Title: Serum lipid patterns of growing pigs during fat absorption
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Title: Serum lipid patterns of growing pigs during fat absorption
Physical Description: 86 leaves : ill. ; 28 cm.
Language: English
Creator: Crum, Roger Clark, 1938-
Copyright Date: 1965
 Subjects
Subject: Swine   ( lcsh )
Livestock   ( lcsh )
Veterinary physiology   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Roger Clark Crum.
Thesis: Thesis (Ph. D.)--University of Florida, 1965.
Bibliography: Bibliography: leaves 81-84.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
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Bibliographic ID: UF00097896
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000406884
oclc - 24683466
notis - ACF3163

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SERUM LIPID PATTERNS OF GROWING PIGS

DURING FAT ABSORPTION






















ROGER CLARK CRUM, JR.










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











UNIVERSITY OF FLORIDA
December, 1965













ACKNOWLEDGEMENTS


The author extends his sincere appreciation to Dr. H. D. Wallace,

chairman of his committee, who has ablely guided and 89sisted in the

preparation of this dissertation. The assistance and cooperation of

the other committee members, Drs. G. E. Combs, J. P. Feaster, J. E.

Moore and J. A. 01son is also deeply appreciated.

Grateful acknowledgement is extended to Drs. F. C. Neal and

J. M. Kling of the Veterinary Science Department for their aid in the

surgical procedures developed for this study.

The aid of Mrs. Evelyn Robbins and Mr. Charles Burgin in conducting

the laboratory phase of the work is recognized with a special note of

thanks. Appreciation is also expressed to Mr. B. R. Cannon and Mr.

L. S. Taylor for their help with the experimental animals.

The author is particularly grateful to his wife, Brenda, for

encouragement and assistance throughout the course of this program.

















TABLE OF CONTENTS


Page
ACKNOWLEDGKENTS ..... .. .. .. .. . .. .. .. ii


LIST OF TABLES . ... ... .. .. .. .. .. .. .. iv


LIST OF FIGURES .. .. ... .. .. ... .. . ... vi


INTRODUCTION. .. .. .. .. .. .. .. .. .. . .. .. 1


LITERATURE REVIEW . . . . . . . 3


CANNULATION OF INTESTINAL LYMPHATIC DUCTS IN SWINE. .. .. 21


EXiPERIMENTAL PROCEDURE. .. ... .. ... .. .. .. 25


RESULTS ANLD DISCUSSION. .. .. .. .. .. .. .. .. .. 4c2


SUMMARY .. .. .. ... ... .. ... .. .. .. 67


APPENDIX. .. .. .. .. ... .. .. .. . . .... 70


LITERATURE~ CITED. .. .. .. .. .. .. .. .. .. . .. 81


BIOGRAPHICAL SKETCH .. .. .. .. . .... .. .. .. 85














LIST OF TABLES


Table Page

1. Mean values of serum lipids in swine reported in the
literature expressed as mg. per cent . . . 7

2. Digestibility coefficients of total dietary fat and the
component fatty acid or triglyceride fed as semi-
purified material (Bayley and Lewis, 1965b). . ... 12

3. Effect of lipid classes on palmitic acid absorption. .. 18

4. Composition of diet self-fed to pigs before experiments. 27

5. Gas chromatographic analysis of lipid materials used in
experiments. ... .. .. .. ... . .. .. 30

6. Standardization of triglyceride determination method:
Recovery results .. .. .. .. . .. 34

7. Standardization of free fatty acid and phospholipid
determination method: Recovery results. .. . 40

8. Repeatability of the analysis method for free fatty acid
and phospholipids. .. .. .. . ... .. .. 41

9. Serum lipid component levels of pigs used in experiments
during control periods . ... .. . .. .. 43

10. Diurnal variation in serum lipid components. . .. 46

11. Effect of blank, palmitic acid and stearic acid on serum
lipid components during absorption . . .. .... 55

12. Effect: of 01eic acid and linoleic acid on serum lipid
components during absorption .. .. . .. .. 56

13. Effect of combined mix 1, combined mix 2 and combined mix 3
on serum lipid components during absorption. . 62

14. Diurnal variations in serum lipid components Individual
observations ... ... .. . .. .. . 71

15. Effect of blanks on serum ~id components during absorption-
Individual observations. . .. .. .. ... 72












LIST OF TABLES
Continued


Table Page

16. Effect of palmitic acid on serum lipid components
during absorption Individual observations. .. .. 73

17. Effect of stearic acid on serum lipid components during
absorption Individual observations . .. .. 74

18. Effect of oleic acid on serum lipid components during
absorption Individual observations ** *. 75

19. Effect of linoleic acid on serum lipid components during
absorption Individual observations . .. .. 76

20. Effect of combined mix 1 on serum lipid components during
absorption Individual observations . .. ** 77

21. Effect of combined mix 2 on serum lipid components during
absorption Individual observations ... .. .. 78

22. Effect of combined mix 3 on serum lipid components during
absorption Individual observations .. . .. 79

23. Effect of triolein on serum lipid components during
absorption Individual observations . . .. .. 80













LIST OF FIGURES


Figure Page

1. Approximate external and internal position of cannula
used for obtaining blood samples. ... .. .. .. .. 28

2. Manifold used on automated analyzer for triglyceride
analysis. ... .. .. .. .. .. ... .. .. 33

3. Manifold used on automated analyzer for free fatty acid
and phospholipid analysis .. .. .. .. .. ... .. 38

4. Serum patterns of free fatty acids plus phospholipids and
triglycerides during a control period . ... .. . 47

5. Effect of the blanks on serum free fatty acids plus phospho-
lipids and serum triglycerides. .. ... .. ... 48

6. Effect of palmitic acid on serum free fatty acids plus
phospholipids and serum triglycerides .. .. ... .. 50

7. Effect of stearic acid on serum free fatty acids plus
phospholipids and serum triglycerides .. .. ... .. 51

8. Effect of oleic acid on serum free fatty acids plus phospho-
lipids and serum triglycerides. . ... ... .. .. 53

9. Effect of linoleic acid on serum free fatty acids plus
phospholipids and serum triglycerides .. .. .. ... 54

10. Effect of a 1:10 ratio of 01eic acid to palmitic acid on
serum free fatty acids plus phospholipids .. .. .. .. 58

11. Effect of a 1:10 ratio of triolein to palmitic acid on serum
free fatty acids plus phospholipids . ... ... .. 59

12. Effect of 1:10 ratios of 01eic acid and triolein to palmitic
acid on serum triglycerides .. .. .. .. . .. ... 60

13. Effect of a 1:5 ratio of 01eic acid to palmitic acid on
serum free fatty acids plus phospholipids and serum trigly-
cerides. 61













INTRODUCTION


Swine have become recognized as having multiple attributes in the

fields of medical science and agriculture. In addition to contributing

to the meat supply they are now utilized in increasing numbers for

biomedical research. Thus the knowledge of their nutritional physiology

becomes important to the researcher from a fundamental interest in the

species and as a source of correlates to human parameters. The impli-

cation of dietary fats in human circulatory and obesity problems has

promoted the investigation of physiological and dietary factors which

may explain the involvement. Statements have been issued by advisory

boards and committees on the proper eating habits which should be

developed for best health. Yet the scientific evidence supporting a

majority of these programs is quite limited. Research efforts are

continually pointed toward determining factors which influence lipid

absorption as well as the basic mechanisms in transfer of lipids from

the lumen of the intestine to the lymph duct. Reports have indicated

that two main factors affect fatty acid absorption: (1) carbon chain

length, (2) degree of unsaturation. Since the chain length of most

natural lipids does not vary extensively, degree of saturation becomes

paramount. Another factor is the ratio of saturated to unsaturated

fatty acids. 01eic acid, particularly, has been suggested to have the

unique characteristic of promoting saturated fat absorption.

The studies reported here were undertaken to determine the effect









of the lipid material being absorbed from the intestine on components

of the serum lipids. This problem was approached through the use of

purified free fatty acids and triglycerides in carefully timed trials

during the period of intestinal absorption. Following the study of

individual lipids, trials were conducted using small proportions of

unsaturated fat in combination with saturated fat.

The sampling technique for blood and subsequent analysis of blood

serum utilized new techniques in the interest of expanding the scope

of present laboratory procedures. Venous cannulas offer the means of

repeated sampling opportunities with a minimum of disturbance to the

animal. Laboratory automation is rapidly expanding the quantity and

quality of laboratory analyses. The analyses conducted in this study

were adapted from recent automated techniques for the determination of

serum triglycerides, free fatty acids and phospholipids.













LITERATURE REVIEW


The research in fat absorption has been conducted by two major

techniques: (1) lymphatic cannulation, (2) feed and fecal analyses

with emphasis on digestibility coefficients. The chapter following

the Literature Review will cover the subject of lymphatic cannulation.

Digestibility studies using swine, chicks and rats are reviewed in

this section with an emphasis on the relationship between carbon chain

length, degree of saturation and absorption value.

Absorption may also be studied by means of changes in the serum

level of specific substances. This procedure cannot be quantitative

since no practical means exist for measuring simultaneous entrance and

removal of a substance in blood. Patterns of materials such as the

serum lipid components can be studied, however, and the work of M~orris

(1954) considered these over 10-hour periods during and following fat

absorption. This work also served to establish significant relation-

ships between the lipid components of the plasma and lymph both during

fat absorption and in the post-absorptive animal. Multiple regressions

were established between plasma and lymph total acids and phospholipids.

These regressions also confirmed that phospholipids were not directly

involved in fat absorption but did not explain the apparent metabolic

significance of the increased levels during the absorption period.

Mead and Fillerup (1957) suggested that the newly absorbed fatty acids

may have been handled in a different manner than endogenous fatty acids.









Through the use of carboxyl-C14i labeled methyl esters of stearic,

01eic and linoleic acid, patterns were established for incorporation

of these materials into serum lipid components. During the first two

hours post-ingestion stearate and 01eate appeared mainly in the

triglyceride fraction. The activity of stearate became progressively

larger in the phospholipid fraction over the next 22 hours. About

50 per cent of the linoleate activity was found in the phospholipid

fraction after 0.5 hours with the remainder of the activity divided

between the sterol and triglyceride fractions. In all cases activity

in the sterol fraction increased over the 24-hour blood sampling period.

At the end of 24 hours the sterol ester fraction comprised 20, 30, and

40 per cent respectively of the stearate, 01eate and linoleate activity.

Mead and Fillerup (1957) suggested that the initial high incorporation

of linoleate in the phospholipid fraction might explain its beneficial

properties in hyperlipemia and associated disorders. Rapid formation

of phospholipids promotes stabilization of serum lipoproteins and sub-

sequently the more rapid oxidation and deposition of the lipid portion.


Serum lipid levels in porcine blood

The relationship between dietary fat and serum or plasma lipid

levels in swine was first: reported by Witz and Beeson (1951) about

20 years after a dietary requirement for fat had been established for

the rat by Burr and Burr (1929). Purified diets containing from 0.06

to 0.12 per cent fat were used in comparison to similar diets" wi4th

5 per cent corn oil added. Weanling pigs weighing 35 to 50 pounds

were maintained on the diets for 86 days. Blood samples were taken

every two weeks and analyzed for plasma fat, cholesterol, vitamin A,









hemoglobin, red blood cells and white blood cells. Plasma fat analyses

were made using a modification of the Babcock Test used in milk fat

determinations. The pigs receiving a lowc fat diet had significantly

lower plasma fat levels than those on a 5 per cent corn oil supple-

mented diet. Pigs on the fat supplemented diet had an average plasma

fat level of 157 mg. per cent while the pigs on low fat diets averaged

129 mg. per cent. A two week period on a repletion diet containing

1.5 per cent corn oil raised the deficient pigs plasma fat level to

153 mg. per cent. White blood cell counts decreased significantly in

the fat deficient group but the other criteria were not significantly

affected by a low fat diet.

Plasma lipid levels have been investigated in several experiments

since the Witz and Beeson (1951) report. Attempts have been made to

correlate plasma lipids with rate of gain (Bowland and Hironaka, 1957),

level of feeding (Irorrow et al., 1963; Anderson and Fausch, 1964),

carcass characteristics (Tremere et al., 1965) and incidence of athero-

sclerosis (Reiser et al., 1959; RowJsell et al., 1960; CallowJay and Potts,

1962). A summary of their determinations on the serum lipids is pre-

sented in Table 1. Bowrland and Hironaka (1957) found no significant

correlation between rate of gain and plasma lipid levels but the

relationship between the levels at 45 kg. and 91 kg. and other carcass

characteristics including backfat thickness and loin eye area suggested

future investigations might consider correlates to estimate body fat

and lean content. Tremere et al. (1965) failed to establish significant

correlations of any consequence between carcass characteristics and

total serum lipids or the components of serum lipids. There did appear

to be a definite relation between weight and the level of serum









triglycerides and phospholipids (Table 1) but this was not discussed.

Morrow et al. (1963) found the apparent diurnal variation in

plasma lipid levels was due mainly to daily temperature variation.

Under controlled environment no definite diurnal pattern was observed.

Reduced environmental temperatures significantly elevated plasmca lipid

levels. No clear effect of fasting could be determined in this study.

Limited feeding, with respect to the pig's normal eating habits, is

a type of intermittent fasting. Anderson and FauJsch (1964) reported a

significant decrease in serum cholesterol and phospholipid levels when

feeding was restricted to two hours access to self-feeders each day.

Sex differences were also evident. Serumn triglyceride levels were not

affected. Boars had significantly lower levels of all plasma lipid

classes than barrows and gilts. There were no significant differences

between arrowss and gilts.

Several surveys on atherosclerosis incidence in swine and their

natural dietary adaptability have promoted the use of the pig in this

area of research. Those which have been designed to study both dietary

influence and the concomiitant influence on the serum lipid are discussed

in this review. Reiser et al. (1959) used miniature swine to study

effects of low fat, saturated fat and unsaturated fat diets with and

without added cholesterol on plasmra lipids and arterial changes. The

saturated fat used was a specially prepared myristoy1-laurin and the

unsaturated fat used was refined cottonseed oil. Cholesterol was added

at a level of 2 per cent. Three-month old pigs were started on the

diets and continued for one year. The results from the terminal serum

lipid analyses are presented in Table 1. During the course of the












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experiment individual pigs were fed 3 gm. of cottonseed oil or

myristoyllaurin per kg. body weight in a special study on the effects

of lipid absorption on serum phospholipids. Although the number of

samples was small several trends were noted. Ingestion of saturated

fat by pigs on lowJ fat or saturated fat diets resulted in a rise in

the serum phospholipid level which returned to the pre-ingestion

within nine level hours. There was no increase in the serum phospho-

lipid level when the unsaturated fat (cottonseed oil) was ingested by

pigs on the saturated fat diets. Reiser et al. (1959) also found no

increase in the serum phospholipids when pigs receiving the unsaturated

fat diets ingested the special feeding of saturated or unsaturated fat.

A similar experiment reported by Calloway and Potts (1962) also used

miniature swine. An adequate purified diet was compared to a purified

diet with protein lowered fromn 22.5 to 13.5 per cent and 5 per cent

corn oil replaced by 25 per cent saturated fats. Included in the

comparison also was the Armny Riation, Individual, Combat (C-ration).

Me~an serum lipid values over a six-month feeding period are presented

in Table 1.

Further studies by RowJsell et al. (1960) used domestic breeds

of swine (Yorkshire and Yorkshire-Landrace crossbreds) to study the

influence of a high butter and a high Egg yolk diet on serum lipids.

Butter or egg yolks replaced 33 per cent of the calories in a standard

16 per cent protein swine ration. Three-month old pigs averaging

100 pounds were used. The pigs were fed the diets for 52 weeks and

blood samples were taken every 8 weeks. No changes were observed in

the serum cholesterol and phosph7olipid levels of the control pigs.

The butter diet increased the serum cholesterol level from 117 agi.








per cent to 321 mg. per cent and serum phospholipids level from 139

mg. per cent to 218 mg. per cent.


Fat digestibiity studies

Thte digestibility responses of the pig to alteration in the fat

composition were first reported by Lloyd and Crampton (1957). The

pigs used in these studies were weaned at two weeks of age. Low-fat

diets were used as control diets while the experimental diets included

20 per cent fats and oils added to the control diets. ?Twenty fats

and oils were tested for digestibility and grouped according to chain

length and degree of saturation. Chain length groups were determined

by saponification values. These values ranged from a high of 258 for

coconut oil to 166 for erucic acid. The digestibilities for each

group were as follows; saponification value over 200 (mean molecular

weight of fatty acid less than 260) 94.4 per cent digestible, 200 to

185 (mean molecular weight between 260 and 285) 91.9 per cent diges-

tible, less than 186 (mean molecular weight more than 285) 78.2 per

cent digestible. Degree of saturation was detennined by iodine num~-ber

which ranged from a low of eight for coconut oil to a high of 188 for

linseed oil. Th~e digestibilities in this group were as follows;

saturated fats (iodine number under 95) 92.3 per cent digestible,

medium saturation fats (iodine number between 95 and 135) 84.3 per

cent digestible, unsaturated fats (iodine number over 135) 89.1 per

cent digestible. A highly significant decrease in digestibility was

observed by Lloyd and Crampton (1957) as chain length increased. No

consistent relationship between degree of saturation and digestibility

was observed in this experiment.









Sewell and Miller (1964) fed purified basal diets containing 0.41

per cent fat which were compared to similar diets with 8 per cent corn

oil, beef tallow and lard added. Fat digestibility of the corn oil

diet was significantly higher than the digestibility of the tallow and

lard diets. Gas chromnatographic analysis of the diets and feces

indicated palmitic and stearic acids absorption values were significantly

lower than values for oleic and linoleic acid.

More detailed experiments have recently been presented by Bayley

and Lewis (1965a, 1965b) Endogenous fat excretion quantity and compo-

sition were studied for several diets. With a standard ration endogenous

fecal fat could not be reduced below 11.5 gm. per day even after ether

extraction of the diet reduced the intake to 3 gm. per day in 900 gm. of

total feed. A purified ration containing 12 per cent cellulose reduced

endogenous fecal fat to 3.8 gm. per day on an intake of 8.2 mgO. of

total fat. Saturated fatty acids comprised 36 per cent of the total

fatty acid intake and 81 per cent of the excretion. Of the saturated

fatty acids, however, 24 per cent were 15 and 17 carbon chain length.

Intake of these fatty acids was less than 1 per cent of the total

intake. Microbial synthesis was assumed to be responsible for the

quantity of excretion. Microbial hydrogenation was also suggested as

responsible for the predominance of saturated fecal fatty acids.

In relation to the endogenous fat problem, Burr et al. (1960)

determined the contribution of the blood lipid pool to endogenous fat

by infusion of materials into the small intestine. The materials used

wiere labeled palmitic acid-1 C14 in a triglyceride emulsion in a

fatty acid-albumin complex and in chyle obtained from thoracic lymph.

Prior to initiating the infusion, the rats were given 0.5 ml. of olive









oil orally for the purpose of serving as a trapping or diluting media

for secretaed lipid and to provide a stimulus for the process of fat

absorption. Burr et al. (1960) suggested the absorptive process may,

in some manner, affect the secretion of lipids into the intestine.

During an infusion period of 2.5 to 3 hours, 4.0 to 4.8 ml. of material

were given. The blood radioactivity stabilized during the third hour

and the rats were sacrificed at the end of the third hour. Analysis

of the intestinal contents indicated up to 11 per cent of the intestinal

lipids originated from the blood lipid pool. This figure may represent

a minimal secretion level if rapid reabsorption occurs and thus gives

only a narrow picture of the true secretion level. An alternative to

this theory suggested that a lipid pool exists in the intestinal mucosa

and a true picture would require inclusion of the size of this pool.

These workers concluded that although significant quantities of material

are secreted into and possibly cycled through the intestine the net effect

is far from minor. This may account for certain discrepancies in the

analysis of apparent and endogenous-corrected digestibility values for

fat. The second experiment of Bayley and Lewis (1965b) supports this

hypothesis. Diets and procedures were described by Bayley and Lewis

(1965a) Three fats and purified forms (70 per cent average) of palmitic,

01eic and stearic acids as free fatty acids and triglycerides were

studied. The fats were soybean oil, beef tallow and a partially hydro-

lyzed mixture of plant and animal fats. Corrected digestibilities for

the fats were 94 per cent for soybean oil, 85 per cent for beef tallow

and 73 per cent for the partially hydrolyzed mixture of plant and

animal fats. These results compare favorably with the general ranges

of digestibility reported by Lloyd and Crampton (1957) and with chick









studies conducted by Renner and Hill (1960) and Feddel et al. (1960).

The use of purified materials produced results s'',ilar to the rat work

by Carroll (1958) and the chick work of Rnenner and Hill (1961b) with

respect to relative absorption values. The results are presented in

Table 2. Th~e digestibilities indicate a pattern in the pig similar to

other species in that unsaturated fatty acids are absorbed to a greater

extent than saturated ones. These results may also be viewed in

support of the Young (1965) hypothesis that palmitic acid or probably

any saturated acid is absorbed according to the molar ratio of 01eic

acid present. Since there is 0.1 to 0.14 moles of 01eic acid per mole

of saturated fatty acid present in the diet containing palmitic and

stearic acid, one would expect approximately 30 to 40 per cent of the

saturated acids to be absorbed in a practical ration. The values of

33 to 64 per cent are within the expected range.



Table 2. Digestibility coefficients of total dietary fat and the
coc~ponent fatty acid or triglyceride fed as semi-purified
material (Bayley and Lewis, 1965b)


Dietary lipid

Digestibility Palm~itic Stearic 01eic
Coefficenta TZa Fb T~a FFb Tiya Fy b


Total Dietary Lipid 77 47 55 60 93 99

Respective Fatty acid 64 33 49 55 98 97


aCorrected for endogenous dietary lipid.
bdinfrmo triglyceride.

c~ed in form of free fatty acid.









Dieta 3ry ratios cf (7 nsrturase to 'a turaed fat Ty acids

Th~ie interest in utilizing added dietary fat in high energy rations

has pr~omoed investigations to determine the optimum ratio of unsaturated

to saturated fatty acids in the diet. Ho~pkins et al,. (1955) fed rats

on defatted commercial diets and purified diets with linoleic acid at a

constant level of 10 per cent of the dietary fat. Thie ratio of 01eic

acid to palmitic plus stearic acid in the fat was varied from 2:7 to

8:1 and provided the remainder of the dietary fat. The ratios were

provided by varying the ratios of coconut oil, corn oil and olive oil.

It was realized that there were minor proportions of other fatty acids

present. The oil mixture was fed as 20 per cent of the diet. Weight

ain~s over a nine-weaek period indicated an optimuim ratio of six parts

01eic acid to three parts of a palmitic and stearic acid mixture. This

ratio was quite close to the comp-osition of rat depot fat. Fecal fat

tended to increase as the ratio of unsaturated fat to saturated fat

increase.

Thle expesriments of Calloway and K~urtz: (1956) were designed to

study effects of hydrogenation of plant oils on digestibility. Attempts

to establish~ multiple reliationships between chain length, degree of

unsastu-ration and glyceride position of fatty acids were made. Purified

diets with 20 per cent fat were fed to rats. Ti~e digestibilities wee

deteLrmined and corrected for endogenous fecal fat through the use of

a fat-free diet. Natural and hydrogenared forms of cottonseed, soybean,

cora, coconut and; palm oil, lard a,;d buttlrjlai: were stucied.Moiu

lards were also rested and are listed below:

1. Cusyra;ced ::d-ognated YLord (13.4j% jiu-~yraLte byr Weigh<)

2, ;ydrTogenated lor-d plUs triburyrin (81:19)








3. Monoglycerides of hydrogenated lard

4. Mannitol esters of lard

5. Mannitol enters of hydrogenated lard


Glyceryl monostearate was tested for comparison to the lard

monoglyceride. Digestibility values varied inversely with chain

length and in aculrvilinear fashion with percent stearic acid. The

slope of the curve changed at 55 per cent stearic acid. Calloway and

Kurtz (1956) estimated maximum stearic acid digestibility to be 30 to

40 per cent. Hydrogenation significantly reduce digesiblities of

all fat except coconut oil. Natural fats were 97 per cent or above

digestible. Hydrogenated butterfat digestibility was reduced to 61 per

cent and the digestibility of the other hydrogenated fats to between

12 and 24 per cent. Butyration of the hydrogenated lard increased its

digestibility from 18 to 35 per cent but the addition of tributyrin

did not affect digestibility. Interesterification of lard with glycerol

to produce monoglycerides increased digestibility from 14 per cent to

34 per cent. There was no practical difference between digestibility

of glycerol and mannitol esters of natural and hydrogenated lard. The

improvement of digestibility by butyration and reduction to monoglycer-

ides of hydrogenated lard emphasizes the importance of structure of the

fat in relation to the absorbability.

Individual fatty acid absorption values were determined for rats

by Carroll (1958). Purified diets were used with fatty acids sub-

stituted for 10 per cent of the dietary glucose. Absorbabilities were

100 per cent for fatty acids with carbon chain length of 10 or less.

The absorption value decreased as carbon chain length increased for the

saturated fatty acids. The absorption value was 86 per cent for lauric









acid, 64 per cent for myristic acid, 44 per cent for palmitic acid,

16 per cent for stearic acid and 7 per cent for behenic acid which

has a 22 carbon chain completely saturated. 01eic acid was 79 per

cent absorbed, again indicating the increased absorbability of

unsaturated fatty acids over saturated fatty acids.

Other dietary factors involved with fat absorption were reported

-f by Feddel et al. (1960). Growing chicks were fed standard broiler
diets w~ith fat substituted for corn and soybean meal. Th itswr


adjusted to maintain a constant ratio of protein to productive energy

when 0 per cent, 10 per cent and 20 per cent levels of fat were used.

No significant difference in fat digestibility was found between the

diets containing 10 per cent and 20 per cent fat. The materials used

and absorbabilities were 92 per cent for safflower oil, 90 per cent for

corn oil, 88 per cent for hog grease (lard) and 65 per cent for beef

tallow. Addition of 0.5 per cent ox bile to the beef tallow increased

the absorption value to 79 per cent. Manipulation of the calcium level

also had a significant effect on beef tallowJ absorption. On a normal

level of calcium (1.24 per cent of the diet) beef tallow was 74 per

cent absorbed. Reducing calcium to 0.33 per cent increased the

absorption to 89.5 per cent and increasing the calcium level to 3.0

per cent decreased absorption to 63.5 per cent. Absorption differences

due to calcium variations reflected the relative formation of unabsorbed

calcium soaps of fatty acids. Ox bile exerted its influence through

increasing the intestinal solubility of beef tallow and its hydrolysis

products. Feddel et al. (1960) did not recognize the solubility of

ox jile and the relation between absorbability and degree of saturation

in their discussion.








The efficiency of fat utilization by growing chicks and adult

hens in terms of metabolizable energy and absorbability has been

reported by Renner and Hill (1960). Efficiencies increased with

respect to age until chicks reached eight weeks of age. The fats

studied were corn oil, lard and tallowJ fed as 17.5 per cent of the diet.

A reference diet without added fat was used to establish endogenous

fecal fat levels for correcting the metabolizable energy and absorba-

bility results. Absorption values were 95 per cent for corn oil, 85

to 92 per cent for lard and 81 per cent for tallow. The variability

of the value for lard was due to a reduced absorption in the adult

hen group which could not be explained. These results followJ a

pattern in that absorption was decreased as the per cent of saturated

fat increased. According to Edwards (1964) corn oil contains 12 per

cent saturated fat, lard contains 36 per cent and tallow contains 48

per cent.

Further studies were conducted by Renner and Hill (1961b) on the

utilization of individual fatty acids substituted for glucose in a low

fat diet at a level of 20 per cent. The absorbabilities of the fatty

acids by the growing chick were 65 per cent for lauric acid, 25 per

cent for mryristic acid, 2 per cent for palmitic, -2 per cent for

stearic acid and 38 per cent for oleic acid. In an experimrent with

adult hens, absorbabilities were 29 per cent for myristic acid, 12 per

cent for palmitic acid, -4 per cent for stearic acid and 94 per cent

for 01eic acid. The hens refused to eat the diet containing 20 per

cent laucric acid. The hens absorbed significantly (PO3.01) more of

the dietary fatty acids than the chicks.

Utilization of natural and hydrolyzed tallow, lard and soybean








oil by growing chicks was studied by Renner and Hill (1961a) in an

effort to define factors other than degree of saturation which may

affect fat absorption. These results show a definite decrease in

absorption of the hydrolyzed material as compared to the natural fats.

Gas chromatographic analysis of dietary versus fecal fatty acid

indicated that most of the decrease in per cent absorption was due to

the saturated fatty acids. It was suggested that the arrangement of

the fatty acids on the glyceride moiety may also be involved in con-

trol of absorption. Lard has a preponderance of the palmitic acid

content in a two-position on the glycerol segment. Samples of lard

were fed as natural lard, partially rearranged lard and completely

rearranged lard. Partial and complete rearrangement were accomplished

by intersterification of the lard with sodium ethylate catalyst.

Absorption of palmitic acid was decreased from 93 per cent in the

natural sample to 85 and 80 per cent respectively in the partial and

complete modification groups. Stearic acid absorption increased from

76 per cent to 80 and 79 per cent in the modified groups. The results

of Renner and Hill (1961a) support the theory that monoglycerides

may have a preferential position in the sequence of fat absorption

regardless of, or in deference to, the chain length and degree of

saturation of the attached fatty acid.

During additional experiments on absorption and metabolizable

energy values of fat, Young (1961) obtained higher absorption values

for hydrolyzed fats than Renner and Hill (19j1b) No clear explana-

tion other than breed or strain differences became evident in this

study or a subsequent one (Young and Artman, 1961). Absorption values

were determined for a number of natural fats and for their hydrolyzed









fatty acids. Although hydrolysis reduced the absorption of all

materials tested, there was significant absorption and utilization

of the saturated fatty acid provided a quantity of unsaturated fatty

acids were present. Several m~ore experiments were conducted to define

the optimum ratio of saturated fatty acids to unsaturated fatty acids

(Young and Garrett, 1963; Young et al., 1963) and the conclusions

were recently discussed by Young (1965). The absorption value for

palmnitic and stearic acids varied considerably between experiments and

experimental diets. However, a uniformly effect of oleic acid has been

noted. Miaxrlimu effect is obtained by a ratio of 1 part palmitic acid

to 0.8 parts 01eic acid. Linoleic acid does not substitute in this

capacity. Various forms of oleic acid studied do possess considerable

Influence as indicated in an experiment using purified diets. The

chicks used in this study had the pancreatic duct ligated and sectioned

preventing pancreatic lipase from entering the intestine. The results

are presented in Table 3.



Table 3. Effect of lipid classes on palmitic acid absorption.



Lipid Added M'olar Ratio Percent Absorption
to Diet (O/2)a Predicted Actual

Palmitic acid 0.10 8 -14
Palzlitic acid + 01eic acid 0.54 32 28
Pamitic acid + m~onolein 0.17 12 48
Palmitic acid + triolein 0.42 25 12



aO-01eic acid, P-Pa~litic acid.









While the absorption value for palmitic acid alone is lower than

expected, the value for palmitic acid plus 01eic acid is well within

the predicted range. Triolein improved absorption slightly but

monolein improved palmitic acid absorption four times greater than

the predicted value. Young relates this improvement to the specific

solubility index of monolein. This index is a measure of the ability

of one lipid to improve the solubility of a more non-polar lipid such

as palmitic acid in a solution. This suggests a requirement for a

quantity of triglycerides in the diet. Lipase hydrolysis would then

produce a quantity of monoglycerides which would aid in the absorption

of the less soluble palmitic and stearic acids. The unsaturated fatty

acids have sufficiently high solubility indexes for absorption without

the aid of monoglycerides.

This review has considered fat absorption from the major aspect

of digestibility. The several experiments discussed here agree on

several points:

1. Absorption of saturated fats varies inversely with carbon

chain length of the fatty acid molecule.

2. The presence of an unsaturated bond increases the absorption

of long chain fatty acids.

3. The position of the fatty acids on the triglyceride moiety

affects the absorption of the fatty acids.

4. 01eic acid possesses a unique and partially undefined ability

to promote the absorption of saturated fatty acids.


Further research would be desirable to define the properties of oleic

acid htmkeit unique in promoting fat absorption. The di,,estibi~lity









work should also be continued in view of the research indicating the

endogenous origin of significant quantities of saturated fatty acids.

R~e-evaluation of digestibility values for fats and oils in respect to

these findings may increase the economic value of some of these

materials.

The research on serum lipids in swine has pertained to the effect

of dietary alterations in relatively long-term studies. This work was

conducted in order to establish correlates to other criteria which were

measured such as incidence of athersclerosis, rate of gain and carcass

characteristics. One short-term study investigated effects of fasting

and environmental temperature on total serum lipids. There are no

reports on the behavior of the porcine serum lipid components during

fat absorption.













CANNULATI-ON OF INTESTINAL LYMPHATIC DUCTS IN SWINE


This chapter presents a resume of the attempts of the author to

locate and utilize the lymphatic system in studying fat absorption

in swine. A brief review on the background of lymphatic cannulation

and the lymph system are also presented.

The lymphatic system has been investigated and proven to be the

major route of transport of lipid materials from the intestinal mucosa

to the blood via the intestinal and thoracic lymph ducts. Studies

have been conducted using the dog (Cain et al., 1947), rat (Bollman

et al., 1948), cat (Mlorris, 1954) and sheep (Lascelles and Morris,

1961; Heath and Morris, 1962) as subjects for the location and cannu-

lation of the thoracic, hepatic and intestinal lymph ducts. In the

experiments to be presented here swine were used as subjects.

Description of the lymphatic system of swine has, until recently,

been limited. Sisson and Grossman (1953) gave a brief discussion of

the anatomical location of certain major lymph ducts. Getty (1964)

and co-workers are presently engaged in a project which, when completed,

will present a detailed description of the complete lymphatic system

of swine. Some of the differences in the location of certain portions

of the lymphatic system have been pointed out by Saar and Getty (1961).

From their preliminary work one may expect to find a great many

differences between swine and the other domestic animals. At present,

however, there are no references on the precise location of the

intestinal and hepatic lymph ducts in swine.








An investigation was initiated with the aid of members of the

Veterinary Science Department to locate and cannulate the intestinal

and lepatic lymph ducts in swine. The pigs used ranged in weight from

10 pounds to 190 pounds. Feed was withheld from the pigs 24 to 36

hours before surgery but free access to water was allowed. The pigs

were anesthetized with sodium pentobarbitala given intravenously,

slowly, until a loss of reflexes was obtained. The amount used varied

considerably with individual animals. One pig was lost due to an over-

dose of anesthetic. Small pigs weighing 9 to 12 pounds required 350

mg. to 450 mg. while larger pigs, 160 pounds, required 1750 mg. to

3000 mg. After a complete loss of reflexes was obtained, the pigs

were placed on an operating table or trough designed to maintain an

animal on its back without rolling to one side. The pigs remained

under anesthesia for up to three hours without major problems. Some

pigs required additional sodium pentobarbitala to maintain adequate

depth of anesthesia. In preparing all pigs for surgery, the abdomen

was washed with a germicide detergent. An incision into the abdominal

cavity was made from the xiphoid cartilage to the umbilicus. Retractors

were used to open the incision and the intestine was covered as much as

possible with saline moistened pads. A 5 to 7 per cent solution of

Evans Blue dye (T-1824) was injected into the duodenum as aid to the

location of the lymph ducts (Bollman et al., 1948; Getty, 1964). After

several minutes, attempts to locate the lymph ducts were made by visual

inspection of the dorsal peritonium, dorsal surface of the pancreas

and caudal area of the liver. A magnifying glass was used as aid in



aNembutal-Sodium, Abbott Laboratories, North Chicago, Illinois.








the inspection. Blunt dissection of these areas also failed to locate

any lumph ducts of consequential size. Several minute ducts were

observed to be stained with the Evans Blue dye indicating it had been

absorbed as expected. At this point in most of the experimental

subjects the areas were stained with blood preventing further attempts

to locate ducts by visual means. In the animals weighing over 80

pounds further attempts were made after sacrificing the animal. The

thoracic duct was easily located in the opened thoracic cavity. Poly-

ethylene tubinga size PE50 (inside diameter 0.58mm, outside diameter

0.97 mrm.) was inserted into the thoracic duct. This duct was followed

back through the aortic hiatus into the abdominal cavity where it

entered the cisterna chyli. In a pig weighing 160 pounds, the

cisterna chyli was about 6 inches to 8 inches long and 2 inches

wide. The cisterna chyli was exposed to its full length and attempts

made to define the source of the many small ducts entering it. The

PE50 tubing was used to follow the ducts back as far as possible and

then a small amount of Evans Blue dye was injected through the tubing

to try and define further the course of the lymphatics. It appeared

that several of the small ducts originated in the intestinal area and

there was no single collecting duct for intestinal and hepatic lymph

as found in other species. In these attempts, fifteen pigs were used.

Ten pigs weighed between 9 and 12 pounds, three pigs weighed between

80 and 120 pounds, and two pigs weighed over 160 pounds.

Further exploratory work would be required to define the lymphatic

system in swine responsible for movement of lymph from the mesenteric



aPolyethylene tubing Intramedic tubing, Clay-Adams, New York, New York.






24



region to the cisterna chyli. It is suggested that a mixture of

the dye and a high fat material be used in dosing the pig prior

to attempting to locate the lymrph ducts. This combination would

serve to distend the ducts with an increased lym~ph flow as well as

to carry a staining material. The use of the larger pigs would seem

to be necessary until the precise locations of the lym~ph ducts were

determined. Rapid location of the cisternla chyli and reverse tracing

of afferent ducts offers the best possibility for elucidating the

route of the lymph ducts.













EXPERIMENTAL PROCEDURE


Animal methods and management

Th~e pigs used in this study were obtained from the University

of Florida Swine Unit hard. All pigs were progeny of a Duroc-Landrace

sowJ and Hampshire boar mating. InitLial weights of the pigs used

ranged from 70 to 130 pounds. Prior to being used for these exper-

iments the pigs were receiving a 16 per cent protein diet. The

composition of the diet is given in Table 4. No ether extracts were

made of the diet but using figures published by Edwards (1964) the

diet was calculated to have 3.17 per cent neutral fat of which 77

per cent was unsaturated. Elevated cages with expanded metal floors

and tops and solid sheet metal sides were used to contain the pigs

during an adjustment period of 12 to 24 hours and the experimental

period. An automatic watering device was attached to the cage and

the cages were placed under a building with good ventilation. Feed

was withheld from the pigs for a period of 12 hours before experiments

were conducted.

Blood samples during the experiment were obtained from a cannula

placed in the marginal ear vein (Figure 1). The cannulation procedure

was developed with aid of the Veterinary Science Department. The

marginal ear vein was clamped off with a hemostat having light pressure

just adequate to stop blood flow. Thie ear was washed with geirm-icide

soup. '=his scr;:bbing of the ear also caused the vein to be quite

distended and aided in the cannulation. A 15 gauge hypodermic needle








was placed in the vein and a 15 inch polyethylene cannulaa, size

PE 90 with an outside diameter of 1.27 mm. and an inside diameter of

0.86 mmn., was inserted through the needle and into the vein and

extended down about 12 inches to the vena cava. The remaining external

portion of the cannula was secured on the ear by means of adhesive tape

which was placed around the cannula and sutured to the ear. The cannula

was filled with a dilute solution of heparin (25 mg. in 100 ml. of 1

per cent NaC1) and sealed with a polyethylene cap forced over the end

of the cannula. As an aid to keeping the cannula free flowing it was

coated with a 4 per cent silicone solution prior to use and air dried.

Blood was obtained with a 20 gauge needle attached to a 10 ml. dispos-

able syringe which was found to be better suited for the operation than

the regular all glass syringe. The 20 gauge needle inserted tightly

into the cannula, also aided the sampling technique. Immediately before

and after sampling the cannula was washed with 0.1 to 0.2 ml. of

heparin solution. Efforts were made to keep this amount to a minimum.

If additional wash solution was required, as in the case of a clot in

the cannula, 1 per cent NaC1 alone was used.

Pigs were used for one to three experiments before being returned

to the herd. At: the time they were returned the cannulas were removed.

No ill effects were subsequently noticed with any of the animals.










aIntramedic tubing, Clay-Adams, Inc., New York, New York.

Siliclad, Clay-Adams, Inc., New York, New York.










Table 4. Composition of diet self-fed to pigs before experiments.



Ingredient Per Cent of Diet


Corn, yellow, ground 77.30

Soybean oilmeal (Solvent process) 20.00

Bonemeal, steamed 1.50

Limestone, ground 0.50

Salt, iodized 0.50

B-vitamin premixa 0.10

Vitamin B-12 premixb 0.05

Trace mineralsc 0.05



aContained 8.0, 14.72, 36.0 and 40 gm. per pound respectively of
riboflavin, pantothenic acid, niacin and choline chloride.

bContained a minimum of .20 mg. of vitamin B-12 per pound.

cContained minerals in the following quantities (%):
MnS04 22.5 ZnS04 10.0
FeSO4 35.0 K2S04 1.7
CuS04 1.9 CaCO3 28.4
00804 0.5
























































Figure 1. Approximate external and internal position of cannula
used for obtaining blood samples.









Dose preparation and procedures

The lipid materials used for dosing the pigs and studying the

serum lipid patterns were prepared as homogenates and given orally.

The amount of lipid to be given was weighed into a beaker. To this

was added 2 gm. of sodium taurocholate, as an emulsifying agent, and

50 ml. of 95 per cent ethanol, as a solvent. The beaker was warmed

until all of the material liquified. The liquid mixture was then

added to 100 ml. of 1 per cent NaC1 in a Waring Blender and homo-

genized for about 1 minute. After removal of the mixture the blender

was washed with an additional 50 ml. of 1 per cent NaC1. This mixture

was given to individual pigs by means of a stomach tube while the pig

was restrained. A large syringe or drench gun was used to force the

dose into the pigs stomach. The dose was followed by a rinse of 100

to 150 ml. of 1 per cent NaC1. Blood samples were taken according to

the following schedule in the experiment: 0:00 time ( Immediately

before dosing), 0:30, 1:00, 1:30, 2:00, 2:30, 3:00, 4:00, 5:00 and

6:00 hours after dosing.


Materials

Lipid materials used in these experiments were obtained front

Nutritional Biochemicals Company., Cleveland, Ohio. These materials

were checked by gas and thin layer chromatography for purity. The

results of the gas chromatography phase are presented in Table 5. No

measurable amounts of unsaturated fatty acids were found in the

saturated fatty acids purchased for use. Thin layer chromatography

indicated only a trace of esterified materials contaminating the

fatty acids and trace amounts of fatty acid esters and free fatty acids

in the triglycerides.
















Material Fatty Acid Percentb
analyzed C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3


Palmitic acid 92.2 7.8
(C16:0)

Stearic acid 12.7 tr. 87.3
(018:0)

01eic acid 1.4 3.5 10.4 tr. 84.7
(C18:1)

Linoleic acid 8.6 2.5 13.8 75.1
(C18:2)

Triolein 4.2 5.6 83.1 7.1
(3C18:1)


Table 5. Gas chromatographic analysis
experiments


of lipid materials used in


aAll materials
Ohio.


were obtained from Nutritional Biochemical Co., Cleveland,


bSeparations obtained with following column and conditions:
6 foot stainless steel column 0.25 inches in diameter
support Gas Chrom P 60/80 mesh
stationary phase 20% diethylene glycol succinate
operating temperatures
column 2100 C.
inlet 2400 C.
detector 2400 C.
nitrogen flow 75 ml. per minute









Extraction methods

Blood samples taken from the ear vein cannulas were immediately

centrifuged for 5 minutes at 350 xg. In addition the samples were

later centrifuged at 1200 xg. for 30 minutes. Serum was removed from

the blood samples and stored at -100 C. for future analysis. Extracts

of the serum were prepared for free fatty acid, triiglyceride and

phospholipid analysis. Glass-stoppered test tubes were used in pre-

paring all extracts. A phospholipid-free extract was prepared by placing

1 ml. of serum on a slurry of approximately 2 gm. of silicic acid and

2 ml. of isopropanol and mixing throughly for about 1 minute. This

mixture was allowed to stand a minimum of 4 hours to fix the phos-

pholipids on the silicic acid. An additional 8 ml. of isopropanol

was added and mixed. The tubes were then centrifuged to separate

the silicic acid. The isopropanol extract was drawn off and stored

at 100 C. A total lipid extract was prepared by blowing 1 ml. of

serum into 15 ml. of a chloroform-methanol mixture (2:1 v/v). The

tubes were vigorously mixed twice during a 30 minute period. After

this time 10 ml. of a 0.02 per cent CaC12 solution was used for

washing the extracts twice according to Folch et al. (1957) A period

of 4 hours for the first wash and 8 hours for the second wash were

allowed for good separation of the aqueous and chloroform phases.

Aspiration of the upper phase after each of the two washings removed

the methanol leaving the serum lipids from 1 ml. of serum in 10 ml.

of chloroform. These extracts were also stored at -100 C.


Triglyceride analysis

The phospholipid-free extract was used in the determination of

serum trilycerides by the semi-automated method of Lofland (1964).








One ml. of the extract was placed in a test tube and saponified

with 0.5 ml. of 0.07 N KOH in 95 per cent ethanol for 20 minutes

at 700 C. The tubes were loosely capped during magnification. The

solvents, isopropanol and ethanol, were evaporated by drawing air

through the sample tubes with a water aspirator while the tubes were

in a 500 C. water bath. The dried samples were redissolved in 3 m1.

of 0.2 N B280 A sampler I module was used in the automated analyzing

apparatusa with a sample leveler and constant volume device attached.

This module replaced the sampler II used by Lofland (1964). A flow

diagram is presented in Figure 2 indicating tube sizes and materials

used. Alternating samples and 0.2 N H2S04 blank were run by the method

at a rate of 40 per hour. Polystyrene cups were used to hold samples

and blanks. The cups were used one time only. This method involved

release of glycerol from the triglyceride moiety by saponification.

The glycerol was dissolved in the 0.2 N H2S04 and fed into the system

by the sampler. Addition of 0.05 M sodium metaperiodate oxidized the

glycerol to formaldehyde and released periodate. The periodate was

reduced to iodide by the addition of 0.5 M sodium arsenite. The

formaldehyde was determined on the oxidation mixture by chromotropic

acid (4,5-dihydroxy-2,7-naphthalene disulfonic acid) in 54.5 per cent

H2SO The 67 per cent H2SO4 was made up by adding 300 ml. of

concentrated H2SO4 to 150 ml. of H20. The colorimetric reaction was

measured at a wavelength of 570 millimicrons and the optical density

was automatically recorded by Bristol recorder. Standard solutions

of triolein in concentrations of 50 to 400 mcg./m1. were used to



aTechnicon Instrurments Corporation, Chauncey, New York.






Page
Missing
or
Unavailable










Table 6. Standardization of triglyceride determination method:
Recovery results



Sample Sample Amt. Std. Added Expected Recovery Percent
No. Reading mcg.,61l. mcg ./m1 mcg ./m1. mcg ./m1 Recove ry


1 90 50 140 103 73.6
2 90 50 140 113 80.7
3 103 50 153 117 76.5
4 103 50 153 117 76.5
5 95 100 195 146 74.8
6 95 100 195 151 77.4
7 110 100 210 178 84.7
8 110 100 210 168 80.0
9 115 100 215 185 86.0
10 115 100 215 185 86.0
11 132 100 232 190 81.8
12 132 100 232 212 91.3
13 142 200 342 300 37.7
14 142 200 342 325 95.0
15 129 300 429 385 89.7
16 129 300 429 405 94.4

Av. 83.5
S.D.a 7.10
S.E.b 1.77



standard deviation
standard error









establish a standard curve for the me hod. Purity of the triclein

was established by thin layer chromatography and the quantity In

the standard solution by weight. Recovery samples were also determirned

and the results presented in Table 6. Although recoveries were lowJer

than desired, the standard error indicates the repeatibility and reli-

ability of the method. Average recovery values for added standard,

in amounts of 50 mcg./m1. to 300 mcg./mll., were 83.5 per cent with a

standard error of 1.77 per cent.

The units of measure in the free fatty acid and phospholipid

analyses and the inability to separate the two components made it

necessary to alter the units of measure in the triglyceride analysis

for discussion purposes. The readings were taken in micrograms per

milliliter (micg./ml.) which could also be read as milligrams per 100

milllitr o seum mg.100ml.. Te mg./100 ml. values were con-

verted to millimoles per liter by using an average molecular weight

of 870 for the triglycerides. Eareafter the millimoles per liter are

referred to as milliequivalents per liter (meq./1.). It is realized

that the triglyzerides do not possess the property of equivalence

In terms of titratable groups. The term is used in order to be more

uiF~orm In referring to the molecular quantity of the components

present.


re '--ty- ac'd -;d ?_oschocl' 7-ia analysis

Th~e analysis for serumT free fatty acids was conducted by the

sermi-auton~ated method of Antonis (1964;) adapted fro;; the work of

Dutcombe (190,3) hie phtoopolipid-free extract was used for this

analyis since the method is sensitive to both free fatty acids and








phospholipids. A 1:60 dilution of the extract was prepared by re-

dissolving a dried aliquot of the isopropanol extract in chloroform.

Efforts were made to keep evaporation to a minimum by lowering the

room temperature and capping and sealing the dilutions rapidly. The

description of the manifoldl used for the automated segment of the

analysis is presented in Figure 3. Chloroform resistant acidflex

tubinga was used where necessary. Alternating samples and chloroform

blanks were run by the method at a rate of 60 per hour. Glass cups

with teflon rings were used to ;iold samples and blanks. The cups

were washed with acetone and air-dried after each use. The sampler

was loaded with cooled samples and covered with an aluminum plate

during operation of the analyzer. The samples dissolved in chloro-

form were mixed by the proportioning pump with an aqueous copper

regent of the following composition:

Aqueous Copper reagent

2M1-triethanolaamine 9 volumes

12.24% (w/v) Cu(N0g)2'3H20 10 volumes

2N-CSS3COOH 1 volume


Thils mixture resulted in formation of copper soaps of fatty acids

which were soluble in chloroform. A separator- on the manifold allowed

the chloroform phase to be resampiled and thea excess chloroformn phase

Gnd aqueous copper reagent to flow into a waste container. The copper

soaps in chloroform~ were m~ixed with 0.2 per cent (w/v) sodium

diethyldithiocar;amatee in buitanol. The optical density of the colored

complex formed was measured at a wcaveleng~th of 440 millimicrons and

the optical density recorded by a B3ristol recorder. A series of









plalitic acid standards ranging from 15 to 90 microeq~uivalents per

liter (meeq./1.) were used to establish a standard curve for the

determination of selran free fatty acids.

The method of Anitonis (1964) was also used for determination of

serum phospholipids in a total serum lipid extract (Figure 3) .

An~tonis (1964) found that the major serum phospholipids, lecithin and

cephlalin, have extinction coefficients similar to the free fatty

acids of serum. Aliquots of the total serum lipid extract were diluted

up to 1:70 for analysis. The materials and methods were identical to

those described for free fatty acid analysis. Standard solutions of

dimyristoy1 cephalin ranging from 9 to 90 microequivalents per liter

were used to establish a standard curve for the method. Since the

total lipid extract contained free fatty acids and phospholipids the

method used a differential means for dete-rmiining phospholipids. Both

series of determinations, free fatty acid and phospholipid, were to be

calculated back to a milliequivalents per liter of serum basis. Values

determlined for samples in the free fatty acid analysis were to be

subverted from the values determined in the phospholipid analysis

leaving a true value for serum phospholipids.

Results from the free fatty acid determination segment were prOVEn

to be completely erroneous and thus have prevented further differentia-

tion of free fatty acids and phospholipids. Attemptsjto determine the

;ource of error we~re marde. Analysis of the isopropanol extract by

thin layer chromotogrs~hy indicated an unidentified compound or group

of comp~ounds located betw~eeni the free fatty acids and triglycerides on

the d~veloped chromzatogram. Air- o;xidaion of the iso~ropanol samplts

wazs considered as a source of contam~inants. Samples were prepared using









































Figure 3. Manifold used on automated analyzer for free fatty acid
and phospholipid analysis.

A. Acidflex pump tubing.
T. Tygon pump tubing.
S. Solraflex pump tubing.
a Mixing coil with glass beads
b Pulse suppressor 0.005" inside diameter.
c Single mixing coil.
d Glass debubbler T.








nitrogen to evaporate the isopropano1 and sealed after dilution under

nitrogen. Ni\o differences wiere found between the analysis of nitrogen-

protected samples and the original samples prepared in air. Due to

this occurrence the samples prepared for phospholipid analysis were

also checked for lipid classes using thin layer chromotography. These

analyses indicated no extraneous compounds, therefore, this group of

analyses were accepted as the determination of serum free fatty acids

plus phospholipid on a milliequivalents per liter basis. This also

explains the necessity of converting the triglyceride data to a mili-

equivalents per liter basis in the results and discussion section in

order to report a meaningful total figure.

A group of recovery samples wlere prepared and analyzed to establish

the validity of this method and results are presented in Table 7.

Average recovery value was 101 per cent with a standard error of 2.63

per cent. In addition, to recoveries, a group of samples were repeated

using different batches of aqueous copper reagent on different days

and the results are presented in Table 8. There was no significant

difference between the groups. These two tests indicate the reliability

of the mecthrod used for the free fatty acid plus phospholipid determnin-

ation and also indicate that the error in separating the two components

was in an area other than chemical analysis. Thec determination of

standard error, standard deviation and application of the t-test

were according to Steele and Torrie (1960) .











Table 7. Standardization of free fatty acid and phospholipid
determination method: Recovery results.



Sample Sample Reading Amnt. Std. Expected Recovery a percent
No. meeq./1. Added a mee./1. meeq./1. Recovery
meeq./1.a

1 50 15 65 60.5 93.1
2 53 30 83 81.0 97.6
3 53 30 83 85.0 102.4
4 33 30 63 60.5 96.0
5 33 30 63 73.0 115.9
6 38 45 83 88.5 106.6
7 38 45 83 79.0 95.2
8 38 45 83 84.0 101.2

Av. 101.0
S.D.b 7.44
S.E.c 2.63



amceq./1. m~icroequivalents per liter
bStandard deviation
cStandard error















FFA+PL'
Sample No. 1 2 Difference

1 0.70 0.63 0.07
2 0.66 0.66 0.00
3 0.42 0.46 -0.04
4 0.63 0.63 0.00
5 0.66 0.63 0.03
6 0.63 0.66 -0.03
7 0.70 0.66 0.03
8 0.63 0.53 0.10
9 1.30 1.19 0.11
10 0.63 0.77 -0.14
11 0.63 0.63 0.00
12 0.60 0.49 0.11
13 1.61 1.44 0.17
14 2.03 1.89 0.14
15 1.54 1.19 0.35

Sum of differences 0.90
Mean difference (a) 0.06
SE of a (sa) 0.29
t 4df 2.07 NSb



aFree fatty acids plus phospholipids-milliequivalents per liter serum.
bNot significant.


Table 8. Repeatability of the
and phospholipids.


analysis method for free fatty acids













RESULTS AND DISCUSSION


The serum lipid analyses which are presented in this report

should be considered from the points of origin of the materials

analyzed. In terms of quantity, the free fatty acids have the lowest

level normally encountered. It has been shown that the adipose tissue

serves as the major source of free fatty acids in the fasting or post-

absorptive animal. The liver is considered to be the major source of

serum phospholipids and also a source of triglycerides in the post-

absorptive animal. During the period of fat absorption the lymphatic

system transports lipid containing chylomicrons from the intestinal

area to the blood system. The chylomicrons are comprised of approxi-

mately 90 per cent triglyceride, 8 per cent phospholipid and 2 per cent

protein, minor lipid and non-lipid materials. Since the liver, adipose

tissue and other organs to a lesser extent are actively removing the

chylomicrons from circulation, full appreciation of the extent of fat

absorption can not be observed through blood or serum analysis. This

more complete picture would require the use of a lymphatic cannula

for collection of the chylomicrons of the lymph. However, some increase

may be observed in the serum lipid level during the fat absorption

period. The major interest in this study concerned the behavior of

the serum lipid components during the fat absorption period. Serum

lipid patterns were determined after giving the pigs oral doses of

individual fatty acids and triglycerides. Combinations of these materials

were also studied. The establishment of baselines for the components of











Table 9. Serum lipid component levels of pigs used in experiments
obtained during -jctrol periods.



Pig Nro. Weight Triglycerides FFA+PLb Total lipids
(lb.) mg./100 ml. meq./1.c meq./1.c meq./1.c


93 111 104 1.20 0.58 1.78
37 72 111 1.28 0.77 2.05
308 89 137 1.58 1.02 2.60
176 76 1L4 1.66' 0.70 2.36
122 96 127 1.46 0.61 2.07
93a 89 154 1.77 1.77 3.54
155a 81 138 1.59 1.84 3.43
30 72 113 1.30 1.54 2.84
181 113 84 0.97 2.36 3.33
141 91 81 0.93 1.94 2.87
325 125 82 0.94 1.17 2.11
144 124 1.43 1.33 2.76
69 61 65 0.75 0.81 1.56
329 128 89 1.02 1.00 2.02
138 103 88 1.01 1.63 2.64

Av. 110 1.26 1.27 2.53
S.D. 27.2 0.31 0.55 0.61
S.E. 7.0 0.08 0.14 0.16


average over 24; hour period.
Drree fatty acid plus phospholipids


- milliequivalents per liter of serr~..


c;illiequivalenits per liter of seruim.









serum lipids in swine serum was also of interest.

Due to the small numbeiir of animals used in each of the experiments

and the extent of the biological variability, statistical analysis of

the data were not considered feasible. Biological variability has also

limited the conclusions or inferences which may be drawn from the

study. From the extent of the variation encountered in these experiments

several factors should be mentioned. Thie pigs selected for this study

were taken from a very genetically uniform group. They were maintained

in similar environmental conditions for the period the experiments

were conducted. It would then appear that the variation between pigs

is such that larger numbers will be required to conduct future research

of this type. The two to four pigs used in each of the experiments to

be discussed in this report were not adequate for critical analysis of

the results. These observations have also been viewed in the light

of the reports of similar work in the literature. No more than four

animals are reported to have been used on each treatment. In several

cases, strong inferences are drawn from results with only two animals.

It is the author's opinion that these results should have been tempered.

The comparisons to be ma~de between the experiments conducted for this

study and the reports from the literature should be viewed with these

points regarded.

In order to eliminate bias and carryover effects, the experiments

were conducted inl a random pattern. Somce pits wJere used in several

experiments but were given a 48 hour recovery period with a single feeding

of 3 to 5 pounds of the control diet. The control levels of all animals

used in this study are presented in Table 9 along with the standard

deviation among pigs. The diurnal variation was also investigated with









two pigs and the data presented in Table 10 and Figure 4. As N:orrom

et al. (1963) previously noted, the variation in levels may be related

to environcmental te33pErture. Eis experient involved eight pigs and

results were uniformC. Th hih itial reading here may be due to

exciting the pigs during the first handling or they m~ay have still been

in an absorptive state from recent eating since they had free access

to self-feeders. Th.e maximum~; levels after initial reading occurred at

7:00 AX which would have been ac the lowest environmental temperature

enoun~trere and the lowe1st readings at 6:00 P.H. which would be during

thetighest termperature period. Both components measured followed

similar patterns with the free fatty acids plus phospholipids (FFA+PL's)

show3~inT more variation than triglycerides (TG's). Since it was not

possible to use controlled ernvironm~ent in these studies attempts were

made to conduct the exp-erit-ents during the midday period from 9:CO A.M.i

to 3:00 ?.M:. These times were not varied more than 2 hours. Th~is

placed the experimental period at a time after the initial rise had

occurred and before the maxizum1 temperature would have a significant

effect on the results.





Th~e blank studies were ieoigned~ to e'lriminate the e~ffct~s of the

dosin media from th xeieta eut.Tree pigs w~ere used in

this exeien. Lo~sin3 ZiXtures given: the pigs welre identical to

exeietlG mituesecethy contained no test l~ipi. Results
fro cis ra r presrnted in Figure 5 and the average data for the

exercen n abe11 urn che; first our after dosing there Twas

a ~ ~ ~ ~~'- fr1 ntesrm?12's. Trc, 1 co 3 hours after dosing FT~d r's

varied less than 0.; r~ac../1. fro naeaeo e.1 Serum
















Time of Day TGa FF+5 oa


12:00 A.MI. 2.20 1.72 3.92
3:00 P.M. 1.56 1.62 31
6:00 P.MI. 1.36 1.58 2.94
9:00 P.M. 1.53 1.70 3.23
12:00 P.M. 1.80 1.90 3.70
7:00 A.M~. 1.74 2.40 4.14
9:00 A.MI. 1.58 1.72 3.30

Average 1.68 1.81 3.49




aTriglycerides milliequivalents per liter of serum.
bFree fatty acids plus pnlospholipids milliequivalents per liter of
serum.


Table 10. Diurnal variation in scrum lipid components.







2.60


2.40


2.20


2.00


1.80


1.60


1.40


0


2.40


2.20


2.00


1.80


1.60


1.40


1.20


0


Free Fatty Acids
plus Phospholipids


Triglycerides


12:00 N. 6:00 P.M. 12:00 M. 6:00 A.M. 12:00 NT.

Time of Day



Figure 4. Serum patterns of free fatty acids plus phospholipids
and triglycerides during a control period.










Triglycerides


1.20


1.00


0.80


0.60 a/ os


0.40


0.20




4-1 Free Fatty Acids plus PhospholiI
1.60


1.40


1.20


0.80


0.60 .122

176


0.20


0.2



0 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours).


Figure 5. Effect of the blanks on serum free fatty acids plus
phospholipids and serum triglycerides.


pids








TG's showed a slight rise but no major change during the 3-hour

experimental period. The three pigs used did not exhibit similar

TG's patterns during the experiment, therefore no effects of blanks

could be noted. In addition one pig received a blank dose with 5 gm.

of oleic acid added. No differences were noted between the FFA+PL's

pattern of this animal and those receiving the standard blank dose.

These results indicated that the addition of small amounts of a fatty

acid may be used without having a specific effect on the fatty acid

of major interest.

The two pigs selected at random for the palmitic acid experiment

had the highest control level of serum lipids observed among all pigs

used in all experiments. No explanation other than chance selection

could be found for the high levels. Administration of 25 gm. of palmitic

acid in the dosing media resulted in a marked increase in the FFAtPL's

level between 0.5 hours and 4 hours after dosing (Figure 6). Both pigs

showed erratic patterns after 2 hours and through the rest of the

experiment.

The TG's were again too erratic to note any pattern or effect of

palmitic acid. It does appear from these results that the slight

hyperlipemic state of pigs used in this experiment did not preclude

an elevation of the FFA+PL's level following the dose of palmitic acid.

The FFA+PL's level had returned to a near control level by 4 to 5 hours

after dosing in both pigs. The average data for both components

during the experiment are presented in Table 11.

The administration of 25 gm. of stearic acid to the two pigs used

in this experiment produced two quite different sets of results

(Figure 7). This fact eliminates the useful contribution of this







2.80
Free Fatty Acids plus
Phospholipids
2.60


2.40


2.20 ~ \/93


2.00





1.60


1.40




I I\ Triglycerides
2.20


2.00 C /3


er 1.80


S1.60


1.40 Y \ lss


1.20




O 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours).


Figure 6. Effect of palmitic acid on serum free fatty acids plus
phospholipids and serum triglycerides.







2.20


Free Fatty Acids plus Phospholipids


2.00


1.80


1.60


1.40


1.20



S1.00
/323 )(
0.80 3


0.60




;I; I .Triglycerides
2.00


0.1.80


t" 1.60


1.40


1.20


1.00




0 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours) .


Figure 7. Effect of stearic acid on serum free fatty acids plus
phospholipids and serum triglycerides.









experiment. One pig gave no response to the dose of stearic acid.

This pig was later used on an experiment testing oleic acid, again

giving no response. Both the FFA+PL's and the TG's were elevated

between 1 and 4 hours after dosing. Since stearic acid has been

found to be very poorly absorbed by several species in other experiments

the limited data obtained here cannot be used to support or dispute the

findings.

A more consistent response was obtained when four pigs were dosed

with 25 gm. of 01eic acid (Figure 8. If the pig no. 325 which gave

no response to stearic or oleic acid is eliminated, a relatively con-

sistent pattern may be noted in the FFAFPL's. An elevation in FFA+PL's

of 1.0 to 1.8 meq./1. for a period of 1 to 2 hours was observed. The

FFA+PL's level peaked between 2 and 4 hours after dosing and had

returned to near control level 6 hours after dosing. The TG's were

relatively stable for the first 2 hours after dosing. There was a

slow rise in the level between 2 and 4 to 5 hours after dosing.

Due to an experimental failure, only one animal completed the

experiment which tested triolein. These data have not been presented

in a figure but may be found in Table 23. There was a definite response

in the FFAHPL's level. An elevation of 1.2 meq./1. was sustained from

2.5 to 4.5 hours after dosing.


Combined lipid experiments

In order to further evaluate the effects of lipid absorption on

serum lipid components, experiments using a saturated fatty acid plus*

a small quantity of mono-unsaturated fatty acid was conducted.

However, the results obtained from this group of experiments were








2.60 Free Fatty Acidspl s pu


2.40


2.20


2.00


1.80


1.60


1.40


1.20


1.00

bC
0.80


0.60




Triglycerides
1.40 3


1.00


0.80


0.60 /




0 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours) .


Figure 8. Effect of 01eic acid on serum free fatty acids plus
phospholipids and serum triglycerides.













Free Fatty Acids plus Phospholipids


1.20


1.00


0.80


0.60


0.40


0.20


0


1.80


1.60


1.40


1.20


1.00


0.80




0 1:00 2:00 3:00 4:00

Time after dosing (hours) .


Figure 9. Effect of linoleic acid on serum free
phospholipids and serum triglycerides.


5:00 6:00


fatty acids plus

















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Lipid Used 01eic Acid (C18:1) Linoleic Acid (C18:2)d

Timea Tb FAPc Tal Tb FFA+tPLC Total



0:00 0.98 0.74 1.72 1.17 0.93 2.10
0:30 1.4 0.76 1.80 1.04 0.52 1.56
1:00 1.00 0.84 1.84 1.04 0.k0 1.46
1:30 1.18 1.22 2.40 1.18 0.69 1.87
2:00 1.10 1.51 2.61 1.16 0.61 1.77
2:30 1.12 1.60 2.72 1.28 0.40 1.68
3:00 1.06 1.74 2.80 0.46
4:00 1.22 1.38 2.60 1.52 0.74 2.26
5:00 1.04 0.91 1.95 1.53 0.60 2.13
6:00 1.14 0.86 2.00 1.38 0.77 2.15



aTime elapsed after dosing pig.
bTriglycerides milliequivalents per liter of serum.
cFree fatty acids plus phospholipids milliequivalents per liter of
serum.

d33 gm. of 75% linoleic acid.


Table 12. Effect of 01eic acid and linoleic
components during absorption.


acid on serum lil id








too variable to be of value in studying serum lipid patterns. Some

of the observations on these experiments follow but must be viewed

with caution due to this variability.

The first experiment (M1) of this series used a 1:10 ratio of

oleic acid to palmitic acid. As noted in the previous experiments

on the blank doses, the administration of 5 gm. of 01eic acid in

itself did not apparently alter the serum lipid pattern. The second

experiment (M2) used a similar ratio of triolein to palmitic acid.

Three pigs were used in this experiment. In the third experiment (M3)

the ratio of 01eic to palmitic acid was increased to 1:5 by adding 5 gm.

of oleic acid to 25 gm. of palmitic acid. The results of these

experiments are presented in Figures 10, 11, 12 and 13.

In the M1 experiment, all pigs had similar control levels of

FFA+PL's. All pigs responded to the dose through elevations of 1.0

to 1.2 meq./1. in the FFA+PL's level during the first hour. After the

first hour the patterns were irregular and different for each pig,

making further deductions impossible. The three pigs used in the M2

experiment had different control levels and also responded differently.

Two of the three pigs showed marked elevation of the FFA+PL's during

the experiment. The M3 experiment produced results from the two pigs

used which seemed to be in opposition at each time period measured,

thus preventing a meaningful interpretation.

The TG levels in all of the combined lipid experiments did not

exhibit any apparent pattern or degree of change with respect to the

dosing material. The average data for both serum lipid components

are presented in Table 13.

The results obtained in this series of experiments must be








Free Fatty Acids plus Phospholipids


3.80


3.60


3.40


3.20


3.00


2.80


2.60


2.40


2.20


2.00


1.80


1.60


1.40


1.20


1.00


r309
I
r
I
I


0122








6:00


3:00 4:00 5:00


0 1:00 2:00


Time after dosing (hours) .


Figure 10. Effect of a 1:10 ratio of 01eic acid to palmitic acid on
serum free fatty acids plus phospholipids.













Free Fatty Acids plus Phospholipids


3.00


2.80


2.60


2.40


2.20


2.00


1.80


1.60


1.40


1.20


1.00


0.80


308


0 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours) .



Figure 11. Effect of a 1:10 ratio of triolein to palmitic acid
on serum free fatty acids plus phospholipids.











1.20 Triglycerides


1.00 .* *---"









0.40


0.20

0 2.5 gm. triolein plus 25 gm. palmitj


Triglycerides
S2.00


Ei1.80


1.60


1.40


S1.20


1.00


0.80

2.5 gm. 01eic acid plus 25 gm. palt

0 1:00 2:00 3:00 4:00

Time after dosing (hours) .


Figure 12. Effect of 1:10 ratios of 01eic acid
acid on serum triglycerides.


ic acid.


1 308

:ic ac2. 0&d

5:00 6:00


and triolein to palmitic








Free Fatty Acids plus Phospholipids


2.80


2.60


2.4018


2.20


2.00


1.80


1.60


1.40


1.20


S1,00


0.80


0

" Triglycerides
1.20


1.00


0.80

0 5 gm. 01eic acid plus 25 gm. palmitic acid.

O 1:00 2:00 3:00 4:00 5:00 6:00

Time after dosing (hours).


Figure 13. Effect of a 1:5 ratio of oleic acid to palmitic acid
on serum free fatty acids plus phospholipids and serum
triglycerides.

































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interpreted with several facts in mind. An elevation in the FFA+PL's

level cannot be attributed to either the free fatty acid or the phospho-

lipid component categorically. Previous evidence presented by Morris

(1954) and Rampone (1960) suggests the rise in lymph phospholipids,

and subsequently in blood lipids, may be accounted for principally from

endogenous sources. Significant changes in serum free fatty acids

during fat absorption have not been reported. Changes in serum free

fatty acids induced by epinephrine injection or ingestion of materials

stimulating ephinephrine release are of short duration in the pig

(Cunningham and Friend, 1964).

The experiments of Morris (1954) and Rampone (1960) are weak in

terms of the number of animals used in each experiment. Morris used

up to 15 cats and rabbits for establishing post-absorptive level and

relationships between plasma and lymph lipid components but during

the absorption period only three animals were used for each experiment.

This fact would seem to limit the value of the regressions established

in view of the variabilities encountered in this study. Rampone also

used four or less dogs on each experiment in his studies. Separately,

the data from the work of both men lack the desired replication but

the inferences drawn in both reports are similar and tend to become

more substantial. The work by Cunningham and Friend (1964) used

8 to 12 pigs on each of two experiments and obtained small standard

errors for the observations.

The apparent changes in the FFAtPL's level observed in this series

experiments was probably due to the phospholipid component. Phospholipids

have been suggested as a stabilizing element in chylomicrons and

lipoprotein complexes (Mead and Fillerup, 1957). The differences









observed in the magnitude and rate of elevation of the FFA-tPL's level

between individual lipids and combinations of lipids in this study may

be influenced by two factors. The first: factor would be the r-ate of

absorption of the lipid from the intestine which would influence the

rate of appearance of the chylomicrons in the serum. The second factor

would be the amount of the lipid which was absorbed. These should be

the major factors involved but a third factor may be the specific fatty

acid which is being absorbed. As Young (1965) has shown, the saturated

fatty acids require a solubilizing influence for absorption. The same

saturated fatty acids may require an increased level or ratio of

endogenous phospholipids to be transported from the intestinal mucosa.

It is also possible that the inherent lower absorption of the saturated

fatty acids masks the effect of these materials on the observed

FFA+PL's level.

Reiser et al. (1959) noted an increase in the serum phospholipid

level only when doses of saturated fat were given to pigs. No response

was observed when unsaturated fats were given. These results are also

derived from small numbers of animals. Some of Reiser's trials were

limited due to missing data during the course of the trial. Another

criticism would be the lack of attention given to the animals selected

for the trials. A large percentage of Reiser's pigs died from

respiratory ailments. No mention of the state of health of the

animals was given. As previously reviewed, Mead and Fillerup (1957)

found larger quantities of saturated fatty acids than unsaturated

fatty acids going into the phospholipid component. Linoleic acid

was considered to promote more rapid formation of lipoprotein complex,

as well as their more rapid distribution and disappearance from the









serum. The depression obtained in the linoleic acid experiment

of this study may be explained by the more rapid dispersion of serum

lipids during the fat absorption period. However, similar limits

to those suggested for this study were encountered. Only two to four

rats were used for each experiment and considerable variability was

noted.

The increased levels of FFA+PL's when 2.5 gm. of 01eic acid or

triolein were added to doses of palmitic acid would tend to support

the hypothesis of Young (1965). The triolein should have a double

influence by releasing oleic acid and producing monolein. Both of

these materials significantly increased palmitic acid absorption in

Young's studies.

The lack of apparent changes in the TG's level is puzzling.

Since the chylomicrons are 90 per cent triglyceride, one would expect

to see marked alteration in the TC's level during the period of fat

absorption. Th~e erratic patterns make any deductions more difficult.

Hiavel and Goldfien (1961) found a rapid removal of the chylomicrons

by the liver and, to lesser extent, by the adipose tissue. Since the

pig has a greater relative quantity of adipose tissue than other animals,

the quantity of lipids removed by this tissue from circulation would

be expected to be greater. Future observations on the fatty acid

composition of the TG's during fat absorption would aid in the

clarification of this problem.

Since the between animal variation has been a major factor in

preventing the useful analysis of data obtained some suggestions might

be in order to aid in future experiments of this type. The four fatty

acids tested in the first series of experiments would work into a latin








square design using four pigs with the advantage of having each animal

subjected to each treatment. The comparisons developed from this type

of design could also be repeated with different pigs to aid in sub-

stantiating the results. Whether this type of design would be used

or not the evidence presented here definitely presents the require-

ments for more animals to be used in the experiments.

These experiments have produced information on the behavior of

serum lipid components during the period of fat absorption. The

results indicate a need for more information in several areas for proper

evaluation of observations. A major advance would be the ability to

separate the free fatty acid and phospholipid components. More

positive statements on the behavior of these components could be made

rather than the inference attempted at this time. Basic information is

needed on the composition of the phospholipids in swine serum in the

post-absorptive state and the absorptive state. The techniques

developed in this study can be used to further evaluate the dietary

lipids through observations on the serum lipid components. This

evaluation will, in turn, assist both the animal scientist and the

biomedical researcher in understanding the broader implications of

changes in dietary composition.













SUMMARY


A series of experiments were conducted with growing pigs to

determine the effect of individual and combined fatty acids and

triglycerides on serum lipid components. Blood samples were obtained

by means of a venous cannula at intervals during the period of fat

absorption. Control studies and blank dose studies (dosing media

without lipid) were conducted to establish baselines for the experi-

ments. Palmitic, stearic, oleic and linoleic acid and triolein were

studied in individual experiments. 01eic acid and palmitic acid in

ratios of 1:10 and 1:5 and triolein and palmitic acid in a ratio of

1:10 were also examined in the experiment series. Analysis of the

serum extracts were conducted with automated analyzing equipment.

Control levels of serum triglycerides (TG's) averaged 1.26

meq./1 .1 0.08 (110 mg. per cent 7 mg.), serum free fatty acids

plus phospholipids (FFAC-PL's) averaged 1.27 meq./1. 0.14 and total

serum lipids measured in the control periods averaged 2.53 meq./1.

0.16. The blank doses and 5 gm. 01eic acid did not cause appreciable

changes in the pattern of lipid components in the serum.

All lipids were given in 25 gm. amounts added to a dosing media

of ethanol, sodium taurocholate and 1 per cent NaC1. The variability

encountered between pigs on the experiments has severely limited the

conclusions which may be drawn at this time. Pigs dosed with palmitic

and 01eic acid showed apparent increases in the serum FFA+PL's from

1 to 4 hours after dosing. The level returned, in most cases, to









pre-dose levels. Pigs dosed with linoleic acid showed an apparent

decrease in FFA+PL's over the same period as increases had been

noted with palmitic and 01eic acid. Stearic acid and triolein

experiments were of limited value because only a single pig completed

each experiment. The TG values for all experiments were too variable

to show any consistent change in the pattern during the course of the

experiments.

The results from the combined lipid experiments were also quite

variable. The marked increase in FFA+PL's was noted in the experiments

using 1:10 ratios of 01eic acid and triolein. The elevation occurred

in both cases during the first 2 hours of the experiment after which

the pattern became erratic and no further consistent change could be

determined. The third experiment which used a 1:5 ratio of 01eic

acid to palmitic acid, was also inconclusive because of lack of

agreement between the serum patterns of the two animals used. No

consistent pattern in the TG's were noted in the combined lipids

section of this study.

The changes noted in the FFA+IPL's level in the study are suggested

to be related to the phospholipid component. In addition it is also

suggested that this increase may be due to endogenous phospholipid

rather than directly related to the lipid being absorbed during the

experiment. The phospholipid may be involved as a stabilizing element

in lipoprotein complexes and chylomicrons. The absorption of saturated

fatty acids would be expected to require more stabilizing elements

since it has been proven in previous reports that saturated fatty acids

require a solubilizing influence of an unsaturated fatty acid.

Inadequate data were obtained in the combined lipid section of this





69


study to relate to these findings.

The lack of an alteration in the TG's pattern in all experiments

may relate to rate of removal from the blood by the liver and the

more extensive quantity of adipose tissue in the pig.

From the results of this study, the need for more animals per

experiment becomes evident. A means of designing experiments to

remove as much individual variation as possible is suggested for

future experiments. This study has produced some new and interesting

data on changes in serum lipid component patterns. The techniques

presented may be used to further the work in fat absorption and its

influences on the physiological status of the body compartments.






































APPENDIX











Table 14. Diurnal variation in serum lipid components Individual
observations.



Pig No. 93 155
d ba FAPb oa
Time of Day TG FFM+PL Total TG FAL Tol


12:00 A.M. 2.48 1.47 3.95 1.91 1.96 3.87
3:00 P.M. 1.59 1.75 3.34 1.54 1.50 3.04
6:00 P.M. 1.44 1.68 3.12 1.28 1.47 2.75
9:00 P.M. 1.61 1.61 3.22 1.45 1.78 3.23
12:00 P.M. 1.79 1.92 3.71 1.81 1.89 3.70
7:00 A.M. 1.76 2.20 3.96 1.72 2.59 4.31
9:00 A.M. 1.72 1.75 3.47 1.43 1.68 3.11

Average 1.77 1.77 3.54 1.59 1.84 3.43



aTriglycerides milliequivalents per liter of serum.
bpree fatty acids plus phospholipid milliequivalents per liter of serum.











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Table 16. Effect of palmitic acid on serum lipid components during
absorption Individual observations.



Pig No. 93 155

Timea Tb FFA+PLC Ttl Tb FFM+PLC Total



0:00 1.71 1.75 3.46 1.41 1.68 3.09
0:30 2.37 1.47 3.84 1.66 1.72 3.38
1:00 2.00 1.61 3.61 1.32 2.24 3.56
1:30 1.87 1.92 3.79 1.69 2.56 4.25
2:00 2.14 2.10 4.24 1.52 2.31 3.83
2:30 2.00 2.31 4.31 1.35 2.14 3.49
3:00 2.10 2.03 4.13 1.54 2.80 4.34
4:00 2.02 2.21 4.23 1.42 1.89 3.31
5:00 1.78 1.65 3.43
6:00 2.03 2.03 4.06



aTime elapsed after dosing pig.
bTriglycerides milliequivalents per liter of serum.
cFree fatty acid plus phospholipid milliequivalent per liter of serum.










Table 17. Effect of stearic acid on serum lipid components during
absorption Individual observations.



Pig No. 144 325

Tma b c b FtPc oa
Time TG FFA+PL Total TG FAP oa


0:00 1.43 1.33 2.76 1.03 0.91 1.94
0:30 1.49 0.98 2.47 1.03 0.98 2.01
1:00 1.29 1.08 2.37 0.90 0.94 1.84
1:30 1.55 2.14 3.69 0.85
2:00 1.55 1.68 3.23 0.63
2:30 2.05 1.64 3.69 0.89
3:00 1.28 1.40 2.68 0.88 0.56 1.44
4:00 1.40 1.12 2.52 1.12 0.63 1.75
5:00 1.36 1.12 2.48 0.98 0.91 1.89
6:00 1.55 0.94 2.49



aTime elapsed after dosing pig.
bTriglycerides milliequivalents per liter of serum,
cFree fatty acid plus phospholipid milliequivalent per liter of serum.















OOOHNNHHOH



II







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dd o BBd
O OO Ne
ad eme me~O~nll







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U OMMHMH 0 0 .E
a 3A
PIT
o II IOI I a,
vl II I IOIOv
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Table 19. Effect of linoleic acid on serum li id components during
absorption Individual observath.



Pig No. 93 37

T8 bF~~L c b c
Time TG FFPAPL Total TG FAP Tol


0:00 1.22 1.12 2.34 1.13 0.74 1.87
0:30 1.15 0.51 1.66 0.92 0.54 1.46
1:00 1.07 0.46 1.53 1.00 0.35 1.35
1:30 1.23 0.77 2.00 1.14 0.62 1.76
2:00 1.23 0.52 1.75 1.09 0.70 1.79
2:30 1.38 0.44 1.82 1.18 0.37 1.55
3:00 0.49 0.42
4:00 1.50 0.45 1.95 1.53 1.02 2.55
5:00 1.30 0.62 1.92 1.74 0.58 2.32
6:00 1.32 0.84 2.16 1.44 0.70 2.14



aTime elapsed after dosing pig.
bTriglycerides milliequivalents per liter of serum.
cFree fatty acid plus phospholipid milliequivalent per liter of serum.
dPigs received 33 gm. of 75% linoleic acid.











mom-en mmwde






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Table 22. Effect of combined mix 3 on serum li lid components during
absorption Individual observations.



Pig No. 181 141


Time TGb FFAtPLc Total TGb FF~ oa


0:00 0.97 2.73 3.70 0.93 2.13 3.06
0:30 0.92 2.00 2.92 1.22 1.58 2.80
1:00 0.86 2.84 3.70 1.04 1.78 2.82
1:30 1.00 1.26 2.26 1.17 1.72 2.89
2:00 0.93 1.22 2.15 0.98 2.59 3.57
2:30 1.08 1.86 2.94 1.13 1.15 2.28
3:00 0.85 2.42 3.27 0.93 0.88 1.81
4:00 1.18 0.84 2.02
5:00 1.24 0.84 2.08
6:00 1.07 1.12 2.19



aTime elapsed after dosing pig.
bTriglycerides milliequivalents per liter of serum.
cFree fatty acid plus phospholipid milliequivalent per liter of serum.
dPigs received 25 gm. palmitic acid and 5.0 gm. 01eic acid.
















Pig No. 30


Timea TGb FFA+PLc Total


0:00 1.38 1.40 2.78
0:30 1.06 1.03 2.09
1:00 1.04 1.68 2.72
1:30 1.26 1.75 3.01
2:00 1.04 1.47 2.51
2:30
3:00 0.98 2.34 3.32
4:00 0.83 2.80 3.63
5:00 0.89 1.33 2.22
6:00 0.92 1.64 2.56


lipid components during absorption -


Table 23. Effect of triolein on serum
Individual observation.


aTime elapsed after dosing pig.
bTriglycerides milliequivalents per


liter of serum.


cFree fatty acid plus phospholipid milliequivalent per liter


of serum.














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BIOGRAPHICAL SKETCH


Roger Clark Crum, Jr., was born October 22, 1938, in Eldorado,

Union County, Arkansas. He was raised on a family farm and attended

elementary and high school at Humphrey, Arkansas. In September of

1956 he entered the University of Arkansas and was graduated with a

Bachelor of Science in Agriculture in June, 1960. During this time

he held an ESSO Four-H Club Scholarship.

In January, 1961, he entered Mississippi State University where

he majored in Animal Nutrition and received the Master of Science in

Agriculture in May, 1962. During this period of study he was granted

a graduate assistantship.

In 1962 he was employed as a summer research assistant in the

Nutrition and Metabolic Disease Section of the research division of

the Upjohn Company, Kalamazoo, Michigan.

In September, 1962, he was granted a research assistantship from

the University of Florida where he has continued hiis studies in

Animal Nutrition with a minor in Biochemistry. He is now a candidate

for the degree of Doctor of Phiilosophy in Animal Nutrition.

Hle is a member of the American Society of Animal Science and

Amierican Oil Chemist Society. He has been elected to the honor

societies of the Sigma Xi and Gamma Sigma Delta and the social frater-

nity of Alpha Gamma Rho.

He was married to the former Brenda Ingram of Lavaca, Arkansas,

in June of 1961.













This dissertation was prepared under the direction of the

chairman of the candidate's supervisory committee and has been

approved by all members of that committee. It was submitted to

the Dean of the College of Agriculture and to the Graduate Council,

and was approved as partial fulfillment of the requirements for

the degree of Doctor of Philosophy.


December 18, 1965



eanan College of Agriculture





Dean, Graduate School

SUPERVI IRY COMMITTEE:



Chairman




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