Essentiality of arginine in total parenteral nutrition of the rat

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Essentiality of arginine in total parenteral nutrition of the rat
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Moore, Linda Fay
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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
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Includes bibliographical references (leaves 215-224).
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by Linda Fay Moore.
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Vita.

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ESSENTIALITY OF ARGININE IN TOTAL
PARENTERAL NUTRITION OF THE RAT
















By


LINDA FAY MOORE


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


UNIVERSITY OF FLORIDA


1989













ACKNOWLEDGEMENTS


The author wishes to thank Dr. Harry S. Sitren for his

support throughout this research. In addition, she wishes

to thank all of the members of the supervisory committee,

Drs. R.S. Shireman, P.R. Borum, R.P. Bates, T.G. Baumgartner

and M.S. Kilberg, for their advice and support during this

project.

A special thank you goes to Dr. Thomas G. Baumgartner

for his instruction in compounding TPN formulas and

arrangement for the use of the IV Prep room at Shands

Hospital; and to Gerald Schmidt for compounding the first

TPN diet.

The author wishes to thank the surgical residents, Drs.

Samuel Mahaffy, Mark Pesa, Lee Ellis, Michael Bryant and

Suzanne Klimberg, who participated as members of the

surgical team for catheterization of the rats. In addition,

a thank you goes to Drs. W.W. Souba, Suzanne Klimberg and

Rabih Salloum who collaborated in the determination of blood

flows.

The author wishes to thank Virginia Wiley for

performing the amino acid analyses, Walter Jones for his

help with graphics and Juanita Bagnall for her laboratory

assistance.

A special thank you goes to Dr. Rachel Shireman for her

advice and support throughout the author's student tenure.















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS..................................... ii

LIST OF FIGURES ....................................... v

LIST OF TABLES...... ............................... ..... x

LIST OF ABBREVIATIONS................................. xiii

ABSTRACT............................................... xv

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

LITERATURE REVIEW.... ................................. 4

Total Parenteral Nutrition........................ 4
Hyperammonemia in TPN............................. 4
Role of Arginine in Prevention of Hyperammonemia.. 6
Urea Cycle... ................................... .. 7
Source and Fate of Arginine, Ornithine and
Citrulline....................................... 13
Orotate as Arginine Deficiency Indicator.......... 24
Measurement of Nutrient Requirement or Adequacy... 28

MATERIALS AND METHODS .................................. 31

Materials and Equipment............................ 31
Animals........... ............... ................ 31
Diets................. ....................... .... 32
Lactalbumin Control Diet...................... 32
TPN Diets of Arginine Supplementation......... 33
Reagents... .. .. ..... ...... ............. ....... 34
Spectrophotometer............................... 35
Technicon AutoAnalyzer.......................... 35
Urinary Metabolite Analysis...................... 35
Urine Collection and Sample Preparation......... 35
Urinary Metabolite Analysis..................... 36
Urinary Urea Nitrogen.......................... 36
Creatinine......... ........................... 36
Urinary Total Nitrogen........................ 36
Orotic Acid........................ ............. 37
Citric Acid .................................... 37


iii








Page

Amino Acid Analysis............................... 38
Sample Preparation............................. 38
Plasma........................................ 38
Hydrolysis of Lactalbumin..................... 39
TPN Amino Acid Solution ....................... 39
Analysis. ...................................... 39
Physiological Samples......................... 39
Hydrolysis Sample............................. 39
Urea Cycle Enzyme Activities....................... 40
Sample Preparation............................... 40
Urea Cycle Enzyme Analysis...................... 40
Carbamoyl Phosphate Synthetase-I.............. 41
Argininosuccinate Synthetase................... 41
Arginase........................................... 41
Protein Determination........................... 42
Miscellaneous Analytical Methods................... 42
Organ Moisture.................................. 42
Organ Total Nitrogen............................ 42
Dietary Nitrogen................................ 43
Serum Urea Nitrogen............................ 43
Technique for TPN.................................. 43
Catheterization. ....... ......................... 43
Administration of Total Parenteral Nutrition.... 45
Experimental Protocols............................ 47
Development of a TPN Model for Determining
Arginine Adequacy.............................. 47
Establishment of Arginine-Adequacy in TPN....... 49
Assessing Urea Cycle Enzyme Activities under
TPN............................................. 51
Determination of Arteriovenous Amino Acid
Differences .... ................................ 52

RESULTS AND DISCUSSION................................ 54

Development of a TPN Model for Determining
Arginine Adequacy................................ 54
Determination of Arginine Adequacy in TPN......... 105
Assessing Urea Cycle Enzyme Activities under
TPN....................................... ........ 127
Determination of Arteriovenous Amino Acid
Differences...................................... 137

SUMMARY AND CONCLUSIONS............................... 152

APPENDIX I DIETARY COMPOSITIONS.................... 157

APPENDIX II METHODS................................ 168

REFERENCES............................................. 215

BIOGRAPHICAL SKETCH.................................... 225














LIST OF FIGURES


figure page


1. Enzymes Involved and Cellular Compartmental-
ization of the Urea Cycle........................ 8


2. Average Daily Urinary Orotic Acid Excretion of
Rats Administered a TPN Formula Supplemented with
Either 4.7% Arginine or 0% Arginine per Total
Amino Acids for Four Days......................... 57



3. Average Daily Urinary Citric Acid Excretion of
Rats Administered a TPN Formula Supplemented with
Either 4.7% Arginine or 0% Arginine per Total
Amino Acids for Four Days......................... 58


4. Average Daily Urinary Urea Nitrogen Excretion of
Rats Administered a TPN Formula Supplemented with
Either 4.7% Arginine or 0% Arginine per Total
Amino Acids for Four Days......................... 59


5. Average Daily Urine Volume of Rats Administered a
TPN Formula Supplemented with Either 4.7% Arg-
inine or 0% Arginine per Total Amino Acids for
Four Days....................................... 61


6. Average Daily Urinary Creatinine Excretion of Rats
Administered a TPN Formula Supplemented with
Either 4.7% Arginine or 0% Arginine per Total
Amino Acids for Four Days......................... 62


7. Average Body Weight Gain of Rats Orally-Fed One
of Two Levels of Arginine, then, One of Two Levels
of Glutamine Supplemented TPN Formula and Controls. 66








Figure


8. Average Daily Urine Volume of Rats Orally-Fed One
of Two Levels of Arginine, then, One of Two
Levels of Glutamine Supplemented TPN Formula and
Controls............................. ..... ..... ... 72


9. Average Daily Orotic Acid Excretion of Rats
Orally-Fed One of Two Levels of Arginine, then,
One of Two Levels of Glutamine Supplemented TPN
Formula and Controls ............................. 73

10. Superimposing Urinary Orotic Acid Excretion Data
of Rats Orally-Fed One of Two Levels of Arginine,
then, One of Two Levels of Glutamine Supplemented
TPN Formula........................................ 75


11. Average Daily Urinary Citric Acid Excretion of
Rats Orally-Fed One of Two Levels of Arginine,
then, One of Two Levels of Glutamine Supplemented
TPN Formula and Controls......................... 77


12. Average Daily Urinary Urea Nitrogen Excretion of
Rats Orally-Fed One of Two Levels of Arginine,
then, One of Two Levels of Glutamine Supplemented
TPN Formula and Controls.......................... 79


13. Average Daily Urinary Total Nitrogen Excretion of
Rats Orally-Fed One of Two Levels of Arginine,
then, One of Two Levels of Glutamine Supplemented
TPN Formula and Controls.......................... 80


14. Average Daily Urinary Creatinine Excretion of Rats
Orally-Fed One of Two Levels of Arginine, then,
One of Two Levels of Glutamine Supplemented TPN
Formula and Controls.............................. 82


15. Average Daily Urine Volumes of Rats Administered
a TPN Formula Supplemented at One of Two Levels of
Arginine, then, One of Two Levels of Glutamine and
Controls............................................ 92











16. Daily Urinary Orotic Acid Excretion of an Animal
to Which a TPN Formula Supplemented to 3.23%
Arginine, then, 5.36% Glutamine was
Successfully Administered Throughout the Twenty-
Two-Day Infusion ................................. 94


17. Average Daily Urine Volumes of Rats Administered
a TPN Formula Supplemented to Either 3.27%
Arginine or 5.3% Glutamine and Controls........... 98


18. Average Daily Urinary Orotic Acid Excretion of
Rats Administered a TPN Formula Supplemented to
Either 3.27% Arginine or 5.3% Glutamine and
Controls............................................ 99


19. Average Daily Urinary Citric Acid Excretion of
Rats Administered a TPN Formula Supplemented to
Either 3.27% Arginine or 5.3% Glutamine and
Controls.............. ........................... 101


20. Average Daily Urinary Urea Nitrogen Excretion of
Rats Administered a TPN Formula Supplemented to
Either 3.27% Arginine or 5.3% Glutamine and
Controls........................................... 103


21. Average Daily Urinary Creatinine Excretion of Rats
Administered a TPN Formula Supplemented to Either
3.27% Arginine or 5.3% Glutamine and Controls..... 104


22. Average Daily Urinary Orotic Acid Excretion of
Rats Administered a TPN Formula Supplemented With
Graded Levels of Arginine and Controls............ 111


23. Dose Response Curve of Urinary Orotic Acid
Excretion to Percent Arginine Supplementation for
Rats Administered a TPN Formula Supplemented with
Graded Levels of Arginine Between Twenty-Four and
One Hundred and Twenty Hours of Supplemented
Infusion......................................... 113


vii








Figure


24. Regression Line of Urinary Orotic Acid Excretion
versus Percent Arginine Supplementation for Rats
Administered a TPN Formula Supplemented to Either
2.64%, 3.42% or 5.14% Arginine per Total Amino
Acids in the TPN Formula.......................... 114


25. Average Daily Urinary Urea Nitrogen Excretion of
Rats Administered a TPN Formula Supplemented With
Graded Levels of Arginine and Their Respective
Controls........................................... 115


26. Average Daily Urinary Total Nitrogen Excretion of
Rats Administered a TPN Formula Supplemented with
Graded Levels of Arginine and Their Respective
Controls........................................... 116


27. Ratio of Average Daily Urinary Excretion of Orotic
Acid to Urea Nitrogen of Rats Administered a TPN
Formula Supplemented with Graded Levels of
Arginine and Their Respective Controls............ 119


28. Ratio of Average Daily Urinary Excretion of Orotic
Acid to Total Nitrogen of Rats Administered a TPN
Formula Supplemented with Graded Levels of
Arginine and Their Respective Controls............ 120


29. Average Daily Urinary Creatinine Excretion of Rats
Administered a TPN Formula Supplemented with
Graded Levels of Arginine and Their Respective
Controls........................................... 122


30. Average Urinary Citric Acid Excretion of Rats
After a Three-Day Surgical Recovery Period, a
Four-Day Arginine-Devoid TPN Administration and
a Five-Day Administration of a TPN Formula
Supplemented with Graded Levels of Arginine and
Controls.......................................... 124


31. Average Daily Urinary Orotic Acid Excretion of
Rats Administered Either 0% Arginine or 4.5%
Arginine and Controls............................. 131


viii


Page








Figure Page


32. Average Daily Urinary Urea Nitrogen Excretion of
Rats Administered Either 0% Arginine or 4.5%
Arginine and Controls............................. 132


33. Average Daily Urinary Total Nitrogen Excretion of
Rats Administered Either 0% Arginine or 4.5%
Arginine and Controls............................ 133


34. Average Daily Urinary Creatinine Excretion of
Rats Administered Either 0% Arginine or 4.5%
Arginine and Controls............................. 135


35. Manifold for the Determination of Urinary Orotic
Acid .............................................. 172


36. Manifold for the Determination of Urinary Urea
Nitrogen............................................ 175


37. Manifold for the Semi-Automated Determination of
Kjeldahl Nitrogen................................ 179

38. Manifold for the Determination of Urinary
Creatinine ........................................ 181














LIST OF TABLES


Table Page


1. Growth Rate in Animals Supplemented with
Arginine, Citrulline or Ornithine................. 14


2. Tissue Distribution of Urea Cycle Enzyme
Activities............................ ........... 16


3. Body and Organ Weights after Ninety-six Hours of
Total Parenteral Nutrition........................ 55


4. Urea Cycle Enzyme Activities After Ninety-six
Hours of Total Parenteral Nutrition................ 64


5. Body Weights at Selected Intervals for Rats
Orally-Fed a Total Parenteral Nutrition Diet...... 67


6. Food Consumption as Expressed by Nitrogen Intake
for Rats Orally-Fed a Total Parenteral Nutrition
Diet.............................................. 69


7. Organ Weights for Rats Orally-Fed a Total
Parenteral Nutrition Diet Containing Either 1.61%
or 5.36% Glutamine and Control.................... 71


8. Urea Cycle enzyme Activities for Rats Orally-Fed
a Total Parenteral Nutrition Diet Containing
Either 1.61% or 5.36% Glutamine and Control........ 84


9. Plasma Amino Acid Concentrations for Rats Orally-
Fed a Total Parenteral Nutrition Diet Containing
Either 1.61% or 5.36% Glutamine and Control........ 85








Table Page


10. Body and Organ Weights for Rats Administered Total
Parenteral Nutrition Diets Supplemented to Two
Levels of Arginine and Two Levels of Glutamine
and Control................................... 91

11. Urinary Orotic Acid Excretion for Rats
Administered Total Parenteral Nutrition Diets
Supplemented to Two Levels of Arginine and Two
Levels of Glutamine and Control................... 93


12. Final Body and Organ Weights for Rats Administered
a Total Parenteral Nutrition Diet Containing
Either 3.27% Arginine or 5.35% Glutamine.......... 96


13. Body Weights and Organ Weights for Rats
Administered Graded Levels of Arginine by Total
Parenteral Nutrition.............................. 107


14. Moisture and Total Nitrogen Content of Organs
from Rats Fed Either 2.64% Arginine or 5.14%
Arginine Supplemented Total Parenteral Nutrition
and Control........................................ 109


15. Summary of Plasma Amino Acid Concentrations for
Rats Fed Graded Levels of Arginine by Total
Parenteral Nutrition and Controls................. 125


16. Body Weight Gain and Organ Weights for Rats
Administered 0% Arginine or 4.5% Arginine by
Total Parenteral Nutrition and Their Respective
Controls................................ ... ......... 129


17. Urea Cycle Enzyme Activities for Rats Administered
0% Arginine or 4.5% Arginine by Total Parenteral
Nutrition and Their Respective Controls........... 136


18. Portal and Renal Vein Blood Flow for Rats on
Total Parenteral Nutrition or Lactalbumin
Control............................................ 138








Table


19. Arteriovenous Amino Acid Differences Between the
Carotid and Hepatic Vein of Rats Administered a
TPN Formula Containing Either 0% Arginine or 4.5%
Arginine to Total Amino Acids and Controls......... 141

20. Arteriovenous Amino Acid Differences Between the
Carotid and Portal Vein of Rats Administered a
TPN Formula Containing Either 0% Arginine or 4.5%
Arginine to Total Amino Acids and Controls........ 142

21. Arteriovenous Amino Acid Differences Between the
Carotid and Renal Vein of Rats Administered a
TPN Formula Containing Either 0% Arginine or 4.5%
Arginine to Total Amino Acids and Controls......... 143

22. Extraction Coefficient for Glutamine, Citrulline,
Ornithine and Arginine in the Liver and Kidney of
Rats Administered Either 0% or 4.5% Arginine TPN
and Controls..................................... 144

23. Amino Acid Flux for Glutamine, Citrulline,
Ornithine and Arginine in the Portal and Kidney
Veins of Rats Administered Either 0% or 4.5%
Arginine TPN and Controls......................... 145

24. Plasma Amino Acid Concentration in the Carotid
Artery of Rats Administered a TPN Formula
Containing Either 0% Arginine or 4.5% Arginine
to Total Amino Acids and Controls.................. 146

25. Plasma Amino Acid Concentration in the Hepatic
Vein of Rats Administered a TPN Formula Containing
Either 0% Arginine or 4.5% Arginine to Total Amino
Acids and Controls................................ 147

26. Plasma Amino Acid Concentration in the Portal
Vein of Rats Administered a TPN Formula Containing
Either 0% Arginine or 4.5% Arginine to Total Amino
Acids and Controls................................. 148

27. Plasma Amino Acid Concentration in the Renal
Vein of Rats Administered a TPN Formula Containing
Either 0% Arginine or 4.5% Arginine to Total Amino
Acids and Controls............. ................. 149


xii














LIST OF ABBREVIATIONS


Terms

AGPA L-z-amino-,/-guanidinopropionic acid
BSA bovine serum albumin
BW body weight
CP carbamoyl phosphate
CTAB Hexadecyltrimethylammonium bromide
d-d-H20 distilled-deionized water
DAMO 2,3-butanedione monoxime
GSA glutamate-2-semialdehyde
i.d. inside diameter
MOPS 3-[morpholino]propanesulfonic acid
NAG N-acetylglutamate
o.d. outside diameter
PAH para-amino hippurate
P-5-C pyrroline-5-carboxylate
q.s. quantus suffa; bring to volume
rpm revolutions per minute
TCA trichloroacetic acid
TN total nitrogen
TPN total parenteral nutrition
TRIZMA (Tris-(hydroxymethyl) aminomethane
UUN urinary urea nitrogen


Enzymes and Cofactors

A arginase
ADP adenosine diphosphate
AL argininosuccinate lyase
AMP adenosine monophosphate
AS argininosuccinate synthetase
ATP adenosine triphosphate
CL citrate lyase
CPS-I carbamoyl phosphate synthetase-I
EDTA ethylenediaminetetraacetic acid
MDH malate dehydrogenase
NAD+ nicotinamide adenine dinculeotide
NADH reduced nicotinamide adenine dinucleotide
OAT ornithine aminotransferase
OCT ornithine carbamoyl transferase
PEP phosphoenolpyruvate
U units of activity


xiii










Units of Measure


molecular weight
kilogram
milligram
millimole
molar
milliequivalents
liter
milliliter
day
minute
centimeter
micron or micrometer


g
Mg
Mmol
mM
mOsmol
dL
AL
hr
sec
mm
nm


grams
microgram
micromole
millimolar
milliosmols
deciliter
microliter
hour
second
milimeter
nanometer


Amino Acids


alanine
arginine
asparagine
aspartate
citrulline
cysteine
glutamine
glutamate
glycine
histidine
isoleucine
amino acid


Leu
Lys
Met
Orn
Phe
Pro
Ser
Thr
Trp
Tyr
Val
TAA


leucine
lysine
methionine
ornithine
phenylalanine
proline
serine
threonine
tryptophan
tyrosine
valine
total amino acids


xiv


Mw
Kg
mg
mmol
M
mEq
L
mL
d
min
cm
jm


Ala
Arg
Asn
Asp
Cit
Cys
Gln
Glu
Gly
His
Ile
AA














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ESSENTIALITY OF ARGININE IN TOTAL
PARENTERAL NUTRITION OF THE RAT

By

Linda Fay Moore

December 1989

Cochairmen: Harry S. Sitren and Rachel S. Shireman
Major Department: Food Science and Human Nutrition

Total parenteral nutrition (TPN) is a nutritional

management technique for individuals who are unable to

tolerate food by the oral or enteral route. A rare

complication of TPN is hyperammonemia which is unrelated to

organ dysfunction and there is clinical evidence that

sufficient arginine in the TPN formulation alleviates and

prevents recurrence of hyperammonemia. The etiology of TPN-

associated hyperammonemia is unclear; it may be due to

inadequate supply of arginine and/or one of its precursors

to the liver or kidney for urea synthesis or to insufficient

urea cycle enzyme activity.

This research established an appropriate rat TPN model

for the study of arginine-related urea cycle insufficiency.

Young adult rats (250 g) were maintained on TPN containing

approximately 1.6, 2.5, 3.2 and 4.8% arginine after a 4-day








arginine-free infusion. The formula also contained

dextrose, fat emulsion, vitamins and minerals and provided

290 mg N and 60 non-N Kcal/day. During the arginine-free

period, urinary excretion of orotate increased 20-fold,

urinary citrate increased 6-fold and urinary urea and total

N doubled. Inclusion of arginine in the TPN formula lowered

urinary orotate to control levels within 48 hours and

urinary urea and total N within 96 hours with all formulas

except 1.6%. The plasma urea cycle amino acids, arginine

and citrulline were depressed in all TPN groups after 5 days

of supplemented infusion. Regression analysis suggested an

arginine requirement of 4.1% arg (R2=0.825, p<.001) which

resulted in an orotate excretion of < 100 Ag/day. This

compares with a reported need of 6-8% to prevent orotic

aciduria in rats on semi-purified diets. Urea cycle enzyme

activities of carbamoyl phosphate synthetase,

argininosuccinate synthetase and arginase were elevated in

the liver during an arginine-devoid TPN infusion relative to

the enzyme activities during a 4.5% arginine-supplemented

period or in control rats. Arginine supplementation

resulted in increased kidney arginase activities relative to

control and arginine-devoid groups. Portal and renal blood

flows were elevated 1.4 and 1.3-fold, respectively, in TPN-

administered rats relative to controls.


xvi














INTRODUCTION


Total parenteral nutrition (TPN) is a nutritional

management technique for individuals who are unable to

tolerate food by the oral or enteral route. An uncommon,

but potentially fatal, complication of TPN is hyperammonemia

which can be unrelated to hepatic or renal insufficiency.

This type of hyperammonemia is indicative of compromised

nitrogen disposal mechanisms, the etiology of which has been

ascribed to an arginine deficiency in the TPN formulation.

Arginine is not considered an essential amino acid for

humans according to the Recommended Dietary Allowances

(National Research Council, 1980), but is thought perhaps to

e limiting under special circumstances of infant parenteral

feeding as reported by Heird et al. (1972). Arginine

requirements have not been established for TPN, nor have

arginine metabolism and urea cycle activities been studied

under TPN.

Nitrogen intake in excess of requirements is disposed

primarily by the synthesis and urinary excretion of urea.

Only the liver contains sufficient quantities of all five of

the urea cycle enzymes to detoxify excess nitrogen and thus,

the liver is the major site for urea synthesis. However,










the liver does not release arginine in any appreciable

amounts to the systemic circulation. Although the kidney

also contains all five of the urea cycle enzymes, the major

role of these enzymes in the kidney is to supply arginine to

peripheral tissues (Featherston et al., 1973). Although the

etiology of TPN-associated hyperammonemia has been ascribed

to a dietary deficiency in arginine, alterations in urea

cycle enzyme activities and/or the supply of arginine

precursors to the liver or kidney may also play a role and

may lead to the requirement for arginine during TPN. Thus,

urea cycle enzyme activities and precursor supply to the

liver and kidney should be evaluated.

Adult rats have also been shown to be sensitive to

arginine deficiency, which is revealed by urinary excretion

of orotic acid, a metabolite which is present in abnormally

high amounts and is an indicator of urea cycle

insufficiency. The rat is a well established model for the

study of arginine metabolism (Milner, 1985; Visek and

Shoemaker, 1986) and has been used extensively as a TPN

model since 1972 (Steiger et al., 1972).

The specific objectives of this research were to

1. Develop an appropriate model to assess arginine

metabolism and adequacy for young adult rats on TPN.

2. Determine the adequacy of arginine for young adult

rats on TPN.

3. Determine carbamoyl phosphate synthetase (CPS-I),










argininosuccinate synthetase (AS) and arginase (A) enzyme

activities in the liver and argininosuccinate synthetase

(AS) and arginase (A) enzyme activities in the kidney of

arginine deficient and arginine sufficient rats on TPN.

4. Measure arteriovenous amino acid differences across

the liver and the kidney in arginine deficient and

sufficient rats on TPN.

Arginine adequacy was assessed as the ability of

arginine replacement in a TPN formulation to decrease and

maintain low urinary orotate excretion, an index of urea

cycle sufficiency. Arginine adequacy studies yielded

information on the appropriate arg/total amino acid ratio in

the TPN formulation that provided the rat adequate arginine

for optimal operation of the urea cycle as well as for

peripheral protein synthesis. Urea cycle enzyme activities

provided information on the capacity of the liver to

detoxify ammonia as well as the capacity of the kidney to

supply arginine to peripheral tissues during TPN. Amino

acid difference data provided information on arginine

utilization and arginine precursor metabolism in the from

the gut, liver and kidney.














REVIEW OF THE LITERATURE


Total Parenteral Nutrition


Total parenteral nutrition (TPN) has been in clinical

practice since the late 1960s. Total parenteral nutrition

is a feeding technique which provides all or nearly all of

the nutritional needs of an individual intravenously

(Reinhardt et al., 1977). This definition of TPN differs

from that of intravenous hyperalimentation which Rhoads et

al. (1981) defines as the intravenous administration of

nutrients in amounts substantially in excess of maintenance

requirements and is therefore designed for correction of

deficits.



Hyperammonemia in TPN



Total parenteral nutrition-associated hyperammonemia or

excessive blood ammonia concentrations is unrelated to

metabolic complications. In humans, hyperammonemia is

defined as blood ammonia levels greater than 250 Ag/dL

(Seashore, 1980). Total parenteral nutrition-associated

hyperammonemia was at one time ascribed to the use of








5
protein hydrolysates made from fibrin or casein in which as

much as 8 to 10% of the available nitrogen in the product

was derived from free ammonia as a by-product of the protein

processing method (Dudrick et al., 1972). However,

hyperammonemia has also been experienced by patients

receiving TPN solutions comprised of crystalline L-amino

acids in which free ammonia levels of the TPN solutions are

low (Heird et al., 1972). For example, ammonia levels in

L-amino acid solutions range from 135-600 Ag/dL depending

upon manufacturer (Heird et al., 1972; Caldwell et al.,

1977; Seashore, 1980) versus 20,000-36,000 gg/dL in 3.0%

protein hydrolysates (Heird et al., 1972).

Although the etiology of TPN-associated hyperammonemia

is still unclear, Johnson et al. (1972) was one of the first

to demonstrate that hyperammonemia was directly proportional

to the amount of protein hydrolysate infused under TPN. In

this study, an infusion rate between 256 to 611 mg N/kg body

weight/day (1.60-3.89 g amino acids/kg BW/day) of either

fibrin or casein hydrolysate resulted in blood ammonia

levels of 103 to 304 jug NH3-N/dL in infants between 2 and

135 days of age at the initiation of TPN (9323 gg NH3-N/dL

is normal for the neonatal period). A supplement of 350

mg/day of arginine (2.01 mmol/day) did not improve the

hyperammonemia. In contrast to this study, Heird et al.

(1972) demonstrated that in their patients receiving a TPN

solution of approximately 2.5 g/kg body weight/day of










synthetic L-amino acids, hyperammonemia could be corrected

within as little as 4 hours after the administration of 2.0

mmol arginine-glutamate/kg BW or 3 mmol arginine-HCl/kg BW

to the daily infusate.



Role of Arginine in Prevention of Hyperammonemia



Although not specifically investigated, the ability of

arginine to decrease blood ammonia levels has been

attributed to the role of arginine in the urea cycle.

Arginine participates directly in the urea cycle by two

different modes of action. First, it is the substrate for

arginase which forms urea and ornithine, a waste product and

urea cycle intermediate, respectively. Secondly, arginine

has been shown in vitro to have a stimulatory effect on the

enzyme N-acetylglutamate synthetase (Kawamoto et al., 1982)

which is independent of de novo protein synthesis (Kawamoto

and Tatibana, 1983). The product of this enzyme reaction,

N-acetylglutamate (NAG) is a specific positive allosteric

effector for carbamoyl phosphate synthetase-I (CPS-I), the

first rate limiting enzyme for urea synthesis. Low CPS-I

activity may result in insufficient capacity to remove

excess ammonia. Thus, a deficiency of arginine can lead to

hyperammonemia by two distinct routes both of which result

in decreased capacity to incorporate ammonia into urea.














Figure 1 depicts the complete urea cycle which consists

of five enzymes: carbamoyl phosphate synthetase-I (CPS-I),

ornithine carbamoyl transferase (OCT), argininosuccinate

synthetase (AS), argininosuccinate lyase (AL) and arginase

(A). All five of these enzymes are present in the liver and

are compartmentalized between the mitochondria and the

cytosol. Carbamoyl phosphate synthetase-I and OCT are

located in the mitochondria and catalyze the conversion of

NH4* and HCO3" to carbamoyl phosphate and ornithine and

carbamoyl phosphate to citrulline, respectively. Powers-Lee

et al. (1987) demonstrated that liver CPS-I and OCT are both

loosely associated with the inner mitochondrial membrane

which may serve as an organizing factor for the two enzymes.

This finding supports the observed channeling of

extramitochondrial ornithine to OCT in isolated mitochondria

(Cohen et al., 1987). Citrulline is transported into the

cytosol where AS catalyzes the condensation of citrulline

plus aspartate to form argininosuccinate which AL cleaves to

arginine and fumarate. Finally, A cleaves arginine to urea

and ornithine. Urea then diffuses out of the liver via the

blood and ornithine is transported into the mitochondria to

initiate another round of the urea cycle. Although CPS-I is

technically the committing step for the urea cycle, AS is

actually considered the rate limiting step in the cycle









Amino 8
Acids

Cytosol Glutamate



Urea Glutamate


Se 2 ATP
Arginine Ornithine b42 ADP
Fumarate Corboamoyl
phosphate
b
I P,
Argininosuccinate


AMP + PP1 Ma. c
SCitrulline
ATP
Aspra Mitochondrion
Asportate
T3
Glutamate
T,
Amino Acids

Enzymes
a = carbamoyl phosphate synthetase I
b = ornithine carbamoyl transferase
c = argininosuccinate synthetase
d = argininosuccinate lyase
e = arginase
1 = transamination to a-ketoglutarate
2 = glutamate dehydrogenase
3 = transamination to oxaloacetate

Figure 1. Enzymes Involved and Cellular
Compartmentalization of the Urea Cycle.










because of its lower total activity compared to CPS-I

(Schimke, 1962a; Briggs and Freedland, 1977; Snodgrass and

Lin, 1981). Thus, the urea cycle functions in the liver to

remove two molecules of ammonia and one molecule of carbon

dioxide while converting them to urea, a less toxic and more

easily excreted form of nitrogen, relative to ammonia. The

first molecule of ammonia arises in the mitochondria from

the oxidative deamination of glutamate by glutamate

dehydrogenase whereas the second molecule arises in the

cytosol from aspartate via the transamination of

oxaloacetate.

Urea synthesis has two regulatory mechanisms. Long-

term regulation is achieved by induction of the urea cycle

enzymes themselves. Urea cycle enzymes have been shown to

increase in activity during conditions causing increased

protein catabolism such as starvation, consumption of a high

protein diet or corticosteroid treatment (Briggs and

Freedland, 1977). Amino nitrogen arising from the

utilization of amino acids as an energy source is excreted

largely as urea (Schimke, 1962a). There is a differential

effect on the excretion of urea during either consumption of

a protein-free diet or starvation even though both result in

extensive loss of total liver protein and other tissue

components. During prolonged fasting (7 days in rats),

energy is derived from body protein stores and results in

increased urea excretion. During consumption of a protein-










free diet, energy is derived from dietary carbohydrate and

fat with a resultant decrease in urea excretion. Schimke

(1962b) showed that consumption of a protein-free diet

decreased total urea cycle enzyme activity 20-50% whereas

fasting increased total urea cycle enzyme activity by 300%.

Muramatsu and Ashida (1962) showed that diets of differing

protein quality fed at the same rate resulted in higher

liver arginase activities in rats given higher quality

protein diet, but was accompanied by a lower urinary urea

excretion. Abrupt change from a 30% protein diet to either

a 15 or 60% protein diet adaptively altered urea cycle

enzymes within 8 days. Animals changed to the 60% protein

diet showed dramatic increases in CPS-I, OCT AND AL within 4

days, and reached a maximum increase by 8 days. Arginase

specific activity increased more slowly than the other

enzymes, but also reached maximum increase by 8 days. These

four enzymes declined in specific activity to approximately

one-half of the peak maxima by the end of the 28 day study.

Decreasing the protein content to 15% showed an opposite

effect. Carbamoyl phosphate synthetase-I, OCT and AL

showed a slight decline in specific activity which leveled

out by 8 days. Arginase specific activity showed a slight

but steady decline over the entire 28 day study period.

Schimke (1962a) concluded that the quantitative relationship

between the hepatic content of urea cycle enzymes and daily

intake of protein suggest that the rate of urea synthesis is








11
increased or decreased by alterations in the content of the

enzymes rather than by changes in substrate concentration.

Although an increase requirement for urea synthesis could

presumably be met most economically for the organism by

increasing the activity of only that enzyme in least

abundance (AS), these data show there is a comparable

increase in all urea cycle enzymes. Snodgrass and Lin

(1987) recently reconfirmed increased activities in the

first four urea cycle enzymes in livers of rats fed

arginine-deficient diets. However, cultured rat liver cells

or hepatoma cells cultured as a monolayer in arginine-

deficient medium did not show higher activities for these

first four enzymes. The authors postulated that the

arginine requirement may be very low in the resting cells of

the monolayer and may be provided by proteolysis which

occurs normally. Boyce et al. (1986) showed that the human

AS gene is controlled by a 5' flanking sequence promoter

region which is controlled by an arginine-mediated

repression mechanism. It is not known if arginine itself or

a repressor protein made on another gene under the influence

of arginine binds to the promoter region. CPS-I, OCT and AL

expression may have similar arginine-mediated repression

controlled promoter regions.

Short-term regulation of urea synthesis appears to be

mediated by mitochondrial concentration of NAG.










N-acetylglutamate acts as a positive allosteric effector

that influences the monomer-dimmer association-dissociation

of CPS-I (Cohen, 1981). Stewart and Walser (1980) measured

liver concentration of NAG and CPS-I activity after

intraperitoneal administration of amino acids in large

doses. Blood alpha amino nitrogen rose immediately after

injection, peaked at 15 minutes, then dropped rapidly. The

activity of CPS-I in intact mitochondria increased rapidly

after injection and peaked at 30 minutes post-injection.

The maximum activity attained was 40 nmol/min/mg

mitochondrial protein, in contrast to a pre-injection

activity of 9 nmol/min/mg mitochondrial protein. The

increase in CPS-I was directly proportional to the increase

of mitochondrial NAG concentration which in turn was

dependent on the amino acid load injected. Both NAG

concentration and CPS-I activity were maximized at a load of

3 g amino acids/kg BW.

Glucagon, a gluconeogenic hormone, has been shown to

increase intramitochondrial concentrations of NAG (Hensgens

et al., 1980). Citrulline production in isolated hepatic

mitochondria from glucagon-injected rats was equivalent to

control hepatic mitochondria incubated with NAG. Thus, the

conclusion was that glucagon-induced urea production is

mediated by the increased intramitochondrial NAG

concentration, which in turn increased citrulline synthesis.








13

Thus, glucagon may be important in the cellular coordination

of proteolysis and urea synthesis.



Source and Fate of Arginine. Ornithine and Citrulline



Arginine has two important physiological roles: 1) as a

urea cycle intermediate for the disposal of ammonia and 2)

as a constituent of proteins. Dietary arginine is not

considered an essential amino acid in adult man (Rose et

al., 1954, National Research Council, 1980), dog (Rose and

Rice, 1939) or rat (Wolf and Corley, 1939). Nonessentiality

of arginine for these adult mammals was determined by

nitrogen balance and absence of weight loss while consuming

an arginine devoid diet. Thus it is assumed that endogenous

synthesis of arginine by these adult mammals adequately

meets their needs. However, Kennan and Cohen (1961)

presented evidence suggesting that adult rats have a limited

capacity for arginine biosynthesis beyond their needs and

that the urea cycle operates at near capacity for ammonia

detoxification. Arginine is considered essential for

optimal growth in many young animals, however. Growing dogs

(Czarnecki and Baker, 1984), cats (Morris et al., 1979) and

rats (Borman et al., 1946) have all been shown to require an

exogenous arginine source to support optimal weight gain

and/or prevent hyperammonemia (Table 1). Nonureotelic










Table 1. Growth Rate in Animals Supplemented with Arginine,
Citrulline or Ornithine.



Growth Rate (g/day)

animal +Arg -Arg +Cit -Arg+Orn


Rata 4.1 4.4 1.6

Kittenb 24.8 N.D. -6.5
20.4 20.9 N.D.

Dogc 189.1 61.3 -11.3

Chickd 5.3 5.4 2.6

Rainbow Troute +++ +++ +



N.D. Not determined
a Purified amino acid diet with arginine at 1.12% of the
diet; isonitrogenous replacement of arginine with citrulline
or ornithine. Milner and Visek, 1975.
bPurified amino acid diet with arginine previously
demonstrated as adequate (1.16% of total AA mix); equimolar
replacement of arginine with citrulline or ornithine.
Morris et al., 1979.

cPurified amino acid diet with arginine at 0.4% of diet.
Isomolar replacement with citrulline or ornithine.
Czarnecki and Baker, 1984.

d1% supplement of arginine, citrulline or ornithine to basal
diet which contained 0.74% arginine. Klose and Almquist,
1940.
eEquimolar replacement of the 1.2% arginine supplement which
is in addition to the arginine present in the 29% casein
basal diet. Actual growth rate not given, but orn is
significant at p < .05. Chiu et al., 1986.










animals such as chicks (Klose and Almquist, 1940) and

rainbow trout (Chiu et al., 1986) have also been shown to

require exogenous arginine to support optimal weight gain

(Table 1). Arginine acts anaplerotically in the urea cycle

and logically, either citrulline or ornithine should be able

to substitute for arginine in this capacity. It has been

shown in the rat (Milner and Visek, 1975), kitten (Morris et

al., 1979) and dog (Czarnecki and Baker, 1984) that both

citrulline and ornithine can substitute for arginine in its

ability to prevent or alleviate hyperammonemia. Hoogenraad

et al. (1985) provided evidence that 105 g Wistar rats fed

for 10 days a 30% L-amino acid diet containing an equimolar

replacement of ornithine for arginine had an equivalent

growth rate compared to rats on the control diet containing

5.7% arginine of the total amino acids. The ability of

ornithine to replace arginine in promoting adequate growth

in young rats has not been corroborated by other

investigators however, and it is generally accepted that

only citrulline can substitute for arginine in providing an

equivalent growth rate in some, but not all, species. In a

growing animal, increased protein synthesis for lean body

tissues comprises a large percentage of the increased weight

gain. In an adult animal, however, weight gain reflects a

greater deposition of fat stores and protein synthesis is

required only for maintenance or repair.










Table 2. Tissue Distribution of Urea Cycle Enzyme
Activities.



Activity (umole/hour/g wet tissue)

Tissue CPS-I OCT AS AL A Ref.


Liver 340

Kidney 4.43

Brain 4

Pancreas 3

Spleen 0

Lung

Thymus

Skeletal
Muscle

Adrenal

Blood

Testicle

Intestinal
Mucosa


13,400 175

78 12

0 3

2,[0.29]

0,{<0.22)


269

43.2

6.9


[<0.22]


[0.67]


0.32

2.60


35,000[40,140] a,d

2240[2,520] b,d

105[48] b,d

(1,440} c,e,d

[0] c,d,e

150 d

54 d

0 d,e


d,e

d

e

e,d


[4,560]


Value in [] is second reference; value in () is third
reference.

a. Schimke, 1962.
b. Ratner, 1976.
c. Tamir and Ratner, 1963.
d. Greengard et al., 1970.
e. Raijman, 1974.










Arginine is the only urea cycle intermediate which is

naturally found in dietary sources, as citrulline and

ornithine are not incorporated into proteins. The only

known endogenous source of arginine is its biosynthesis

utilizing the urea cycle enzymes (Windmueller and Spaeth,

1981). Table 2 lists the tissue distribution for the urea

cycle enzymes. Liver is the only tissue known to contain

adequate quantities of all five of the urea cycle enzymes to

effectively convert ammonia to urea as a nitrogen disposal

organ. As can be seen in Table 2, kidney is the only

extrahepatic tissue which also contains the full complement

of urea cycle enzymes. However, general agreement is that

the kidney does not significantly, if at all, contribute to

ammonia disposal via urea synthesis. Only those tissues

with sufficient AS and AL activities can synthesize arginine

from citrulline and only those tissues containing sufficient

CPS-I and OCT activities can synthesize arginine from

ornithine. Thus, while the kidney has sufficient capacity

to synthesize arginine starting with citrulline, it has

limited capacity to synthesize arginine starting from

ornithine due to its low CPS-I and OCT activity.

Rogers et al. (1972) and Featherston et al. (1973) have

both shown the significant role the kidney plays in

supplying arginine for both hepatic and extrahepatic protein

synthesis. Skeletal muscle contains 13.4% arginine making

it the most abundant amino acid in that tissue (Souba et








18

al., 1985b). Radioisotopic studies using either guanidino-

4C arginine or ureido-14C citrulline at two levels of

dietary protein consumption (8% or 80% casein) indicated

that isotope incorporation into TCA-precipitatable proteins

in liver, muscle, spleen and kidney differed depending on

dietary protein level and isotope source (Rogers et al.,

1972). The label from either source is retained to a

greater extent in the tissue proteins of the lower dietary

protein group. Label incorporation into tissue proteins

from 4C-citrulline was greater in all tissues at the 80%

dietary protein consumption level; however, it was only

slightly greater in the liver. At the lower protein

consumption level, incorporation into the kidney was over

three times greater from citrulline than from arginine. It

was, however, nearly equal in muscle and spleen and was 1.5

times lower in the liver. Thus, these data indicate that

dietary arginine is incorporated into peripheral proteins

dependent upon the protein level in the diet and that

citrulline is an important precursor to arginine for

subsequent protein synthesis both in the liver as well as

for extrahepatic tissues. In a subsequent study,

Featherston et al. (1973) showed that there was little or no

label incorporation into muscle from anephretic animals from

14C-citrulline, thus confirming the role of the kidney in

arginine synthesis from citrulline for extrahepatic protein

synthesis.










Apart from urea and protein synthesis, arginine is

utilized for the biosynthesis of creatine and polyamines.

Creatine acts as an ATP regeneration mechanism in the

skeletal muscle by accepting the high energy phosphate from

ATP in times of muscle quiescence and subsequently

transferring that high energy phosphate to ADP to regenerate

ATP during muscle contraction. In creatine biosynthesis,

arginine transfers its guanidino group to glycine to form

the intermediate guanidoacetate which is subsequently

methylated to form creatine. Ornithine is formed as a

product from the guanidino transfer. Arginine is purported

to be the source of ornithine in polyamine biosynthesis via

the action of arginase (Tabor and Tabor, 1984; Pegg, 1986).

Ornithine can be synthesized in any tissue containing

arginase (Table 2). It is also synthesized as a by-product

of guanidino-group transfers such as in the guanidoacetate

intermediate for creatine synthesis. Ornithine has also

been shown to be synthesized in the small intestine from

glutamate; the reaction is presumably catalyzed by the

enzyme ornithine aminotransferase, OAT (Henslee and Jones,

1982). Ornithine aminotransferase catalyzes the reversible

reaction of ornithine plus a-ketoglutarate to glutamate-s-

semialdehyde (GSA) plus glutamate with the in vivo

equilibrium thought to favor glutamate formation. Although

there was no direct evidence for conversion of glutamate to

ornithine by OAT, Henslee and Jones (1982) showed










incorporation of 14C into ornithine from radiolabeled

glutamate by an OAT enriched mitochondrial preparation.

Rogers and Phang (1985) measured pyrroline-5-carboxylate

synthase (P-5-C synthase) activity in rat small intestine

and further substantiated the evidence for a biosynthetic

pathway of ornithine synthesis from glutamate. The

postulated glutamate to ornithine pathway involves P-5-C

synthase catalyzed conversion of glutamate to GSA which in

turn becomes the substrate for OAT. Ornithine is produced

by the reversible OAT reaction.

Some evidence has been presented indicating that

ornithine may be synthesized from glutamine. Lund and

Wiggins (1986) presented evidence indicating that during

urea synthesis, ornithine and NAG concentrations increase in

isolated hepatocytes when incubated with glutamine. The

pathway postulated for ornithine synthesis from glutamine in

rat liver is similar to the bacterial system in which there

is sequential conversion of N-acetylglutamate phosphate to

N-acetylglutamate semialdehyde to N-acetylornithine to

ornithine. This is only a postulation as the enzymes

involved in this conversion have not been identified in rat

liver. It is also postulated that glutamine is degraded by

a liver specific, allosterically controlled glutaminase in

this conversion of glutamine to ornithine.

Ornithine is of course utilized in the urea cycle as

the immediate precursor to citrulline. Ornithine is also










utilized in polyamine biosynthesis. The function of

polyamines is still obscure. However, there is agreement

that they are essential for cell growth (Pegg, 1986) and are

most ubiquitous in actively dividing or metabolizing cells

and are associated with protein synthesis (Tabor and Tabor,

1984). Ornithine decarboxylase is the committing step for

polyamine biosynthesis and catalyzes the decarboxylation of

ornithine to yield putrescine (1,4-diaminobutane), the first

in a series of three polyamines. Although ornithine is

consumed at the committing step, there appears to be

substantial interconversion between the three polyamines,

putrescine, spermidine and spermine catalyzed by spermidine

or spermine N'-acetyltransferase, respectively (Pegg, 1986);

thus, the true ornithine demand for polyamine synthesis is

unknown.

Endogenous citrulline is derived from ornithine and

carbamoyl phosphate condensation catalyzed by OCT. Only

those tissues with adequate CPS-I and OCT activity have the

capacity to synthesize citrulline from ornithine. Thus the

liver and small intestine are the two sources of citrulline

biosynthesis. Work by Drotman and Freedland (1972) using

perfused isolated rat liver indicated that with excess

ammonia and ornithine, there was a net export of citrulline.

This led to the hypothesis that the liver supplied

citrulline for renal synthesis of arginine for extrahepatic

tissues. This hypothesis has been refuted by a series of










experiments indicating that the small intestine is the

source for citrulline (Windmueller and Spaeth, 1974, 1978,

1980, 1981). Windmueller and Spaeth (1974) provided

evidence from isolated rat small intestine that citrulline

and ornithine are released into the venous blood from U-

14C-glutamine uptake which accounted for 6% and 0.3% of the

labeled carbon, respectively. Calculating the percent

release from glutamine-N, approximately 34% was released as

citrulline. In a subsequent study, Windmueller and Spaeth

(1978) presented data showing that 4.7% and 1.3% of the

carbon backbone of citrulline and ornithine, respectively,

are derived from glutamine uptake by the small intestine in

vivo. Nitrogen extraction from glutamine uptake by perfused

intact small intestine was shown to be approximately 28% for

citrulline and 1% for ornithine (Windmueller and Spaeth,

1980). In a subsequent study, they determined that the

kidney extracted from circulating blood approximately 84% of

the intestinally-released citrulline and returned

approximately 75% of that citrulline as arginine

(Windmueller and Spaeth, 1981). Hoogenraad, et al. (1985)

demonstrated that selective inhibition of intestinal OCT of

young rats on an arg-deficient diet resulted in a rapid and

complete inhibition of growth. Additionally, serum

citrulline decreased nearly 2-fold, arg decreased 4.6-fold

whereas ornithine increased 2.7-fold. This evidence

demonstrates the role intestinally derived citrulline has in








23
maintaining arginine homeostasis. Liver and intestinal OCT

appear to have different regulatory mechanisms which are

apparently related to the respective physiological roles.

Wraight et al. (1985) fed rats either a 15% or 60% protein

diet after adapting the animals to a 30% protein diet.

After 7 days of the experimental diet, liver OCT activity

was increased; however, intestinal OCT activity was

moderately but statistically decreased in the 60% protein

group relative to the 15% protein group. The authors

postulated that liver OCT activity was increased in response

to an increased demand for nitrogen disposal whereas the

decreased intestinal OCT activity was in response to a

decreased requirement to provide citrulline precursor to

maintain arg homeostasis. Windmueller and Spaeth (1981)

provided circumstantial evidence for an extraintestinal,

extrahepatic citrulline source. This was demonstrated by

the absence of circulating blood citrulline depletion when

the intestine alone or the intestine plus an additional

organ (spleen, stomach, liver or pancreas) was excluded from

the circulation. Small intestinal synthesis of citrulline

from glutamine could be critically compromised for the TPN

patient due to the decreased circulating glutamine as a

result of the underlying conditions indicating the necessity

for TPN (Souba, 1985). Hughes and Dowling (1980)

demonstrated marked hypoplasia (denoted by decreased mucosal

mass) and hypofunction (denoted by decreased histologically










measured mucosal thickness) within 3 days of parenteral

feeding of rats. Unfortunately, it is not known if this

hypoplasia and hypofunction is a result of decreased

exogenous nutrition, decreased intestinal blood supply

including a decrease of its normal respiratory fuels, or

alteration in hormonal stimulation of the intestinal tract.

Thus arginine homeostasis may be compromised in the TPN

administered animal or man since exogenous nutrition to the

gut is eliminated.

The fate of citrulline is for arginine biosynthesis by

the two step pathway utilizing AS and AL. The two tissues

containing adequate amounts of these two enzymes are the

liver and kidney. Windmueller and Spaeth (1981) provided

evidence that citrulline is not sequestered from the

arterial supply by the liver. Their radiolabeled studies

substantiated earlier arteriovenous amino acid difference

evidence that the liver does not take up citrulline or

ornithine (Coulson and Hernandez, 1968). Windmueller and

Spaeth (1981) determined that 86% of the citrulline released

by the small intestine is taken up by the kidney and that

75% of that is returned to the venous supply as arginine.



Orotate as Arqinine Deficiency Indicator


Milner and Visek (1973) were among the first to provide

experimental evidence that feeding rats an arginine-










deficient diet leads to increased urinary orotic acid

excretion. Since that report, many reports on the positive

correlation between increased urinary orotate excretion and

arginine deficiency and/or ammonia toxicity have been made.

The hypothesis for increased orotate synthesis is that

carbamoyl phosphate (CP) is synthesized in excess of its

capacity to become incorporated into citrulline; hence, CP

is exported into the cytosol where it is utilized as the

substrate for aspartate carbamoyl transferase and ultimately

synthesized into orotate (Visek and Shoemaker, 1986).

Orotate, a precursor for pyrimidines, is synthesized in

excess of pyrimidine synthesis requirements and thus is

excreted in the urine. The correlation between orotic

aciduria and arginine deficiency and/or ammonia toxicity is

well documented. In the more recent reviews (Milner, 1985;

Visek and Shoemaker, 1986; Zieve, 1986), orotic aciduria is

suggested as an biochemical index for arginine deficiency or

ammonia toxicity.

However, the explanation for orotic aciduria is not

accepted by all researchers. Barbul (1986), does not agree

that the hypothesis adequately accounts for the involvement

of the cytosolic enzyme, carbamoyl phosphate synthetase-II

which synthesizes CP for pyrimidine biosynthesis. Barbul

does not agree that the published evidence supports the

hypothesis of a metabolic shunting of ammonia from one

carbamoyl phosphate synthetase system to the other (CPS-I to










CPS-II). However, Natale and Tremblay (1969, 1974) and

Tremblay et al. (1977) have presented substantial evidence

that CP derived from CPS-I is indeed incorporated into

orotate and that incorporation is substantial even under

physiological conditions. Natale and Tremblay (1969)

demonstrated in isolated rat liver mitochondria that

carbamoyl phosphate is freely permeable to the mitochondrial

membrane. In a subsequent study Natale and Tremblay (1974)

demonstrated under optimal conditions for citrulline

synthesis, approximately one-half of the carbamoyl phosphate

synthesized was exported to the extramitochondrial medium.

Tremblay et al. (1977) showed in liver slice preparations

that over 80% of the NaH14CO3 incorporated into orotic acid

at physiological levels of ammonia and ornithine was ammonia

dependent rather than glutamine dependent. They

demonstrated that 5 mM ammonia did not stimulate synthesis

of glutamine in the liver slice reaction medium.

Additionally, glutamine added to liver slice preparations

stimulated the incorporation of NaH14COO3 into orotic acid;

however, this stimulation could be accounted for by the

generation of ammonia by glutaminase and was blocked by the

addition of ornithine. These authors concluded that CPS-I

is the major source of CP in the biosynthesis of hepatic

orotate under physiological conditions as well as ammonia

intoxication. Tremblay et al. (1977) also demonstrated

that tissues with low or no CPS-I activities such as kidney










and spleen were not stimulated to produce 14C-orotate from

labeled bicarbonate under the same conditions in which liver

slices produced nearly 20 times the normal amount. Arashima

and Matsuda (1972, as referenced by Tremblay et al., 1977)

documented a clinical case in which an infant with only 13%

CPS-I activity (compared to normal) exhibited congenital

hyperammonemia without concomitant orotic aciduria.

Although orotic aciduria and hyperammonemia result in

many species fed an arginine-deficient diet, there is not a

similar response in adult humans orally-fed an arginine-

deficient diet (Carey et al., 1987). These authors showed

that urinary orotate excretion actually fell in adult humans

fed an arg-deficient, isonitrogenous diet. The urinary

orotate excretion returned to normal levels when the

nitrogen content of the arg-deficient diet was doubled.

Urinary urea and blood ammonia levels stayed within normal

values and increased in response to total nitrogen load.

The significant finding from this experiment was a 60% fall

in postprandial plasma arginine concentration following an

arginine-deficient meal. The conclusion by these authors

was that the biochemical indices which are useful in animal

experiments to determine arginine requirement or

essentiality do not indicate that there is a requirement for

arginine in orally-fed adult humans. Only in TPN patients

has there been a reported incidence of a biochemical index

for arginine requirement indicating that insufficient










arginine was supplied to the patient (Heird et al., 1972).

The 1980 Recommended Dietary Allowances (National Research

Council, 1980) reports that although arginine is not

required for infants or adults, it may become limiting for

humans in special circumstances and references the 1972

Heird article. Thus, only in TPN may arginine become

required in humans.



Measurement of Nutrient Requirement or Adequacy



The protocol for establishing protein and amino acid

requirements in the past has been based on nitrogen balance

studies. Recently, however, this practice has been

questioned (Millward and Rivers, 1986; Young, 1986). When

fed in a meal-type fashion (versus continuous as in TPN), it

is well established that protein deposition in the

postprandial state followed by mobilization in the

postabsorptive state occurs in the adult (Millward and

Rivers, 1986). The amplitude of this circadian variation

also appears dependent on the protein intake. Thus,

establishment of indispensable amino acid and protein

requirements must account for this dependance and

variability. In an opinion article published by V.R. Young

(1986), radioactive isotope tracer studies were cited which

indicated that as the indispensable amino acid is gradually

decreased, there are significant changes in amino acid










metabolism which appear to be linked to the physiological

requirement of the host.

The level of urinary orotic acid excretion has been

suggested as an indicator of arginine deficiency in the rat

(Milner, 1985; Visek and Shoemaker, 1986; Carey et al.,

1987). Therefore, in the rat, dietary arginine adequacy is

indicated by the absence of excessive orotic aciduria. No

absolute value has been assigned to the level of urinary

orotic acid excretion which is considered excessive or

indicative of an arginine deficiency. Hassan and Milner

(1979) reported orotic acid excretion levels around 100 gg/d

in control rats weighing 180-220 g and consuming a 14%

L-amino acid diet. It has been shown that orotic acid

excretion can increase by as much as 49 times the control

level when arginine is removed from the diet of a previously

well nourished rat (Milner and Visek, 1975). It has also

been shown that orotic acid excretion varies with both

protein intake and ratio of arginine to total protein

(Milner and Visek, 1978). At the highest protein intake

(35%) and 5.22% arginine/total protein, urinary orotate

excretion was approximately 500 Ag/day. The same level of

protein intake and 10.44% arginine/total protein decreased

urinary orotate excretion to less than 200 gg/day. At 14%

total protein, both 5.22% and 10.44% arginine/total protein

supplementation levels yielded a urinary orotate excretion

level of less than 300 Ag/day. Schimke (1962a, 1962b)








30

provided evidence that urea cycle enzyme activities are

dependent on both dietary energy and protein intake levels.

In the clinical situation, energy requirements and nitrogen

load are tailored to each individual patient and may be

altered every few days depending upon metabolic parameters

(personal communication, T.G. Baumgartner). Thus, to

properly determine arginine requirements and utilization

under TPN, one must account for energy supply, nitrogen load

and arginine/total amino acid ratio.














MATERIALS AND METHODS


Brief descriptions of assays and methodologies are

presented in this section. Detailed descriptions for

dietary formulations and assay or methodological procedures

are presented in Appendices I and II, respectively.



Materials and Equipment

Animals

Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc.,

Indianapolis, IN) from 225-275 g were used throughout all

experiments. The animals were individually housed in

suspended stainless steel wire mesh cages, 24-hour lighting

was provided and the room temperature was maintained between

70F and 750F. Unless otherwise noted, the animals received

a lactalbumin-based semipurified control diet containing

approximately 0.29 g nitrogen and 60 non-nitrogen calories

per day with water provided ad-libitum during the one week

adaptation period. Weight gain was monitored at least three

times per week and those animals gaining 3-5 g/day were

assigned to an experimental regimen.










Diets

Lactalbumin Control Diet. The lactalbumin diet was

formulated to meet dietary requirements recommended by the

National Research Council (1978) as well as to be

isonitrogenous and isocaloric to the TPN diets. Ten

kilograms of diet were formulated for each experiment

according to the chart in Appendix I and mixed according to

the following procedure. Lactalbumin (United States

Biochemical Corp., Cleveland, OH) was first supplemented

with L-arginine-HCl to provide 6.5 g arg per 100 g total

lactalbumin protein. This diet will be referred to

hereafter as the 'supplemented lactalbumin diet'. This

supplementation level was determined to be necessary from

data obtained from experiments conducted during the

"Development of a TPN Model for Determination of Arginine

Adequacy" phase of this work. The lactalbumin control diet

used in those experiments was formulated exactly as

described here except the lactalbumin was not supplemented

with arginine. The appropriate amount of supplemented

lactalbumin plus the remaining dry ingredients were added to

the mixing bowl of a commercial mixer (Univex Mixer,

Universal Industries) and thoroughly mixed for 20 minutes.

The corn oil was added slowly and the entire diet was mixed

an additional 20 minutes. The mixed diet was placed in a

plastic-lined 5 gallon container and closed with a tight-










fitting lid. The diet was stored at 4C and portions were

removed for daily use.

TPN Diets for Arginine Supplementation. Daily nitrogen

and caloric requirements were calculated based on equations

developed by Tao et al. (1979) for TPN-fed rats. The amino

acid formulation was also derived from data presented by Tao

et al. (1979). Amino acid solutions were prepared for each

experiment under a laminar flow hood located in the Food

Science Building. Distilled-deionized water (d-d-H20) which

was passed through a 0.45 micron filter (Millipore

Milli-QT Water System, Millipore Corp., Bedford, MA) and

sterilized flasks were used to prepare the amino acid

solutions. L-amino acids were purchased either from Sigma

Chemical Company (St. Louis, MO) or Fisher Scientific

Company (Pittsburgh, PA). The base amino acid solution was

warmed slightly on a heated stir plate and stirred

continuously to allow dissolution of all amino acids. Each

amino acid was added in the stated mixing order (Base Amino

Acid Solution, Appendix I) and allowed to dissolve prior to

subsequent addition. The solution was cooled and then

filtered through a 0.22 micron filter (Pall Cardioplegia

Plusm Filter with Solution Spike, Pall Biomedical Products

Corp., East Hills, NY) into evacuated sterilized containers

(Kendall McGaw Laboratories, Inc. Irvine, CA). Arginine

solutions were prepared in a similar manner; the formulation

is listed in Appendix I.










TPN formulas were prepared by adding the amino acid

solution, arginine solution (if necessary), dextrose,

macroelements (except calcium) and sterile water for

injection into appropriately sized evacuated, sterile

containers (Kendall McGaw Laboratories, Inc. Irvine, CA).

(Name and source of commercial solutions used to compound

the TPN diets are listed in Appendix I.) The solutions were

compounded under a laminar flow hood in the Pharmacy

Services IV-Prep room at Shands Hospital, Gainesville, FL,

sealed with parafilm, boxed in the original shipping carton

and stored at 4*C until use.

Commercial solutions for IV injection of calcium

gluconate, lipid, vitamins and trace elements were added

daily prior to hanging the TPN bottle. The TPN bottle was

allowed to come to room temperature, weighed and wrapped in

foil just prior to hanging. Commercial IV administration

tubing (Travenol Laboratories, Inc., Deerfield, IL) was used

to deliver the TPN formulas and was replaced every two days.

Care was taken to avoid contamination of the administration

lines during handling.



Reagents

Except for the solutions used for TPN diets, all

chemicals used were reagent grade and solutions were

prepared using distilled, resin deionized (Ultrapure mixed

resin, Barnstead Sybon Corp., Boston, MA) water (d-d-H,0).










Chemicals were purchased either from Fisher Scientific Co.

or Sigma Chemical Co.



Spectrophotometer

Spectrophotometric analysis were performed using a

Perkin-Elmer Lambda 3A UV/VIS Spectrophotometer. Urea cycle

enzyme activities for one experiment were determined using a

Coleman Jr. II Spectrophotometer, Model 6/20 to avoid

possible 14C contamination of major laboratory equipment.



Technicon AutoAnalyzer

A Technicon AutoAnalyzer I (Technicon Instruments

Corp., Chauncey, NY) was used to determine concentrations of

the urinary metabolites orotic acid, urea nitrogen, total

nitrogen and creatinine. A description of the AutoAnalyzer

system of operation is listed in Appendix II.



Urinary Metabolite Analysis



Urine Collection and Sample Preparation

Twenty-four-hour urine collections were made under 1.0

mL of 6 N H2SO4 to inhibit microbial growth and maintain

ammonia in an ionized state. Collections were made daily

between 09:00 and 10:00 hours. Urine volume was measured,

diluted to 100 mL with d-d-H20, mixed thoroughly and

filtered through Whatman # 1 filter paper into 39 mL (115










mm x 23 mm) flat-bottomed disposable polypropylene tubes

(Sarstedt, West Germany). Dilution of urine was made to

normalize the volume of urine samples and to avoid volume

calculations when determining metabolite concentrations.

Prepared urine samples were frozen and stored at -20C for

analysis.



Urinary Metabolite Analysis

Urinary Urea Nitrogen. Urinary urea nitrogen (UUN) was

determined according to the colorimetric Technicon

AutoAnalyzer method N-lc which is a modification of the

procedure described by Marsh et al. (1965). The method is

based on the direct reaction of urea and diacetyl monoxime

(2,3-butanedione monoxime) in the presence of ferric ion and

thiosemicarbazide under acidic conditions.

Creatinine. Urinary creatinine was determined

according to the colorimetric Technicon AutoAnalyzer method

N-llb which is a modification of the Folin and Wu procedure

as described by Oser (1965). The method is based on the

chromogenic product produced by creatinine condensation with

picric acid in a very alkaline solution.

Urinary Total Nitrogen. A semi-micro-Kjeldahl

technique adapted from procedures reported by Ferrari (1960)

and Technicon AutoAnalyzer method 31-69A was used to digest

the samples and convert the nitrogenous compounds to

ammonium acid sulfate. Urine samples were oxidized in a










digestion mixture consisting of sulfuric acid and catalyst.

Cooled samples were diluted with d-d-H2O to 2% H2SO4 just

prior to ammonia determination. Ammonia was determined on

the Technicon AutoAnalyzer using a modification of the

Berthelot reaction in which indophenol is formed by the

reaction of ammonia, phenol and hypochlorite.

Orotic Acid. A combination of the manual methods

described by Stajner et al. (1968) and Rogers and Porter

(1968) were adapted to run on the AutoAnalyzer. The basis

of the method is that orotic acid reacts with bromine water

to form 5,5'-dibromobarbituric acid. This compound is

reduced to barbituric acid in the presence of 5% ascorbic

acid which then reacts with p-dimethylaminobenzaldehyde

(Erlich's reagent) to form a yellow chromophore.

Citric Acid. Two methods were used to determine the

concentration of urinary citrate. The initial method was a

modification of the spectrophotometric method outlined by

Camp and Farmer (1967). This method is based on the

formation of pentabromoacetone from citric acid and color

development with thiourea. This was a tedious method due to

the extraction of the pentabromoacetone into a heptane layer

and subsequent color development in the heptane extract.

Thus, a second method which used citrate lyase was adopted

(Nielsen, 1976). The method, used without modification, is

based on the conversion of citrate to oxaloacetate and

acetate by citrate lyase coupled with the conversion of










oxaloacetate to malate by the NADH-dependent enzyme malate

dehydrogenase.



Amino Acid Analysis


Sample Preparation

Plasma. Whole blood samples were collected in

heparinized syringes and kept on ice until all samples were

collected. Samples were then placed in 15 mL conical glass

tubes. Spin-Quik polypropylene beads (Omni-Tech., Inc.,

Santa Monica, CA) were added to the tubes to aid in plasma

separation. The tubes were centrifuged (International

Centrifuge Model UV, International Equipment Co., Needham,

MA) for 10 min at 1500 rpm and the plasma was removed from

the packed cells in 500 PL aliquots to obtain an approximate

volume. Plasma was deproteinized using 50 mg sulfosalicylic

acid per mL plasma. Samples were vortexed just enough to

mix the plasma and sulfosalicylic acid and then centrifuged

for 10 min at 2000 rpm. The clear supernatant was removed,

placed in Micro-centrifuge tubes (Fisher Scientific) and

stored at 0C until analysis. Samples were analyzed on a

Beckman 119CL using a physiological column.

Hydrolysis of Lactalbumin. A lactalbumin sample from

the control diet was hydrolyzed according to the procedure

of Roach and Gehrke (1970). Approximately 30 mg of

lactalbumin was weighed into 20 x 150 mm Kimax culture tubes










fitted with Teflon-lined caps. Hydrolysis was performed

with 6 N HC1 at 145C for 2 hrs. The hydrolyzed solution

was cooled to room temperature and neutralized with 6 N

NaOH. The neutralized solution was adjusted to pH 2.2 +

0.05 with concentrated HC1, centrifuged to sediment any

particulate and the supernatant was stored at 0C in Micro-

centrifuge tubes until analysis. Samples were analyzed on a

Beckman 119CL using a hydrolysate column.

TPN Amino Acid Solutions. An amino acid solution for

the arginine-devoid TPN diet and each of the four arginine-

supplemented TPN diets was made according to the formulation

outlined in Appendix I. The solutions were stored in Micro-

centrifuge tubes and stored at 0C until analysis. The

solutions were analyzed on a Beckman 119CL using a

hydrolysate column.



Analysis

Physiological Samples. Analyses were performed using a

Beckman 119CL Amino Acid Analyzer (Beckman Instruments, Palo

Alto, CA) equipped with an integrator (Hewlett Packard Model

3390A data system) and a single 6 x 460 mm column packed

with Beckman W-3P resin.

Hydrolysis Samples. Analyses were performed using the

Beckman 119CL equipped with a 6 x 460 mm single-column

packed with Beckman W-3 resin (hydrolysate column). The

signal was recorded on a Hewlett Packard integrator.










Urea Cycle Enzyme Activities



Sample Preparation

Whole liver and both kidneys were excised from

anesthetized animals and placed in ice cold d-d-H2O and kept

on ice until homogenization for enzyme analyses, but no

longer than two hours post-excision. Approximately 0.5 g of

liver or kidney tissue was homogenized for 20 sec in 19

volumes of cold water using a Polytron tissue homogenizer

(Brinkmann Instruments, Inc., Westbury, NY). Liver samples

were taken from a central location from both the left

lateral lobe and the medial lobe. Kidney samples were

obtained from both kidneys from a cross-section of a

sagittal dissection excluding the inner medullary region.

No further dilution of the homogenate was necessary to

determine the activities of CPS-I or AS; but liver

homogenate was diluted 25-fold and kidney homogenate was

diluted 5-fold with a 0.5% bovine serum albumin (BSA)

solution to measure arginase activity.



Urea Cycle Enzyme Analyses

The procedure used for determining the three urea cycle

enzyme activities was that outlined by Nuzum and Snodgrass

(1976). The composition of the three incubation mixtures is

listed in Appendix II. Carbamoyl phosphate synthetase-I and

argininosuccinate synthetase require the supplementary










enzymes ornithine carbamoyltransfrease (OCT) and

argininosuccinase (AL), respectively. These two enzymes are

not readily available commercially, thus, were isolated as a

crude enzyme preparation from beef liver obtained at

slaughter from the Animal Science Department, University of

Florida, according to the procedures outlined by Marshall

and Cohen (1972) and Ratner (1970), respectively. All

assays were incubated at 370C for the specified time.

Carbamoyl Phosphate Synthetase-I. Only liver tissue

was analyzed for CPS-I activity as the activity in kidney is

reportedly very low. The product of the enzymatic assay,

citrulline, was assayed spectrophotometrically according to

the procedure outlined by Boyde and Rahmatulah (1980).

Activity was expressed as Amol citrulline produced per hour

per mg protein or g liver.

Arqininosuccinate Synthetase. Both liver and kidney

tissues were analyzed for AS activity. The product of the

assay, urea, was analyzed spectrophotometrically according

to the procedure outlined by Nuzum and Snodgrass (1976).

Activity was expressed as gmol urea produced per hour per mg

protein or g tissue.

Arqinase. Liver homogenate was diluted 25-fold and

kidney homogenate was diluted 5-fold with a 0.5% bovine

serum albumin (BSA) solution before initiation of the assay

for arginase. The product of the assay, urea, was measured

according to the procedure outlined by Nuzum and Snodgrass










(1976). Activity was expressed as Amol urea produced per

hour per mg protein or g tissue.



Protein Determination

Tissue homogenate samples were analyzed for protein

content according to the procedure of Lowry et al. (1951)

using BSA as the standard.



Miscellaneous Analytical Methods



Organ Moisture

Organ moisture was determined by freeze drying the

excised, weighed and frozen tissues. Freeze drying was

accomplished in a Virtis shelf-type freeze dryer (The Virtis

Company, Gardner, NY) by evacuating the chamber to 30-60 Am

at a temperature of 500C. Moisture was determined after

approximately 48 hrs of freeze drying time when no further

loss in weight was observed.



Organ Total Nitrogen

The semi-micro-Kjeldahl method was used to determine

the total nitrogen content of organs. The freeze-dried

tissue was ground to a fine powder with a mortar and pestle.

Digestion of the sample and subsequent determination of

ammonia were described previously.










Dietary Nitrogen

Total nitrogen content was determined on unsupplemented

lactalbumin, supplemented lactalbumin, supplemented

lactalbumin diet and all TPN diets by the semi-micro-

Kjeldahl method as described previously.



Serum Urea Nitrogen

A combination of two methods for the determination of

serum urea as described by Crocker (1967) and Foster and

Hochholzer (1971) was developed for routine use in our

laboratory. The combined spectrophotometric method takes

advantage of the diacetyl monoxime-thiosemicarbazide color

development with urea in the presence of ferric ion and

antipyrine in a slightly acidic solution.



Technique for TPN



Catheterization

Animals were briefly anesthetized with Halothane U.S.P.

(Halocarbon Laboratories, Inc., Hackensack, NJ) and

maintained with ketamine-HCl (Ketaset, Veterinary Products

Bristol Laboratories, Syracuse, NY) plus 0.1% acepromazine

malate, injection, 10 mg/mL (TechAmerica Group, Inc. Elwood,

KS) at a dose rate of 10 mg ketamine-HCl/100 g body weight

(BW) intramuscularly. Surgical tools, dressings and

catheters were sterilized by autoclaving before use.








44
Animals were shaved on the neck above the right jugular vein

and back between the dorsal scapulae. The skin was washed

with 70% alcohol. A small incision (2.0-2.5 cm) was made in

the neck exposing the right external jugular vein. The vein

was occluded distally with 5-0 silk suture, then a venotomy

was performed and a 0.020" i.d. x 0.037" o.d. SialasticR

catheter (Dow Corning Corp, Medical Products, Midland, MI)

was inserted and guided through the jugular vein into the

superior vena cava for a distance of approximately 3 cm from

insertion site. The tip of the catheter resided + 2 mm from

the right atrium. The catheter was secured in place

proximal to the insertion site using 5-0 silk suture and one

drop of cyanoacrylate resin ("super" glue). The other end

of the catheter was tunneled subcutaneously and exited

dorsally at the midscapular region. A plastic cup assembly

was centered around the catheter and sutured to the skin of

the back. The incision in the neck region was closed with 9

mm wound clips (Clay Adams, Div. of Becton Dickinsen & Co.,

Parsippany, NJ).

Surgeries were performed using a three to four person

team. One to two persons catheterized the animals. Another

person tunneled the catheter to its external site, sutured

the cup to the back of the neck and closed the ventral

opening. The final person anesthetized the animals,

prepared them for surgery, connected them to the TPN

administration set-up and administered post-operative care








45

to the animals. Generally, a surgical resident assigned to

our laboratory performed both the catheterizations and

securing of the catheter and assembly cup. (Names of all

residents who assisted me with this research are listed in

the acknowledgements.) I anesthetized the animals,

connected them to the TPN administration set-up and gave

post-operative care.



Administration of Total Parenteral Nutrition

The catheter exiting dorsally from the animal was

connected to a segment of medical grade 0.020" i.d. x 0.060"

o.d. polyvinyl transmission line (Tygon Microbore Tubing,

Norton Performance Plastics, Akron, OH) via a 22 gauge

stainless steel nipple. The line was threaded through a

tightly wound metal coil which was attached distally to an

infusion swivel (Instech Laboratories, Inc., Plymouth

Meeting, PA). The metal coil and swivel assembly were

attached to the cup assembly secured to the back of the

animal via a tight fitting matching cap. This system

allowed the animal complete freedom to move about in the

metabolic cage. The infusion swivel was attached to a

segment of approximately 330 cm of 0.020" x 0.060" medical

grade polyvinyl transmission line. This transmission line

was attached to 0.025" i.d. silicon manifold pump tubing

(Fisher Scientific) via a 20 gauge stainless steel nipple.

Up to six manifold pump tubings were connected to a stream








46

divider with 22 gauge nipple connectors at the outlet and a

13 gauge nipple connector at the inlet. The inlet was

fitted with a short piece of silicon tubing into which a 16

gauge blunt needle and hub were inserted. The TPN formula

was delivered through commercial IV administration tubing

(Travenol Laboratories, Inc., Deerfield, IL) and connected

to one stream divider. A variable speed peristaltic

cassette pump (Manostat, New York, NY) equipped to deliver

TPN for up to 18 rats was used to infuse the formula.

Approximately 24 hours prior to animal surgery, all formula

delivery tubings and stainless steel connectors were

autoclaved, hung and flow rates adjusted with sterile

saline.

A 3-day post-surgery recovery period was allowed during

which the animals were infused with sterile saline at

approximately 15 mL/day. They were allowed to consume the

supplemented lactalbumin diet in the same quantity as during

the adaptation period. During the third day post-surgery,

the flow rate was gradually increased to 55 mL/day. An

arginine-devoid TPN solution was initiated on the fourth day

and per os feeding stopped. Four days later, an arginine-

supplemented solution was started and administered for an

additional five days. Tap water was provided ad-libitum

throughout the entire period. Rats were observed several

times daily for such things as separation of catheter from

transmission line, infiltration of diet into the neck area










or dislodgement of catheter. Animals which had become

infiltrated or had the catheter dislodge were removed from

the experiment; animals with disconnected catheters were

lightly anesthetized, reconnected to the TPN transmission

line and allowed to continue on the experiment.



Experimental Protocols



Arg-devoid or arg-supplemented diets and/or time

periods will be referred to as -Arg or +Arg, respectively.

All percentages of arg in this text will refer to the

percentage expressed as g arg/100 g total amino acids (TAA).

The few experiments utilizing gln will be referred to in the

same manner.



Development of a TPN Model for
Determining Arqinine Adequacy




Four separate experiments were conducted to establish

an appropriate protocol to determine arg adequacy in TPN.

An initial experiment was conducted to establish the

effect of a -Arg TPN infusate on urinary metabolites and on

urea cycle enzyme activities. All animals were adapted at

least one week to the laboratory environment and were

provided a 21% protein laboratory ration and water ad-

libitum. Two groups of animals were catheterized and diet










infusion was begun as soon as the animals recovered from

anesthesia. One group of animals was infused with a -Arg

diet and the other was fed a +Arg diet. Diet composition is

outlined in Appendix I as 'Preliminary TPN Diet'. Animals

were infused for four complete days. Daily urine

collections were made and metabolites measured. On the

fifth day, animals were sacrificed, organs were excised and

weighed and urea cycle enzyme activities measured.

A second experiment was conducted in which the TPN diet

was fed orally in a 30 mL drinking bottle to the animals to

establish the effect of route of administration on urinary

metabolites and urea cycle enzyme activities. The animals

were adapted to the laboratory environment at least one week

and were provided a 21% protein laboratory ration and water

ad-libitum. The TPN diet was formulated as though it would

be administered intravenously. Half of the volume was given

between 09:00 and 10:00 hrs and the second half was given

between 17:00 and 18:00 hrs. Glutamine (gln) was

incorporated into this experiment to establish preliminary

data on the ability of amino acids known to supply arg

precursors to replace arg in a TPN formulation. In a

sequential manner, two levels of +Arg and two levels of

isonitrogenous replacement of arg by gln were fed for five

days, each after a 3-day -Arg period. There were three

groups. A control group received an unsupplemented

lactalbumin which was isonitrogenous and isocaloric to the










TPN diets. A second group received the higher level of

supplementation of first 3.27% arg and then 5.38% gin. The

third group received the lower level of supplementation of

0.81% arg and then 1.35% gin. Unsupplemented lactalbumin

controls were pair-fed isonitrogenously throughout the

entire experiment. Daily urine collections were made and

urinary metabolites measured. At the termination of the

+gln period, animals were sacrificed, organs excised and

weighed and urea cycle enzyme activities measured.

The next two experiments were parenteral infusion

studies using the same design as the oral feeding study.



Establishment of Arginine Adequacy in TPN



Rats were adapted to the laboratory for one week and

fed the supplemented lactalbumin control diet at a rate of

0.29 g nitrogen/day. Rats gaining weight at a rate of at

least 3 g/day were randomly assigned to either a TPN diet or

the control diet.

For those animals assigned to a TPN diet, catheters

were inserted, saline was infused, they were allowed the

post-surgery recovery and given four days of the -Arg TPN

infusion, as described previously. One of four levels of

+Arg diet was then infused over the next five days. The

four calculated levels of arginine supplementation were 1.6,

2.5, 3.2 and 4.8%. The lactalbumin fed control animals were










also housed in individual metabolic cages and remained on

the supplemented lactalbumin diet at the rate of 0.29 g

N/day.

Twenty-four-hour urine collections were made as

described earlier on both control and operated animals

beginning on the first day post-surgery. Urine analysis

included orotate, urinary urea, creatinine, citrate and

total nitrogen. Urinary orotate concentrations were

determined at least every other day to verify the presence

of orotic aciduria and to follow the return of orotic acid

excretion to more normal levels.

The experiment was terminated by exsanguination of the

animals via the inferior vena cava under anesthesia.

Approximately 7-10 mL of venous blood were collected in

heparinized syringes; the liver, kidneys, spleen, small

bowel, heart and lungs were excised. Catheter placement and

general status of catheter entry site were checked and

observations were recorded. The mesentery was removed from

the small bowel at the time of excision. Upon excision,

organs were immediately placed in ice cold saline, with the

exception of liver and kidney used for urea cycle enzyme

activity determination. Once all organs had been excised

they were blotted dry and weighed.

A one-way analysis of variance was performed on organ

weights and urinary metabolites to determine differences

between groups. An adequate supplementation level was








51
determined as that level of supplementation which decreased

and maintained an orotic acid excretion of <100 pg/day

within 48 hours. This was determined from regression

analysis of urinary orotate vs. % arg over the linear

portion of the curve (Robbins et al., 1979).



Assessing Urea Cycle Enzyme Activities under TPN



After acclimatization to the laboratory, rats assigned

to TPN-feeding were catheterized, allowed to recover and

administered the -Arg diet. Arginine was supplemented at a

level of 4.5% arg as determined from the results of the

protocol to establish arginine adequacy. Arg-supplemented

lactalbumin-fed animals served as controls and began the

experiment at the time of catheterization of the TPN-fed

animals. Liver CPS-I, AS and A and kidney AS and A

activities were determined in one-half of the animals in

both groups at the end of the arg-devoid period. The same

enzyme activities were determined on the remaining animals

in both groups at the end of the arg-supplemented period.

Enzyme activities were determined within two hours of

excision, as outlined previously.

Twenty-four-hour urine collections were made and

urinary metabolites analyzed. Organs were excised and

weighed at the termination of the experiment. A one-way

analysis of variance was performed on organ weights and










enzyme activities to determine differences between arg-

devoid and arg-supplementation feeding.



Determination of Arteriovenous Amino Acid Differences



Animals were adapted, and those assigned to TPN-

feeding were catheterized, allowed to recover and

administered -Arg diet. Arg was supplemented at the level

of 4.5% arg as determined from the results obtained from the

protocol to establish arg adequacy. Supplemented

lactalbumin-fed animals served as controls and began the

experimental period at the time of catheterization for the

TPN-fed animals. Blood flow rates and plasma amino acid

concentrations in the hepatic, portal and left renal veins

and the carotid artery were determined at the end of the

arg-devoid period in one-half of the animals of both groups

and at the end of the arg-supplemented period in the

remaining animals. Blood flow rates were determined

according to the method established by Welbourne et al.

(1986). Animals were anesthetized and a tracheotomy was

performed. TPN rats were continued on diet infusion after

being anesthetized. The carotid artery was catheterized and

0.1 mL of 1000 USP units/mL heparin (Elkins-Sinn, Inc.

Cherry Hill, NJ) was infused. Needle catheters (25 gauge)

were inserted into the portal, hepatic and left renal veins.

A 30 gauge needle catheter was inserted into one of the










tertiary branches of the mesenteric vein. An infusion of

14C labeled para-aminohippurate (14C-PAH) was initiated and

continued 45 min to allow time for the label to reach steady

state concentrations at a rate of 0.039 mL/min. At the end

of the infusion period, approximately 0.8 mL of blood was

slowly withdrawn from each vessel in the following order:

hepatic, portal, renal and carotid. An aliquot of 0.3 mL

whole blood was precipitated with 0.3 mL of 10% perchloric

acid and centrifuged. An aliquot of 500 gL of the

supernatant was placed into a scintillation vial and 20 mL

ScintiVerseM II were added. The samples were counted in a

Beckman LS2800 scintillation counter (Beckman Instruments,

Inc., Fullerton, CA) for 10 minutes at channel settings

corresponding to 14C pulses. An aliquot of the infusate was

also prepared for counting in the same manner as the blood.

Flow rate was calculated as follows: (cpm infusate/(cpm vein

- cpm artery) x infusate flow rate x (1/BW/100g). The

remaining whole blood was prepared for amino acid analysis

as described previously.














RESULTS AND DISCUSSION


This section will be divided according to the

objectives for this dissertation research. The results of

each objective will be discussed individually followed by a

summary discussing the overall results of the dissertation

research.



Development of a TPN Model for
Determining Arginine Adequacy


Four experiments were performed to accomplish this

objective. The first experiment was a preliminary one which

established the effects of either a +Arg of 4.7% arg or a

-Arg TPN diet on rats. Since this was a preliminary

experiment, animals not needed for other experiments

conducted in our laboratory were used even though the

initial body weights were higher than those expected to be

used for future experiments. A total of eight rats were

catheterized and TPN administration began immediately. Six

rats completed the four-day experiment; two animals became

infiltrated with diet and had to be removed from the

experiment. Table 3 lists the body and organ weights at the

termination of this experiment. Final body weight was











Table 3. Body and Organ Weights after 96 Hours of 4.7%
Arginine or 0% Arginine TPN.


4.7% Arg
(Ave+SD)


0% Arg
(AveSD)


Final Body Weight (g)

379.67 + 15.58


358.67 + 20.07


Weight Gain (g/d)


-0.50 + 0.54


Organ Weight (g/100g BW)

Liver 5.08 + 0.24

Kidney 0.91 + 0.05

Spleen 0.53 + 0.20

Lung 0.73 + 0.13

Heart 0.36 + 0.04


0.83 + 1.50


3.88

0.84

0.37

0.62

0.35


+ 0.32

+ 0.07

+ 0.08

+ 0.09

+ 0.03










greater in the +Arg group due to a heavier initial weight.

Despite the heavier final weight, the +Arg group lost an

average of 0.5 g per day whereas the -Arg group gained an

average of 0.83 g per day. This difference in weight gain

may have been due to individual animal response to surgical

recovery and adaptation to TPN which was confounded by the

short duration of the experiment. Statistical differences

were not determined due to the short duration of the

experiment and movement of the rats to balance the groups on

Day 2. Except for heart weight, all organs were heavier in

the +Arg group relative to the -Arg group. Liver and spleen

exhibited the greatest differences of 23.6 and 30.2%,

respectively. Figure 2 shows the daily urinary orotate

excretion for both groups in this experiment. Orotate

excretion nearly doubled in the -Arg group within 24 hrs of

TPN with a peak orotate excretion of approximately 1400

Ag/day on Day 3, relative to the +Arg group. Urinary

citrate excretion shows a steady increase in citrate

excretion in the -Arg group relative to the +Arg group

throughout the duration of the experiment (Figure 3). Peak

citric acid excretion in the -Arg group occurred on Day 4

and was four-fold greater than the +Arg group. Figure 4

showed the urinary urea nitrogen (UUN) excretion data for

this experiment. There was little difference in UUN

excretion between the two groups during the first two days









2500


- ZUUU









0
S1750


1500

S 1250 -
u / \

1000 -\










0
5' 750 /

"J 500o


Q5o -----------I-------.e,---..-----------


1 2 3 4

Experimental Day








Figure 2. Average Daily Urinary Orotic Acid Excretion of
Rats Administered a TPN Formula Supplemented with Either
4.7% Arginine or 0% Arginine per Total Amino Acids for Four
Days.



















01


'-I



L
C


C
L.


2,000


1,750


1.500


1,250


1,000


750


500


250 -"I


0 -i----- ------------!
1 2 3 4

Experimental Day








Figure 3. Average Daily Urinary Citric Acid Excretion of
Rats Administered a TPN Formula Supplemented with Either
4.7% Arginine or 0% Arginine per Total Amino Acids for Four
Days.









250


Z





0

E
0

o
0


L.
D
0
*L


200





150





100





50


01 --
1 2 3 4

Experimental Day









Figure 4. Average Daily Urinary Urea Nitrogen Excretion of
Rats Administered a TPN Formula Supplemented with Either
4.7% Arginine or 0% Arginine per Total Amino Acids for Four
Days.










of infusion. The +Arg group showed a slight, but steady

increase in UUN from days three to four, relative to the

-Arg group. Total daily urine volume is depicted in Figure

5. The +Arg group excreted slightly more urine on all days

except the first, relative to the -Arg group. Urinary

creatinine (Figure 6) was slightly elevated in the +Arg

group relative to the -Arg group throughout the entire

experiment. The coefficient of variation (CV) for

creatinine excretion of individual rats varied between 3.7

and 9.9% with one animal having a CV of 18.3%. Data from

this experiment for urinary orotate and citrate concur with

data presented by Milner and Visek (1975) for characteristic

urinary metabolite response in rats due to an arginine-

deficient diet. Urinary creatinine and total urine volume

followed the same pattern as data presented by Milner et al.

(1974). Both of these parameters were depressed in the -

Arg group. In humans, there is a normal variation in daily

creatinine excretion of 4 to 8%; stress (emotional) and

trauma can increase this variation by 5 to 10% and 20 to

100%, respectively (Heymsfield et al. 1983). Fisher (1965)

reported urinary creatinine CV between 10 and 20% for rats

fed a 15% protein stock diet. Variations of urinary

creatinine within normal limits have been used as a

verification for completeness of urine collection. The

creatinine CV of individual rats from this experiment

indicated that complete urine collections were obtained.








85


N%
-j
E


0
E
03
Z


65
60
55
50
45
40
35
30
25
20
15
10
5
0


I


*4.7X kg

a OX kg


Experimental Day








Figure 5. Average Daily Urine Volume of Rats Administered a
TPN Formula Supplemented with Either 4.7% Arginine or 0%
Arginine per Total Amino Acids for Four Days.


MOMMIr-


%*X0

















-0J


E


C
0

C

V
L-
0
Q)



C
L


11


10 i-i
1 2 3 4

Experimental Day









Figure 6. Average Daily Urinary Creatinine Excretion of
Rats Administered a TPN Formula Supplemented with Either
4.7% Arginine of 0% Arginine per Total Amino Acids for Four
Days.










Only urinary urea excretion in this experiment contrasted

with data presented by Milner and Visek (1975).

Urea cycle enzyme activities measured in this

experiment are listed in Table 4. Liver urea cycle enzymes

were all elevated after 96 hours of -Arg TPN infusion

relative to the +Arg group. Liver enzyme activities in the

-Arg group were 194%, 148% and 141% greater than the

activities in the +Arg group for CPS, AS and A,

respectively. Ha and Milner (1979) measured liver urea

cycle activities in rats fed orally an arginine-deficient

diet and reported a 164% increase in CPS activity relative

to the control group. However, AS and A activities were not

significantly increased by the arg-deficient diet relative

to controls.

Kidney urea cycle enzyme activities were depressed in

the -Arg group relative to the +Arg group. Kaysen and

Strecker (1973) reported that arginase activity in the liver

was approximately 30 times greater than the activity in the

kidney. The liver:kidney arginase activity ratio in this

experiment was 14.1 for the +Arg group and 29.6 for the -

Arg group. The renal medulla and cortex were separated and

arginase activities were measured in the cortical region.

Snellman et al. (1979) compared renal arginase activities in

rats fed either a 6% or 21% protein diet. They reported a

renal arginase activity increase of 327% and 50% in the

cortical region and the outer medullary region,










Table 4. Urea Cycle Enzyme Activities After 96 Hours of
4.7% Arginine or 0% Arginine TPN



4.7% Arg 0% Arg
Enzyme (mean + SEM) (mean SEM)


Liver Enzyme Activity*

CPS 1.52x107 + 0.3x107 2.95x107 + 0.7x107

AS 33.98 + 3.5 50.13 + 6.4

A 6.06x104 + 0.7x104 8.55x104 + 1.7x104


Kidney Enzyme Activity*

AS 34.78 + 7.7 26.28 + 3.4

A 4.30x103 + 0.7x103 2.89x103 + 1.7x103


A*mol product/hr/g tissue
CPS = carbamoyl phosphate synthetase
AS = argininosuccinate synthetase
A = arginase










respectively, in rats on the low protein diet. Total

dietary protein was equivalent in this experiment; thus, the

increased kidney urea cycle enzyme activities in the +Arg

group may be due to increased substrate supply.

The conclusion of this experiment was that TPN-

administered rats have a similar response to an arg-

deficient diet as rats orally-fed an arg-deficient diet.

Thus, this animal protocol provides an adequate model to

study arginine metabolism in parenteral nutrition.

The second experiment was designed to determine the

effect of route of administration as well as alternate

substrate supply on arginine metabolism when compared to

TPN-administered experiments. Thus, the TPN diet was

orally-fed. The experiment utilized three groups of seven

animals each. The first group received an orally-fed TPN

diet supplemented first to 0.78% arg and then 1.61% gln,

each after a three-day -Arg period. The second group

received an orally-fed TPN diet supplemented first to 3.23%

arg and then 5.36% gln, again, after a three-day -Arg

period. The third group served as a control group and was

pair-fed isonitrogenously an unsupplemented lactalbumin

diet.

Figure 7 shows the growth curve of the three groups

over the course of the experimental period. Table 5 shows

the average body weights of the three groups initially as

well as at the transition between supplementation levels of









400


01

-c

-o


0


200 -1J L ---141iii- 1
-3 -1 0 1 2 3 4 5 6 7 8


9 101112 13 14 1516


Experimental Day






Figure 7. Average Body Weight Gain of Rats Orally-Fed One
of Two Levels of Arginine, then, One of Two Levels of
Glutamine Supplemented TPN Formula and Controls.










Table 5. Body Weights at Selected Intervals for Rats
Orally-Fed a TPN Diet.



Low Level High Level Control
Time Period (g, mean + SD)


Initial 270.00 + 6.0a 270.71 + 18.38 263.29 + 11.58

Adaptation 313.14 + 20.7a 319.86 + 29.4a 326.57 + 13.08

First -Arg 308.14 + 12.5a 308.29 + 22.0' 313.29 + 10.1a

+Arg 317.43 + 12.7a 314.28 + 20.8a 329.14 + 8.2a

Second -Arg 325.57 + 13.6a 321.57 + 26.2a 335.86 + 97.a

+Gln 334.14 + 12.1b 335.42 + 23.8b 360.43 + 10.5a


Except initial body weight, all weights given are those at
the end of the time period specified. Initial refers to
arrival to the laboratory, adaptation refers to the end of
the adaptation period, first and second -Arg refer to the
end of the -Arg periods, +Arg refers to 0.78% arg for the
low level and 3.23% arg for the High Level and +Gln refers
to 1.61% gin for the Low Level and 5.38% for the High Level.

Values without common superscript within a row differ by
p<0.05.








68

the diets. There were no statistical differences in average

body weight between the three groups upon arrival at the

laboratory, at the end of the ad-libitum-fed adaptation

period or during the first -Arg and +Arg phase of the

experiment. However, there was a trend toward a decreased

body weight in both orally-fed TPN diet groups relative to

the control group. By the end of the +Gln period, there was

a difference (p < 0.05) between the control animals and the

two orally-fed TPN diet groups, but no difference in body

weight between the two orally-fed TPN diet groups. All

three groups lost weight during the first -Arg period due to

low food consumption; but did not lose weight during the

second -Arg period (Table 5 and Table 6). During the first

-Arg period, food consumption (expressed as N intake) in the

control animals was statistically less relative to the two

orally-fed TPN diet groups (Table 6). The difference in

food consumption was concluded to be due to a lack of

adaptation to the diet which resulted in an initial refusal

of the semipurified diet. This refusal lead to the body

weight loss during the initial -Arg period. Except on Days

12 and 13, there was no difference (p < 0.05) in food

consumption between the two orally-fed TPN diet groups. The

weight loss during the first -Arg period for these two

groups was concluded to be due to a lack of adaptation to an

isonitrogenous diet as well as a response to the -Arg diet.











Table 6. Food Consumption as Expressed by Nitrogen Intake
for Orally-Fed TPN Diet Rats.



Low Level High Level Control
Day (mg N, mean + SD)


+ 45.8a

+ 27.8a'b

+ 8.6a'b

+ 5.0a'b

+ 3.7a

+ 1.9a

+ 2.4a

+ 29.9a

+ 31.1a

+ 35.3a

+ 43.18

+ 35.1b

+ 12.0b

+ 3.4a

+ 31.1a

+ 39.5a


252.00

281.14

282.14

287.14

281.57

284.00

280.29

267.71

262.00

243.57

274.71

268.29

283.86

267.14

273.14

268.43


25.3a

8.5a

6.78

2.7a

3.2a

3.9a,b

8.8a

29.3a

32.2a

71.0a

9.8a

30.6a

3.4a

22.1a

21.9a

23.6a


150.14 +

259.57 +

266.71 +

280.57 +

283.56 +

277.86 +

277.86 +

268.43 +

244.57 +

244.57 +

251.57 +

286.57 +

276.43 +

257.00 +

241.14 +

272.29 +


Days 1-3 were first -Arg diet; Days 4-8 were +Arg at 0.78%
arg in Low Level and 3.23% arg in High Level; Days 9-11 were
second -Arg diet; Days 12-16 were +Gln at 1.61% gln in Low
Level and 5.38% gin in High Level. Control was fed
unsupplemented lactalbumin control diet.

Values without common superscript within a row differ by
p<0.05.


230.57

273.43

276.14

283.86

285.57

285.71

286.43

266.14

248.29

249.86

252.43

237.57

265.86

279.00

260.71

247.43


56.3b

12.6b

13.8b

3.6b

5.0a

8.8b

8.8a

9.7a

6.4a

1.18

9.3a

2.0a

9.1a

26.8a

38.3a

13.3a










Table 7 lists the organ weights at the termination of

the experiment. There were no differences (p < 0.05) in

organ weights, except spleen weight, due to diet. Spleen

weight was lower (p < 0.05) in both orally-fed TPN groups

relative to the control group.

Feeding the rats a TPN diet orally increased the urine

output (Figure 8). Only Day 1 differed from the pattern of

no statistical difference between the two orally-fed TPN

diet groups and these two groups differed from the control

group. On Day 1, the two orally-fed TPN diet groups

excreted more urine than controls, however, all three groups

were different (p < 0.05) from each other.

Figure 9 shows the daily urinary orotate excretion

levels. All groups exhibited orotic aciduria throughout the

entire experiment, although this was not realized until

further experiments had been conducted. Overall, there was

not a clear trend in dietary influence on orotate excretion.

Standard deviations within a group were very large and the

coefficient of variation (CV) ranged from 12 to 148%. It

was learned from subsequent experiments the urinary orotate

excretion of rats, within a dietary group, not receiving

adequate arginine had extremely large variations. During

the first -Arg period, the orally-fed TPN diet groups tended

toward higher orotate excretion by the second and third days

of -Arg feeding, as expected. As a result of the large CV,

there were no differences (p 0.05) between any of the










Table 7. Organ Weights for Rats Orally-Fed a TPN Diet
Containing Either 1.61% or 5.36% Glutamine and Control.



1.35% Gln 5.38% Gln Control
Organ (mean + SD, g/100 g BW)


Liver 3.57 + 0.2a 3.79 + 0.2a 3.54 + 0.2a

Kidney 0.58 + 0.05a 0.59 + 0.04a 0.60 + 0.04a

Spleen 0.21 + 0.02b 0.22 + 0.03b 0.26 + 0.04a

Lung 0.47 + 0.03a 0.46 + 0.05a 0.46 + 0.02a

Heart 0.37 + 0.02a 0.37 + 0.03a 0.36 + 0.02a

GI* 2.59 + 0.25a 2.70 + 0.32a 2.51 + 0.18a


Small intestine from stomach to cecum with mesentery
intact.

Values without a common superscript within a row differ by
p<0.05.









60
56
S52
\48
44
40
( 36
E 32
3 28
0 24
> 20
I 16
*- 12
D 8
4
0


Experimental Day


-Arkg


+Ark/+Gln


0


E

:3


L.
DI


9 10 11 12 13 14 15 16
Experimental Day


Figure 8. Average Daily Urine Volume of Rats Orally-Fed One
of Two Levels of Arginine, then, One of Two Levels of
Glutamine Supplemented TPN Formula and Control.


wo"M ow









25000
"0 0.78X kg
s 22500 --
3 20000 C.23X kg
S17500 / Unupplemented
U / Lactobumin Control
< 15000 / /
.2 12500
0 10000
0 7500

b 5000
*E 2500

1 2 3 4 5 6 7 8
Experimental Day
-Arg +Arg/+Gn

S25000 1.61
1.1iX Gln
S22500 -
5.36X Gin
S20000
--e -
17500 Unupplemented
*RU Lactolbumin Control
<15000 -
.2 12500
0
7500
0 5000
ZC 2500

9 10 11 12 13 14 15 16
Experimental Day
Figure 9. Average Daily Orotic Acid Excretion of Rats
Orally-Fed One of Two Levels of Arginine, then, One of Two
Levels of Glutamine Supplemented TPN Formula and Control.










groups on Day 2, although the orotate excretion was higher

in the orally-fed TPN diet groups. On Days 1 and 3, all

three groups were statistically different from each other.

On Days 5 and 6, the 0.78% arg group excreted more orotate

than the other two groups, however, only on Day 6 was this

difference significant (p < 0.05). There were no

differences in orotic acid excretion between any of the

groups on Days 7 and 8. During the second -Arg period, the

orally-fed TPN diet groups did not display the expected

increase in orotic acid excretion. These two groups

excreted less (p < 0.05) orotic acid at the end of the

second -Arg period relative to the last day of the first

-Arg period. After initiation of +Gln, the two orally-fed

TPN diet groups generally excreted more orotic acid than the

control animals. However, only on Days 12 and 16 were the

excretion values different (p < 0.05) from controls. At

this point in the research project, it was not known if rats

fed a TPN diet were capable of excreting orotate at levels

similar to those reported in the literature as 'control'

levels for animals fed a purified diet. Thus, no target

orotate excretion level had been chosen for an indication of

adequate arginine supplementation. Figure 10 superimposes

the urinary orotate data for the two phases of this

experiment. The increase in orotic acid excretion during

the first, but not the second -Arg period was concluded to









__ 6_


10,000



3.000



1,000


S0.78% Arg
S 300 --)

>, 3.23% Arg
b ---
3 100
.C 2.12% Gin


5.36% Gin
30
-Akg +Arg/+Gln

10 ------
1 2 3 4 5 6 7 8

Experimental Day









Figure 10. Superimposing Urinary Orotic Acid Excretion of
Rats Orally-Fed One of Two Levels of Arginine, then, One of
Two Levels of Glutamine Supplemented TPN Formula.








76

be partially due to feeding a 21% protein laboratory ration

ad-libitum during the adaptation period.

Figure 11 depicts the daily urinary citrate excretion.

Excretion levels are elevated in all groups on Day 1 and in

the orally-fed TPN diet groups on Day 2, relative to the

remainder of the experiment. However, after the second day

of the experiment, citrate excretion was fairly uniform

throughout the entire experiment within all groups. Citrate

excretion in control rats was slightly elevated during the

first five days of the experiment. There was no difference

(p < 0.05) in excretion between the groups for Days 3

through 5. For the remaining +Arg period, the orally-fed

TPN diet groups were not statistically different from each

other, but were higher than controls. During the second

phase of this experiment, citrate excretion was

statistically higher in the orally-fed TPN groups relative

to the control group, except Days 11, 14 and 16. There was

a large variation in citrate excretion on Day 11 in both the

orally-fed TPN diet groups and this variation probably led

to lack of statistical difference between the three groups

on this day. There was no difference in citrate excretion

by a paired-t analysis of the data from the end of the

various periods for either of the orally-fed TPN diet

groups. However, there was a statistical difference in

citrate excretion shown by the paired-t analysis for control

animals between the two -Arg periods and between the +Arg









500
-o
450

S4001

S350o

< 300,
u
250

0200

L. 150
0
100



50


50
-0
N450
3400

. 350
U
< 300

S250

200

b 1501

C 100
D3


+Arg/+Gln


10 11 12 13 14


15 16


Experimental


Day


Figure 11. Average Daily Urinary Citric Acid Excretion of
Rats Orally-Fed One of Two Levels of Arginine, then, One of
Two Levels of Glutamine Supplemented TPN Formula and
Control.


1 2 3 4 5 6 7 8
Experimental Day


-Arg


50 L
9


I


r


w










and the first -Arg period, but not for the +Gln and second

-Arg period.

Urinary urea nitrogen (UUN) excretion for this

experiment is shown in Figure 12. There was a steady

decline in UUN for the first five days of the experiment for

all groups, and thereafter, UUN excretion remained fairly

constant. Control animals excreted a greater amount of UUN

relative to the two orally-fed TPN diet groups. Except Days

1, 2 and 6, control animals excreted statistically higher

amounts of UUN, relative to the two orally-fed TPN diet

groups. There was no difference (p : 0.05) in UUN excretion

between the two TPN diet groups, except all groups were

statistically different from each other on Days 2 and 6. On

the first day, the control and 3.23% arg group differed

statistically (p < 0.05) in UUN excretion. There was a

difference in paired-t analysis for all groups between the

two -Arg periods and between the +Arg and first -Arg period.

However, there was no difference in paired-t analysis for

UUN between the +Gln and second -Arg period.

Urinary total nitrogen for this experiment is shown in

Figure 13. Urinary total nitrogen followed the same pattern

as UUN. There was a decline in total nitrogen excretion for

the first five days and a relatively constant excretion

level thereafter. Except for Day 2, control rats excreted

more total nitrogen relative to the two orally-fed TPN

groups. The difference was statistically greater after
















Z
C-





0
q)


0
C
*-
0.


1 2 3 4 5 6 7
Experimental Day


S-krg


35C
325
30C
275
25C
225
200
175
150
125
100
75
50
25
0


+Arg/+Gln


9 10 11 12 13 14 15 16
Experimental Day
Figure 12. Average Daily Urinary Urea Nitrogen Excretion of
Rats Orally-Fed One of Two Levels of Arginine, then, One of
Two Levels of Glutamine Supplemented TPN Formula and
Control.


I I


"0

0'
E

Z
0




C
L


1.61%X Gn
---
s.36X Gan

) Umpplnmented
Loctabumin Control
--
pI





I I


)









325 -
"300 -
P 275
E 250
225-
z 200
S175 "



o1075 -
LC 50
S25
0 --- -- i --- ---
1 2 3 4 5
Experimental
-Arg Ar
350
325 -
N 300
o 275
E 250
225
200
0 175
0 150 -


.S75
C. 50
S25

9 10 11 12 13
Experimental
Figure 13. Average Daily Urinary Total
of Rats Orally-Fed One of Two Levels of
of Two Levels of Glutamine Supplemented
Control.


6 7 8
Day
9/+Glnmo


14 15 1
Day
Nitrogen Excretion
Arginine, then, One
TPN Formula and








81

Day 2. Except Day 2, there was no statistical difference in

urinary total nitrogen excretion between the two orally-fed

TPN diet groups. There was a difference in urinary total

nitrogen (paired-t analysis, p < 0.05) for the orally-fed

TPN diet animals between the two -Arg periods, the +Arg and

the first -Arg period and the +Gln and second -Arg period.

There was a difference (p < 0.05) in urinary total nitrogen

for the control animals during the two -Arg periods and the

+Arg and first -Arg period. However, there was no

statistical difference between the +Gln and second -Arg

period for controls.

Daily urinary creatinine excretion is shown in Figure

14. Urinary creatinine excretion is fairly uniform

throughout the entire experimental period. Except on the

last day of the first phase of the experiment (Day 8), there

was no statistical difference in urinary creatinine

excretion between the three groups. On Day 8, the control

group excreted a greater amount (p : 0.05) of creatinine

relative to the two orally-fed TPN diet groups. The second

phase of the experiment showed some statistical differences

in creatinine excretion, however, the trend of significance

was not uniform. On Days 12, 13, 14 and 16, the two orally-

fed TPN diet groups were not statistically different from

each other and except for Day 14, they were different from

the control group. On Days 12, 14 and 16 the creatinine

excretion level of these two groups was less than that of










N
Ob

E
0


*E-
a
0


0
a
C
*
1L


1 2 3 4 5 6 7 8


-.. -krg


-o

0

E

C
a,


0


a
C
*


Experimental Day
+Arg/+Gln


9 10 11 12 13 14 15 1
Experimental Day
Figure 14. Average Daily Urinary Creatinine Excretion of
Rats Orally-Fed One of Two Levels of Arginine, then, One of
Two Levels of Glutamine Supplemented TPN Formula and
Control.


w-~ ~








83

the controls. However, on Day 13 these two groups excreted

more creatinine than controls. The absolute difference in

excretion was never more than 4.2 mg/d, thus there may not

be a significant physiological difference in the creatinine

excretion levels between any of these three diets.

Individual animal creatinine excretion CV was between 9.5

and 13.7 mg/d. There was no difference in creatinine

coefficient of variation between any of the animals, except

the one with the lowest variation in excretion level. Thus,

it can be concluded that urine samples were adequately

collected.

Urea cycle enzyme activities were measured at the

termination of the experiment and activities are listed in

Table 8. Liver AS activity was elevated, but not

significantly (p < 0.05), in the orally-fed TPN diet groups

relative to the control group, and the 1.61% gln group was

elevated relative to the 5.36% gln group. Liver A activity

was significantly depressed (p < 0.05) in the orally-fed TPN

diet groups relative to the control group. The 1.61% gln

group was depressed to a greater extent than the 5.36% gln

group, but not significantly (p 0.05). Kidney AS activity

was depressed (p 0.05) in both the orally-fed TPN groups

relative to the control groups. There was no statistical

difference in kidney A activity among any of the diets.

Plasma amino acids were measured at the termination of

the experiment and are listed in Table 9. Serine, glycine










Table 8. Urea Cycle Enzyme Activities for Rats Orally-Fed a
TPN Diet Containing Either 1.61% or 5.36% Glutamine and
Control.



1.61% Gln 5.36% Gin Control
Enzyme (mean + SD)


Liver Enzyme Activity*

CPS (xl06) 3.38 + 2.0 nd nd

AS 56.75 + 15.8a 43.81 + 12.58 46.72 + 27.2a

A (xl05) 6.24 + 4.2b 9.74 + 1.1b 14.00 + 4.5a

Kidney Enzyme Activity*

AS 10.83 + 5.2b 17.15 + 3.3b 53.32 + 21.4a

A (x103) 8.68 + 9.48 2.50 + 0.6a 3.94 + 1.3a


Amol product/hr/g tissue


CPS = carbamoyl phosphate synthetase
AS = argininosuccinate synthetase
A = arginase

Values without a common superscript within a row differ by
p<0.05.