONS
Five experiments were carried out to investigate the
bioavailability of various tocopherol sources in ruminants as well as
the effect of mode of administration. Experiment I was conducted to
investigate the bioavailability of d-a-tocopherol and dl-a-tocopherol
suspended in sesame oil solution (200 mg/ml) and administered
intramuscularly at a rate of 40 mg/kg body weight. In all sheep given
the various vitamin E sources by intramuscular oil depot injection, a
delayed increase in plasma a-tocopherol levels occurred. Sheep
administered d-a-tocopherol and slaughtered after 360 hr had a larger
(P<.01) area under the plasma concentration time curve (AUC) than
sheep injected with d-a-tocopherol or dl-a-tocopherol and killed 240
hr post dosing. A higher plasma C maxima (P<.01) was found in the d-
a-tocopherol group than in the others. Pancreatic, hepatic, lung,
spleen and muscle a-tocopherol concentrations were higher (P<.05) for
this group than for controls. Tendencies for higher (P>.05) tissue a-
tocopherol concentrations were found for the d-a-tocopherol
administered group (360 hr) than in the other vitamin E treated sheep.
Experiment II assessed the bioavailability of four tocopherol
sources provided orally of either (1) dl-a-tocopherol, (2) dl-a-
tocopheryl acetate, (3) d-a-tocopherol or (4) d-a-tocopheryl acetate
in crossbred yearling wethers at a level of 400 IU/day/sheep for 28 d.
Higher a-tocopherol concentrations (P<.01) were found in the heart and
120

121
lung of the sheep provided with d-a-tocopherol than the other forms.
In all diets, pancreas contained the highest concentration followed by
the liver and spleen. Muscle and adipose tissue were found to have
the lowest (PC.05) concentration of a-tocopherol. Sheep provided d-a-
tocopherol had a larger (P<.05) area under the plasma a-tocopherol
concentration time curve than the other groups. Following the
provision of 400 IU's/d of the various tocopherol sources to sheep, it
was found that d-a-tocopherol was more potent than its acetate or the
1-isomeric forms of vitamin E.
In experiment III beef cows were provided a daily oral dose of
1000 IU of various tocopherol sources for 28 d to investigate plasma
and tissue a-tocopherol concentrations. Supplementation over a 1 to 7
d period of time with the various vitamin E preparations increased
(P<.01) plasma a-tocopherol concentration above baseline values, d-a-
tocopherol and its corresponding acetate ester increased plasma a-
tocopherol concentration quicker (P<.05) than the racemic products
with the greatest response observed with d-a-tocopherol. There was a
wide distribution of a-tocopherol concentration in the tissues.
Across all treatments, the highest a-tocopherol concentrations were
found in the adrenal gland and liver with the lowest observed in
muscle and thyroid tissue. Higher a-tocopherol concentrations were
found in the adrenal gland, kidney (P<.05), liver and lung following
d-a- than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered
to sheep intraruminally (100 mg/kg b.w.) or cattle (50 mg/kg b.w.) in
a single oral dose. The sheep data showed that the C maxima plasma a-

122
tocopherol concentration had a tendency (P>.05) to be higher in the
dl-a-tocopherol than in the ester-dosed group. A higher (P<.05)
plasma a-tocopherol tolerance curve area was found in the dl-a-
tocopherol than in its ester form, as well as a quicker (P<.05) time
(h) for maximum plasma a-tocopherol concentration. Data from cows
revealed a greater (P<.05) plasma tolerance area following the
administration of dl-a-tocopherol than its acetylated form.
Experiment V was designed to determine plasma and tissue a-
tocopherol concentrations in sheep following a 5 g dose of dl-a-
tocopherol (water based) injected intraperitoneally followed by serial
slaughterings. A significant time effect for all tissues was noticed,
while the peak for a-tocopherol concentrations was observed 3 d post
dosing. A varied rate of uptake of a-tocopherol was noticed with the
liver, lung and spleen showing a pronounced uptake while the muscle
showed the least. This study provided an indication of when a plateau
in a-tocopherol concentration had been reached for each individual
tissue. Studies reported in this dissertation indicated that blood
plasma and tissue a-tocopherol concentrations were influenced by
various sources of vitamin E as well as by mode of administration and
its type of vehicle. Higher indexes of bioavailability were observed
in favor of natural a-tocopherol than synthetic a-tocopherol in sheep
and cattle dosed with equivalent amounts (IU). Sheep given the
vitamin E preparations by intramuscular oil depot injection resulted
in a delayed increase in plasma and tissue a-tocopherol concentration.
Intraperitoneal injection of dl-a-tocopherol (water base)
resulted in a substantial and quick rise in plasma and tissue a-

123
tocopherol levels. Comparing the currently accepted biological
potencies (IU/mg) provided by the National Formulary (1985) for the
various tocopherol sources to the estimate values for ruminants are as
follows respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37). The results
obtained from these experiments suggest that the current accepted
biological potencies of the various tocopherol sources may be invalid
for ruminants.
Experiments reported in this dissertation show that the natural
form of a-tocopherol had a higher bioavailability than did the
synthetic, though the reason for this remains unresolved.
Further research in ruminants should be directed towards
identifying the potential presence of specific binding proteins in
tissues of ruminants which have been shown to be fairly specific for
the natural stereoisomer in other species.

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BIOGRAPHICAL SKETCH
Nicholas Hidiroglou was born in December 1961 in Kapuskasing,
Ontario. He attended local schools and graduated from Confederation
High School in 1980. He entered Macdonald College of McGill
University in September 1980 and was awarded the degree of Bachelor of
Science in Agriculture in 1983. In January 1984, the author entered
graduate school at the University of Florida and completed a Master of
Science degree in animal science in May 1986. He enrolled in a Ph.D.
program in 1986 and is currently a candidate for the degree of Doctor
of Philosophy in animal science. The author has accepted a Research
Associate position at East State Tennessee University in Johnson City.
139

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L.R. McDowell, Chairman
Professor of Animal Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Douglas Bates
Assistant Professor of Animal
Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Kermit Bachman
Associate Professor of Dairy
Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Rachel Shireman
Associate Professor of Food
Science and Human Nutrition
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
August 1989
Dean, Graduate School



113
observation for several other variables. The effect it has on the
results is noted in the tables. The main effect of its omission was a
decrease in the variances and an increase in the values at d 3. The
result is, the contrast tests shift the plateau to d 6 for the neck,
leg, heart and adrenal which seems quite reasonable studying the
means. As well, the increase at d 28 becomes significant (P<.05) for
the adrenal. At 28 d, the skeletal muscles and kidney had the lowest
values among the various tissues, while the adrenal ranked first
followed by the pancreas.
The means for the ratio of the tissue to plasma (t/p) levels are
given in Table 7.4 and the associated analysis of variance in Table
7.5. The observations at d 3 did not fail the Tietjen and Moore test
(P>.05) for any variable. However, its effect has been noted in the
tables. Here there is no effect (P>.05) over time for the neck and
heart. While the overall test is not significant (P>.05), the
contrast of d 3 to later times indicates d 3 values were depressed for
these two tissues. The leg data show a similar pattern although the
overall time effect was significant in this case. After a significant
increase, the kidney data appear to achieve a plateau by d 6, the
spleen by d 10. Liver and lung data also show a significant increase
and than a decrease (P<.01). Although the tests indicate a plateau is
reached, the means suggest that a decrease may continue to d 28. The
adrenal and pancreas data show a significant time effect, but without
a plateau.
In Table 7.6, summary statistics are given for the four animals
whose plasma values were recorded throughout the experiment. These


70
ranked lower (P<0.05). Lowest (P<0.05) concentrations of a-tocopherol
were observed in the muscle and adipose tissue (Figure 4.1).
Table 4.3 (and Figure 4.2) shows that the relative retention time
(minutes) of the trimethylsilyl esters (TMSi) obtained from purified
a-tocopherol tissues samples are very similar to that obtained from
the TMSi a_tocopherol standard.
Mass spectra of the gas chromatographic peak corresponding to the
a-tocopherol standard (TMSi) (Figure 4.3) and that corresponding to
the peak with the same retention time obtained for the pancreas, for
example, are identical, confirming the presence of a-tocopherol in
these biological extracts.
The peak obtained at mass/charge 502 represents the molecular ion
of a-tocopherol TMSi (430 a-tocopherol + 72 trimethylsilylether). The
fragmentation pattern reveals peaks at mass/charge 277, resulting from
the loss of the side chain (C^H-^) and at mass/charge 237 due to the
cleavage of the chroman structure accompanied by hydrogen
rearrangement and the loss of a CH3-C=CH fragment (Nair and Luna,
1968).
Blood Plasma
Analysis of blood plasma a-tocopherol concentrations summarized
in Table 4.4 show that diet and tissue source were significant
(P<0.01) but not the interaction diet x day. Higher (P<0.01) a-
tocopherol plasma concentrations were observed in sheep supplemented
with d-a-tocopherol on days 9, 15, 17 and 25 (Figure 4.4) than in the
other three groups. Also, in sheep fed d-a-tocopherol, the most


5
Figures 2.2 and 2.3 show that vitamin E exists as eight plant-
derived compounds that have a common 6-chromanol ring structure but
that differ in the side chain and number of methyl groups. The four
tocols have a phytol side chain while the four trienols having trans
double bonds at 3', 7' and 11' of the phytol side chain. The methyl
substituents occur in the ring at positions 5,7,8 (a-), 5,8 (B-), 7,8
(y), and 8 (6-) in both the tocol and trienol series.
The tocols contain three asymmetric carbons specifically at the 2
position (Fig. 2.4) in the ring and in the 4' and 8' position of the
side chain, thus giving a total of eight possible optical isomers.
The epimeric configuration at the 2 position is apparently dominant in
determining biological activity.
FIGURE 2.4. ASYMMETRIC CARBONS OF TOCOPHEROLS


CHAPTER 1
INTRODUCTION
Existence of an anti-sterility vitamin was brought to light in
the early 1920s when evidence was obtained that female rats reared on
a diet containing all the vitamins known at that time failed to
reproduce, although they were apparently normal in other respects
(Evans and Bishop, 1922; Mattill and Coulin, 1920; Sure, 1924).
Although the rats would mate and conceive, pregnancy invariably was
terminated by fetal death followed by resorption. The missing factor
was characterized as a vitamin by Evans and Bishop (1922) and
designated vitamin E. Evans et al. (1927), who found that the
unsaponifiable fraction of wheat germ oil was a convenient raw
material for chemical investigation, and isolated (1936) from wheat-
germ oil two alcohols, a- and 6-tocopherol, both of which showed
vitamin E activity.
The name tocopherol originated from the Greek tocos (childbirth),
phero (to bear) and ol (alcohol). Two years later, its structure was
elucidated (Fernholz, 1938), and shortly thereafter it was synthesized
(Karrer et al., 1938). During the period 1930-1950, multiple varied
deficiency disorders of animals were reported to be cured by vitamin
E. Work by Schwarz and Folz (1957) led to the identification of
selenium as the factor other than vitamin E that could also prevent
the degeneration of liver in rats. Dose of selenium required for
complete protection against liver degeneration was about one
1


-TOCOPHEROL fig/ml PLASMA cr-TOCOPHEROL pg/ml PLASMA
96
FIGURE 6.1. PLASMA TOCOPHEROL CONCENTRATION (uG/ML) IN SHEEP
FOLLOWING ADMINISTRATION OF A SINGLE MEGADOSE (100 MG/KG
BODY WEIGHT) OF (A) DL-ot-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL


4
Tocopherols Chemical Name
R1 R2 R3
a-Tocopherol
CH
CH-
CH-
5,7,8-Trimethyltocol
3-Tocopherol
CH-
H 3
CHo
5,8-Dimethyltocol
Y-Tocopherol
H 3
CH-
CH-
7,8-Dimethyltocol
3-Tocopherol
H
H 3
CH3
8-Methyltocol
(a)Tocopherois
FIGURE
2.2.
STRUCTURAL
FORMULAS OF TOCOPHEROLS
Tocotrienols
Chemical Name
R1
R2
R3
a-Tocotrienol
CH-
CHo
CH-
5,7,8-Trimethyltocotrienol
3-Tocotrienol
CHo
H 3
CH-
5,8-Dimethyltocotrienol
Y-Tocotrienol
H 3
CH
CH-
7,8-Dimethyltocotrienol
6-Tocotrienol
H
H 3
CHo
8-methyltocotrienol
FIGURE 2.3. STRUCTURAL FORMULAS OF TOCOTRIENOLS


125
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65
Instrumentation
The chromatographic apparatus consisted of an M 6000 pump and a
WK septumless injector (Waters Associates, Inc., Milford, Mass., USA).
A Perkin-Elmer 650-150 fluorescence spectrophotometer, equipped with a
microflow cell unit, was used for quantitation. Wavelength settings
were 295 and 330 nm for excitation and emission, respectively. The
column as a Bondapak C18 (3.9 mm x 30 cm) of 10 pm particle size
purchased from Waters Associates (Milford, Mass., USA). The mobile
phase was a solvent system consisting of methanol and water in a 97:3,
with a flow rate of 3 ml/minute. All solvents used were HPLC grade.
Mass Spectrometry in Combination with Gas Chromatography
Further identification of a-tocopherol in various tissues was
carried out by a combination of a Hewlett-Packard (HP) gas
chromatograph 5790-mass spectrometer 5970A series with a data station
(HP-9000). The mass spectrometer performs three basic functions.
First, molecules are subjected to bombardment by a stream of high-
energy electrons, converting some of the molecules to ions. The ions
are accelerated in an electric field. Second, the accelerated ions
are separated according to their mass-to-charge ratio in a magnetic
field or electric field. Finally the ions with a particular mass-to-
charge ratio are detected by a device which is able to count the
number of ions which strike it. The detector's output is amplified
and fed to a recorder (Roboz, 1968). Eluates from the HPLC were


121
lung of the sheep provided with d-a-tocopherol than the other forms.
In all diets, pancreas contained the highest concentration followed by
the liver and spleen. Muscle and adipose tissue were found to have
the lowest (PC.05) concentration of a-tocopherol. Sheep provided d-a-
tocopherol had a larger (P<.05) area under the plasma a-tocopherol
concentration time curve than the other groups. Following the
provision of 400 IU's/d of the various tocopherol sources to sheep, it
was found that d-a-tocopherol was more potent than its acetate or the
1-isomeric forms of vitamin E.
In experiment III beef cows were provided a daily oral dose of
1000 IU of various tocopherol sources for 28 d to investigate plasma
and tissue a-tocopherol concentrations. Supplementation over a 1 to 7
d period of time with the various vitamin E preparations increased
(P<.01) plasma a-tocopherol concentration above baseline values, d-a-
tocopherol and its corresponding acetate ester increased plasma a-
tocopherol concentration quicker (P<.05) than the racemic products
with the greatest response observed with d-a-tocopherol. There was a
wide distribution of a-tocopherol concentration in the tissues.
Across all treatments, the highest a-tocopherol concentrations were
found in the adrenal gland and liver with the lowest observed in
muscle and thyroid tissue. Higher a-tocopherol concentrations were
found in the adrenal gland, kidney (P<.05), liver and lung following
d-a- than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered
to sheep intraruminally (100 mg/kg b.w.) or cattle (50 mg/kg b.w.) in
a single oral dose. The sheep data showed that the C maxima plasma a-


TOCOPHEROL Wml PLASMA)
75
Diet 1 : dlatocopherol
Diet 2 : dla-tocopherol acetate
Diet 3 : da-tocopherol
DAY
FIGURE 4.4.
SEES SS5SS 0F SHEEP


71
TABLE 4.3. RETENTION TIME OF a-TOCOPHEROL FROM GAS-
CHROMATOGRAPHY/MASS SPECTROMETRY.
Relative retention time (minutes)
Substance
TMSia
Standard oi-tocopherol
4.1
Pancreas
3.9
Liver
3.8
Heart
3.9
Spleen
3.9
Lung
3.9
aTMSi; Trimethylsilyl ether.


131
Karrer, P., and 0. Isler. 1941.35. U.S. Patent 2, 411, 969.
Kasparek, S. 1980. Chemistry of tocopherols and tocotrienols. In
Vitamin E: A Comprehensive Treatise (L. J. Machlin, ed.), pp. 7-
65. Marcel Dekker, New York and Basel.
Kayden, H. J., and M. G. Trabel. 1987. Vitamin E absorption,
lipoprotein incorporation and transfer from lipoproteins to
tissues. In: 0. Hayaishi and M. Mino (Eds.) Clinical and
Nutritional Aspects of Vitamin E. pp 129-138. Elsevier Science
Publications, New York.
King, J. M., and D. C. Maplesdon. 1960. Nutritional muscular
dystrophy in calves. Can. Vet. J. 1:421.
Kitabchi, A. E., and J. Wimalasena. 1982. Specific binding sites for
D- -tocopherol on human erythrocytes. Biochim. Biophys. Acta.
684:200.
Klatskin, G., and D. 0. Molander. 1952. The absorption and excretion
of tocopherol in Laenner's cirrhosis. Clin. Invest. 31:159.
Koch-Weser, J. 1974. Bioavailabiity of drugs. New Engl. J. Med.
291:233.
Krishnamurthy, S., and J. G. Bieri. ^963. The absorption storage and
metabolism of alpha-tocopherol- C in the rat and chicken. J.
Lipid Res. 4:330.
Lannek, M. 1962. The ethiology of nutritional muscular dystrophy in
swine. Nord. Veterinaer Med. 14 (Suppl.):2, 19.
Lehninger, A. L. 1975. Biochemistry. The molecular basis of cell
structure and function, pp. 302-306. Worth Publishers, Inc.,
New York, NY.
Leibovitz, B. E., and B. V. Siegel. 1980. Aspects of free radical
reactions in biological systems. Aging J. Gerontol. 35:45.
Lundberg, W. 0. 1962. Mechanisms. _Ir^ Symposium on Foods: Lipids
and their Oxidation. The Second in a Series of Symposia on Foods
held at Oregon State University (H. W. Shultz, E. A. Day, and R.
0. Shinnhuber, eds.) 2:31-50. Avi Publishing Company, Westport,
CT.
Machlin, L. J. 1984. Vitamin E. Li Handbook of Vitamins,
Nutritional, Biochemical and Clinical Aspects, pp. 99-145.
Marcel Dekker, New York.
Machlin, L. J., E. Gabriel, and M. Brin. 1982. Biopotency of a-
tocopherols as determined by curative myopathy bioassay in the
rat. J. Nutr. 112:1437.


18
declining rapidly. Most of the tocopherol was bound to the structural
components of the liver cells, primarily mitochondria and microsomes.
Vitamin E is transported in the blood via lipoproteins. A rapid
exchange among lipoprotein classes occurs with vitamin E after it
enters the circulation via the chylomicrons. Since plasma tocopherol
level is correlated with the total plasma lipid content, low density
lipoprotein (LDL), the most common lipoprotein in human plasma,
carries most of the plasma vitamin E. There is also a rapid exchange
between tocopherol in the erythrocyte membrane and lipoproteins such
that approximately 20% of the plasma tocopherol concentration is
carried by red blood cells. Delivery to other tissue cells appears to
be in association with the receptor-mediated uptake of LDL (Traber and
Kayden, 1984).
Type and composition of diet influence absorption of vitamin E
from the gut. Pudelkiewicz and Matterson (1960) reported that only
about one-third of the d-ci-tocopherol in alfalfa is available to
chicks. The poor utilization is attributed to a fat-soluble compound
in alfalfa that acts antagonistically to a-tocopherol decreasing its
availability. An antagonistic relationship also exists between
absorption of vitamin E and unsaturated fatty acids (Bunyan et al.,
1968).
Generally, in animals, the uptake of vitamin E from the small
intestine is lowered when tocopherol is fed in an oily form. The
uptake is facilitated by bile salts (Simon et al., 1956).
When tocopheryl acetate ester was fed, most fecal tocopherol
appeared as free tocopherol; however, a small portion appeared in the


61
administration of d-a-tocopherol, tissue levels of <*-tocopherol were
higher than those of control. In some cases there were large
individual variations in tissue a-tocopherol concentrations which made
comparisons difficult between corresponding tissues in the various
groups. It is apparent that of the tissues examined the depot fat is
the preferred storage tissue for vitamin E. According to Machlin et
al. (1979) adipose tissue tocopherol is not readily available upon
metabolic demand. Tocopherol concentrations in the liver of control
were low indicating low dietary intake. Rammel and Cunliffe (1983)
using HPLC reported that levels of 2 mg of a-tocopherol per kg of
fresh liver were observed in clinically normal sheep. According to
these workers a-tocopherol hepatic levels less than 2 mg/kg become
health problems. In deer, Meadows et al. (1982), also using HPLC,
observed liver a-tocopherol concentrations of 5 yg/g wet tissue.
In conclusion, the results showed a delayed increase in plasma o-
tocopherol concentration in sheep administered IM two vitamin E
preparations. A more sustained increment in plasma a-tocopherol was
observed in sheep IM-injected with d-a-tocopherol than dl-a-
tocopherol. Following d-a-tocopherol IM administration, higher tissue
a-tocopherol levels occurred in sheep killed 360 hr after dosing than
in those of control sheep.
Summary
The bioavailability of two vitamin E preparations (d-a-tocopherol
and dl-a-tocopherol) suspended in sesame oil solution and administered
intramuscularly was evaluated in sheep. In sheep administered d-a-


57
In Group 3 (IM d-a-tocopherol injected sheep killed 240 hr after
dosing) there was also a more sustained delivery than in group 1 (t
maximum 191>151). This resulted in a marginal increase in C max but
no increase in AUC was noted. This may be due to the fact that
initial values in Group 3 were very low compared to the other groups
(Cq=0.71). It was noted that relative to initial values the increase
in vitamin E concentration in Group 3 sheep was much higher than for
Group 1 and approached the relative increase for Group 2 (C
max/Co=2.63).
The AUC measurement of Group 3 was comparable to that of group 1.
This may have been due largely to the higher initial value in this
group. The elimination rates of the three groups appeared to be very
similar (no differences in Cmax/C^,).
Tissue Concentrations of Vitamin E
Sheep receiving d-a-tocopherol and slaughtered 360 hr after
dosing had higher (P<0.05) pancreatic, hepatic, lung, spleen and
muscle a-tocopherol levels than the control group (Table 3.2). There
also was higher a-tocopherol concentration (P<0.05) in the pancreas in
sheep of Group 2 than Group 3. In the lung, brachiocephalicus muscle,
pancreas and spleen of sheep slaughtered 240 hr after dosing, higher
a-tocopherol concentrations were found in the d-a-tocopherol than dl-
a-tocopherol administered animals. In these tissues a-tocopherol
concentrations in Group 3 had a tendency to be higher than those of
Group 1 sheep, a-tocopherol concentrations in all groups were higher
(P<0.01) in the adipose and adrenal than in other tissues analyzed.


15
tocopherol levels, which suggested that intestinal uptake or plasma
transport is more efficient for a- than y-tocopherol (Jonsson et al.,
1981). Tocopherol esters are hydrolyzed prior to absorption, and both
bile and pancreatic juice are necessary for absorption to proceed
(Gallo-Torres, 1970). These facts support the idea that free
tocopherol is absorbed by diffusion from a mixed micelle of fatty
acids, monoacyl glycerols, bile salts and acids, cholesterol and other
fat-soluble vitamins. Maximal absorption occurs at the junction of
the upper and middle thirds of the small intestine (Hollander et al.,
1975). After crossing the brush border into intestinal mucosal cells,
tocopherol is not re-esterified but is incorporated as the alcohol
into chylomicrons in mammals and enters the plasma via the lymphatic
system (Behrens et al., 1982).
Desai et al. (1965) showed, in confirmation of studies by Weber
et al. (1962) with rats, that 1-a-tocopherol was absorbed as well as
or better than the d-form of the vitamin. It appeared therefore that
the differences in biopotency must be due to differences in retention
whereby the d-epimer is retained much better than the 1-epimer in the
blood and perhaps in other tissues of the body. The results indicated
the existence of an active carrier of d-a-tocopherol in the blood and
tissues which has a greater affinity for the d-epimer than for 1-a-
tocopherol. Recently it has been reported that specific binding
proteins exist for a-tocopherol in the cytosol and nuclei of rat liver
tissue (Catignani et al., 1977; Guarnieri et al., 1980 Prasad et al.,
1980) as well as in human erythrocytes (Kitabachi and Wimalasena,
1983) which are fairly specific for the natural stereoisomer. The


LIST OF FIGURES
FIGURE PAGE
2.1 STRUCTURAL FORMULA OF a-TOCOPHEROL 3
2.2 STRUCTURAL FORMULAS OF TOCOPHEROLS 4
2.3 STRUCTURAL FORMULAS OF TOCOTRIENOLS 4
2.4 ASYMMETRIC CARBONS OF TOCOPHEROLS 5
2.5 BIOSYNTHESIS OF THE RING STRUCTURE OF TOCOPHEROLS AND
TOCOTRIENOLS 9
2.6 STRUCTURE OF TOCOPHERYL QUINONE 11
2.7 STRUCTURE OF a-TOCORED 12
2.8 STRUCTURE OF a-TOCOPURPLE 12
2.9 SCHEMATIC REPRESENTATION OF THE PEROXIDATION OF
UNSATURATED FATTY ACIDS 45
3.1 PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE I.M.
INJECTION OF D-a-TOCOPHEROL SUSPENDED IN STERILE
SESAME OIL 55
4.1 a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 69
4.2 IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/
MASS SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD,
(B) a-TOCOPHEROL IN PANCREAS 72
4.3 MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A)
AND a-TOCOPHEROL IN THE PANCREATIC TISSUE (B)
FOLLOWING HPLC COLLECTION 73
4.4 a-TOCOPHEROL CONCENTRATIONS IN THE BLOOD PLASMA OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS. 75
5.1 a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE
SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS 87
viii


ABUNDANCE
90
MASS/CHARGE
FIGURE 5.2. MASS SPECTRAL SCANNING OF THE a-TOCOPHEROL STANDARD
(A) AND IN BLOOD PLASMA (B) FOLLOWING HPLC COLLECTION
AND TLC.


24
lesions, especially in the very young (Gullickson, 1949). However, a
high placental susceptibility to vitamin E deficiency as noted in rats
and sows, apparently in mice and perhaps in rabbits, also appears to
exist in cows according to Trinder et al. (1969). These authors noted
a high frequency of placenta retentions (19%) in non-brucellic cattle
in areas where muscular dystrophy in lambs was widespread. Intra
muscular injections of vitamin E (680 IU) and potassium selenate (15
mg) one month before parturition markedly reduced the frequency of
retention. Administrations of potassium selenate only were less
effective, and earlier injections (7 weeks prepartum) ineffective.
A Variety of Disorders Due to Vitamin E Deficiency in Domestic Animals
Vitamin E deficiency causes various disorders in most animal
species. An exhaustive description would be tedious and time
consuming. Observations will be limited to three frequently affected
species: chickens, lambs and calves.
Chickens
Encephalomalacia. Clinical signs of nutrition-related
encephalomalacia are exclusively nerve-related (Dam, 1944). The first
manifestation is ataxia. Next, there may be clonic contractions and
especially trembling. The chicken keeps its head tucked under, as if
rolled into a ball; then paralysis sets in and it falls down in
lateral decubitus. Death is the usual outcome of this clinical form
which entails 20 to 80% losses, depending on the intensity of the
deficiency and the susceptibility of the subjects. Lesions are
significant because they make it possible to distinguish this disorder


35
Of all the test systems, the erythrocyte hemolysis test (EHT) is
of special importance. This test can be done not only as a pure in
vitro variant but also as an iin vivo-in vitro form. The EHT provides
a means of indirect measurement of the human vitamin E status and
correlates well with the tocopherol concentrations in blood plasma.
Individuals with decreased tocopherol concentration are characterized
by an increased hydrogen peroxide-induced sensitivity of the
erythrocytes to hemolysis in the blood serum.
The most commonly used hemolytic agent is hydrogen peroxide.
Washed erythrocytes are incubated in a hydrogen peroxide solution
(about 2%) over a period of 3 hr. Hemolysis liberates hemoglobin
during incubation; correction for this hemoglobin is made by
determining the amount of hemoglobin after incubation with distilled
water which corresponds to 100% hemolysis (without addition). The
hemolysis result obtained with hydrogen peroxide is given as a
percentage.
The relative activity of d-a-tocopherol and d-y-tocopherol in the
in vitro EHT is tabulated in Table 2.4 (Brubacher and Weiser, 1968).
Erythrocytes from rats which were given a vitamin E-deficient diet
were incubated in a test tube together with the various tocopherols
and then hemolyzed with dialuric acid. The tocopherol esterified with
acetic acid is inactive in this test. D-ct-tocopherol has again been
shown to be biologically superior to d-a-tocopherol.
The hemolysis test was originally conceived as an i^ vitro test
but was later modified. This modified test differs from the above-
mentioned in^ vitro test in that the vitamin E active substance is


25
from another neural disease of viral origin in chickens, infectious
encephalomyelitis.
Characteristics of nutrition-related encephalomalacia (Adamstone,
1947), are (1) discrete hepatic lesions; the liver is often pale,
degenerated, speckled with sanguinous suffusions; (2) intestinal
hemorrhagic lesions, occurring mainly around the duodenum and
involving the muscularis; and (3) cerebral lesions, more specific,
macroscopically detectable in some cases, but always visible upon
histological examination.
Upon opening the cranium, the cerebellum is edematized, covered
with hemorrhagic patches, and of very poor consistency. Microscopic
examination confirms the edema and hemorrhages, and in addition makes
it possible to detect changes in Purkinje cells, which appear
retracted, with granulous cytoplasm and pycnic nuclei.
In viral encephalomyelitis, the nerve lesions are different from
the encephalomalacia with perivascular lymphocytic infiltration. In
addition, lymphocytic infiltration can be detected in the pancreas,
the succenturiate ventricle and the muscularis of the gizzard.
The distinction between these two diseases is an important one,
since in encephalomalacia the responsible agent is the feed, whereas
in encephalomyelitis it is the breeder, since contamination of the
chickens occurs jt_ ovo.
Exudative diathesis Exudative diathesis appears in chickens at
the same age as does encephalomalcia and is due to a vitamin E
deficiency associated with a deficiency in sulfur amino acids (Bunyan
et al., 1962). It is characterized by the appearance of subcutaneous


82
Blood Collection
Blood samples (15 ml) were obtained via jugular venipuncture on d
0, 1, 7, 14 and 28 of the feeding period and plasma was analyzed for
a-tocopherol concentration. Samples were always collected in the
morning before vitamin E treatments were provided. Blood samples were
centrifuged at 1,000 x g for 15 min at 4C immediately after sampling,
and plasma was stored at -20C until analyzed for a-tocopherol
concentration.
Tissue Collection
On the last d of the experiment, cows were shipped to a local
abattoir and slaughtered the following d. At that time, 10-50 g
samples were collected from ten different tissues: heart, thyroid,
liver, kidney, adrenal, pancreas, spleen, lung and neck muscle. These
samples were frozen within 2 hours and stored at -20C until analyzed
for a-tocopherol concentration using high pressure liquid
chromatography.
Analytical Method
Quantification of a-tocopherol in tissues was performed by HPLC
using a fluorescence detector as described by McMurray and
Blanchflower (1979b) following tissue homogenization and heptane
extraction by the method of Burton et al. (1985) and Ingold et al.
(1987). Serum samples were prepared for HPLC analysis according to
the method of McMurray and Blanchflower (1979b). Identification and
quantification of a-tocopherol were by comparison of retention times


19
esterified form (Krishnamurthy and Bieri, 1963) indicating that
hydrolysis of the ester took place in the digestive tract.
Rosenkrantz et al. (1951) observed that approximately 8% of ex-
tocopherol given to human subjects was recovered as tocopherol
quinone, suggesting the oxidation of tocopherol in the human digestive
system.
No tocopherol or tocopheryl quinone was excreted in rat urine
although large doses of vitamin E were administered (Klatskin and
Molander, 1952). McArthur and Watson (1939) failed to detect any
tocopherol in cow urine. Simon et al. (1956) reported that about 18%
of tocopherol intake appeared in the urine of the rabbit and man after
intravenous administration of isotopic vitamin E. Urinary radio
activity was not attributed to a-tocopherol or tocopheryl quinone but
to two polar metabolites identified as an acid and its lactone which
were subsequently named tocopheronic acid and tocopheronolactone
(Green et al., 1967). Work by Chow et al. (1967) confirmed the
findings of Simon and associates.
Krishnamurthy and Bieri (1963) reported that only 0.5% of total
activity was in the urine when the rat was sacrificed 24 h after oral
14
administration of C a-tocopherol. In a long-term experiment, the
same authors found that the activity in the urine was very low.
Furthermore, no significant amount of vitamin E was lost in the form
of CO2.
Almost all studies on the pharmacological properties of vitamin E
used doses between 200 and 800 mg/day. John Bieri, a leading American
expert on vitamin E, had the following to say on dosage: "Nothing


11
boiling point at 0.1 atm 210-210C. Tocopheryl esters are more stable
to oxygen but cannot function as anti-oxidants in this form.
Metabolic Degradation Products of Tocopherol
The chemistry of the oxidation of tocopherols is highly complex
and has been reviewed by Kasparek (1980). Complete chemical
degradation of the tocopherol molecule occurs upon treatment with
chromic acid and potassium permanganate, while milder oxidation by
nitric acid, silver nitrate, ferric chloride, auric chloride, ceric
sulphate and nitrogen dioxide leads to formation of a-tocopheryl
quinone (Fig. 2.6). With nitric acid and ferric chloride, a-tocored
(Fig. 2.7) and ot-tocopurple (Fig. 2.8) are also formed.
3
FIGURE 2.6. STRUCTURE OF TOCOPHEROL QUINONE


TOCOPHEROL Wg FRESH TISSUE)
69
Diet I : dl-otocopherol
Diet 2 : dl-otocopherol acetate
c
Diet 4 : d-o-tocopherol acetate
TISSUE TYPE
FIGURE 4.1. a-TOCOPHEROL CONCENTRATIONS IN THE VARIOUS TISSUES OF
SHEEP SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS


62
tocopherol and killed after 360 hr (Group 2), there was a higher
(P<0.01) bioavailability as assessed by the area under the plasma
concentration-time curve, than for sheep killed 240 hr after injection
with d-a-tocopherol (Group 3) or dl-a-tocopherol (Group 1). In the 3
groups (5 sheep each) the highest blood plasma a-tocopherol increment
occurred in the d-a-tocopherol injected sheep. For sheep injected
with d-a-tocopherol and slaughtered at 360 hr (Group 2) the
pancreatic, hepatic, lung, spleen and muscle tocopherol concentrations
were higher (P<0.05) than in the control group. Also in group 2 there
was a tendency for higher tissue tocopherol concentrations than in the
other vitamin E treated sheep (Group 1 and 3).


134
Peak, I. R., and J. G. Bieri. 1971. Alpha and gamma-tocopherol in
the rat: In vitro and in vivo tissue uptake and metabolism. J.
Nutr. 101:1615.
Pehrson, B., and J. Hakkarainen. 1986. Vitamin E status of healthy
Swedish cattle. Acta. Vet. Scand. 27:351.
Plack, P. A., and J. G. Bieri. 1964. Metabolic products of alpha-
tocopherol in the livers of rats given intraperitoneal injections
of alpha-tocopherol. Biochim. Biophys. Acta 84:729.
Porta, E. A., and W. S. Hartroft. 1969. Lipid pigments in relation
to aging and dietary factors (lipofuscins). _In Pigments in
Pathology (M. Wolman, ed.), pp. 191-235. Academic Press, New
York.
Prasad, K. N., D. Gaudreau, and J. Brown. 1981. Binding of vitamin E
in mammalian tumor cells in culture. Proc. Soc. Exp. Biol. Med.
166:167.
Pryor, W. A. 1970. Free radicals in biological systems. Scientific
American 223:70.
Pryor, W. A. 1971. Free radical pathology. Chem. Engin. News June
7:34, 41.
Pudelkiewick, W. J., and L. D. Matterson. 1960. A fat-soluble
material in alfalfa |£iat reduces the biological availability of
tocopherol 5 methyl- C-succinate. J. Biol. Chem. 221:797.
Pudelkiewicz, W. J., L. D. Matterson, L. M. Potter, L. Webester, and
E. P. Singsen. 1960. Chick tissue storage bioassay of alpha-
tocopherol. Chemical analytical techniques and relative
biopotencies of natural and synthetic alpha-tocopherol. J. Nutr.
71:115.
Rammell, C. G., and B. Cunliffe. 1983. Vitamin E status of cattle
and sheep. 2. Survey of liver from clinically normal cattle and
sheep for a-tocopherol. N. Z. Vet. J. 31:203.
Raychaudhur, C., and I. D. Desai. 1971. Ceroid pigment formation and
irreversible sterility in vitamin E deficiency. Science
173:1028.
Rigdon, R. H., T. M. Fergusson, and J. Couch. 1962. Muscular
dystrophy in the chicken. A pathology study. Texas Rpts. Biol.
Med. 20:446.
Roboz, J. 1968. Introduction to Mass Spectrometry, Instrumentation
and Techniques, pp. 1-20. Interscience Publishers, John Wiley
and Sons, Inc., New York, NY.


BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
BY
NICHOLAS HIDIROGLOU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


52
silage (20%). The trial was carried out in three successive periods
(2 months each), with each period utilizing one group of five sheep.
Sheep were administered respective tocopherol treatments
intramuscularly. The parenteral vitamin E preparations, suspended in
sesame oil (200 mg/ml) were administered slowly at 40 mg/kg body
weight into the muscle of the crural area of each leg. Treatments
with respective slaughter times are as follows:
Group 1 dl-a-tocopherol at 240 h
Group 2 d-a-tocopherol at 360 h
Group 3 d-a-tocopherol at 240 h.
A group of six wethers originating from the same flock, was used as a
control (Group 4).
Blood samples were taken from the jugular vein of treated sheep
at specific time intervals after dosing ranging from zero to 360 h.
The plasma was separated from the cellular blood components in a
refrigerated centrifuge at 4C and stored at -20C until assayed for
a-tocopherol. Tissue samples were taken from all sheep after
slaughter and stored at -20C until analyzed for a-tocopherol content.
Feed samples were taken during the experiment for vitamin E analysis.
Analytical
High pressure liquid chromatography (HPLC) was used for plasma a-
tocopherol determination (McMurray and Blanchflower, 1979a). The HPLC
system consisted of an M 6000 pump and W.K. septumless injector
(Waters Associates Inc., Milford, Mass., USA) and Waters y Bondapak
C^2 (10 ym) column (3.9 x 300 mm) coupled to a Perkin-Elmer 650-105


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
L.R. McDowell, Chairman
Professor of Animal Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Douglas Bates
Assistant Professor of Animal
Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Kermit Bachman
Associate Professor of Dairy
Science


17
vitamin E from the absorptive cell thus requires several stages. In
mammals, it must first pass though the lateral or basal plasma
membrane of the cell, then through the basal lamina before entering
the fluid of the lamina propria. From this location the vitamin
enters the capillaries of the lymph and is transported in the
chylomicrons (Dobbins, 1975). Apparently only small amounts of
tocopherol are transported from the intestine via the portal vein in
mammals, whereas all of the tocopherol absorption in birds occurs via
the portal vein directly to the liver (Machlin, 1984).
The absorption of orally fed vitamin E follows the pattern of
lipids in general and of fat soluble vitamins in particular (Desai et
al., 1965; Wiss et al., 1962). The specific site of absorption is not
well established. The small intestine is thought to be the major site
of absorption for tocopherol even though some absorption takes place
from the stomach of nonruminants and the rumen of ruminants (Blaxter
and Brown, 1952; Roles, 1967). The presence of vitamin E in both the
blood and lymph of animals suggests that absorbed vitamin E can be
transferred by either the blood or lymphatic route (Roles, 1967).
Wiss et al. (1962) were also able to establish a mathematical
relationship between the logarithms of tocopherol intake and plasma
and liver concentration in chickens fed high doses of d-ot-tocopheryl
acetate (2,000 to 20,000 IU/kg of feed). Using [C^]-dl-a-tocopheryl
acetate, they demonstrated that maximal liver concentration was
reached only after several hours, and persisted longer than the
synthetic antioxidant ethoxyquin (6-ethoxy-l,2-dihydro-2,2,4-
trimethylquinoline), which attained a maximum within 30 min before


16
exact nature of the binding of a-tocopherol in the cell is an elusive
problem which needs investigation. Experiments by Desai and Scott
(1965) and Scott (1965), comparing the oral administration of d- and
1-a-tocopheryl acetates in the presence of graded levels of dietary
selenium, indicated that selenium is involved in some unknown way in
the retention of d-a-tocopherol in plasma. It remains to be
determined if the differences in plasma levels of d- and 1-epimers of
a-tocopherol are the result of differences (1) in rate of excretion,
(2) in rate of destruction, (3) in the affinity of the epimers for
specific carriers, or (4) in chemical activity influenced by
structural configurations.
Gallo-Torres (1970) reported the obligatory role of bile for the
intestinal absorption of vitamin E into the lymph of rats. Only
negligible amounts of radioactivity could be detected in the thoracic
duct lymph when both bile and pancreatic juice were absent from the
duodenum.
The lipid-bile micelle structure is required to transport the
fat-soluble vitamin E across the "unstirred water layer" which
represents the aqueous phase in the intestinal lumen immediately
adjacent to the brush border of the microvilli. Vitamin E is
absorbed, together with free fatty acids, monoglycerides, and other
fat-soluble vitamins, by penetrating the epithelial cell through the
apical plasma membrane of the absorptive cells in the brush border.
Studies on absorption and transport of a-tocopherol by Davies et al.
(1971) showed that, for normal utilization of absorbed a-tocopherol,
normal lipoprotein transport mechanisms are involved. Transfer of


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
By
NICHOLAS HIDIROGLOU
August 1989
Chairman: Dr. L. R. McDowell
Major Department: Animal Science
This research was designed to investigate the bioavailability of
various tocopherol sources for ruminants. In experiment I, d-a-
tocopherol and dl-a-tocopherol suspended in sesame oil were administered
intramuscularly in sheep at a level of 40 mg/kg body weight (b.w.).
Sheep administered d-a-tocopherol and killed after 360 hr resulted in a
higher (P<.01) bioavailability than for sheep injected with d-a-
tocopherol or dl-a-tocopherol and sacrificed 240 hr after dosing. A
more sustained increment in plasma a-tocopherol was observed with d-a-
tocopherol than dl-a-tocopherol.
In experiment II, sheep were provided with 400 IU/d of either d-a-
tocopherol, dl-a-tocopherol or their corresponding acetate forms for 28
d. Higher concentrations of a-tocopherol were observed in tissues of
sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed
d-a-tocopherol, the area under the plasma a-tocopherol time curve (AUC),
was larger as compared to other forms of vitamin E supplementation.
x


117
values were typical of those recorded for all the animals up to the
time of their slaughter.
Discussion
Liver and spleen were characterized by a high accumulation of
vitamin E. Similar results were observed in the liver and spleen of
rats following IP dosing with a large amount of vitamin E (Peake and
Bieri, 1971). This route of administration did not cause any
alteration in the metabolism of vitamin E. Indeed, Black and Bieri
(1964) demonstrated that following IP dosing of rats with ^C-d-a-
tocopherol the radioactivity recovered in the livers was an unchanged
-tocopherol. There is an increase in the intensity of exchange by
the tissues, metabolism and elimination from the body when this
vitamin is given to sheep IP.
The important uptake of vitamin E by the sheep liver supports the
concept that the liver is a target for vitamin E action. Vitamin E is
delivered to the liver via the portal vein and returned to the rest of
the body by the hepatic vein. According to Kayden and Traber (1987),
there must be mechanisms in the liver for the regulation of tocopherol
incorporation into lipoproteins, its storage in the liver and for its
excretion. When a massive dose of vitamin E was given to sheep IP,
there was a peak 3 d post dosing in the vitamin E concentration in all
tissues and then a decline. The greatest rate of loss occurred in the
hepatic tissue, where the loss was proportional to its concentration
in vitamin E. Bieri (1972) reported that all tissues (except depot


84
carrier gas flow rate of .5 cc/min, which was connected to a series
mass selective detector. Mass spectra were obtained at an electron
beam energy of 70 eV and an accelerating voltage of 1,800 volts.
Molecular ions ranging from 50 to 600 mass/charge were monitored
continuously.
Statistical Method
Analysis of variance (Steel and Torrie, 1980) was used to
estimate the difference in a-tocopherol concentration among similar
time blood samplings as well as among corresponding tissues in the
four treatment groups. An additional two way analysis was carried out
to test groups and d for blood plasma and for groups and tissue types.
Comparisons were made by using Duncan's multiple range test (Duncan,
1985). Statistical models used in the experiment were as follows.
For the one-way analysis: Y_ = p + g^ + e_, where g^ is effect of
th th
i group; Y = p + d^ +£ij> where d^ is effect of i d. For two-
way analysis: Y. .. = p + g. + t. + e. where g. is effect of i^
ljk 6i j ijk 6i
group and t^ is effect of d.
Results
Plasma
Supplementation over a 1 to 7 d period of time with the various
vitamin E preparations increased (P<.01) plasma a-tocopherol
concentration above the baseline level (Table 5.2). Plasma a-
tocopherol concentrations at 1 d were higher (PC.01) than the original
baseline values in the d-a-tocopherol group but not for


32
activity in the biological system. The 2R, 4'R, 8'R configuration,
which is identical to the naturally occurring form, has the highest
activity (100% relative biological activity). The configuration in
the C-2 position is particularly important. It is noted that in all
the disastereomer pairs the 2S form has less activity than the 2R
form. The greatest difference in activity is noted in a comparison
between the 2S, 4'R, 8'R-a-tocopheryl acetate and the 2R, 4'R,8'R-a-
tocopheryl acetate, which is identical to the naturally occurring
form. The relative activity is only 21 to 31% for the SRR form. The
SSS isomer exhibits the surprisingly high relative activity of 60%
compared to the RRR isomer.
Observations on the biological activity of stereoisomer mixtures
vary. Ames (1979) showed on the basis of 19 tests that the activity
of all-rac-a-tocopheryl acetate does not exceed the calculable
activity. By contrast, Weiser and Vechi (1982) found in two
experiments that stereoisomer mixtures had a synergistic effect in the
rat resorption sterility test.
Erythrocyte hemolysis test
This test is based on the protection of erythrocytes by vitamin E
against hemolysis induced by dialuric acid or hydrogen peroxide. Rats
weighing about 100 g are fed a diet free of vitamin E for a depletion
period of 3 to 4 weeks. A sample of blood is then taken from each rat
and tested for hemolysis induction by addition of dialuric acid. Only
animals showing a degree of hemolysis of 96 to 99% are retained in the
test. In a preliminary trial with five animals per dose, dosages of
vitamin E are determined which will reduce the hemolysis to a range of


TABLE OF CONTENTS
PAGE
DEDICATION
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES viii
ABSTRACT x
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 REVIEW OF LITERATURE 3
Chemistry of Vitamin E 3
Absorption and Storage of Vitamin E 12
The Metabolism of Vitamin E 20
Vitamin E Deficiency 22
Biological Activity of Vitamin E 28
Role of Vitamin E in the Respiratory Chain 41
Intervention of Vitamin E in Hematopoiesis 42
Biological Role of Vitamin E 43
Role of a-Tocopherol as a Chain Breaking
Antioxidant 49
CHAPTER 3 PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP
FOLLOWING INTRAMUSCULAR ADMINISTRATION IN AN OIL
CARRIER 51
Introduction 51
Experimental 51
Analytical 52
Statistical Analysis 53
Results 54
Discussion 59
Summary 61
CHAPTER 4 BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS
IN SHEEP 63
Introduction 63
Materials and Methods 63
Results 67
Discussion 76
Summary 77
IV


122
tocopherol concentration had a tendency (P>.05) to be higher in the
dl-a-tocopherol than in the ester-dosed group. A higher (P<.05)
plasma a-tocopherol tolerance curve area was found in the dl-a-
tocopherol than in its ester form, as well as a quicker (P<.05) time
(h) for maximum plasma a-tocopherol concentration. Data from cows
revealed a greater (P<.05) plasma tolerance area following the
administration of dl-a-tocopherol than its acetylated form.
Experiment V was designed to determine plasma and tissue a-
tocopherol concentrations in sheep following a 5 g dose of dl-a-
tocopherol (water based) injected intraperitoneally followed by serial
slaughterings. A significant time effect for all tissues was noticed,
while the peak for a-tocopherol concentrations was observed 3 d post
dosing. A varied rate of uptake of a-tocopherol was noticed with the
liver, lung and spleen showing a pronounced uptake while the muscle
showed the least. This study provided an indication of when a plateau
in a-tocopherol concentration had been reached for each individual
tissue. Studies reported in this dissertation indicated that blood
plasma and tissue a-tocopherol concentrations were influenced by
various sources of vitamin E as well as by mode of administration and
its type of vehicle. Higher indexes of bioavailability were observed
in favor of natural a-tocopherol than synthetic a-tocopherol in sheep
and cattle dosed with equivalent amounts (IU). Sheep given the
vitamin E preparations by intramuscular oil depot injection resulted
in a delayed increase in plasma and tissue a-tocopherol concentration.
Intraperitoneal injection of dl-a-tocopherol (water base)
resulted in a substantial and quick rise in plasma and tissue a-


8
Synthetic dl-a-tocopherol and its esters are prepared from
isophytol. This synthesis yields a mixture of eight isomers. Although
semi-synthetic dl-a-tocopherol is racemic only at carbon 2 as prepared
from natural phytol, samples of this compound may be available for
research though it is not a commercial source of vitamin E.
Biosynthesis
Although conclusive proof of the biosynthetic route is still
lacking, it is probable that the ring structure is formed from tyrosine
by the pathway presented in Figure 2.5 (Whistance and Threlfall, 1967,
1968). Methylation of the ring structure by methyl transfer from
methionine and formation of an initial isoprenoid side-chain from
mevalonic acid completes the formation of a tocotrienol (Griffiths et
al., 1968).
Properties of Vitamin E
Physical properties
Both d-a-tocopherol (RRR) and dl-a-tocopherol (all-rac) are
practically insoluble in water but are almost completely soluble in
oils, fats, acetone, alcohol, chloroform, ether, benzene, and other fat
solvents. All tocopherols are stable to heat and alkali in the absence
of oxygen and are unaffected by acids up to 100C; they are slowly
oxidized by atmospheric oxygen, a process which is increased rapidly by
heat and catalyzed by ferric or silver salts. On exposure to light,
the tocopherols gradually darken. They are not precipitated by
digitonin. The commercial forms of vitamin E are d-a-tocopheryl
acetate and dl-a-tocopheryl acetate.


77
forms of vitamin E (Dunkley et al., 1967). As suggested in humans
(Week et al., 1952), it could be that the physiological mechanism
effecting hydrolysis in the ruminant duodenum of tocopherol esters is
less effective than in rats. It is reasonable to assume that plasma
tocopherol levels in this experiment reflect the relative efficacy of
various forms of vitamin E on tissue storage. Indeed, as was reported
for humans (Djuetal, 1958), the present data on -tocopherol
concentrations in sheep tissue show a variable pattern which is
largely a reflection of varied bioavailability of the different forms
of vitamin E. It may be concluded that following ingestion of
equivalent amounts (IU) of the various forms of tocopherol by sheep d-
a-tocopherol was more potent than d-ot-tocopheryl acetate or the 1-
isomeric forms of vitamin E.
Summary
Seventeen crossbred yearling wethers were randomly allotted to
four dietary groups that received 400 IU/day/sheep either of (1) dl-a-
tocopherol (five sheep), (2) dl-a-tocopheryl acetate (five sheep), (3)
d-a-tocopherol (four sheep) or (4) d-a-tocopheryl acetate (three
sheep). Blood samples were taken at day 0 and then at frequent
intervals for ct-tocopherol determination. At the end of the 28-day
experiment, animals were killed and various tissues sampled. Higher
concentrations of ct-tocopherol were observed in tissues of sheep fed
d-a-tocopherol than the other tocopherol forms. In sheep fed d-a-
tocopherol, the most important index of bioavailability, the area
under the plasma ot-tocopherol time curve (AUC) (Gibaldi and Perrier,


97
HOUR
. PLASMA TOCOPHEROL CONCENTRATION (vG/ML) IN CATTLE
FOLLOWING ADMINISTRATION OF A SINGLE DOSE (50 MG/KG
BODY WEIGHT) OF (A) DL-ct-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL
FIGURE 6.2


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF VARIOUS VITAMIN E
COMPOUNDS IN RUMINANTS
By
NICHOLAS HIDIROGLOU
August 1989
Chairman: Dr. L. R. McDowell
Major Department: Animal Science
This research was designed to investigate the bioavailability of
various tocopherol sources for ruminants. In experiment I, d-a-
tocopherol and dl-a-tocopherol suspended in sesame oil were administered
intramuscularly in sheep at a level of 40 mg/kg body weight (b.w.).
Sheep administered d-a-tocopherol and killed after 360 hr resulted in a
higher (P<.01) bioavailability than for sheep injected with d-a-
tocopherol or dl-a-tocopherol and sacrificed 240 hr after dosing. A
more sustained increment in plasma a-tocopherol was observed with d-a-
tocopherol than dl-a-tocopherol.
In experiment II, sheep were provided with 400 IU/d of either d-a-
tocopherol, dl-a-tocopherol or their corresponding acetate forms for 28
d. Higher concentrations of a-tocopherol were observed in tissues of
sheep fed d-a-tocopherol than the other tocopherol forms. In sheep fed
d-a-tocopherol, the area under the plasma a-tocopherol time curve (AUC),
was larger as compared to other forms of vitamin E supplementation.


49
suggest that these lipofuscin pigments arise from membrane lipid and
protein fragments formed in the course of their peroxidation phenomena
(Porta and Hartroft, 1969).
Consequences of lipid peroxidation are alterations in structural
(membrane permeability), functional components of the cell and their
component enzymes particularly subcellular partices such as
mitochondrias, microsomes, and lysosomes.
Role of a-Tocopherol as a Chain Breaking Antioxidant
A major biological function of a-tocopherol is to act as a chain
breaking antioxidant, scavenging free radicals, capable of terminating
chain reactions among unsaturated fatty acids (Zalkin and Tappel,
1960).
Chain-breaking antioxidants react directly with radicals. Many
phenols (ArOH) of which vitamin E is an example, can stop a radical
chain reaction because they are able to trap the chain-carrying
peroxyl radicals. The free radical scavenger action of a-tocopherol
depends on the ability of donation of the phenolic hydrogen atom to a
fatty acyl free radical, which resolves the unpaired electron of the
radical so that free radical attack on further molecules of
unsaturated fatty acids is prevented (Burton, 1989)
ArOH + ROO -* R00H + ArO.
The relatively stable phenoxyl radical produced is so unreactive to
attack another polyunsaturated fatty acid moeity or 0£ that instead it
is inactivated by reaction with a second peroxy radical R00 +
ArO" -* inactive products or may in the presence of ascorbate
undergo reduction back to the original phenol (Burton, 1989).


BIOGRAPHICAL SKETCH
Nicholas Hidiroglou was born in December 1961 in Kapuskasing,
Ontario. He attended local schools and graduated from Confederation
High School in 1980. He entered Macdonald College of McGill
University in September 1980 and was awarded the degree of Bachelor of
Science in Agriculture in 1983. In January 1984, the author entered
graduate school at the University of Florida and completed a Master of
Science degree in animal science in May 1986. He enrolled in a Ph.D.
program in 1986 and is currently a candidate for the degree of Doctor
of Philosophy in animal science. The author has accepted a Research
Associate position at East State Tennessee University in Johnson City.
139


50
The latter mechanism is the basis of the so-called synergistic
action of vitamins C and E. The antioxidant breaks the chain of
oxidation and can dramatically reduce the length of the autoxidation
chain and the extent of peroxidative damage. The better the
antioxidant, the greater is the amount of material that is spared from
peroxidative damage. Vitamin E appears to be a factor ensuring the
integrity of cellular membranes protecting their constituent
polyunsaturated fatty acids from peroxidation.


CHAPTER 2
REVIEW OF LITERATURE
Chemistry of Vitamin E
Structure
The term "vitamin E" today applies to a group of related chemical
compounds, the tocopherols and tocotrienols. The structural formula
of a-tocopherol is indicated in Figure 2.1.
FIGURE 2.1. STRUCTURAL FORMULA OF a-TOCOPHEROL
The following tocopherols (Fig. 2.2) and tocotrienols (Fig. 2.3)
are known:
3


115
TABLE 7.5. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG RATIO OF TISSUE TO
PLASMA.
Tissue Overall
time effect
DOF 5
3-28a
versus 0
1
Source of variation
6-28 10-28 15-28
versus 3 versus 6 versus 10
1 1 1
28
versus 15
1
Residual
19
Kidney
0.432*
0.765*
1.128*(**)b
0.021
0.181
0.064
0.138
Liver
4.657**
19.108**
1.090**
0.719(*)
0.414
0.955(*)
0.222
Neck
0.415
0.535
0.836*(NS)
0.676
0.029
0.001
0.165
Leg
0.510*(NS)
0.481
1.081*(N S)
0.611(*)
0.076
0.301
0.153
Heart
0.170
0.002
0.604*(NS)
0.080
0.144
0.020
0.109
Spleen
1.231*(**)
2.527*(**
) 1.567*(**)
0.235
1.042(*)
0.787
0.310
Lung
4.298**
10.924**
0.000
1.663(*)
6.985**
1.919(**
) 0.577
Adrenal
1.189**
3.323**
0.447
0.958*
0.273
0.943*
0.202a
Pancreas
0.739**
1.520**
1.416**
0.368*
0.012
0.326*
0.058a
g
Residual is based on 17 degrees of freedom because of missing observations.
^Significance levels in parentheses note changes which occurred when one outlier
was removed.
* (**)Indicates a significant effect at 5% (1%) level. NS indicates non
significance at 5% level.


133
McCay, B., J. L. Poyer, M. Pfeifer, E. May, and M. Gilliam. 1971. A
function for alpha-tocopherol: Stabilization of the microsomal
membrane from radical attack during TPNH-dependent oxidations.
Lipids 6:297.
Mellors, A., and M. C. McBarnes. 1966. The distribution and
metabolism of alpha-tocopherol in the rat. Brit. J. Nutr.
20:69.
Mervyn, L., and R. A. Morton. 1959. Unsaponifiable fractions of
lipid from normal and diseased human kidney. Biochem. J.
72:106.
Muth, 0. H., J. R. Schubert, and J. E. Oldfield. 1961. White muscle
disease, myopathy in lambs and calves. VII. Etiology and
prophylaxes. Am. J. Vet. Res. 22:460.
Nair, P. 1972. Vitamin E and metabolic regulation, Vitamin E and
its Role in Cellular Metabolism (P. Nair and H. J. Kayden, eds.).
Ann. NY Acad. Sci. 203:53-61.
Nair, P. P., and Z. Luna. 1968. Identification of a-tocopherol from
tissues by combined Gas-Liquid-Chromatography, Mass Spectrometry
and Infrared Spectroscopy. Arch. Biochim. Biophys. Acta.
127:413.
Nair, P. P., H. S. Murty, and N. R. Grossman. 1970. The in vitro
effect of vitamin E in experimental Porphyria. Bioch. et Bioph.
Acta 215 21:112.
Newmark, H., W. Pool, J. G. Bauernfeird, and E. Ritter. 1975.
Biopharmaceutic factors in parenteral administration of vitamin
E. J. Pharm. Sci. 64:655.
Ogihara, T., Y. Nishida, M. Miks, and M. Mino. 1985. Comparative
changes in plasma and RBC a-tocopherol after administrations of
dl-a-tocopherol acetate and dl-a-tocopherol. J. Nutr. Sci. Vit.
31:164.
Oldfield, J. E., J. R. Schubert, and 0. H. Muth. 1963. Implications
of selenium in large animal nutrition. J. Agr. Food Chem.
11:388.
Overman, R.S., J. M. McNeely, M. E. Todd, and I. Wright. 1954.
Effects of vitamin E preparations on plasma tocopherol levels.
J. Clin. Nutr. 2:168.
Pappenheimer, A. M., and M. Goettsch. 1931. A cerebellar disorder in
chicks apparently of nutritional origin. J. Exp. Med. 53:11.


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CHAPTER 6
PLASMA TOCOPHEROL IN RUMINANTS AFTER INGESTING FREE OR ACETYLATED
TOCOPHEROL
Introduction
Administration of a massive dose of vitamin E is one of the usual
methods in the prophylaxes of vitamin E deficiencies (Ogihara et al.,
1985). Overman et al. (1954) reported in humans that a single massive
oral dose of vitamin E is effective in increasing the free tocopherol
plasma level to a significant degree after six hours. This temporary
rise with ensuing drop has been designated as a tolerance curve. The
tolerance curve mirrors the intestinal absorption of vitamin E.
However, on this subject, the data for ruminant animals are sparse.
Indeed, in ruminant species with their unique digestive system and
physiologic differences in metabolic mechanisms, there are only few data
on the bioavailability of the various forms of vitamin E. For this
reason, the objective of the experiment was to gain information on blood
plasma response to ot-tocopherol in sheep or cattle provided with
different chemical forms of tocopherols but with identical weights of
these compounds, namely dl-cx-tocopherol and dl-a-tocopheryl acetate.
Materials and Methods
Trial 1
Ten yearling crossbred wethers weighing 40 to 50 kg were used. All
animals originated from a flock fed a commercial diet that
94


36
TABLE 2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN THE
IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER AND WEISER (1968).
Compound
Relative activity (%)
d-a tocopherol
100
d-y tocopherol
30
administered orally to the experimental animals 2 days before blood
samples are taken, after which the hemolysis test is undertaken. This
test model shows that even the forms which are esterified with acetic
acid exhibit vitamin E activity after they have been liberated by
enzymes in the intestine (Table 2.5). Here again it is the d-a
configuration, both in the free form and in the form esterified with
TABLE 2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR
ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO HEMOLYSIS TEST IN
RATS; EXCERPT FROM BLISS AND GYORGY (1969).
Compound
Relative activity (%)
3 d-a-tocopherol 132.8
4 D-a-tocopheryl acetate 147.4
5 all-rac-a-tocopheryl acetate 100.0 (standard)
6 D-y-tocopherol 21.7
7 D-y-tocopheryl acetate
18.9


86
the other groups (P>.05). To elevate plasma a-tocopherol
concentration, d-a-tocopherol outranked all the other forms at all
sampling dates except at 28 d, where its effect was not different
(P>.05) from d-a-tocopheryl acetate (Table 5.2). D-a-tocopherol
supplementation during the 28 d experimental period resulted in the
highest blood plasma a-tocopherol concentration followed by d-a-
tocopheryl acetate; the racemic mixtures ranked lower (Table 5.2)
despite being dosed at equal units. Blood plasma a-tocopherol
increased continuously from 0 to 28 d (Table 5.2). The difference in
plasma a-tocopherol concentration (yg/ml) between d 28 and d 0 were as
follows (Table 5.2). D-a-tocopheryl acetate = 6.16; d-a-tocopherol =
7.83; dl-a-tocopherol = 4.05; dl-a-tocopheryl acetate = 3.25.
Consequently the relative bioavailabilities of the various tocopherols
vs dl-a-tocopheryl acetate (1.0 IU/mg) could be calculated as follows:
(4.05 x 1.1 IU/mg)
dl-a-tocopherol = 1.37 IU/mg; d-a-tocopheryl acetate
3.25
(6.16 x 1.36 IU/mg) (7.83 x 1.1 x 1.36 IU/mg)
= 2.58 IU/mg; d-a-tocopherol
3.25 3.25
=3.60 IU/mg.
Tissues
There were differences among treatments (P<.01) and types of
tissue (PC.OOOl) in vitamin E tissue concentrations at slaughter 28 d
after supplementation began Fig. 5.1 and Table 5.3). The adrenal
gland and liver concentrations of a-tocopherol were greater (P<.01)
for cattle fed d-a-tocopherol or d-a-tocopheryl acetate than when fed
racemic forms of vitamin E. The a-tocopherol concentrations were


FIGURE PAGE
5.2 MASS SPECTRAL SCANNING OF THE a-TOCOPHEROL STANDARD
(A) AND IN BLOOD PLASMA (B) FOLLOWING HPLC COLLECTION
AND TLC 90
6.1 PLASMA TOCOPHEROL CONCENTRATION (pG/ML) IN SHEEP
FOLLOWING ADMINISTRATION OF A SINGLE MEGADOSE (100 MG/
KG BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL 96
6.2 PLASMA TOCOPHEROL CONCENTRATION (pG/ML) IN CATTLE
FOLLOWING ADMINISTRATION OF A SINGLE DOSE (50 MG/KG
BODY WEIGHT) OF (A) DL-a-TOCOPHERYL ACETATE OR (B)
DL-a-TOCOPHEROL 97
IX


23
widespread effects of vitamin E deficiency on muscles (Hove and
Harris, 1947).
Other species. Monkeys and roosters are also susceptible to
vitamin E deficiency. Stallions, bulls, rams, billy goats, and
perhaps male mice, are not (Baxter and Brown, 1952).
In females
Rodents. In rats, vitamin E deficiency does not appear to affect
the development and functioning of the genital system (estrus,
ovulation, fecundation and nesting), but it has a major impact on
prenatal development, mainly during the days following nesting. The
uterus and the ovaries become covered with brownish yellow pigment
while the basic signs are early embryo mortality followed by
resorption as a result of placental lesions which may sometimes
hemorrhage. When there is a chronic deficiency, the rat may produce
only one or two live young, while the others die (Martin and Moore,
1939). The survivors present serious muscular dystrophy lesions
(Bonetti and Stirpe, 1963). This cause should be kept in mind when a
reduction in fecundity in this species occurs.
Other species. Where there is vitamin E deficiency, feto
placental hemorrhages and some degree of embryo mortality have
occasionally been noted in sows and rats. Surviving piglets develop
poorly and present muscular dystrophy. It may be concluded that sows
are susceptible to vitamin E deficiency, though to less extent than
rats (Lannek, 1962).
The effects on reproduction in cows, ewes, goats and mares are
nil, although these species are very susceptible to muscular dystrophy


7
1938; Fieser et al., 1940) or isophytol (Karrer and Isler, 1941). All
the tocopherols were subsequently synthesized and their structures
elucidated.
TABLE 2.1. NOMENCLATURE FOR TOCOPHEROLS
Description
Configuration
Recommended
Other designation
Natural
2R,4'R,8'R
RRR a-
[d]-a-tocopherol.
Steriochemically uniform
product (only RRR-)
Semisynthetic
2R,4'R,8'R
1 ambo a
dl-a-tocopherol.
Two stereoisomers in the
product (about 50% RRR-a
historical standard)
Synthetic
Mixture of all
All rac-a
[dl]-a-tocopherol.
Eight stereoisomers in
the product (about 12.5%
RRR-a)
Synthetic
2S,4'R,8R
2-epi-a
[ 1 ]-ot-tocopherol.
Steriochemically uniform
product (only SRR-a)
Two principal sources of vitamin E are in commercial use: d-a-
tocopherol and esters (of acetate and succinate) of these compounds.
The acetate esters are prepared chemically by reaction of the alcohol
forms with acetic anhydride; they do not exist in nature. D-a-
tocopherol is largely obtained from natural sources by molecular
distillation. However, some of the d-a-tocopherol is prepared by
further methylation of B-, y-, and 6-tocopherols, or by hydrogenation
of a-tocotrienol.


PAGE
CHAPTER 5 BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN
E IN BEEF CATTLE AS INFLUENCED BY SUPPLEMENTATION
OF VARIOUS TOCOPHEROL COMPOUNDS 79
Introduction 79
Materials and Method 80
Results 84
Discussion 91
Summary 93
CHAPTER 6 PLASMA TOCOPHEROL IN RUMINANTS AFTER INGESTING
FREE OR ACETYLATED TOCOPHEROL 94
Introduction 94
Materials and Methods 94
Results 99
Discussion 102
Summary 105
CHAPTER 7 PLASMA AND TISSUES VITAMIN E CONCENTRATIONS IN
SHEEP AFTER ADMINISTRATION OF A SINGLE
INTRAPERITONEAL DOSE OF dl-ct-TOCOPHEROL 106
Introduction 106
Experimental Procedure 106
Results 109
Discussion 117
Summary 119
CHAPTER 8 GENERAL CONCLUSIONS 120
LITERATURE CITED 124
BIOGRAPHICAL SKETCH 139
IV


100
TABLE 6.1. DL-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES (LEAST SQUARE
MEANS SE) IN SHEEP AFTER A SINGLE ORAL ADMINISTRATION
OF VITAMIN E (100 mg/kg BODY WEIGHT).
Cmax1
Tmax3
(hr1)
Co3
CmaxCo Ct^ Ct/Co
Cmax/Ct
AUC5
(pg/ml)
(pg/ml)
(pg/ml) (pg/ml) (pg/ml)
(pg/ml)
(pg/ml/hr)
dl-a-tocopherol
3.58.22
26.514.04
.421.06
8.9911.13 .731.07 1.801.25
4.951.54
332.18120.27
dl-a-tocopheryl acetate
3.05.20
40.413.61
.411.05
7.6711.01 .591.06 1.501.22
5.361.48
269.63118.13
Significance (P)
<.05
<.05
Maximum plasma concentration.
I
Maximum time (hr).
Initial plasma concentration.
Terminal plasma concentration.
Area under plasma concentration time curve.
Significance tested following analysis of variance.


38
tocopheryl acetate intake. Uptake in the erythrocytes 6 h after oral
administration of d-a-tocopherol was 280% higher than after
administration of the synthetic material. Direct comparison at 24 and
48 h after oral administration indicated that uptakes of the free d-a-
tocopherol in the erythrocytes was 300 and 255% greater than uptake of
the ester. These test results provided impressive support for the
superiority of the natural vitamin E structure as previously shown in
clinical studies.
Liver storage tests
Liver storage tests are based on the observation that amount of
tocopherol stored in the liver of rats and chicks has a linear
relationship to the level of vitamin E consumed.
Pudelkiewicz et al. (1960) used day-old White Plymouth Rock male
chicks fed a vitamin E-deficient diet for 13 days for assay of liver
storage of vitamin E. Smallest and largest chicks are discarded, with
those remaining distributed by weight into groups of eight chicks
each. The tocopherol standards and the unknown materials to be
assayed are then mixed with the basal diet and the chicks in all lots
are pair-fed to the lot eating the least amount of feed over a period
of 14 days. Tocopherol content of pooled livers from each group was
then determined by chemical assay using the Emmerie-Engel reaction
after careful extraction, molecular distillation, and chromatographic
separation of the tocopherols.


64
Sheep were placed in individual metabolism cages 10 days before
administration of vitamin E as an adjustment period. They were
randomly allotted to four dietary groups of five each that consisted
of 400 International Units/day/sheep of either (1) dl-a-tocopherol,
(2) dl-a-tocopheryl acetate, (3) d-a-tocopherol (4) d-a-tocopheryl
acetate, which was mixed with the commercial diet. Appropriate
amounts of the various forms of tocopherols were dissolved in absolute
ethanol prior to diet mixing. In the middle of the experiment, one
sheep from group 3 and two sheep from group 4 stopped eating and were
removed from the experiment.
Blood samples were taken at various intervals by jugular puncture
with plasma separated from the cellular blood components in a
refrigerated centrifuge and stored at -30C until assayed for vitamin
E. All animals were slaughtered after 30 days. Samples from selected
tissues (fat, muscles from the neck and leg, kidney, lung, heart,
spleen, liver, pancreas) were collected and stored at -30C until
analyzed for d-a-tocopherol.
Analytical Methods
Quantitation of d-a-tocopherol in sheep tissues, as well as
plasma samples, was performed by high-pressure liquid chromatography
(HPLC) using a fluorescent detector, following tissue homogenization
and heptane extraction. Identification and quantitation of a-
tocopherol were obtained by comparison of retention time as well as
peak areas with tocopherol standards. Standards were purchased from
Eastman Kodak (Rochester, NY, USA).


132
Machlin, L.J., J. Keating, J. Nelson, M. Brin, R. Flipaki, and 0. N.
Miller. 1979. Availability of adipose tissue tocopherol in the
guinea pig. J. Nutr. 109:105.
MacMahon, M.T., G. Neale, and G. R. Thompson. 1971. Lymphatic and
portal venous transport of alpha tocopherol and cholesterol.
Europ. J. Clin. Invest. 1:288.
Martin, A. J. P., and T. Moore. 1939. Effects of prolonged vitamin E
deficiency in the rat. J. Hyg. 39:643.
Martius, C., and C. Furer. 1963. Ueber die umwandlung des
tocopherols im tierkoerper. Biochem. J. 336:474.
Marusich, W. L., G. Ackerman, and J. G. Bauernfeind. 1967.
Biological efficiency of d- or dl-ct-tocopherol acetate in
chickens. Poult. Sci. 46:541.
Marusich, W. L., G. Ackerman, W. C. Reese, and J. C. Bauernfeird.
1968. Relative activity of D and DL-a-tocopheryl acetate based
on plasma levels. J. Anim. Sci. 27:58.
Mason, K. E. 1954. The tocopherols. Effects of deficiency. A. In
animals. The Vitamins (W. H. Sebrell Jr. and R. S. Harris,
eds.) Vol. Ill, pp. 514, 541. Academic Press, New York.
Mason, I. E., and P. L. Harris. 1947. Bioassay of vitamin E. Biol.
Symp. (Jacques Cattell, ed.), Vol. XII, p. 459.
Mattill, H. A., and R. E. Coulin. 1920. The nutritive properties of
milk, with special reference to reproduction in the albino rat.
J. Biol. Chem. 44:137.
McMurray, C.H., and W. G. Blanchflower. 1979a. Application of a
high-performance liquid chromatographic fluorescence method for
the rapid determination of a-tocopherol in the plasma of cattle
and pigs and its comparison with direct fluorescence and high-
performance liquid chromatography-ultraviolet detection methods.
J. Chromat. 178:525.
McMurray, C.H., and W. G. Blanchflower. 1979b. Determination of ot-
tocopherol in animal feedstuffs using high-performance liquid
chromatography with spectrofluorescence detection. J. Chromat.
176:488.
Meadows, J., G. L. Nickell, J. Baxyer, and S. Kapila. 1982. Trace
substances in environmental health: Proceedings of Univ. of
Missouri Annual Conference. Environ. Health 16:268.
McArthur, C. S., and E. M. Watson. 1939. Isolation of alpha- and
beta-tocopherols and their derivatives. Caad. Chem. 23:350.


14
species. Also, with labile substances like the tocopherols, a simple
estimation of absorption based upon amounts present in the food and
feces may be inaccurate.
These and other metabolic studies have shown that tocopherols are
incompletely absorbed. The amount absorbed seems to depend on the
requirement of the organism (Klatskin and Molander, 1952). Absorption
and elimination also seem to depend on the amount in the diet.
There is a linear relation between the logarithms of ingested and
liver tocopherol in rats (Bolliger and Bolliger-Quaife, 1956).
Griffiths (1960) noticed a linear relation between the logarithms of
serum tocopherol and dietary concentration of vitamin E in chickens.
The same author determined the ratio of pure a-, B-, Y-, and <5-
tocopherol in the livers of growing chicks as 100:41:19:0, but his
findings are contradicted by Gray (1959), who found considerably lower
tocopherol levels in rat plasma at high levels of tocopherol intake
than Griffiths (1960) found in chicken serum.
Work of Dju et al. (1950) with hens included the observation that
a-tocopherol was absorbed to a much greater extent than the y- and 6-
compounds. The serum tocopherol values of two hens which received
weekly supplements of 1.6 g and 2.0 g a-tocopherol were 20.0 and 20.1
mg per 100 ml, but two hens that received weekly supplements of 0.8
and 1.0 g 6-tocopherol had serum tocopherol values of only 1.35 and
2.1 mg per 100 ml. Gamma-tocopherol appears to be only one-third to
one-half as well absorbed as a-tocopherol in formula fed infants and
supplemental vitamin E (1200 IU/day of all-rac-a-tocopherol: 800 mg a-
tocopherol equivalents) in adults significantly reduced plasma Y-


43
aminolevulinic acid (ALA), followed by the condensation of two
molecules of ALA to form porphobilinogen. These two transformations
are catalyzed by two enzymes, ALA-synthetase and ALA-dehydratase,
respectively.
Nair et al. (1970) have shown that these two enzymes are under
the control of vitamin E. The fact that actinomycin D (which inhibits
transcription from DNA to RNA) impeded the ability of vitamin E to
reestablish heme formation in the mitochondria of deficient tissues
suggest that vitamin E might intervene at this level by acting as a
specific repressor of the transcription of the enzymes of ALA (Nair,
1972).
Since heme is not only a constituent of hemoglobin, but also of
myoglobin, cytochromes and various oxidoreduction enzymes, it is
possible to better understand certain aspects of vitamin E deficiency,
namely the decoloration of the muscles in muscular dystrophy due to
rarefaction of the myoglobin. Similarly, the decoloration which
various researchers have observed in the mitochondria of deficient
tissues reflects a rarefaction of the cytochromes, which contributes
to a lowering of cell respiration by disruption of the respiratory
chain at the level of the cytochromes.
Biological Role of Vitamin E
Antioxidant Activity
It has been known for some time that vitamin E is an excellent
fat-soluble antioxidant (Dam, 1957; Tappel, 1962) although the
physiological significance of this trait is only now becoming clear.


30
Resorption sterility test
The fetal resorption test in female rats is the classical means
of conducting the bioassay of tocopherols (Mason and Harris, 1947;
Ames et al., 1963). It measures the all-or-none response to the test
substance by successfully mated vitamin E-deficient female rats in
producing viable offspring.
The resorption sterility test in rats is frequently used in
addition to the hemolysis test. This test system was used to
determine the biological activity of the eight enantiomers of the
synthetic all-rac-a-tocopheryl acetate (Weiser and Vechi, 1982).
The resorption sterility test is based on the fact that female
rats supplied with a vitamin E-deficient diet are unable to carry
their offspring to term. The fetuses die before the end of the
gestation period and are subject to intra-uterine resorption. Weaned
rats are given a vitamin E-free diet for 3 to 4 months at which time
their sterility is assessed by test matings with fertile males;
insemination is checked by vaginal smear for spermatozoa. Different
levels of standard RRR-a-tocopherol and the unknown substances are
added to olive oil (or sometimes to portions of diet) which is
administered to pregnant females through the 5th to 9th days of
pregnancy. If dosing is delayed beyond the 10th to 12th day of
pregnancy, vitamin E is ineffective. The animals are killed on the
19th day, i.e., 2 days before parturition, and examined for living
fetuses and for sites of implantation of fetuses that have been
resorbed. Animals having less than four implantation sites are
discarded, and those having one or more living fetuses are regarded as


6
The tocotrienols possess only one center of asymmetry at C2, in
addition to sites of geometrical isomerism at C^' and Cy'. Thus, a
number of stereo-isomers of the tocopherols and tocotrienols can
exist. Natural -tocopherol was shown conclusively to have the 2R,
4'R, 8'R configuration (Table 2.1). Thus, RRR-a-tocopherol can be
used to denote the natural tocopherol isomer. The epimer of natural
a-tocopherol, i.e., (2S, 4'R, 8'R)-a-tocopherol can be named 2-epi-a-
tocopherol. A mixture of RRR and SRR-a-tocopherol can be obtained
synthetically and is named 2-ambo-a-tocopherol (ambo = Latin for
both). The reduction product of natural -tocopherol is a mixture of
four diastereo-isomeric a-tocopherols and can be called 4' ambo, 8'
ambo-a-tocopherol. Synthetic a-tocopherol from synthetic phytol or
isophytol is a mixture of four racemates in equal proportions and
should be named all-rac-a-tocopherol.
Bieri and Prival (1967) reported that a group of synthetic
tocopherol derivatives exhibited vitamin E activity. Among those
reported were a-, 8-, and y-tocopheramines and N-methyl-8-, and N-
methyl-y-tocopheramines. The tocopheramine derivatives differ from
the tocopherols in that an amino or an N-methyl amino group is
substituted for the hydroxy group on the C-6 of the ring.
Synthesis
Chemical
Karrer et al. (1938) were the first to synthesize a-tocopherol by
condensation of trimethylhydroquinone with phytyl bromide. Phytyl
bromide was soon replaced by the natural phytol (Karrer and Isler,


78
1975) was larger as compared to other forms of vitamin E
supplementation.


56
TABLE 3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN SHEEP AFTER A
SINGLE INTRAMUSCULAR ADMINISTRATION OF VITAMIN E (40 mg/kg).
Vitamin E
Group preparations
Cmax1
(pg/ml)
Tmax3
(hr1)
Co3
(pg/ml)
Cmax/Co
(Pg/ml)
Ct4
(Pg/ml)
Ct/Co Cmax/Ct
(pg/ml) (
AUC5
pg/ml/hr)
1
dl-a-tocopherol
1.76d
151b
1.12c
1.68d
1.47b
1.39d 1.23
286d
2
d-a-tocopherol
2.40c
254c
0.94d
2.68c
1.87a
2.06c
1.35
637c
3
d-a-tocopherol
1.84d
190a
0.71d
2.63c
1.30b
1.82c
1.54
277d
SE
0.23
24.78
0.09
0.21
0.17
0.24
0.15
27
Sheep in Group 1 and 3 were slaughtered 240 d after dosing while sheep in Group
2 were killed 360 h after dosing.
^Maximum plasma concentration.
2
Maximum time (hr).
3
Initial plasma concentration.
Terminal plasma concentration.
3Area under plasma concentration-time curve.
a A
c,d
Means in the same columns with different superscripts differ (P<0.05),
Means in the same columns with different superscripts differ (P<0.01),


101
TABLE 6.2. TRIAL 2: D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES
(LEAST SQUARE MEANS SE) IN CATTLE AFTER A SINGLE ORAL
ADMINISTRATION OF VITAMIN E (50 mg/kg BODY WEIGHT).
Vitamin E
Preparations
Cmax^
(pg/ml)
Tmax3
(hr-1)
Cmax/Co
(pg/ml)
CT3
(pg/ml)
CT
(pg/ml)
Cmax/CT
(pg/ml)
AUC4
(u/ml/hr-1)
dl-a-tocopherol
10.84
35.38
3.88
3.71
1.36
3.04
1995
dl-a-tocopheryl
acetate
9.23
48.61
3.43
3.21
1.23
2.84
1589
SE
0.61
14.89
0.37
0.40
0.43
.39
88
Significance (P)
<.05
Maximum plasma concentration.
I
Maximum time (hr).
Terminal plasma concentration.
Area under plasma concentration time curve.


33
TABLE 2.3. RELATIVE ACTIVITY OF VARIOUS o-TOCOPHEROL STEREOISOMERS
AS DETERMINED BY RESORPTION STERILITY TEST IN FEMALE RATS.
Standard with 100% biological activity:
RRR-a-tocopherol acetate a- Relative activity in relation
tocopherol acetate isomer to the standard (in percent)
2R,4'R,8'R
100
2R,4R,8'S
90
2R,4'S,8'S
73
2S,4'S,8'S
60
2R,4'S,8'R
57
2S,4'R,8'R
31
2S,4R,8'S
37
2S,4'S,8'R
21
all-rac-a
32
(8 enantiomers)
67-77
2-ambo-a
60.2
(2 enantiomers)
73


Ill
Table 7.3. There was a significant time effect for all the tissues.
In most tissues, the tocopherol concentrations recorded at 3 d after
dosing were the largest, but the various tissues responded differently
to vitamin E injection. Examination of the tissue data 3 d postdosing
revealed a striking accumulation of injected vitamin E in the liver.
The lungs had the next highest concentration at that date. The spleen
invariably contained a considerable amount of a-tocopherol 3 d post
initiation. ot-Tocopherol concentrations in the remaining tissues,
also 3 d after dosing, showed a considerable increase but to a smaller
degree. The contrasts over time give some indication of when a
plateau had been reached, but it must be kept in mind that
significance may fail to be achieved because of large variances or
insufficient data. For example, for the kidney the mean of days 15 to
28 is not different (P>.05) from the mean at day 10. This indicates
there is no further significant change from the level reached by day
6, suggesting a plateau has been reached. However, as the means
continue to decrease up to and including day 28, it would seem
inappropriate to draw this conclusion. Similarly, the significance
tests suggest the neck, leg, heart, adrenal and pancreas have reached
a plateau by 3 d. The liver and lung do not appear to have reached a
plateau by 15 d, however. The contrasts suggest the spleen has
reached a plateau by day 10, although the mean decreases on d 28.
There were particularly low values recorded for one animal at d
3. Tietjen and Moore (1972) statistics indicated this value was an
outlier (P<.05) for liver, spleen and lung and it was an extreme


22
observed large amounts of radioactivity in dermal tissue suggesting
that skin may play a role for tocopherol secretion or excretion.
Vitamin E Deficiency
The absence of vitamin E in the diets of animals poses health
problems and affects various functions.
Effects of Deficiency on the Reproductive System
In 1920, Mattill and Conklin showed that a deficiency in vitamin
E led to reproductive disorders and lesions of the genital apparatus
in rats. The data were confirmed in 1922 by Evans and Bishop. As a
result of some premature generalization of these observations, vitamin
E came to be considered as an anti-sterility factor and its
therapeutic use was widely advocated. Now, however, it is apparent
that the role of this factor varies widely among species.
In males
Rodents. In rats, vitamin E deficiency results in testicle
degeneration (Mason, 1954). The gland is reduced to half its normal
size and becomes brownish and flaccid. Degenerative lesions affect
the spermiduct and lead to the complete cessation of sperm production.
Neither Sertoli cells nor the interstitial gland appear to be
affected. Some changes in related glands (seminal vesicles and
prostate) have been reported.
It appears that guinea pigs (Curto, 1966), hamsters and rabbits
are also susceptible, although less markedly so. In rabbits, in
particular, testicle lesions seem minor when compared to the


60
experiments, there is little or no lymphatic flow, and this would
affect the release of the vitamin E preparations which probably was
confined to connective tissue surrounding the muscle (Balard, 1968).
Bille et al. (1976) showed that vitamin D injected by IM oil depot,
into slaughter pigs, had a half-life of about 30-40 days at the
injection site, and that oil granulomata had formed after dosing.
Recently, it was reported (Dickson et al., 1986) that in sheep,
following the IM injection of a vitamin E preparation (5 ml) suspended
in arachis oil (400 mg dl-ct-tocopherol acetate/ml) into quadriceps
muscle, a considerable storage of vitamin E occurred in their lymph
gland (iliac). These workers observed that in the injected sheep a
continuous increase of vitamin E concentration occurred during 3 weeks
(from 76 ug/g to 2687/yg/g lymph gland). On weeks 5 and 6 there were
still high concentrations of vitamin E (1855 pg and 550 pg/g) in the
lymph gland (iliac) of the killed sheep. The results of their
experiment showed that the duration of the experimental period was
relatively short and longer time was needed in order to establish
accurately the various indexes of bioavailability. The planning of
our experiment was based on work carried out on chicks (Marusich et
al., 1967) and in dogs (Newmark et al., 1975) dosed IM with various
forms of vitamin E. In their animals the plasma vitamin E showed its
peak within 24 hr. The difference in initial d-ot-tocopherol plasma
values between the present 3 groups may have been due to different
roughage batches used before the start of the trial. The results
reported herein show that the time of slaughter had a major influence
on tissue vitamin E levels. In sheep slaughtered 360 hr after IM


68
TABLE 4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS IN
TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.
Source
DF
MS
F
P
Model
35
142.76
16.10
0.0001
Error
117
8.86
Corrected total
152
Source
DF
SS
F
P
Diet
3
212.02
7.97
0.0001
Tissue
8
4318.39
60.86
0.0001
Diet x tissue
24
305.90
1.44
0.1049
TABLE 4.2. TISSUE a-TOCOPHEROL
THE
CONCENTRATIONS (pg/g FRESH TISSUE) IN
FOUR DIETS.
Diet
Mean
N
d-a-tocopherol
10.43a
36
d-a-tocopheryl acetate
8.75b
27
dl-a-tocopheryl acetate
7.72b
45
dl-a-tocopherol
7.46b
45
In vertical row, numbers with the same letter are not different
(P>0.05).


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Rachel Shireman
Associate Professor of Food
Science and Human Nutrition
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
August 1989
Dean, Graduate School


29
forms are supplemented in many foods and feeds as dl-a-tocopherol or
as d- and dl-a-tocopheryl acetate or succinates. Bioassays may be
confounded by the levels of antioxidants, prooxidants, selenium, and
other factors which may alter the biological activity of the vitamin.
The biological activity of tocopherols (and of substances with
vitamin E-like activity) can only be assessed with a bioassay
technique which determines the ability of the tocopherol to reverse
clinical signs of vitamin E deficiency. The manifestations of a
deficiency differ markedly in different species. The signs of
deficiency most commonly used in bioassay work are rat sterility
(fetal resorption in the female and testicular atrophy in the male),
rat erythrocyte hemolysis, and muscular dystrophy in a number of
different species. A comprehensive review of bioassay methods for
tocopherols has been reported by Bliss and Gyorgy (1967) and Ames
(1971). Other tests, such as the liver storage, and elevation of
plasma tocopherol, have also been used. These latter methods are not
direct measures of biological activity iji vivo, but they do reflect
the relative absorption of the test compounds as well as their
turnover in the liver or red blood cells. In general, the erythrocyte
hemolysis and tissue storage tests correlate well with the i£ vivo
procedures and may be correlated with alterations in red blood cell
half-life observed in vitamin E-deficient humans. Therefore, although
caution should be used in interpreting results, these methods are
convenient and may be useful.


85
TABLE 5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (pg/ml) IN CATTLE FED VARIOUS
PREPARATIONS OF VITAMIN E.
Item
Day
SE
Group
mean
0
1
7
14
28
Treatment
d-a-tocopheryl acetate
1.49ag
2.46bfg
3.67bef
5.64bde
7.65abd
.71
4.18a
d-a-tocopherol
1.39af
3.82ae
5.25ae
8.41ad
9.22ad
.58
5.61b
dl-a-tocopherol
1.39ag
2.40bfg
3.14bef
3.98be
5.44bcd
.44
3.27c
dl-a-tocopheryl acetate
1.37af
2.65bef
3.10bde
4.02bde
4.62cd
.52
3.16c
SE
.10
.25
.45
.82
.82
.27
Day mean
1.41d
2.83e
3.80f
5.518
6.73h
.31
3 b c
Means in the same column with different letters in their superscripts
differ (PCO.Ol).
d G f 2
Means in the same row with different letters in their superscripts differ
(P<0.01).


LIST OF TABLES
TABLE PAGE
2.1. NOMENCLATURE FOR TOCOPHEROLS 7
2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS 10
2.3. RELATIVE ACTIVITY OF VARIOUS ALPHA-TOCOPHEROL
STEREOISOMERS AS DETERMINED BY RESORPTION STERILITY
TEST IN FEMALE RATS 33
2.4. RELATIVE BIOLOGICAL ACTIVITY OF VITAMIN E COMPOUNDS IN
THE IN VITRO HEMOLYSIS TEST; EXCERPT FROM BRUBACHER
AND WEISER (1968) 36
2.5. RELATIVE BIOLOGICAL ACTIVITY OF TOCOPHEROLS AND THEIR
ESTERS WITH ACETIC ACID IN THE IN VITRO/IN VIVO
HEMOLYSIS TEST IN RATS; EXCERPT FROM BLISS AND
GYORGY (1969) 36
2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL 41
3.1. D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES IN
SHEEP AFTER A SINGLE INTRAMUSCULAR ADMINISTRATION OF
VITAMIN E (40 mg/kg) 56
3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (yg/g wet) OF
SHEEP FOLLOWING A SINGLE INTRAMUSCULAR ADMINISTRATION
(40 mg/kg BODY WEIGHT) OF VARIOUS VITAMIN E
PREPARATIONS 58
4.1. ANALYSIS OF VARIANCE FOR a-TOCOPHEROL CONCENTRATIONS
IN TISSUES OF SHEEP FED DIFFERENT FORMS OF VITAMIN E... 68
4.2. TISSUE a-TOCOPHEROL CONCENTRATIONS (yg/g FRESH TISSUE)
IN. THE FOUR DIETS 68
4.3. RETENTION TIME OF a-TOCOPHEROL FROM GAS-CHROMATOGRAPHY/
MASS SPECTROMETRY 71
4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS
IN BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF
VITAMIN E 74
4.5. AREA (AUC) UNDER THE PLASMA a-TOCOPHEROL CONCENTRATION
CURVE (yg/ml h_i) 74


48
For its part, ozone, a layer of which shelters the planet from
short-wavelength ultraviolet radiation and known to be harmful, also
causes oxidations which are detrimental to biological cell systems.
Study of the chemical composition of the cell membranes shows
that they are formed by an association of lipids and proteins
performing important functions such as electronic phenomena, active
transport, glycolysis, biosynthesis of lipids and proteins, etc.
Lipids are the most important constituent not only of these cell
membranes, but also those of the intra-cellular organelles including
the the mitochondrias and microsomes. Many factors suggest that the
membranes consist of a film of lipids, rich in triglycerides and
phospholipids, between 40 and 65 A in thickness, covered on both sides
by a layer of proteins each about 10 A thick (Lehninger, 1975). The
peroxidation of the lipid constituents of the membranes, which contain
a high proportion of mono and polyunsaturated fatty acids (C16:1 *
C18:1; C18:2; C18:3; and C20:4) disturb their metabolic functions
(Tappel, 1972). The polyunsaturated fatty acid moieties of lipids are
characterized by the presence of two or more carbon-carbon double
bonds which are separated by a methylene-CH2-unit. Methylene units
are very susceptible to attack by radicals as peroxyl and Roo' with an
extremely rapid combination with oxygen to form peroxyl radicals
(Tappel, 1972). Lipid peroxidation of polyunsaturated fatty acids
particularly arachidonic acid results in the formation and
accumulation of lipid peroxidation products known as lipofuscin
pigments (Tappel, 1972). These pigments accumulate in tissues
(myocardium, brain, liver, testicles, etc.) in response to vitamin E
deficiency, oxidative stress and age. Results of chemical analyses


92
Peterson and Hakkarainen, 1986). Following vitamin E administration,
the levels increased progressively for all treatments during the 4 wk
experimental period to the range (4 to 9 yg/ml) reported acceptable
for beef cattle (Bayfield and Mylrea, 1969).
In humans and monkeys, Machlin and Gabriel (1982) reported that
it is advisable to treat subjects with high levels of a-tocopherol for
at least 1 to 3 wk to attain maximal plasma a-tocopherol levels.
In order to evaluate the effectiveness of the various vitamin E
preparations, most studies (Horwitt et al., 1984; Baker et al., 1986)
have relied on serum a-tocopherol concentrations and have paid little
attention to tocopherol concentrations in tissue. In the present
experiment, tissue analysis showed higher concentrations following d-
a-tocopherol or d-a-tocopheryl acetate supplementation than with the
racemic forms. These results suggest strongly that d-a-tocopherol or
its acetate form is more available to bovine tissue than the 1-epimer.
Cattle, like the rat, appear to discriminate well between 2R, 4'R,
8'R, and 2S, 4'R, 8'R a-tocopherol (Ingold et al., 1987).
Dju et al. (1958) reported that, in the human body, there is a
wide variation in the distribution of a-tocopherol in the various
tissues. In the present experiment, highest concentrations were
detected in the adrenal gland and liver. The liver is the major
storage organ for a-tocopherol (Machlin and Gabriel, 1982) and (for a
short time at least) helps maintain plasma tocopherol levels when the
intake of vitamin E becomes inadequate. As indicated earlier, in rats
(Behrens and Madere, 1986), the adrenal gland accumulated more
tocopherol per gram of tissue than other tissues, regardless of the


98
Analytical Method
Plasma samples were prepared for high pressure liquid
chromatography (HPLC) according to the method of McMurray and
Blanchflower (1979). The chromatographic apparatus consisted of a model
6000 pump and WK septumless injector (Waters Associates, Milford,
Mass.). A Perkin-Elmer 650-150 fluorescence spectrophotometer, equipped
with a microflow cell unit, was used for quantification. Wavelength
settings were 295 and 330 nm for excitation and emission, respectively.
The column was a y Bondapak C18 (3.9 mm x 30 cm) of 10 ym particle size
purchased from Waters Associates (Milford, Mass.) The mobile phase, a
solvent system (HPLC grade), consisted of methanol and water in a 97:3
ratio with a flow rate of 3 ml/minute.
Statistical Analysis
The indices of bioavailability were calculated for each individual
sheep or cattle. These indices were: (1) the maximum a-tocopherol
plasma (C max) concentration (peak of plasma a-tocopherol curves); (2)
the time of maximum (t max) a-tocopherol concentration; and (3) the area
(AUC) under the a-tocopherol plasma concentration time curve (Gibaldi
and Perrier, 1975). Statistical analysis was carried out by analysis of
variance (sheep) or covariance (cattle) by the general model procedure
(SAS, 1980). The following model was used for the sheep,
Yij = y+ t. + Ei j;
where y = mean;
ti = ith type i = 1,2
E^j = random error;


LITERATURE CITED
Adamstone, F. B. 1947. Histologic comparison of brains of vitamin A
deficient, vitamin E deficient chicks. Arch. Pathol. 43:301.
Alaupovic, P., B. C. Johnson, Q. Crider, H. N. Bhagaran, and B. J.
Johnson. 1961. Metabolism of alpha-tocopherol and the isolation
of non-tocopherol-reducing substance from animal tissues. Am.
J. Clin. Nutr. 9:76.
Alderson, N. E., G. E. Mitchell, Jr., C. 0. Little, R.L. Warner, and
R. E. Tucker. 1971. Preintestinal disappearance of vitamin E in
ruminants. J. Nutr. 101:655.
Ames, R. S. 1971. Isomers of alpha-tocopheryl acetate and their
biological activity. Lipids 6:281.
Ames, R. S. 1979. Biopotencies in rats of several forms of alpha-
tocopherol. J. Nutr. Res. 109:2198.
Ames, R. S., M. J. Ludwig, D. R. Nelau, and C. D. Robenson. 1963.
Biological activity of an 1-epimer of d-alpha-tocopheryl acetate.
Biochemistry 2:188.
Baker, H., 0. Frank, B. Deangelis, and S. Feingold. 1980. Plasma
tocopherol in man at various times after ingesting free or
acetylated tocopherol. Nutr. Rep. Int. 21:531.
Baker, H., G. J. Handelman, S. Short, L. J. Machlin, H. N. Bhagavan,
E. A. Dratz, and 0. Frank. 1986. Comparison of plasma alpha-
tocopherol and gamma-tocopherol levels following chronic oral-
administration of either all-rac-alpha-tocopheryl acetate or RRR-
alpha-tocopheryl acetate in normal adult male-subjects. Am. J.
Clin. Nutr. 43:382.
Balard, B. E. 1968. Biopharmaceutical considerations in subcutaneous
and intramuscular drug administration. J. Pharm. Sci. 57:357.
Behrens, H., G. Matschullat, and K. Tuck. 1975. Uber die
vertraglichkeit oliger vitaminlosungen beim schaf nach
intramuskularer applikation. Dtsch. Tierarzt. Wschr. 82:1.
Behrens, W. A., J. N. Thompson, and R. Madere. 1982. Distribution of
alpha-tocopherol in human plasma lipoproteins. Am. J. Clin.
Nutr. 35:691.
Bieri, J. G. 1972. Kinetics of tissue a-tocopherol depletion and
repletion. Ann. N. Y. Acad. Sci. 203:181.
124


99
while for cattle the model used was,
Yij = y + CQ + tA + ij
where C = value at time 0.
Results
Trial 1
Following the oral administration of the two vitamin E
preparations to sheep, there was an increase in the plasma a-
tocopherol level (Figure 6.1). The peak of this increase was reached
faster (P<.05) in the dl-a-tocopherol than in its ester form (Table
6.1). The shorter T-maximum observed in the dl-a-tocopherol dosed
group may have contributed to a faster onset of biological action.
The data show that C plasma a-tocopherol concentrations had a
maxima r
tendency (P>0.05) to be higher in the dl-a-tocopherol than in the
ester-dosed group. A direct comparison of the plasma tolerance curve
area (AUC) between the two groups (Table 6.1), showed that the AUC was
higher (P<.05) in the dl-a-tocopherol than in the sheep administered
with dl-a-tocopheryl acetate (Table 6.1).
Maximum plasma vitamin E concentration, as well the tolerance
curve, are dependent chiefly on factors affecting the absorption of
the vitamin from the intestine. Our results showed that the
effectiveness of the absorption indicated by the tolerance curve area
was higher in the sheep dosed with free alcohol form than with the
acetylated group. The higher peak observed in the plasma tolerance
curve in the dl-a-tocopherol group than in its acetate form probably
reflects its higher potency. At the end of the two-wk experimental


123
tocopherol levels. Comparing the currently accepted biological
potencies (IU/mg) provided by the National Formulary (1985) for the
various tocopherol sources to the estimate values for ruminants are as
follows respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37). The results
obtained from these experiments suggest that the current accepted
biological potencies of the various tocopherol sources may be invalid
for ruminants.
Experiments reported in this dissertation show that the natural
form of a-tocopherol had a higher bioavailability than did the
synthetic, though the reason for this remains unresolved.
Further research in ruminants should be directed towards
identifying the potential presence of specific binding proteins in
tissues of ruminants which have been shown to be fairly specific for
the natural stereoisomer in other species.


44
Because of its antioxidant effect, vitamin E is able to entrap free
radicals before they can attack life-supporting cell structures. Free
radicals are, by definition, chemical compounds which contain an odd
number of electrons. Free radicals can be formed in biological
systems through homolytic cleavage of covalently bonded organic
compounds resulting in bond splitting, whereby each fragment retains
one electron of the original bonding pair.
H
R C = C.
Bond splitting as such always gives rise to a pair of radicals.
Radicals can also be formed from a capture of an electron by a
molecule as illustrated below:
O2 + e 0^* .
The formation of superoxide anion radical results in a net negative
charge because of the extra electron in its posession. Capturement of
electrons by molecular is known to occur by its interaction with
flavin oxidoreductase systems (Fridovich, 1976). For a better
understanding of these phenomena, the effect of free radicals upon the
breakdown of an essential fatty acid as described by Leibovitz and
Siegel (1980) is presented in Fig. 2.9.
In a fatty acid, at a carbon atom adjoining a double bond, an
agressive substance (aS) removes hydrogen (I), resulting in the
formation of a fatty acid radical (II). This radical reacts with
oxygen to form a peroxy fatty acid radical (III). This new radical in
its turn removes hydrogen from another essential fatty acid, resulting
in the formation of a hydroperoxy fatty acid (IV) and again in a fatty


Experiment III investigated the concentration of a-tocopherol in
plasma and tissues of beef cows following a daily oral administration of
1000 IU of four tocopherol sources for 28 d. The d-a-tocopherol and its
acetate ester increased plasma tocopherol concentrations faster (PC.05)
than the racemic products, with the greatest response occurring with d-a-
tocopherol. Tissue analyses confirmed that in adrenal gland, kidney,
liver and lung, ct-tocopherol concentrations were higher (PC.05) following
d-a-than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered to
sheep or cattle (100 or 50 mg/kg b.w., respectively) in a single oral
dose. Blood plasma a-tocopherol tolerance curve area was higher (PC.05)
in the dl-a-tocopherol group than in its ester form, as well as quicker
(PC.05) time (h) for maximum plasma a-tocopherol concentration. A
greater (PC.05) plasma tolerance curve area was observed in the cattle
following administration of dl-a-tocopherol than its acetylated form.
In experiment V plasma and tissue vitamin E concentrations were
determined following a 5 g dosage of dl-a-tocopherol intraperitoneally in
sheep, which were slaughtered on d 3, 6, 10, 15 and 28 post dosing.
There was a significant time effect for all tissues, while in most, the
peak for a-tocopherol concentrations was observed at 3 d post dosing.
Rate of uptake of vitamin E varied for different tissues, with liver,
spleen and lung showing a pronounced uptake and muscle showing least.
Comparing the established biological potencies (IU/mg) of the
various tocopherol sources to the estimate values for ruminants are as
follows, respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37).


40
D-a-tocopheryl acetate can be easily obtained in chemically and
isomerically pure form. The relative biological activities of the
tocopherols have been determined in experimental animals and are now
internationally accepted. They are calculated as follows:
1 d-a-tocopherol equivalent (mg)
= d-a-tocopherol x 1
= d-a-tocopheryl acetate x 0.91
= all-rac-a-tocopheryl acetate x 0.67
= d-y-tocopherol x 0.1.
These equivalence values have been disputed (Ames, 1979; Leth and
Sandergaard, 1983), however, in 1983 they were confirmed by a WH0/FA0
Expert Committee (WHO, 1983). Evidently the experts did not consider
it necessary to change the relative biological activities of
tocopherols based on animal experiments. More recent human studies
suggest that the findings on which the conversion factors are based,
i.e., experiments with rats given a vitamin E-deficient diet, should
be regarded as species-specific for the experimental animals.
A comparison of the tocopherol concentrations in human tissues
and cells emphasizes the physiological significance of free RRR-a-
tocopherol relative to other tocopherols and derivatives. The vitamin
E requirement in man is satisfied by the preferential utilization of
d-a-tocopherol in the diet. That d-a-tocopherol possesses the
relatively greatest biological activity has been confirmed by a
multitude of relevant i^n vitro and Tn vivo tests and particularly by
clinical studies. Accordingly, RRR-a-tocopherol is the actual vitamin
E in man.


108
sheep were selected at random and killed at each of days 3, 6, 10, 15
and 28 after dosing.
Five g of dl-a-tocopherol were given intraperitoneally (IP) to
each treated sheep as a single injection in emulsion (Tween 80) with a
final volume of 75 ml. The IP dose was administered using the
technique of Hurter (1987). Blood samples were withdrawn by jugular
puncture at designated times prior to (0) and after vitamin E
administration (3, 6, 10, 15 and 28 d) and collected in heparinized
tubes. Plasma was obtained upon centrifugation at 500 x g for 15 min.
The animals were killed by exsanguination. Portions of kidney, liver,
adrenal, pancreas, skeletal muscles, heart, spleen and lung were
removed. All tissues were rinsed in water and their surfaces dried
with filter-paper. During removal of all tissues, care was taken to
dissect away superfluous adipose tissue. All tissues and plasma were
stored at -20C until analyzed for a-tocopherol concentration.
Analytical Methods
Quantitation of d-a-tocopherol in blood plasma and tissues was
performed by high pressure liquid chromatography (HPLC) using a
fluorescent detector (McMurray and Blanchflower, 1979). The HPLC
consisted of an M6000 pump and WK septumless injector. A Perkin-Elmer
650-150 fluorescence spectrophotometer equipped with a microflow-cell
unit was used for quantification. Wavelength settings were 295 and
330 nm for excitation and emission, respectively. The column was a y
Bondapak (3.9 mm x 30 cm) of 10 ym particle size. Elution was
performed with methanol:water (97:3) solvent with a flow rate of 3


21
Investigations on the metabolites of a-tocopherol in animal
tissues were conducted by several workers with the anticipation that
the nature of the metabolites would provide insight into the mechanism
of action of vitamin E. Simon et al. (1956) isolated and
characterized a metabolite of a-tocopherol from both rabbit and human
urine. This oxidation product was given the common name tocopherol-5-
methyl-C^ succinate. The same authors found that urine contained
mainly metabolized tocopherol, while intact tocopherol accounted for
the majority of the radioactivity in the feces. Mervyn and Morton
(1959) reported the presence of a-tocopheryl quinone in a nephritic
human kidney deficient in vitamin E. Alaupovic et al. (1961) found no
evidence of a-tocopheryl quinone, a-tocopheryl hydroquinone, a-
tocopheroxide, tocopheronic acid or tocopheronolactone in pig and rat
livers. Csallany et al. (1962) identified two new metabolites of a-
tocopherol isolated from the liver of the rat after injection of [5-
methyl-^C] d-a-tocopherol. About 25% of the recovered activity was
unchanged a-tocopherol, about 19% was a-tocopheryl quinone, and about
50% was dl-a-tocopherone, a dimer of oxidized -tocopherol. Csallany
and Draper (1963, 1964) separated and identified the cis-and trans-
isomers of dialpha-tocopherone. Draper et al. (1967) also identified
a trimer of a-tocopherol in the mammalian liver. Krishnamurthy and
Bieri (1963), and Plack and Bieri (1964) indicated that oral and
intraperitoneal administration of a-tocopherol to rats and chicks
gave rise mostly to unchanged tocopherol in the tissues of the tested
animals. Mellors and McBarnes (1966) confirmed the above findings.
Following intravenous injection of H-a-tocopherol, Shiratori (1974)


116
TABLE 7.6. PLASMA TOCOPHEROL (yg/ml) VALUES AND SUMMARIES FOR THOSE ANIMALS
SLAUGHTERED AT 28 DAYS.
Days
1
Animal
2 3
4
Mean
SEM
Day 0
0.58
0.47
0.78
0.48
0.58
0.072
Day 1
6.41
6.09
7.27
3.59
5.84
0.790
Day 2
2.89
3.67
6.64
6.56
4.94
0.972
Day 3
1.95
2.81
4.53
8.13
4.36
1.368
Day 6
1.64
2.34
3.59
2.66
2.56
0.405
Day 10
1.56
1.72
1.87
1.44
1.65
0.094
Day 15
1.44
1.15
a
1.64
1.41
0.142
Day 28
1.88
1.64
2.03
2.27
1.96
0.132
Area^
51.43
52.56
74.77
71.96
62.68
6.199
Maximum
concentration
plasma (yg/ml)
6.41
6.09
7.27
8.13
6.98
0.459
Time to maximum
concentration (d)
1.00
1.00
1.00
3.00
1.50
0.500
c
Elimination
rate
3.41
3.71
3.58
3.58
3.57
0.062
aNo measurement was
taken at
this point
in time.
Total area under the plasma concentration curve.
c
Elimination rate is defined as the maximum/terminal plasma level.


135
Roles, 0. A. 1967. Present knowledge of vitamin E. Nutr. Rev.
25:33.
Rose, C. S., and P. Gyorgy. 1952. Specificity of hemolytic reaction
in vitamin E deficient erythrocytes. Am. J. Physiol. 168:414.
Rosenkrantz, H., A. T. Milharat, and M. Farber. 1951. Counter-
current distribution in identification of the tocopherol
compounds in feces. J. Biol. Chem. 112:9.
SAS. 1980. A user's guide to SAS. SAS Institute Inc., Raleigh, NC.
SAS. 1985. SAS User's Guide: Statistics. SAS Inst., Inc., Cary,
NC.
Schwarz, K. 1958. Liver Function. Iii Symposium on Approaches to the
quantitative Description of Liver Function (R. W. Brauer, ed.),
pp. 13-24. American Institute of Biological Sciences Pub. No. 4.
Schwarz, K. 1961a. A possible site of action for vitamin E in
intermediary metabolism. Amer. J. Clin. Nutr. 9:71.
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Schwarz, K. 1962. Vitamin E, trace elements and sulfhydryl groups in
respiratory decline: An approach to the mode of action of
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Schwarz, K. 1972. Jn_ Vitamin E and its Role in Cellular Metabolism
(P. Nair and H. J. Kayden, eds.). Ann. NY Acad. Sci. 203:45-52.
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Vitam. Horm. 20:621.
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dystrophy activity of the d- and 1-epimers of alpha-tocopherol
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tOCOptofOl pf/ml plxmi
55
FIGURE 3.1. MEAN PLASMA a-TOCOPHEROL LEVELS IN SHEEP AFTER SINGLE
I.M. INJECTION OF D-ct-TOCOPHEROL SUSPENDED IN STERILE
SESAME OIL.


127
Dam, H., and J. Glavind. 1939. Alimentary exudative diathesis, a
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Dam, H., I. Prange, and E. Sandergaard. 1952. Muscular degeneration
(white striations of muscles) in chicks reared on vitamin E
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Davies, T., J. Kelleher, C. L. Smith, and M. S. Losowsky. 1971. The
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Desai, I. D. 1980. Assay methods, .In Vitamin E (L. J. Machlin,
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Desai, I. D., C. R. Barekh, and M. L. Scott. 1965. Absorption of D-
and L-a-tocopheryl acetate in normal and dystrophic chicks.
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Desai, I. D., and M. L. Scott. 1965. Mode of action of selenium in
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Dickson, J., D. L. Hopkins, and G. H. Doncon. 1986. Muscle damage
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63:231.
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deficiency. Vitam. Horm. 20:511.
Diplock, A. T., E. E. Edwin, J. Green, J. Bunyan, and S.
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Ultrastructural changes in the Golgi apparatus in the liver of
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Dju, M. Y., E. E. Mason, and L. J. Filler. 1958. Vitamin E
(tocopherol) in human tissues from birth to old age. Am. J.
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105
(1974) observed higher plasma tocopherol concentrations (1.28 .07
pg/ml) for sheep fed dry feed. It appears that in the ruminant
animal, following a single oral dose of dl-a-tocopherol or its
acetylated form, the terminal values were higher in sheep and cattle
than the initial values. This may be related to a slow saturation of
tissues with both forms of tocopherol. However, no difference was
observed in the elimination rates (C max/CT). In conclusion, the data
in ruminants confirm the higher biological potency of the dl-a-
tocopherol vs. dl-a-tocopheryl acetate.
Summary
Two trials were carried out in order to evaluate the
bioavailability of dl-a-tocopherol and dl-a-tocopheryl acetate
administered to sheep or cattle in a single oral dose. In the first
trial, two groups of five sheep were used. They received 100 mg/kg
body weight of either dl-a-tocopherol or its acetylated form. The
blood plasma tolerance curve area and the maximum plasma concentration
were higher in the dl-a-tocopherol group than in its ester form. The
time (h) for maximum plasma a-tocopherol concentration was quicker
(P<0.05) in the dl-a-tocopherol group than in its ester form. In a
second trial, four heifers received the two forms (50 mg/kg body
weight) in rotation after an appropriate washing period between the
two dosings. Again, a greater plasma tolerance curve area was
observed in the cattle following administration of dl-a-tocopherol
than its acetylated form.


13
identified as free a-tocopherol by isotope dilution. It was concluded
that the vitamin was re-secreted into the intestinal tract from the
blood or via the bile. In confirmation by Krishnamurthy and Bieri
(1963), who administered the vitamin orally, a second increase in
fecal tocopherol was noticed which they did not attribute to
coprophagy since the rats had tail cups. Mellors and Mcbarnes (1966)
demonstrated that no significant amount of either tocopherol or its
metabolites was introduced into the lumen of the rat guts via the bile
or through secretion from mucosal cells. By using a-tocopherol
labelled with radioactive C^, Shantz (quoted by Harris and Ludwig,
1949) remarked that 80% of the vitamin E given in oil solution was
excreted in the feces of rats.
Dju et al. (1950) observed that chickens receiving 1 g of a-
tocopherol/day for a prolonged period eliminated 75% of the ingested
quantity unchanged via the feces by 24 h. In contrast, chickens
receiving a diet supplemented with 17.5 and 35 mg of a-tocopherol/kg
only eliminated about 23% in the feces (Pudelkiewicz and Matterson,
1960).
Information on the absorption and excretion of tocopherols by
farm animals is extremely sparse. In unpublished work with young
calves, Blaxter and Brown (1952) indicated that about 25% of a-
tocopherol added as the acetate to a diet of dried skimmed milk was
excreted in the feces when the daily intake of the ester was from 25
to 100 mg. From such scanty data it is clearly impossible to draw the
conclusion that species differ in their ability to absorb tocopherols,
especially since great variation has been reported for a single


103
the relative less favorable response to tocopherol ester is due to
factors influencing the hydrolysis of these esters.
It seems probable that in cattle, like other mammals, dl-a-
tocopheryl acetate following hydrolysis is split in the intestinal
mucosa and is absorbed as free tocopherol from the gut and passed to
the systemic circulation via the lymphatic system. According to
Tikriti (1969), in cattle the acetylated form of vitamin E is
hydrolyzed in the digestive system, beginning in the rumen, and
appears to be utilized somewhat less efficiently than the free form.
One cannot rule out, as it was suggested by Gallo-Torres et al.
(1971), the possibility of a rate-limiting hydrolytic reaction
occurring in the lumen of the intestine prior to entrance of the
vitamin E into the intestinal wall of the ruminant animal.
Our results showed that T-maxima is much longer in ruminants than
in humans following oral loading with vitamin E. Indeed, Hashim and
Schuttinger (1966) observed that in normal human subjects plasma
tocopherol levels began to rise between 2 and 4 h and peaked at 7.5 h
following oral administration of vitamin E. They also observed that
at 24 h the plasma tocopherol level had declined and that at 48 h
post-absorptive levels were reached. Marusich et al. (1967) reported
that in chickens, following the administration of a single oral dose
(50 IU of dl-<*-tocopheryl acetate), the peak plasma values obtained at
6 h were followed by a steady decline. However, in rats, Marusich et
al. (1968) observed that plasma tocopherol levels peaked at 24 h,
following administration of a single oral dose of dl-a-tocopheryl
acetate.


TABLE PAGE
5.1. COMPOSITION OF THE GRAIN PORTION OF THE DIET FED TO
COWS FOR 28 d 81
5.2. PLASMA CONCENTRATION OF a-TOCOPHEROL (pg/ml) IN CATTLE
FED VARIOUS PREPARATIONS OF VITAMIN E 85
5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (Pg/g FRESH TISSUE)
IN CATTLE FED VARIOUS VITAMIN E PREPARATIONS 88
6.1. DL-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES (LEAST
SQUARE MEANS SE) IN SHEEP AFTER A SINGLE ORAL
ADMINISTRATION OF VITAMIN E (100 mg/kg BODY WEIGHT).... 100
6.2. TRIAL 2: D-a-TOCOPHEROL PHARMACOKINETIC PLASMA VALUES
(LEAST SQUARE MEANS SE) IN CATTLE AFTER A SINGLE
ORAL ADMINISTRATION OF VITAMIN E (50 mg/kg BODY
WEIGHT) 101
7.1. DIET OF SHEEP 107
7.2. MEAN LEVELS OF a-TOCOPHEROL IN TISSUES (pg/g FRESH)
AND PLASMA (pg/ml) AT TIME OF SLAUGHTER 110
7.3. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG TISSUE
VALUES 112
7.4. MEANS OF THE RATIO OF TISSUE TO PLASMA LEVELS OF
a-TOCOPHEROL AT TIME OF SLAUGHTER 114
7.5. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG RATIO OF
TISSUE TO PLASMA 115
7.6. PLASMA TOCOPHEROL (pg/ml) VALUES AND SUMMARIES FOR
THOSE ANIMALS SLAUGHTERED AT 28 DAYS 116
via


39
Muscular degeneration tests
Muscular dystrophy has been used as a means of measuring vitamin
E biopotency in rabbits (Hove and Harris, 1947; Fitch and Diehl, 1965)
and chicks (Scott and Desai, 1964; Desai and Scott, 1965).
In chickens, muscular degeneration is evaluated directly after 3
to 4 weeks on deficient or supplemented diets. The breast muscle is
examined and scored from 0 to 4, depending on the severity of the
lesion. Indirect measure of muscular degeneration, such as
creatinuria and levels of plasma aspartate aminotransferase and lactic
dehydrogenase, have also been used in ducks and rabbits. The time of
onset of creatinuria or the extent of muscular dystrophy, assessed on
a scale, are both used as biological measures, and the effect on these
parameters of standard RRR-a-tocopherol or a test material is assessed
as the basis of the assay. A method based on the reversal of muscular
degeneration in the rat, using plasma pyruvate kinase levels as an
index, appears to be quite reliable, rapid, and sensitive (Machlin et
al., 1982).
D-a-Tocopherol Equivalents
A basis for establishing the biological activity of vitamin E
preparations is necessary to ensure uniformity of dosage. In 1978 the
International Union of Nutritional Sciences (IUNS), therefore,
recommended that d-a-tocopherol be used as the standard compound and
that the biological activity be specified in activity equivalents
(Fromming, 1984).


74
TABLE 4.4. ANALYSIS OF VARIANCE OF a-TOCOPHEROL CONCENTRATIONS IN
BLOOD PLASMA OF SHEEP FED DIFFERENT FORMS OF VITAMIN E.
Source
DF
MS
F
P
Model
66
2.99
4.64
0.0001
Error
217
0.64
Corrected total
283
Source
DF
SS
F
P
Diet
3
44.79
23.09
0.0001
Tissue
16
225.13
11.13
0.0001
Diet x tissue
47
40.53
1.33
0.0883
TABLE 4.5. AREA (AUC)
UNDER THE PLASMA a-TOCOPHEROL CONCENTRATION
CURVE (pg/ml h_1).
Diet
AUC (mean SE)
d-a-tocopherol
102.4 8.13a
d-a-tocopheryl acetate
90.0 9.44b
dl-ot-tocopheryl acetate
75.0 7.31c
dl-a-tocopherol
67.3 7.31d
In vertical row, numbers with the same letter are not different
(P>0.05).


104
The fact that the biological response to dl-a-tocopherol was
greater than with its acetylated form agrees with the results reported
in humans (Baker et al., 1980). These workers observed that following
administration of a single oral dose (800 mg) of dl-a-tocopheryl
acetate, plasma d-a-tocopherol peaked (50% from the initial value) 8 h
after ingestion. The same dose of free tocopherol induced a greater
rise (60% from the original value) in 5 h.
It seems that dl-a-tocopherol serves more pharmacological action
when ingested in amounts many times the generally recognized
nutritional requirements than its esterified form. The results
demonstrated that T max was faster following oral dosing of the free
than acetylated form. This suggests that the hydrolysis rate of
esterified tocopherol to the physiologically active free tocopherol is
quite slow.
In this study, the area (AUC) under the a-tocopherol plasma
concentration time curve undoubtedly was influenced by the amount of
vitamin E activity again. Indeed, the higher vitamin E potency of dl-
a-tocopherol (1.1 IU/mg) compared to 1.0 IU/mg, for the acetate form
probably contributed to the tendency of a higher plasma vitamin E peak
as well as the greater AUC.
In the present experiment, initial plasma tocopherol
concentrations (2 to 3 yg/ml) from hay-fed cows were in the range of
values observed by Pehrson and Hakkarainen (1986) and Hakkarainen et
al. (1987). The initial value of a-tocopherol in the plasma of sheep
fed a commercial diet was very low. In grain-fed sheep, Caravagi
(1969) reported plasma concentrations of .89 .16 SD yg/ml. Storer


109
ml/min. All solvents used for the HPLC mobile phase or for the
extraction were HPLC or pesticide grade. Tissue samples for vitamin
E determination were prepared according to the method of Burton et al.
(1985).
Statistics
One-way analysis of variance (ANOVA) was performed on the
logarithms of the tissue values and the tissue to plasma ratios.
Logarithms were taken to stabilize the variances which increased with
increasing concentrations. To further study the effect of time, five
contrasts were examined (SAS Institute, 1985). Untransformed means
have been presented with approximate standard errors based on the
analysis of the logarithms. Residuals were examined and an outlier
was identified using Tietjen and Moore's statistic (Tietjen and Moore,
1972). The changes which occurred when this outlier was removed from
the analysis are noted.
Plasma values were also analyzed by analysis of variance. This
analysis was a one-way ANOVA of time within the animal. Here also, a
transformation to logarithms was necessary and the time effect was
examined by several contrasts.
Results
Mean levels of dl-a-tocopherol in tissues and plasma at the time
of slaughter are given in Table 7.2. The accompanying analysis of
variance of the logarithms of these values at slaughter is given in


88
TABLE 5.3. TISSUE a-TOCOPHEROL CONCENTRATIONS (yg/g FRESH TISSUE) IN CATTLE FED
VARIOUS VITAMIN E PREPARATIONS.
Dietary
form
Tissue
mean
d-
Tissue
a-tocopheryl
acetate
d-a-toco-
pherol
dl-a-toco-
pherol
dl-a-tocopheryl
acetate SE
pg/g fresh tissue
Adrenal gland
38.41a
38.07a
27,91b
28.75b
2.68
33.28d
Heart
20.43a
18.71a
18.43a
16.12a
1.42
18.42f
Kidney
12.07a
13.11a
8.81b
12.28a
1.00
11.56h
Liver
24.20ab
27.00a
15.67c
19.88bc
1.79
21.68e
Lung
15.73a
16.52a
10.64b
15.04a
1.08
14.48
Muscle
5.29a
5.79a
5.70a
5.79a
.46
5.64j
Spleen
17.95a
15.79ab
11.76b
13.88ab
1.38
14.848
Thyroid
3.40b
4.43ab
5.63a
4.35ab
.46
4.45J
SE
1.82
1.63
1.10
1.10
.80
Group mean
17.18a
17.42a
13,03b
14.53b
.57
Means in the same row with different letters in their superscripts differ
(P<0.05).
d,e,f,g,h
Means in the same column with different letters in their superscripts
differ (PC0.0001).


47
In the last few years, much research has been done concerning
free radicals and into how various substances act to inhibit them
(Cheeseman et al., 1984).
Free radicals are involved in two types of biological phenomena.
Some appear in normal cell reactions, others in abnormal reactions and
in processes associated with the aging of organisms. All biological
oxidations do not necessarily give rise to such radicals. The
mechanisms by which they are produced are numerous as they result from
many types of reactions. These reactions include the peroxidation of
lipids, in the course of which, under certain conditions increase
depending upon the rate of formation of free radicals. It has also
been shown that some processes, whether enzymatic or not, produce
reactions of the same type when some of the cell constituents released
are exposed to the direct action of oxygen in a free environment or in
the absence of inhibiting agents. Some researchers, such as Pryor
(1970), do not rule out the possibility that radicals may escape an
electron transport chain, then causing rapid and destructive
oxidations.
It may seem paradoxical, as Pryor (1971) points out, that oxygen,
which cells need to release energy from food, is so reactive that it
manages to trigger, alongside the usual processes, some undesirable
parallel reactions oriented toward actions detrimental to the
phenomena essential to life. In the course of such oxidative
reactions, it causes damage to the cells by producing unfavorable
modifications in the structure of their membranes, in the membranes of
the intracellular organelles and in the endoplasmic reticulum.


9
CH2CHNH2COOH
Phe
OH
ch2cooh
O-glucose
homogentisic
acid glucoside
OH
1
Tyr
CH2COCOOH
p-hydroxyphenyl
Oh pyuvate
OH
homoarbulin
O-glucose
FIGURE 2.5. BIOSYNTHESIS OF THE RING STRUCTURE OF
TOCOPHEROLS AND TOCOTRIENOLS


CHAPTER 4
BIOAVAILABILITY OF VARIOUS VITAMIN E COMPOUNDS IN SHEEP
Introduction
The biological activity of the various tocopherol compounds has
been established from experiments with laboratory animals. However,
biopotency evaluation of different forms of vitamin E based on a
specific animal model (i.e., rat antisterility test) may not be valid
for other species. Biopotencies based on this test do not correlate
with human data. Recently Baker et al. (1986) reported that human
assay data confirmed the currently accepted biopotencies of 1.0 IU/mg
and 1.36 IU/mg for dl-tocopheryl acetate and d-a-tocopheryl acetate,
respectively. There are little data available for ruminants on the
absorption and body distribution of vitamin E. In view of the unique
digestive and physiological system in ruminants, the present
investigation determined bioavailability of the various forms of
vitamin E in sheep fed over a relatively long-term study (28 days).
Materials and Methods
Initially, twenty crossbred yearling wethers weighing 60 to 70 kg
were used. All animals originated from a flock fed a commercial mix
that consisted of 56.45% corn meal, 16.5% (44% CP) soybean meal, 25%
cottonseed hulls, 1% trace mineral salt, dicalcium, 1% monocalcium
phosphate, and .037% of vitamin A and D^. They were fed this diet
throughout the experiment.
63


ABUNDANCE
72
MASS/CHARGE
FIGURE 4.2. IDENTIFICATION OF a-TOCOPHEROL BY GAS-CHROMATOGRAPHY/MASS
SPECTROPHOTOMETRY (A) a-TOCOPHEROL STANDARD, (B) a-
TOCOPHEROL IN PANCREAS


59
Discussion
Blood plasma vitamin E data showed that in sheep dosed with
various vitamin E preparations, the movement of the active free ex-
tocopherol from the site of the IM injection to the blood stream was
quite slow. In all sheep given the vitamin E preparation by IM oil
depot injection, a delayed increase in plasma a-tocopherol levels
occurred. D-a-tocopherol IM administration proved to be more potent
than dl-a-tocopherol. The length of the experimental period had an
effect on the bioavailabilities index. The IM dosed sheep with d-a-
tocopherol and slaughtered 360 hr later had a bioavailability much
higher than in sheep dosed similarly but killed 240 hr after dosing.
During this 360 hr period a gradual but sustained response occurred.
Overman et al. (1954) reported that IM administration of vitamin
E is ineffective in humans, regardless of the type of compound and the
type of vehicle (oil or water) used. However, according to Caravaggi
et al. (1968), IM injection of vitamin E is the best route for
administration of this vitamin. These workers observed that in sheep,
following IM injection of a commercial preparation of d-a-tocopherol
acetate in aqueous suspension, the plasma a-tocopherol reached its
peak 8 hr after its administration. The slow release of the IM
injected vitamin E preparation in oil carrier could be due to myositis
and inflammation of the lymphatic vessels (Bille et al., 1976),
through which the vitamin E molecule is absorbed (Blomstrand and
Forsgren, 1968; Machlin et al., 1979). It is also known (Voffey and
Coutrice, 1956) that exercise influences the flow of fluid along the
lymphatic vessels. When an animal is at rest, as in these


58
TABLE 3.2. a-TOCOPHEROL LEVELS IN THE TISSUES (p g/g wet)1 OF SHEEP FOLLOWING
A SINGLE INTRAMUSCULAR ADMINISTRATION (40 mg/kg BODY WEIGHT)
OF VARIOUS VITAMIN E PREPARATIONS.
Groups:
Vitamin E preparation:
Mode administration:
Time after dosing (hr)
dl-a-tocopherol
240
d-a-tocopherol
360
d-a-tocopherol
240
Control
Tissues:
Adrenal
11.524.29ab
13.621.31ab
12.491.99ab
17.6416.36ab
Depot fat
10.521.73ab
17.851.69ab
16.822.52ab
12.84H ,68ab
Muscle vastus medial
4.200.55b
7.750.60a
4.921.01b
3.88i0.94b
Muscle:
brachiocephalicus
2.740.41d
6.261.13c
5.770.84c
3.46i0.70d
Heart
3.360.40ab
5.331.03ab
3.840.37ab
5.76H .59ab
Kidney
2.870.58ab
6.100.93ab
4.570.99ab
3.44i0.91ab
Lung
3.080.71e
7.521.10c
5.460.76d
2.91l0.23e
Liver
4.270.37a
5.921.39a
4.180.48a
2.22i0.36b
Pancreas
3.040.56b
6.941.00a
6.341.22a
4.32i0.50b
Spleen
2.720.56b
6.081.04a
5.6511.04a
3.30i0.54b
Least square means SE.
3 b
Means in the same rows with different superscripts differ (P<0.05).
c d G
Means in the same rows with different superscripts differ (P<0.01).


76
important indice of bioavailability, the area under the plasma a-
tocopherol concentration time curve (AUC), was larger (P<0.05) as
compared to dl-a-tocopheryl acetate or dl-a-tocopherol supplemented
sheep (Table 4.5). No statistical difference (P>0.05) was observed in
the maximum o-tocopherol concentration (pg/ml) between the four
groups. They were 3.6 0.69 (SE) (dl-a-tocopherol) and 4.3 0.69
(dl-a-tocopheryl acetate), 5.11 0.77 (d-a-tocopherol) and 4.8 0.89
(d-a-tocopheryl acetate). Also, no difference on the time (days) of
maximum concentration (P > 0.05) was observed. They were 18.2 3.24
(SE) (dl-a-tocopherol), 14.8 3.24 (dl-a-tocopheryl acetate), 11.5
4.30 (d-a-tocopherol) and 22.3 4.96 d-ar-tocopheryl acetate. The
elimination curve rates (concentration maximum/concentration terminal)
in the four groups were very similar. They were 1.6 0.20 (SE) (dl-
a-tocopheryl acetate), 1.8 0.22 (d-a-tocopherol) and 1.7 0.25 (d-
a-tocopheryl acetate) and 1.4 0.20 (dl-a-tocopherol).
Discussion
Paucity of bioavailability data from various forms of tocopherol
in sheep limits any comparison with the present data. Biological
response of sheep to dietary intake of various forms of vitamin E was
greater with the d-a-tocopherol than with its esterified form or the
isomers. Indeed, the area plasma time curves, which provide a proper
measure of relative availability for physiological function, and
tissue a-tocopherol concentrations were highest in the d-a-tocopherol
supplemented group. This indicates that in the ruminant body there
may be a difference in the transfer rate between natural and unnatural


91
the loss to the cleavage of the chroman structure accompanied by
hydrogen rearrangement and the loss of a CH^-C=CH fragment.
Discussion
The data show that, in cattle, a greater elevation of plasma
levels of -tocopherol occurred over pre-treatment levels after
ingestion of the naturally occurring d-a-tocopherol than its ester
forms or the 1-epimer of -tocopherol. These results agree with those
of Baker et al. (1980) who reported in humans that plasma -tocopherol
was considerably higher when the free tocopherol was orally
administered rather than its acetate ester. According to Machlin and
Gabriel (1982), the higher blood plasma -tocopherol attained
following supplementation with -tocopherol compared to -tocopheryl
acetate suggests that hydrolysis of the acetate may be a limiting
factor when high levels of the vitamin are administered. Harris and
Ludwig (1949) reported lower blood plasma a-tocopherol levels in
humans dosed with the nonesterified rather than esterified forms.
This is related to a diet-mediated oxidative destruction of the free
form in the gut prior to its absorption. This does not seem to be the
case with ruminants. Indeed, the stability of d-a-tocopherol in
ruminal liquor is high (Astrup et al., 1974b). Vitamin E is not
metabolized or oxidized to any great extent in the gastrointestinal
tract of cattle (Astrup et al., 1974a).
Plasma pre-treatment -tocopherol concentrations were low.
Levels less than 1.5 yg/ml in blood plasma were considered by several
workers as potentially hazardous for cattle (McMurray and Rice, 1982;


95
consisted of corn meal, 56.45%; soybean meal (44% CP), 16.5%;
cottonseed hulls, 25%; trace mineral salt, 1%; monocalcium phosphate,
1%; and .037% of vitamin A and D^. They were continued on this diet
throughout the experiment. Sheep were placed in individual metabolism
cages 10 days before administrations of vitamin E as an adjustment
period. They were then randomly assigned to two groups of five sheep
each. Each animal received a single intraruminal dose of either (1)
dl-o-tocopherol (i mg = 1.10 IU) or (2) dl-a-tocopheryl acetate (1 mg
=1.0 IU) at a rate of 100 mg/kg body weight.
Trial 2
Four crossbred dairy heifers (averaging 250 kg) were used in a
crossover design with two 20 d periods. They were given ad libitum
access to hay and water during the study. In the first period, two
heifers were administered orally with dl-a-tocopherol and two with dl-
a-tocopheryl acetate. The single dose given for each component was 50
mg/kg body weight. The end of the first period was followed by a
three-wk washing period. Then the treatment was reversed and the
procedures were repeated in period 2.
Blood Samples
Blood samples were withdrawn from the jugular vein in heparinized
vacutainers and immediately were centrifuged. They were collected
before vitamin E administrations and at precise intervals (Figures 6.1
and 6.2). Plasma was separated, frozen and stored at -20C until
analyzed for a-tocopherol.


37
acetic acid, that possesses the highest biological activity. Although
the test method is not precise enough to differentiate d-a-tocopherol
from its acetate, the two forms with the natural configuration are
nevertheless significantly superior to the synthetic all-rac-a-
tocopheryl acetate.
Plasma and erythrocyte concentration test
Vitamin E acts as an antioxidant in biological membranes (Dam,
1957). Therapy is designed to achieve a high tocopherol concentration
in these membranes.
In test rats which were reared on a vitamin E-deficient diet,
free d-a-tocopherol was compared with synthetic all-rac-a-tocopheryl
acetate to determine their effect in producing changes in vitamin E
concentration in plasma and in erythrocytes after intravenous or oral
administration (Ogihara et al., 1985). The tocopherol concentration
in the red blood cells was investigated as an indicator of the
physiologically available tocopherol in biological membranes.
Although intravenous administration of all-rac-a-tocopheryl
acetate produced a higher tocopherol level in plasma than RRR-a-
tocopherol 6 h after infusion, the concentration in the erythrocytes
at the same time increased by 100% after infusion of RRR-a-tocopherol
compared to the response when all-rac-a-tocopheryl acetate was
infused.
Differences between the natural, free vitamin E and the synthetic
acetate ester were even more pronounced in these test animals after
oral administration. Plasma concentration 6 h after oral
administration of d-a-tocopherol was 40% higher than after all-rac-a-


137
Tikriti, H. H., F. A. Burrows, Jr., A. Weisshaar, and R. L. King.
1968. Utilization of tocopherol-acetate by the lactating dairy
cow. J. Dairy Sci. 51:979.
Traber, M. G., and H. J. Kayden. 1984. Vitamin E is delivered to
cells via the high affinity receptor for low density lipoprotein.
Am. J. Clin. Nutr. 40:747.
Trinder, N., C. D. Woodhouse, and C. P. Renton. 1969. The effect of
vitamin E and selenium on the incidence of retained placentae in
dairy cows. Vet. Rec. 85:550.
United States Pharmacopeia, Twentieth Revision (U.SP XX). 1980.
United States Pharmacopeial Convention, Inc., Rockville, MD.
United States Pharmacopeia. 1985. National Formulary. USP XXI. pp.
1118-1120.
Vasington, F. D., S. M. Reichard, and A. Nason. 1960. Biochemistry
of vitamin E. Vitam. Horm. 18:43.
Weber, F. 1983. Digestion and absorption of nutrients. Int. J. Vit.
Nutr. Res. Supp. No. 25:55.
Weber, F., U. Gloor, and 0. Wiss. 1962. Fett seife. Anstrichmittel
64:1149.
Week, E.F., F. J. Sevigne, and E. M. Ellis. 1952. The relative
utilization of alpha-tocopherol and alpha-tocopheryl acetate by
humans. J. Nutr. 46:353.
Weiser, H., and M. Vechi. 1982. Stereoisomers of alpha-tocopheryl
acetate. II. Biopotencies of all eight stereoisomers,
individually or in mixtures as determined by rat resorption-
gestation tests. Int. J. Vit. Nutr. Res. 52:351.
Whistance, G. B., and D. R. Threlfall. 1967. Biosynthesis of
phytoquinones. An outline of the biosynthetic sequences involved
in terpenoid quinone chromanol formation by higher plants.
Biochem. Biophys. Res. Commun. 28:295.
Whistance, G. B., and D. R. Threlfall. 1968. Biosynthesis of
phytoquinones: Biosynthetic origins of the nuclei and satellite
methyl groups of plastoquinone. Tocopherols and
tocopherolquinones in maize shoots, bean shoots and iug leaves.
Biochem. J. 109:577.
Wimalasena, J., M. Davis, and A. E. Kitabchi. 1982. Characterization
and solubilization of the specific bindings sites for D-a-
tocopherol from human erythrocyte membranes. Biochem. Pharmacol.
31:3455.


Experiment III investigated the concentration of a-tocopherol in
plasma and tissues of beef cows following a daily oral administration of
1000 IU of four tocopherol sources for 28 d. The d-a-tocopherol and its
acetate ester increased plasma tocopherol concentrations faster (P<.05)
than the racemic products, with the greatest response occurring with d-a-
tocopherol. Tissue analyses confirmed that in adrenal gland, kidney,
liver and lung, a-tocopherol concentrations were higher (P<.05) following
d-a-than dl-a-tocopherol supplementation.
In experiment IV, dl-a-tocopherol and its ester were administered to
sheep or cattle (100 or 50 mg/kg b.w., respectively) in a single oral
dose. Blood plasma a-tocopherol tolerance curve area was higher (P<.05)
in the dl-a-tocopherol group than in its ester form, as well as quicker
(P<.05) time (h) for maximum plasma a-tocopherol concentration. A
greater (P<.05) plasma tolerance curve area was observed in the cattle
following administration of dl-a-tocopherol than its acetylated form.
In experiment V plasma and tissue vitamin E concentrations were
determined following a 5 g dosage of dl-a-tocopherol intraperitoneally in
sheep, which were slaughtered on d 3, 6, 10, 15 and 28 post dosing.
There was a significant time effect for all tissues, while in most, the
peak for a-tocopherol concentrations was observed at 3 d post dosing.
Rate of uptake of vitamin E varied for different tissues, with liver,
spleen and lung showing a pronounced uptake and muscle showing least.
Comparing the established biological potencies (IU/mg) of the
various tocopherol sources to the estimate values for ruminants are as
follows, respectively: d-a-tocopherol (1.49, 3.60); d-a-tocopheryl
acetate (1.36, 2.58); dl-a-tocopherol (1.1, 1.37).
xi


26
edema which deforms the head and neck and gives the feet a greenish
tint, hence its other name of "green foot disease" (Dam and Glavind,
1939).
Lesions are also edematous and affect the entire subcutaneous
connective tissue. This tissue has a gelatinous aspect and presents
hemorrhagic spots, in particular on major muscle masses and in the
periarticular area. In addition, whitish muscular dystrophy patches
are found fairly regularly in the gizzard area.
Muscular dystrophy. Muscular dystrophy appears around the age of
one month. It takes the form of locomotor troubles and weight loss
and entails considerable mortality. Lesions affect striated muscle
fibers which present zenkerism. They are closely comparable to those
of myopathy-dyspnea in calves and muscular dystrophy in lambs.
Muscular dystrophy in chickens appears to be due to a deficiency of
both vitamin E and selenium.
Lambs
This deficiency is also associated with clinical manifestations
appearing in areas where the soil is selenium-poor (Muth et al.,
1961). Winter lambs born of ewes which are more or less underfed pay
the heaviest consequence, particularly where ewes have two or three
young, whose requirements cannot easily be met (Oldfield et al.,
1963).
Clinical signs begin inconspicuously with listlessness; sick
lambs dislike moving about, and have trouble following the rest of the
flock. However, the appetite remains normal. Gradually, the gait
changes; steps become smaller, posteriors are stiff and splayed, and


102
period, the terminal plasma tocopherol levels in both groups were
higher (P<.05) than the original values.
Trial 2
A few hours after oral dosing in both groups of cattle there was
an increase in plasma a-tocopherol levels (Figure 6.2). The free form
was absorbed with higher effectiveness than its ester form. This is
shown by its greater plasma tolerance curve area and its tendency to
higher maxima concentration and speedier t maxima appearance. There
was significantly less utilization of the ester form versus the
alcohol form. In the present experiment, the system of rotation used
in administering the two vitamin E doses minimized the individual
variations.
Discussion
In the present experiment, the plasma persistence curve was
mainly used to provide a criterion of availability of vitamin E for
physiological function following oral dosage to ruminants with two
isomeric forms.
The present results show that in the ruminal environment, despite
a strong reducing system, the dl-ot-tocopherol was preserved
sufficiently to induce a superior persistence curve area response to
that of identical weight of the esterified form. It is possible that
the degree of hydrolysis could be an important factor in the shape of
the tolerance curve. As the stability of free tocopherol is known to
be inferior to that of tocopherol esters, it may be hypothesized that


112
TABLE 7.3. ANALYSIS OF VARIANCE MEAN SQUARES OF THE LOG TISSUE VALUES.
Tissue Overall
time effect
DOF 5
3-28a
versus 0
1
Source of variation
6-28 10-28 15-28
versus 3 versus 6 versus 10
1 1 1
28
versus 15
1
Residual
19
Kidney
5.036**
17.612**
6.424**
0.687*
0.286b
0.171
0.152
Liver
14.382**
59.183**
8.517**
2.350**
0.566(*)
1.294*(**) 0.235
Neck
1.413**
6.709**
0.312(*)
0.019
0.004
0.019
0.212
Leg
1.527**
6.907**
0.187(*)
0.009
0.028
0.503
0.210
Heart
2.466**
11.353**
0.484(**)
0.162
0.238
0.091
0.117
Spleen
7.057**
24.121**
7.422**
1.368(*)
1.276(*)
1.097
0.413
Lung
11.984**
43.916**
2.144(**)
3.898*(**)
7.572**
2.388(**
) 0.574
Adrenal
4.522**
20.818**
0.647(*)
0.086
0.398
0.658(*)
0.149c
Pancreas
0.468**
2.063**
0.083
0.003
0.000
0.169
0.063c
aMean of
treatments
3, 6, 10,
15 and 28
(3-28) are
compared
to control
group 0.
Significance levels in parentheses note changes which occurred when one outlier
was removed.
c
Residual is based on 17 degrees of freedom because of missing observations.
* (**)Indicates a significant effect at 5% (1%) level.


80
different chemical forms of vitamin E by sheep. That work was
extended to cattle with the purpose of comparing plasma and tissue a-
tocopherol concentrations following supplementation of various
tocopherol compounds.
Materials and Method
Animals
Twenty-four Charolais-Hereford crossbred beef cows ranging from 2
to 10 years of age were culled 6 months after calving from the herd of
the Nappan Experimental Farm, N.S., Canada. These unbred cows were
stratified by age into four groups, and the six animals within each
group were randomly assigned to four dietary tocopherol preparations.
The treatments were d-o-tocopheryl acetate, d-a-tocopherol, dl-a-
tocopherol and dl-a-tocopheryl acetate. Cows received daily 1,000 IU
(a dosage above a physiological level) of their respective vitamin E
preparation. This amounted to 735 mg of d-a-tocopheryl acetate, 671
mg of d-a-tocopherol, 900 mg of dl-a-tocopherol and 1000 mg of dl-a-
tocopheryl acetate, respectively. Each vitamin E preparation was
diluted in alcohol to 3 ml and mixed with 25 g of dry molasses. All
cows were individually fed 2 kg of average quality grass hay (8.7% CP,
35% ADF, 54% TDN, .51% Ca, .23% P, .02 ppm Se and 12 ppm vitamin E)
and had ad libitum access to a barley-soybean mixture (Table 5.1) with
no added a-tocopherol. The vitamin E-molasses mixture was prepared
daily and was topdressed on the grain portion for each individual cow.
The experiment lasted 28 days after which time the cows were
sacrificed at commercial facilities.


31
positive, while rats with no live young are negative. The vitamin E
activity of the test substance is determined by calculating the
percentage of rats in each test group that showed a positive response
and by plotting the calculated probits against dose (or log dose) to
derive the 50% fertility dose. The ratio of the 50% fertility dose of
the standard to the unknown substance is the relative vitamin E
biopotency of the test substance. It will be clear from the foregoing
that the fetal resorption test is tedious and time consuming and that
meaningful results can be achieved only by carrying out a large number
of tests on any given test substance, but it must still be regarded as
the final reference point in assessing vitamin E biopotency of an
unknown substance.
This test model is species-specific in its validity. The test
results cannot be directly applied to man, but because this model can
be standardized it can be used for comparisons of structure and
activity relationships. As in any other specific animal model, its
validity limits must be taken into account. The results obtained for
relative activity of the free, easily oxidizable tocopherols vary
greatly in this model from one team of investigators to another
(different laboratories) since the free tocopherols are easily
inactivated by other components in the diet of the experimental
animals. Knowledge of this interference dates back to the 1940s, and
consequently this model is used mainly to test and compare acetylated
tocopherols.
The results tabulated in Table 2.3 show that it is the
configuration in the phytol side chain which decisively affects the


107
TABLE 7.1. DIET OF SHEEP3
Ingredient
(%)
Corn meal
56.45
Soybean meal
16.50
Cottonseed hulls
25.00
Trace mineral salt*3
1.00
Dicalcium, monocalcium phosphate
1.00
Vitamins A, D^c
0.037
2
Analyzed for vitamin E and Se (8 ppm and .235 ppm, respectively).
^Provided, per kilogram of diet: 2.1 g NaCl, .22 mg I, .088 mg Co,
9 mg Fe, .725 mg Cu, 2.625 mg Mn and 8.75 mg Zn.
Q
Provided, per kilogram of diet: 5,000 IU vitamin A and 500 IU
vitamin D.


ACKNOWLEDGMENTS
The author wishes to express his sincere gratitude to Dr. L. R.
McDowell, chairman of the supervisory committee, for his guidance,
patience and friendship throughout the doctoral program. The author
gratefully acknowledges the assistance and time provided by other
members of the supervisory committee, including Drs. Douglas Bates,
Joseph Conrad, Kermit Bachman and Rachel Shireman.
Deep appreciation is extended to Dr. Keith U. Ingold of the
National Research Council of Canada (NRCC) for providing laboratory
facilities. The author is especially indebted to Dr. Graham W. Burton
of the NRCC for his guidance, patience, and friendship. The technical
assistance of Ann Webb, Dave Lindsay, Ewa Lusztyk of the NRCC is
deeply appreciated. Many special thanks are extended to Dr. L.
Arvanitis for his continual support and friendship throughout my
graduate program.
Assistance of fellow graduate students, Oswaldo Balbuena, Pablo
Cuesta, Edmundo Espinoza, Akhmad Probowo, Rodrigo Pastrana, Libardo
Ochoa and Roger Merkel, during blood and tissue collections is
appreciated. Special gratitude is expressed to Rodrigo Pastrana and
Roger Merkel for continual support during tissue collections from
animals.
Sincere appreciation is expressed to Mrs. Pat French for typing
iii
this dissertation.


138
Wiss, 0., R. H. Bunnell, and U. Gloor. 1962. Absorption and
distribution of vitamin E in the tissue. Vitam. Horm. 20:441.
World Health Organization (WHO). 1983. Expert Committee on
Biological Standardization, 33rd Report. World Health
Organization Technical Report Series 687, Geneva.
Yoffey, J. M., and F. C. Coutrice. 1956. Lymphatics, lymph, and
lymphoid tissue. 2nd Ed. Harvard University Press, Cambridge,
MA.
Zalkin, H., and A. L. Tappel. 1960. Studies of the metabolism of
vitamin E action. Arch. Biochem. Biophys. 88:113.


114
TABLE 7.4. MEANS OF THE RATIO OF TISSUE TO PLASMA LEVELS OF cx-TOCOPHEROL AT
TIME OF SLAUGHTER.
0
3
Time (day)
6 10
15
28
SEMa
Kidney
1.82
5.18
(5.78)
2.65
3.08
2.56
2.04
.20M
(.20M)b
Liver
1.97
36.84
(45.60)
21.80
17.02
16.57
8.26
.25M
(.21M)
Adrenal
3.72
7.95
(8.34)
7.84
16.15
9.89
17.00
. 24M
(.25M)
Pancreas
18.36
5.29
(5.37)
7.73
10.94
9.74
14.62
. 12M
(.13M)
Neck
2.74
1.37
(1.58)
1.42
2.09
2.35
2.32
. 21M
(.21M)
Leg
2.81
1.36
(1.63)
1.48
2.08
3.09
2.03
.21M
(.18M)
Heart
6.09
4.24
(4.73)
5.43
7.40
6.35
5.60
. 17M
(.17M)
Spleen
5.13
21.50
(27.01)
11.16
12.53
11.16
4.96
. 30M
(.24M)
Lung
4.54
29.29
(38.15)
42.54
47.52
15.82
6.07
.43M
(.31M)
3
The standard error of the mean is based on the analysis of variance on the
transformed scale and is approximate. It varies with the mean, M, and has been
expressed in terms of it.
^The values in parentheses are the results when one outlier was removed.


-TOCOPHEROL, pg/g FRESH TISSUE
87
40n
n
DL-a-TOCOPHEROL
D-a-TOCOPHEROL
30-
.
D
I
20-
5
-
5 S 5
s S 5
s 2
D
10-

D D

c D
0-
1 1 IT I 1 1 1 1 1 1 1
40-]
DL-a-TOCOPHERYL ACETATE 5
+-i
,
D-a-TOCOPHERYL ACETATE
30-
T
-
20-
x 5
5 S
-
5 5
5
5 *
10-
-


D 0
0-1
THY MUS KID LUN SPL HEA LIV ADR THY MUS KID LUN SPL HEA LIV ADR
TISSUE TYPE
FIGURE 5.1. a-TOCOPHEROL CONCENTRATIONS IN THE TISSUES OF CATTLE
SUPPLEMENTED WITH DIFFERENT VITAMIN E COMPOUNDS


This work is dedicated to my parents,
Dr. and Mrs. Hidiroglou, for their
continual support and encouragement
throughout the doctoral program.


no
TABLE 7.2. MEAN LEVELS OF ct-TOCOPHEROL IN TISSUES (pg/g FRESH) AND PLASMA
(yg/ml) AT TIME OF SLAUGHTER.
0
3
Time (da
6
y)
10
15
28
SEMa
Kidney
0.96
28.07
(33.09)b
8.25
6.80
5.46
3.96
. 21M
(.19M)
Liver
0.97
212.61
(270.01)
69.26
37.20
34.42
15.71
. 26M
(.17M)
Adrenal
1.70
42.25
(47.67)
24.59
35.51
20.21
33.86
.21M
(.20M)
Pancreas
11.36
28.50
(31.59)
23.19
24.40
20.74
28.60
. 11M
(.12M)
Neck
1.47
7.65
(9.24)
4.54
4.57
5.24
4.49
. 25M
(.23M)
Leg
1.56
7.55
(9.38)
4.59
4.52
6.63
3.96
.25M
(.19M)
Heart
2.90
23.55
(27.82)
16.84
16.40
14.73
11.03
. 18M
(.16M)
Spleen
2.73
125.20
(160.60)
35.12
27.93
25.13
9.74
. 35M
(27M)
Lung
2.09
164.85
(216.34)
128.75
102.66
33.22
12.19
.43M
(.25M)
Plasma
0.56
5.43
(5.97)
3.13
2.23
2.22
1.96
. 14M
(.14M)
Q
The standard error of the mean is based on the analysis of variance on the
transformed scale and is approximate. It varies with the mean, M, and has been
expressed in terms of it.
bThe values in parentheses are the results when one outlier was removed.


12
FIGURE 2.7. STRUCTURE OF a-TOCORED
FIGURE 2.8. STRUCTURE OF a-TOCOPURPLE
Absorption and Storage of Vitamin E
Mechanism of absorption is similar to that of other fat soluble
vitamins (Weber, 1983). Its absorption is closely associated with
that of fat, and is accelerated by the presence of bile.
Excess tocopherol is eliminated in the feces. Simon et al.
(1956) observed that rabbits receiving a single dose of 10 to 15 mg 5-
methyl-C^-d-a-tocopherol succinate eliminated 65% of the dose via the
feces in 3 days and 80% in 6 days; 90% of the radioactivity was


93
source of vitamin E supplementation. In general, the apparent
biopotency of various vitamin E sources for cattle are comparable to
results reported for humans by Horwitt et al. (1984).
Summary
Twenty-four crossbred beef cows were used to investigate the
concentration of (-tocopherol) in plasma and tissues following oral
administration of four tocopherol sources. Animals were assigned to
the following treatments: dl-a-tocopherol, d-a-tocopherol, dl--
tocopheryl acetate and d-a-tocopheryl acetate. Animals received a
daily oral dose of 1,000 IU of the respective tocopherol treatment for
28 d and then were slaughtered. Blood samples were collected on d 0,
1, 7, 14 and 28 for tocopherol concentration assays, and samples from
ten different tissues were collected from slaughtered cows.
Identification of -tocopherol in tissues was confirmed by HPLC
retention times and by comparison of mass spectra with that of ot-
tocopherol standards. The d-ot-tocopherol and its acetate ester
increased plasma tocopherol concentration faster than the racemic
products with the greatest response occurring with d-a-tocopherol.
Across all treatments, the highest -tocopherol concentrations were
noted in the adrenal gland and liver with the lowest found in muscle
and thyroid tissue. Tissue analyses confirmed that in adrenal gland,
kidney, liver and lung, -tocopherol concentrations were higher
following d- than dl-a-tocopherol supplementation.


34
approximately 20 to 80% for both the standard and the test
preparations. For the bioassay, two or three doses are selected from
within this range for the standard and for each sample to be assayed.
Each dose is dissolved in 0.2 ml olive oil and administered orally by
stomach tube. After 40 to 44 hr the percent erythrocyte hemolysis is
again measured. The use of dialuric acid as a hemolytic agent for
erythrocytes derived from vitamin E-deficient animals was introduced
by Rose and Gyorgy (1952), and later improved by Friedman et al.
(1958).
As an alternative to dialuric acid, hydrogen peroxide may be used
as the hemolytic agent (Gyorgy et al., 1952). It should be noted that
there is no spontaneous hemolysis iri vivo in vitamin E-deficient
animals. However, the rat hemolysis test is not a direct measure in
vivo of the biopotency of a vitamin E-active substance, since the test
is carried out on blood derived from rats given a vitamin E-deficient
diet. Furthermore, the results obtained need to be interpreted with
great caution since the period of giving the deficient diet needs only
be quite short before a positive test can be obtained, and the animal
would not necessarily show overt signs of vitamin E deficiency such as
sterility. The test is thus regarded by many as a measurement of the
vitamin E content of the blood rather than as an assessment of the
vitamin E status of the animal. With this difficulty in mind,
however, the test provides a means of assessing the probable vitamin E
activity of an unknown substance and has been found to agree well with
the results obtained by fetal resorption (Harris and Ludwig, 1949) and
chick liver storage (Pudelkiewicz et al., 1960).


CHAPTER 3
PLASMA AND TISSUE LEVELS OF VITAMIN E IN SHEEP FOLLOWING
INTRAMUSCULAR ADMINISTRATION IN AN OIL CARRIER
Introduction
Vitamin E is a biological antioxidant that has been used
pharmacologically in young sheep and cattle for the prevention or
treatment of conditions associated with nutritional muscular dystrophy
(Jenkins et al., 1972). Assessments of the vitamin E status in the
ruminant animal treated with various tocopherol preparations have
relied on serum or plasma tocopherol concentrations (Jenkins et al.,
1970). To our knowledge there is no information on the effects of
various forms of vitamin E preparations on sheep tissue tocopherol
levels. The purpose of the present investigation was to assess the
bioavailability in sheep of two forms of vitamin E (d-a- and dl-a-
tocopherols) administered intramuscularly using a serial blood plasma
analysis of a-tocopherol concentration. In addition, the a-tocopherol
levels were determined in a few selected tissues.
Experimental
Animals
Yearling crossbred wethers weighing 40-50 kg were used. All
animals originated from a flock that was born and raised in total
confinement (Heaney et al., 1980). The animals were fed ad libitum, a
diet that consisted of providing grass (40%), hay (40%), and corn
51


10
The melting point of RRR-a-tocopherol is 2.5 to 3.5C; thus at
room temperature, the compound is a viscous yellow oil which is
soluble in aprotic solvents. Optical rotations of these tocopherols
are very small and depend on the nature of the solvent. Table 2.2
gives the specific rotations of the spectra of natural tocopherols and
tocotrienols in ethanol which show maxima in the range of 292-8 nm;
infrared spectra show OH (2.8 to 3.0 pm) and CH (3.4 to 3.5 pm)
stretching and a characteristic band at 8.6 pm.
TABLE 2.2. SPECIFIC OPTICAL ROTATIONS OF NATURAL TOCOPHEROLS
Compound
Solvent
25
a
546.1
a-Tocopherol
Ethanol
+0.32
Benzene
-3.0
6-Tocopherol
Ethanol
+2.9
Benzene
+0.9
Y-Tocopherol
Ethanol
+3.2
Benzene
-2.4
6-Tocopherol
Ethanol
+3.4
Benzene
+1.1
Other properties include a molecular weight of 430.69; boiling
point at 0.1 atm (used in molecular distillation) of 200 to 220C;
density 0.95 at 25C in reference to water at 4C, and a refractive
index in sodium light spectrum at 20C of 1.5045.
8-Tocopherol is a viscous, pale yellow oil; absorption maxima of
297 nm; E.1% = 86.4.
1 cm


ABUNDANCE
73
FIGURE 4.3. MASS SPECTRAL SCAN OF THE a-TOCOPHEROL STANDARD (A) AND
a-TOCOPHEROL IN THE PANCREATIC TISSUE (B) FOLLOWING
HPLC COLLECTION


45
I.
II.
III.
R CH = CH CH2 R
Electron,
Proton
R CH = CH CH R ^
R CH = CH CH R
0-0-
R CH = CH CH2 R
R CH = CH CH R -
IV. R CH = CH CH R
I
0 OH
FIGURE 2.9. SCHEMATIC REPRESENTATION OF THE PEROXIDATION
OF UNSATURATED FATTY ACIDS


2
five-hundredth that of vitamin E. Biological potencies of various
sources of vitamin E have been established primarily through the rat
fetal resorption assay (Mason and Harris, 1947). From this assay the
following biological potencies were established: 1 mg of dl-a-
tocopheryl acetate =1.0 IU; 1 mg dl-a-tocopherol = 1.1 IU; 1 mg d-a-
tocopheryl acetate = 1.36 IU; 1 mg d-a-tocopherol = 1.49 IU.
Recently, considerable effort has been made to determine the
biological activity of various vitamin E compounds in species other
than rats, and specifically humans (Horwitt et al., 1984; Baker et
al., 1986).
In ruminants, although much is known about clinical signs of
vitamin E deficiency (Rice and McMurray, 1982), very little attention
has been given to the different potencies of various vitamin E
compounds. This dissertation is particularly concerned with the
biological activity of various vitamin E compounds in ruminants as
well as the effect of mode of administration and its type of vehicle.


67
Results
Tissues
Diet and tissues were highly significant (P<0.0001) in their
vitamin E concentrations, but diet x tissue interaction was not
significant (P > 0.05) (Table 4.1). Higher a-tocopherol
concentrations (P<0.01) were observed in the tissues of sheep fed the
d-a-tocopherol than other vitamin E compounds (Table 4.2). Analysis
of variance for each individual tissue of the four diets showed
differences (P<0.01) only for heart, kidney and lung (Figure 4.1).
Higher concentrations (pg/g fresh tissue) of a-tocopherol were
observed in the sheep's heart fed d-a-tocopherol (14.74 1.64 SE)
than in the d-a-tocopheryl acetate (9.65 1.64 SE), dl-a-tocopheryl
acetate (8.47 2.84 SE) or dl-a-tocopherol (6.20 2.50 SE)
supplemented sheep. Also, in d-a-tocopherol fed sheep, higher
concentrations were observed in lung (12.13 2.58 SE) than in d-a-
tocopheryl acetate (8.99 1.51 SE) or dl-a-tocopheryl acetate (6.14
1.16 SE) or dl-a-tocopherol (6.40 1.91 SE) supplemented sheep. In
kidney, higher a-tocopherol concentrations were found in the d-a-
tocopheryl acetate (7.20 0.27) than d-a-tocopherol (5.81 0.67 SE)
or dl-a-tocopheryl acetate (4.78 1.84) or dl-a-tocopherol (4.11
0.63). However, analysis of variance did not show a difference (P >
0.05) among corresponding tissues among treatments. In all diets,
pancreas contained the highest concentrations, followed by liver and
spleen. Immediately after these tissues, heart, lung and kidney were


46
acid radical which can react further. Once this chain reaction has
started, it continues until all unsaturated compounds have been broken
up, self-quenching occurs, or the radical is entrapped by an
antioxidant (Chow, 1979).
Autoxidation eventually results in some form of degradation of
the material affected. For example, foodstuffs such as corn oil,
butter and margarine become rancid. The process is generally
represented by four elementary reactions:
Initiation: production of R* (1)
Propagation: R. + 0^ R00' (2)
ROO. + RH R00H + R' (3)
Termination: ROO. + ROO. Molecular products (4)
In this scheme, RH represents a lipid molecule and R. the carbon-
centered radical derived from it by removal of a hydrogen atom.
(Propagation can also occur by addition of the peroxyl radical to a
carbon-carbon double bond.) These reactions can be prevented by
antioxidants (Lundberg, 1962).
R00. + XH2 R00H + XH.
XH. + XH. X + XXH2
XH = antioxidant
At the cellular level, autoxidation is clearly undesirable because it
threatens the integrity, protective and organizational properties of
the membranes which enclose cells and organelles, thus placing the
survival of the cell itself in jeopardy.


83
and peak areas with tocopherol standards. Selenium and a-tocopherol
in the feed were determined, respectively, by the method of Hoffman et
al. (1968) and McMurray and Blanchflower (1979a).
Instrumentation
The chromatographic apparatus consisted of a M 6,000 pump and WK
septumless injector. A Perkin-Elmer 650-150 fluorescence
spectrophotometer equipped with a microflow cell unit was used for
quantification. Wavelength settings were 295 and 330 nm for
excitation and emission, respectively. The column was a P Bondapak
C18 (3.9 mmx 30 cm) of 10 pm particle size. The mobile phase was a
solvent system (HPLC grade) consisting of methanol and water (97:3)
with a flow rate of 3 ml/min.
Mass Spectrometry in Combination with Gas Chromatography
Further confirmation of -tocopherol in various tissues was
carried out by a combination Hewlett-Packard (HP) gas chromatograph
5790-mass spectrometer 5970A series with a data station (HP-9000).
Eluate from the HPLC was purified for a-tocopherol by TLC on silica
gel using cyclohexane-diethylether (4:1 v/v) as the solvent.
Authentic standards of d-a-tocopherol as well as tissue samples were
silylated after HPLC as described by Ingold et al. (1987) before
injection onto the gas chromatograph/mass spectrometer. The
tocopherol trimethylsilyl ethers were injected (3 pi) into an ultra-1
(0V 101 methyl silicone) capillary column (12-m x 2-mm i.d.),
maintained at 280C with an injector temperature of 350C and a helium


119
Summary
Blood plasma and tissue vitamin E were determined in twenty-five
crossbred sheep following intraperitoneal injection of dl-a-
tocopherol. Five sheep were used as controls (no treatment and killed
at d 0). The remaining 20 sheep were administered intraperitoneally
with 5 g of dl-a-tocopherol. From these twenty vitamin E dosed sheep,
four were slaughtered at each of d 3, 6, 10, 15 and 28 after dosing.
There was a significant time effect for all on a-tocopherol
concentrations for all tissues. In most tissues, the peak for ot-
tocopherol concentrations was observed at 3 d post dosing. There was
a varied rate of uptake for different tissues examined. Three d post
IP dosing, a large uptake of vitamin E by the liver was noticed, and
this supports the concept of the hepatic tissues as a target organ for
vitamin E action. Also at d 3, a pronounced uptake was observed in
the spleen and lung. Vitamin E concentrations in the remaining
tissues at d 3 post dosing showed also a considerable increase, but to
a lesser degree than those in liver, spleen and lung. A decline of
vitamin E concentration in all tissues occurred after d 3. This
study, through the use of contrasts over time (d), provided some
indication of when a plateau in vitamin E concentrations had been
reached for each individual tissue following an IP dosing.


136
Shiraturi, T. 1974. Uptake, storage, and excretion of chylomicra-
bound ^H-a-tocopherol by the skin of the rat. Life Sci. 14:929.
Simon, E. J., A. Eisengart, L. Sundheim, and T. Milharat. 1956a. The
metabolism of vitamin E. II. Purification and characterization
of urinary metabolites of alpha-tocopherol. J. Biol. Chem.
221:807.
Simon, E. J., E. S. Gross, and A. T. Milharat. 1956b. The metabolism
of vitamin E. 1. The absorption and excretion of d-a-tocopheryl
5 methyl C1 succinate. J. Biol. Chem. 221:797.
Simon-Schmoss, R. S., I. A. Reimann, and V. Boehlau. 1984. Vitamin
E-therapie: Zur frage der resorption von vitamin E. Notabene
Midici 14:793.
Slater, E. C. 1958. The possible role of vitamin E in respiratory-
enzyme systems. Proc. of the Fourth Int. Congr. of Biochemistry,
Vienna. Vol. XI: Symposium XI. I.U.B. Symp. Series 13:316.
Sternberg, I., and E. Pascoe-Dawson. 1959. Metabolic studies in
arteriosclerosis: I. Metabolic pathway of C1 labelled alpha-
tocopherol. Can. Med. Assoc. J. 80:266.
Storer, G. B. 1974. Fluorometric determination of tocopherol in
sheep plasma. Bioch. Med. 11:71.
Sure, B. 1924. Dietary requirements for reproduction. II. The
existence of a specific vitamin for reproduction. J. Biol. Chem.
58:693.
Tappel, A. L. 1962. Vitamin E as the biological lipid antioxidant.
Vit. Horm. 20:493.
Tappel, A. L. 1972. Vitamin E and free radical peroxidation of
lipids. 2ll Vitamin E and its Role in Cellular Metabolism (P.
Nair and H. J. Kayden, eds.). Ann. NY Acad. Sci. 203:12-28.
Thompson, N.J., and M. Hidiroglou. 1983. Effect of large oral and
intravenous doses of vitamins D~ and Do on vitamin D in milk. J.
Dairy Sci. 66:1638.
Tietjen, G. L., and R. H. Moore. 1972. Some Grubbs-type statistics
for the detection of several outliers. Technometrics 14(3):583.
Tikriti, H. H. 1969. The metabolism of vitamin E in the lactating
dairy cow in relation to oxidized flavor in milk. Ph.D.
dissertation. University of Maryland, College Park, MD.


CHAPTER 7
PLASMA AND TISSUE VITAMIN E CONCENTRATIONS IN SHEEP AFTER
ADMINISTRATION OF A SINGLE INTRAPERITONEAL DOSE OF dl-a-TOCOPHEROL
Introduction
There have been remarkably few systematic investigations on the
disposition of pharmacologic doses of vitamin E in sheep. Massive
doses in sheep can be given orally, intramuscularly (IM) or
intravenously (IV) (Hidiroglou and Karpinski, 1987). Bioavailability
of vitamin E after intramuscular (IM) administration has recently been
reported (Hidiroglou and McDowell, 1987).
A potential alternative to IV or IM dosing could be the delivery
of vitamin E into the peritoneal space. Since no studies have been
published on the disposition kinetics of vitamin E in sheep plasma and
tissues following administration of a single intraperitoneal (IP) dose
of vitamin E, such a study using dl-a-tocopherol was undertaken.
Experimental Procedure
Animals
Twenty-five clinically normal, one-year-old, crossbred wethers
were used in the study. The sheep weighed from 30 to 35 kg at the
beginning of the experiment. A standard diet (Table 7.1) and water
were available ad libitum. Five sheep were selected at random and
slaughtered at d 0 as the control with no treatment. Thereafter, 4
106


66
purified for a-tocopherol by thin layer chromatography on silica gel
using cyclohexane-diethylether (4:1 v/v) as the solvent.
Authentic standards of d-a-tocopherol, as well as tissue samples,
were silylated before injection onto the GC/MS (Ingold et al.f 1987).
The tocopherol trimethylsilyl ethers were injected (3 yl) into a
12 m x 2 mm i.d. ultra-1 (OV 101 methyl silicone) capillary column
maintained at 280C with an injector temperature of 350C and a helium
carrier gas flow rate of 0.5 cc/min, which was connected to a series
mass-selective detector. Mass spectra were obtained at an electron
beam energy of 70 eV and an accelerating voltage of 1800 volts.
Molecular ions ranging from 50 to 600 mass/charge were monitored
continuously.
Statistical Methods
Indices of bioavailability of various preparations of vitamin E
were performed by analysis of variance. These indices were: 1) the
maximum a-tocopherol plasma concentration (peak of plasma a-tocopherol
concentration curve), 2) the time of maximum (t max) a-tocopherol
concentration, and 3) the area (AUC) under the a-tocopherol plasma
concentration time curve. Analysis of variance was also used to
estimate the difference in the vitamin E concentration between
corresponding tissue in the four groups, as well as among tissues.


126
Burton, G. W. 1989. Antioxidant action of carotenoids. J. Nutr.
119:109.
Burton, G. W., A. Webb, and K. U. Ingold. 1985. A mild, rapid, and
efficient method of lipid extraction for use in determining
vitamin E/lipid ratios. Lipids 20:29.
Caravaggi, C. 1969. Vitamin E concentrations in the serum of various
experimental animals. Comp. Biochem. Physiol. 30:585.
Caravaggi, C., M. W. Gibbons, and F. Wright. 1968. The appearance of
tocopherol in the blood of sheep after intramuscular injection of
a-tocopheryl acetate. N.Z.J. Agrie. Res. 11:313.
Catignani, G. L., and J. G. Bieri. 1977. Rat liver a-tocopherol
binding protein. Biochim. Biophys. Acta. 497:349.
Cheeseman, K. H., G. W. Burton, K. U. Ingold, and T. F. Slater. 1984.
Lipid peroxidation and lipid antioxidants in normal and tumor
cells. Toxicol. Pathol. 12:235.
Chernick, S. S., J. G. Moe, G. P. Rodnan, and K. Schwarz. 1955. A
metabolic lesion in dietary necrotic liver degeneration. J.
Biol. Chem. 217:829.
Chow, C. K. 1979. Nutritional influence on cellular antioxidant
defense systems. Amer. J. Clin. Nutr. 32:1066.
Chow, C. K., H. H. Jjaper, A. S. Csallany, and H. Ch^ji. 1967. The
metabolism of c alpha-tocopheryl quinone and C alpha-
tocopheryl-hydroquinone. Lipid 2:390.
Corwin, L. M., and K. Schwarz. 1960. Maintenance of a ketoglutarate
and succinate oxidation in E-deficient liver homogenates by
alpha-tocopherol, a tocopherol metabolite, menadione and
diphenylphenylenediamine. Nature (London) 186:1048.
Csallany, A. S., and H. H. Draper. 1963. The structure of a dimeric
metabolite of D-alpha tocopherol isolated from mammalian liver.
J. Biol. Chem. 938:912.
Csallany, A. S., and H. H. Draper. 1964. Cis-trans-isomers of dl-
alpha-tocopherone. J. Biol. Chem. 239:574.
Curto, G. M. 1966. Fat soluble vitamins in veterinary science. Riv.
Zootec. 39:200.
Dam, H. 1944. Studies on vitamin E deficiency in chicks. J. Nutr.
27:193.
Dam, H. 1957. Influence of antioxidants and redox substances on
signs of vitamin E deficiency. Pharmacol. Rev. 9:1.


54
Results
Tocopherol Content of Roughages
Average tocopherol content of roughage at the time of consumption
were 39, 69 and 34 (mg/kg DM) for maize-silage, grass-silage and hay,
respectively. The only forms of vitamin E found in hay and grass-
silage were a-tocopherol. In maize-silage a-tocopherol was, on the
average about half the total tocopherol and was equal to the sum of a
+ 3 tocopherols (26%) and 6 tocopherol (24%).
Blood Plasma a-Tocopherol
After IM administration of d-a-tocopherol preparation, a lag
period was noted in the sheep until vitamin E increased in the
systemic circulation (Fig. 3.1; Group 2). There was no clear evidence
of an elimination phase in the data. In all 3 groups there was a
suggestion of a plateau near the peak. It would appear as though the
system reached an equilibrium at or near peak levels.
In Group 2 (IM d-a-tocopherol injected sheep killed 360 hr after
dosing) the most important index of bioavailability (Koch-Weser,
1979), the area under the plasma concentration-time curve (AUC) was
the largest (P<0.01) compared to the other 2 groups (Table 3.1). This
appeared to be due to a more sustained release into the plasma (t max
much larger), resulting in a higher delivery of the vitamin. Maximum
concentration (C max) was much higher (P<0.01) in group 2 than in the
others.


53
fluorescense spectrophotometer set at 295 and 330 nm for excitation
and emission, respectively. The column was eluted with methanol:water
(97:3) at 3 min/ml. All solvents were HPLC or pesticide grade. Blood
plasma (2 ml) samples were precipitated with ethanol (2 ml), and
extracted with hexane (4 ml). Tissue samples were saponified and
extracted with hexane (Thompson and Hidiroglou, 1983).
Identification and quantitation of the a-tocopherol was
accomplished by comparison of retention time and peak areas with d-a-
tocopherol standard. Tocopherol determination in roughage was
performed according to the techniques of McMurray and Blanchflower
(1979b).
Statistical Analysis
The indexes of bioavailability of the various preparations of
vitamin E were determined by analysis of variance. These indexes
were: 1) the maximum a-tocopherol plasma (C max) concentration (peak
of plasma a-tocopherol concentration curve), 2) the time of maximum (t
max) a-tocopherol concentration, and 3) the area (AUC) under the a-
tocopherol plasma concentration time curve (Balard, 1968; Koch-Weser,
1974). Differences in the vitamin E concentration between
corresponding tissues in the four groups were determined by analysis
of variance.


CHAPTER 5
BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS
INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS
Introduction
Biopotencies of various tocopherol compounds are generally
provided by the following relationship according to the National
Formulary: 1 mg dl-a-tocopheryl acetate = 1.0 IU; 1 mg dl-a-
tocopherol = 1.1 IU; 1 mg d-a-tocopheryl acetate = 1.36 IU; 1 mg d-a-
tocopherol = 1.49 IU. These values have been based largely on small
animal bioassay (rat anti-sterility assay). Horwitt (1980) and
Horwitt et al. (1984) reported that in humans, on a mg basis, d-a-
tocopheryl acetate was 2.16 times more potent than dl-a-tocopheryl
acetate following a single oral dose. However, Baker et al. (1986)
confirmed in humans following a continuous oral dosing that 1 mg of
dl-a-tocopheryl acetate (all-racemic-a-tocopheryl acetate) has a
biopotency of 1.0 IU whereas d-a-tocopheryl acetate (2R, 4'R, 8'R-a-
tocopheryl acetate) has a biopotency of 1.36 IU (28 d). Horwitt
(1980), Horwitt et al. (1984) and Baker et al. (1986) used the
elevation of plasma a-tocopherol as a measure of bioavailability.
Although much is known about clinical signs of deficiency in
calves and lambs (Rice and McMurray, 1982) very little attention is
given to the different potencies of various vitamin E forms in cattle.
The present authors (Hidiroglou and McDowell, 1987; Hidiroglou et al.,
1988) have undertaken a series of studies on the utilization of
79


89
lower in the kidney (PC.05) and lung (PC.01) of the cattle
supplemented with dl-a-tocopherol than for cattle fed the other
treatments. Higher concentrations (PC.05) of a-tocopherol were
observed in the thyroid of cattle fed dl-a-tocopherol than in that of
the cows receiving d-a-tocopheryl acetate, but the a-tocopherol
concentrations were higher (PC.05) in the spleen of the group fed d-a-
tocopheryl acetate than in that of the dl-a-tocopherol group. No
difference (P>.05) was found for heart and muscle as a result of
feeding the various forms of vitamin E.
Across all the treatments, the adrenal gland contained the
highest a-tocopherol concentration, followed generally by the liver or
heart; the lowest concentrations were observed in the thyroid and
muscle tissues (PC.05) (Table 5.3 and Figure 5.1).
Identity of a-tocopherol in tissues was confirmed by comparing
the mass spectra of the a-tocopherol standard to those obtained by
collecting the eluates from the HPLC and introducing it into the gas
chromatography/mass spectrometry system following silylation. A good
spectral match was obtained for the sampled compared with the a-
tocopherol standard spectra. Mass spectra of the gas chromatographic
peak corresponding to the a-tocopherol standard (TMSi; Fig. 5.2) and
that corresponding to the peak with the same retention time obtained
for the plasma, for example, are identical, confirming the presence of
-tocopherol in those biological extracts. The peak obtained at
mass/charge 502 represents the molecular ion of a-tocopherol (TSMi:
430 for a-tocopherol + 72 for trimethylsilyl ether). The
fragmentation pattern reveals peaks at mass/charge 277 resulting from


128
Draper, H. H., A. S. Csallany, and M. Chiou. 1967. Isolation of a
trimer of alpha-tocopherol from mammalian liver. Lipids 2:43.
Dunkley, W. L., M. Ronning, A. A. Fianke, and J. Robb. 1967.
Supplementing rations with tocopherol and ethoxyquin to increase
oxidative stability of milk. J. Dairy. Sci. 50:492.
Evans, H. M., and K. S. Bishop. 1922. On the existence of a hitherto
unrecognized dietary factor essential for reproduction. Science
56:650.
Evans, H. M., G. 0. Burr, and T. L. Althausen. 1927. Antisterility
vitamin, fat-soluble E. Mem. Univ. Calif. 8:1.
Evans, H. M., 0. H. Emerson, and G. A. Emerson. 1936. The isolation
from wheat germ oil of an alcohol, alpha-tocopherol, having the
properties of vitamin E. Science 64:350.
Fernholz, E. 1938. On the constitution of alpha-tocopherol. J.
Amer. Chem. Soc. 59:1054.
Fieser, L. F., M. Tishler, and N. L. Wendler. 1940. The method used
for vitamin K synthesis also applicable for the synthesis of a-
tocopherol. J. Am. Chem. Soc. 62:996.
Fitch, C. D., and J. F. Diehl. 1965. Metabolism of 1-alpha
tocopherol by the vitamin E deficient rabbit. Soc. Exp. Biol.
Med. Proc. 119:553.
Fridovich, I. 1976. Oxygen radicals, hydrogen peroxide, and oxygen
toxicity. Free Radicals in Biology (W. A. Pryor, ed.), pp.
239-277. Academic Press, New York.
Friedman, L., W. Weiss, F. Wherry, and 0. L. Kline. 1958. Bioassay
of vitamin E by the dialuric acid hemolysis method. J. Nutr.
65:143.
Fromming, K. H. 1984. Moglichkeiten der synthese, probleme der
standardiezung und die galenische veranbeitung von vitamin E.
Pharm. Ztg. 129:1558.
Gallo-Torres, H. E. 1970. Obligatory role for bile for the
intestinal absorption of vitamine E. Lipids 5:379.
Gallo-Torres, H. E. 1982. Transport and metabolism. In: L. J.
Machlin (Ed.) Vitamin E. pp 193-267. M. Dekker, New York.
Gallo-Torres, H. E., and N. Miller. 1971. Tissue uptake and
metabolism o| d,]^-^- l^-a-tocopheryl nicotinate and d,L-a-
tocopheryl-1 2 H^-acetate following intravenous
administration. Intf J. Vit. Nutr. Res. 41:339.


41
The United States Pharmacopoeia (USP) (1980) in Table 2.6 shows
the recommended weight/unit relationships for six tocopherols:
TABLE 2.6. USP WEIGHT/UNIT RELATIONSHIPS OF TOCOPHEROL.
1 mg all-rac-o-tocopherol = 1.1 units
1 mg all-rac-a-tocopheryl acetate = 1 unit
1 mg all-rac-a-tocopheryl succinate = 0.89 unit
1 mg RRR-a-tocopherol = 1.49 units
1 mg RRR-a-tocopheryl acetate = 1.36 units
1 mg RRR-a-tocopheryl succinate = 1.21 units
Role of Vitamin E in the Respiratory Chain
It has long been known that the hepatic necrosis that accompanies
vitamin E deficiency is preceded by a reduction of cell respiration
shown on cross sections of prenecrotic liver (Chernick et al., 1955).
Vitamin E supplementation immediately reestablishes respiration, both
in vivo and in vitro (Schwarz, 1955). Selenium is also effective, but
only in^ vivo, which seems to indicate that a different mechanism is at
work (Schwarz, 1972).
Cell respiration takes place at the level of the mitochondria,
through the respiratory chain, which consists of a series of hydrogen-
transport systems which intervene in an order determined by their
oxidoreduction potentials. Energy released by this hydrogen transport


129
Gallo-Torres, H. E., F. Weber, and 0. Wiss. 1971. The effect of
different dietary lipids on the lymphatic appearance of vitamin
E. Int. J. Vit. Nutr. Res. 41:504.
Gibaldi, M., and 0. Perrier. 1975. Pharmacokinetics. 1st ed.
Marcel Dekker Inc., New York, NY.
Gibaldi, M., and 0. Perrier. 1982. Pharmacokinetics. 2nd ed.
Marcel Dekker Inc., New York, NY.
Gray, D. E. 1959. Metabolic effects of a-tocopherol acetate. II.
Influence of a-tocopherol acetate on cholesterol and phospholipid
synthesis in rat liver homogenates. Vitam. Horm. 5:19.
Green, J., A. J. Diplock, I. Bunyan, E. E. Edwin, and D. McHale.
1967. Ubiquinone and the function of vitamin E. Nature 19:318.
Griffiths, T. W. 1960. Studies in the requirements of the young
chick for vitamin E: The effects of different sources and levels
of dietary starch on gain in weight and body vitamin E storage.
Brit. J. Nutr. 14:269.
Griffiths, W. T., D. R. Threlfall, and T. W. Goodwin. 1968.
Observations on the nature and biosynthesis of terpenoid quiones
and related compounds in tobacco shoots. Biochem. J. 5:124.
Guarnieri, C., F. Flanigni, and C. M. Caldarera. 1980. A possible
role of rabbit heart cytosol tocopherol binding in the transfer
of tocopherol into nuclei. Biodhem. J. 190:469.
Gullickson, T. W. 1949. The relation of vitamin E to reproduction in
dairy cattle. Ann. N. Y. Acad. Sci. 52:256.
Gyorgy, P., G. Cogan, and C. S. Rose. 1952. Availability of vitamin
E in the newborn infant. Proc. Soc. Exptl. Biol. Med. 81:536.
Hakkarainen, J., B. Pehrson, and J. Tyopponen. 1987. Blood vitamin
E, selenium and glutathione peroxidase concentrations in heifers
fed either on grass or on winter feed. J. Vet. Med. 34:508.
Harris, P. L., and M. L. Ludwig. 1949. Vitamin E potency of alpha
tocopherol and alpha tocopherol esters. J. Biol. Chem. 180:611.
Hashim, S. A., and G. R. Schuttinger. 1966. Rapid determinations of
tocopherol in macro and microquantities of plasma. Am. J. Nutr.
18:137.
Heaney, D. P., L. Ainsworth, T. R. Batra, P. S. Fiser, A. J. Hackett,
G. A. Langford, and A. J. Lee. 1980. A. R. I. Tech. Bull. No.
2, Agrie. Can. Anim. Res. Centre. Ottawa, Ontario.


81
TABLE 5.1. COMPOSITION OF THE GRAIN PORTION OF
THE DIET FED TO COWS FOR 28 da
Ingredient
% as-fed basis
Barley
90
Soybean meal
7.5
Calcium carbonate
1.0
Dicalcium phosphate
1.0
Vitamin premix^
.25
Trace mineral saltc
.25
g
Analyzed for vitamin E and selenium
(8 ppm and .235 ppm, respectively).
^Provided, per kilogram of diet:
5,000 IU vitamin A and 500 IU vitamin
D.
c
Provided, per kilogram of diet:
2.1 g NaCl, .22 mg I, .088 mg CO,
9 mg Fe, .725 mg Cu, 2.625 mg Mn
and 8.75 mg Zn.


28
affected by trembling, and die of either pulmonary edema or heart
failure.
In pasture calves, the syndrome appears when they are released in
the spring. They begin by running and jumping, but suddenly one or
two may slow down and stand still, with a stiff, awkward stance. Then
they lie down and are unable to get up. In this case also, heart and
respiratory disorders lead to swift death (King and Maplesdon, 1960).
Vitamin E-selenium lesions are identical with those of lambs,
both macro- and microscopically. From the foregoing it will appear
that vitamin E deficiency appears to lead to two major types of
syndromes: (1) degenerative muscular lesions, common to a number of
species and generally related to selenium deficiency, and (2) vascular
lesions (edema and hemorrhages), well known in poultry, affecting
mainly nervous and subcutaneous connective tissues.
Biological Activity of Vitamin E
Biological activities of the various tocopherols are different.
Extensive and very complex studies on several experimental animal
species have been conducted to determine the biological activity of
vitamin E components which have been described in the literature for
many years.
Methods of Assay
Determination of biologically active vitamin E is complicated by
many interrelated factors. Chemical analyses must contend with the
fact that eight different tocopherols occur in nature, and synthetic


118
fat) contain a labile pool of a-tocopherol, which mobilizes rapidly,
and a fixed component which is retained for long periods.
The high concentrations of vitamin E in the liver, spleen and
lung, 3 d after initiation of the IP administration to sheep are in
agreement with the results of Gallo-Torres (1971) in rats where these
organs contained high levels of vitamin E following parenteral
administration of this vitamin. In the lung very high concentrations
of vitamin E could be attributed to this organ's abundant blood
supply. The high tissue/plasma (t/p) ratio observed in the adrenal
and pancreas of sheep provided with IP vitamin E characterizes the
intensity of metabolism in these organs. Tikriti et al. (1968)
observed in dairy cows that, following parenteral administration of
radiotocopherol, pancreas and adrenal were relatively high in vitamin
E activity. The heart t/p indicated an intermediate vitamin E
activity while in the muscle the t/p was low. This might indicate
that the muscle has a different mechanism of action concerning
handling of vitamin E than other organs.
Gallo-Torres (1982) noted that information on the fate of vitamin
E after parenteral administration is scarce. The present work
provided data on the fate of a massive single IP dose of vitamin E in
the body of sheep, indicating a differential handling by the various
organs. Judging from the results of vitamin E distribution, it
appears that the IP method is a convenient route of vitamin E
administration to sheep.


CHAPTER 8
GENERAL CONCLUSIONS
Five experiments were carried out to investigate the
bioavailability of various tocopherol sources in ruminants as well as
the effect of mode of administration. Experiment I was conducted to
investigate the bioavailability of d-a-tocopherol and dl-a-tocopherol
suspended in sesame oil solution (200 mg/ml) and administered
intramuscularly at a rate of 40 mg/kg body weight. In all sheep given
the various vitamin E sources by intramuscular oil depot injection, a
delayed increase in plasma a-tocopherol levels occurred. Sheep
administered d-a-tocopherol and slaughtered after 360 hr had a larger
(P<.01) area under the plasma concentration time curve (AUC) than
sheep injected with d-a-tocopherol or dl-a-tocopherol and killed 240
hr post dosing. A higher plasma C maxima (P<.01) was found in the d-
a-tocopherol group than in the others. Pancreatic, hepatic, lung,
spleen and muscle a-tocopherol concentrations were higher (P<.05) for
this group than for controls. Tendencies for higher (P>.05) tissue a-
tocopherol concentrations were found for the d-a-tocopherol
administered group (360 hr) than in the other vitamin E treated sheep.
Experiment II assessed the bioavailability of four tocopherol
sources provided orally of either (1) dl-a-tocopherol, (2) dl-a-
tocopheryl acetate, (3) d-a-tocopherol or (4) d-a-tocopheryl acetate
in crossbred yearling wethers at a level of 400 IU/day/sheep for 28 d.
Higher a-tocopherol concentrations (P<.01) were found in the heart and
120


42
system (which corresponds to oxidation of the substrate) is partially
stored in the form of adenosine triphosphate (ATP). Determination of
the P/0 ratio (number of atoms of inorganic P which disappear when one
atom of oxygen is reduced), which is generally equal to 3, provides a
measure of cellular respiratory activity.
One of the links in the respiratory chain is ubiquinone (or
coenzyme Q) which intervenes between the flavin-nucleotide and
cytochrome system. The structure of ubiquinone is similar to that of
a-tocoquinone, a metabolite of a-tocopherol which, along with the
corresponding hydroquinone can form a hydrogen transport system
comparable to that of ubiquinone.
In the anemias observed when vitamin E deficiencies occur in
primates, an improvement is obtained upon administration of ubiquinone
as with vitamin E, thereby indicating the similarity of action for the
two substances.
It is likely that vitamin E intervenes in the respiratory chain,
and thereby contributes to the metabolic equilibrium of cells
(Schwarz, 1972).
Intervention of Vitamin E in Hematopoiesis
Anemia, which accompanies vitamin E deficiency in primates, and
the decoloration of the muscles in muscular dystrophy in many species
(resulting from rarefaction of the myoglobin) have led to research
into the possible role of this vitamin in heme synthesis.
Heme synthesis results from a series of transformations starting
with glycine and succinyl-CoA, leading to the formation of 6 -