Efficacy of ornithine-alpha-ketoglutarate (OKGA) as a dietary supplement in growing rats

C’hcol Nufnrwn (1991) 10: 155-161 0 Lonpman Group UK Ltd 1991

Efficacy of ornithine-alpha-ketoglutarate a dietary supplement in growing rats M. JEEVANANDAM,

M. R. ALI, L. RAMIAS

Trauma Center, St. Joseph’s 85013, USA (Correspondence

(OKGA) as

and W. R. SCHILLER

Hospital and Medical Center, 350 West Thomas Road, Phoenix, and reprint requests to M.J.)

This work was presented in part at the Annual FASEB Meeting held in Washington, 1990: FASEB Journal 4: A806, (Abst 3126); 1990.

Arizona

DC, April 1-5,

ABSTRACT-New substrates of potential benefit to critically ill patients receiving traditional nutritional support have been suggested to meet organ or tissue specific needs. The addition of an anabolic stimulus during nutritional support therefore appears to be a reasonable adjunct to augment protein synthesis. The purpose of this investigation was to evaluate the efficacy of the neutral salt ornithine alphaketoglutarate (OKGAI as a dietary supplement to promote growth in young rats by enhancing protein metabolism. A group of 16 male Sprague-Dawley rats (150-1709) were housed in individual metabolic cages and after dark-light cycle adaptation were fed ad iibitum an oral liquid diet for 7 days. Half of the animals were given the control diet and the other half was fed a test diet. This isonitrogenous test diet contained the control diet with 2.3% of nitrogen (N) replaced by N from OKGA. Daily weight, food intake and urinary excretions of N, creatinine, urea, erotic acid, polyamines and amino-acids were determined. At the end of 7 days of free-feeding, the rats were sacrificed and blood was collected for free amino-acids. Rats fed the OKGA supplemented diet consumed 16% more diet, retained 11% more nitrogen and gained 15% more weight. The accelerated protein metabolism is reflected in the changes in plasma and urinary free amino-acid levels. Enhanced protein anabolism is evident from the increased urinary excretion of polyamines in the OKGA fed rats. The increased ratio of urinary urea N to total N and the decreased erotic acid excretion in OKGA fed rats suggests thata NH4+ was efficiently diverted through urea cycle. It is concluded that in growing rats, supplementing isonitrogenous diet with OKGA significantly stimulates food intake compared to controls. This results in better weight gain and improvement in protein metabolism.

lin and growth hormones ( 1, 2. 3), thereby promoting intracellular amino-acid transport and protein synthesis. Administration of adjuvant OKGA after surgical trauma reduces the intracellular depletion of glutamine (4), and improves skeletal muscle protein synthesis and nitrogen economy (5). The anabolic effect of OKGA may partly be linked to the diversion of ornithine to the biosynthesis of polyamines which play an important role in the regulation of cell growth and tissue protein synthesis (6). The 2:l molar combination of L-ornithine and alphaketoglutaric acid modifies amino-acid metabolism and hormonal patterns in a way which is not observed when they are administered separately (7, 8), suggesting that the simultaneous administration of ornithine and alphaketoglutarate is necessary for the expected anabolic effects.

Introduction

Enteral nutrition has evolved into an effective means of providing nutrients to malnourished hospitalised subjects. While current nutritional support may prevent further protein loss during severe illness or injury, exogenous nutrient supply by itself will not stimulate protein synthesis significantly, nor will it produce notable protein accrual during short-term use. The addition of an anabolic stimulus during intensive nutritional therapy appears to be a rational adjunct to augment protein synthesis that may eventually lead to accrual of structural and functional proteins resulting in growth. It has been demonstrated that the ornithine salt of alphaketoglutaric acid (OKGA) is a powerful stimulator of hormonal release. particularly insu155

156 ORNITHINE-ALPHA-KETOGLUTARATE

AS A DIETARY SUPPLEMENT

An OKGA supplemented diet may lead to an altered utilisation of the diet, stimulating growth rate in healthy animals. In this report, the benefits of the preferential consumption of OKGA supplemented isonitrogenous diet in the course of freefeeding young growing rats are investigated. Materials and methods Sixteen male, young Sprague-Dawley rats (Ace Animals, Inc., Boyertown, PA, USA) weighing between 150-170gm were housed in individual metabolic cages (Plas-Labs, Lansing, MI, USA) located in our temperature-regulated vivarium with controlled 12h light-dark cycles. They were adapted to ad libitum oral feeding and water for 3 days. The animal research facility at St. Joseph’s Hospital and Medical Center is accredited by the American Association of Accreditation of Laboratory Animal Care. The research protocol was reviewed and approved by the Institutional Animal Care and Use Committee and we had adhered to the ‘Guide for the Care and Use of Laboratory Animals’ (NIH Publication No. 85-23 rev.). On day 0 after taking morning weight, and ad libitum feeding of a casein-based liquid diet was started and continued for 7 days. Polyamine-free liquid diet was chosen for convenience since the management of feeding will be uniform and the intake estimation will be more accurate. Animals had free access to water throughout the experiment. Food intake was determined daily in the morning by weighing food containers at the beginning and at the end of each 24h period. Urine output was collected over the span of 08.OOh08.OOh for the determination of 24h excretion of total urinary nitrogen (N), creatinine, urea, erotic acid, polyamines and amino-acids. Half of the animals were given the control oral liquid diet (no. F1259, Bio-Serv Inc., Frenchtown, NJ, USA) which had 6Smg N and 1 kcal energy per ml corresponding to 6.18 mgN/g diet. The mean daily consumption of a 15Og rat was 80ml. The other half of the animals were fed a test oral liquid diet. This isonitrogenous test diet contained the above control diet from which 146mg N/L was replaced by N from OKGA (Cetornan from Laboratories J. Logeais, Paris, France). Each g of OKGA, which is a neutral, monohydrate salt of 2moles of the basic amino-acid L-ornithine and lmole of alphaketoglutaric acid, has 131mg N. The test diet therefore had 2.3% N replaced by OKGA. Considering the higher metabolic rate of rats, on the basis of body weight, this dose of

OKGA for the rat (79.7mg OKGA-N/kg/d) corresponded to twice the normal 20g OKGA dose (37.4mg OKGA-N/kg/d) recommended daily for the enteral route in a 70kg man (9). Each rat received a daily supplementary dose of 429 pmole of L-ornithine and 215 Fmole of alphaketoglutaric acid. After 7 days of oral free-feeding, the rates were sacrificed on the morning (08.00h-08.30h) of the 8th day and blood was collected by cardiac puncture. The total daily urinary nitrogen was determined by using a Chemiluminescence Digital Nitrogen Analyzer (Antek Instruments, Inc., Houston, Texas, USA). Urine concentrations of urea and creatinine were determined using standard procedures with a Micro Centrifugal Analyzer (Multistat Plus, Instrumentation Laboratory, Lexington, Mass., USA). Plasma and urine concentration of individual free amino-acids were determined by the automated ion-exchange method using an amino-acid analyser (Model 7300, Beckmann Instruments, Inc., Palo Alto, CA, USA) as described and used by us in other reports (10). For the determination of 3-methylhistidine the rat urine samples were hydrolysed with 3N HCl for 12-14h at 110°C and then analysed as above using the amino-acid analyser and a shortened program. The polyamines, putreseine (PU), spermidine (SD) and spermine (SM) in the urine were determined after deproteinisation by 50% sulphosalicilic acid, separation by the ion-exchange technique in an amino-acid analyser (Model 7300, Beckman Instruments, Inc., Palo Alto, CA, USA), post-column derivatisation with 0-phthaldehyde and detection by fluorometer. This assay procedure was validated and used by us previously (11). Urinary erotic acid was determined using an improved spectrophotometric method (12). Daily N retention was calculated by subtracting urinary N excretion from dietary N intake. Because faecal output was small and constant, N losses from this source were considered small and negligible and were not included in nitrogen balance calculations. Inclusion of a constant value of 56.6% of urinary nitrogen as faecal output (13) would not have appreciably altered the conclusions from this study. All results are reported as Mean f SEM. Significances of differences between variables were calculated with non-paired Student’s t-test (14) and the correlations were calculated by linear regression. A p value of 0.05 or less was considered statistically significant.

CLINICAL

NCTRITION

157

Cumulative, Voluntery Food Intake and Nitrogen Excretion in Growing Rats 800

GKGA

600 i.. . No .~~ccn I ... .. .. ... .. ...

400 ,..._..__.._...:

1 200 .___...._._____:

. .._._..........

,..___.._______:

;..............:

0 8Od -

600 WGA I ~......._._____. I; . . . . . . . . . . . . . . . ’ No OKGA

400 . . . . ..__...___.

..............I: *

200 .._...._......_ 0

1

_..__.,.__.._.:

. . . . .._.......*

I 2

1 3

I 4

I 5

I 6

I 7

Days of Feeding Fig. 1. Cumulative, voluntary ornithine-alpha-ketoglutarate

food consumption and nitrogen excretion in growing rata fed an oral liquid cher with or without (OKGA). The results are normalised to a rat of initial 150g.

Results

Food consumption and N excretion data, normalised to a 15Og rat are shown in Figure 1. Water and food were freely accessible to both sets of rats. During the 7-day period, the rats fed the OKGA supplemented diet consumed 16% more food and excreted 40% more N than the control diet fed rats. Body weight changes during the study period are shown in Figure 2. Rats receiving the supplemented diet grew better and this became apparent after only 4 days of feeding the diet. The day-to-day changes in the mean body weight, food intake and urinary excretory data are given in Table 1. The increased intake of food on the very first day of feeding liquid diet resulted in the increase of nitrogen and other excretory products. The altered protein metabolism due to OKGA supplemented diet is seen in the increased retention of N, and increased excretion of polyamines. The mean daily changes in the nutritional parameters over the 7 days of ad libitum feeding in the growing rats are given in Table 2. The extra retention of 30mg N per day due to OKGA may account fully for the increase of l.Og of body weight as lean body mass. The proportion of total N excreted as urea N was increased from 78% to 84% in the OKGA supplemented rats. This observation, along with their reduced erotic acid

CN

C

excretion, suggests that the ammonia N seems to be diverted preferentially through the urea cycle by the additional OKGA. The excretion of 3methylhistidine, when corrected for muscle mass, is the same (0.5 & 0.1 kmol/mg creatinine) for both groups of animals. Urinary polyamines, PU (Fig. 3) and SD, are significantly increased in OKGA fed rats. The excretion of spermine (SM) is low and hence not

,A _,_A-.

1.30 , 2

3

4

5

2

T

6

Days of Feeding

Fig. 2. Mean daily body weight of rats fed a duet supplemented with (OKGA) or without (no OKGA) ornithine-alpha-ketoglutarate. The results are normalised to a rat of initial 15Og.

_0

7

158 ORNITHINE-ALPHA-KETOGLUTARATE

AS A DIETARY SUPPLEMENT

0.025) increased in OKGA fed rats, indicating the increased production of PRO. Similar trends are seen in all urea-cycle amino-acids (ARG, ORN, CIT). The fact that the supplemented ornithine is efficiently metabolised by the tissues can be inferred from its low daily excretion (0.2% of the ingested dose) and unchanged plasma levels. Plasma PHE level and its ratio to plasma TYR are similar in both groups of rats. However, the daily urinary excretion of PHE and TYR are increased by 60% and 200% respectively in OKGA fed rats. The ratio of plasma TRP to LNAA, the large neutral amino-acids (PHE + TYR + VAL + LEU + ILE) is 0.18 in control rats compared to 0.14 in OKGA fed rats. The changes in gluconeogenic amino-acids (ALA, GLY, SER, GLN, GLU) are not significant.

Fig. 3. Cumulative putrescine excretion in ratio with or without OKGA.

Discussion

reported. Increased involvement of polyamines in the OKGA fed rats indicates the enhanced cellular activity of protein metabolism (11) and hence, perhaps, the accelerated growth. The effect of dietary OKGA supplementation on the plasma and urinary levels of individual essential (EAA) and non-essential (NEAA) free amino-acids is summarised in Table 3. Although the circulating plasma PRO levels are similar in both groups of rats, the urinary excretion is significantly (p = Table 1 Effects of feeding OKGA supplemented 1

Days A. 1) 2) 3)

Without OKGA: Body weight (g) Daily food intake (g) Daily excretion Nitrogen (mg N) Urea (mg N) Creatinine (mg) Putrescine (pmole) Spermidine (kmole) Urine volume (ml)

B. 1) 2) 3)

With OKGA supplement: Body weight (g) Daily food intake (g) Daily excretion Nitrogen (mg N) Urea (mg N) Creatinine (mg) Putrescine (pmole) Spermidine (Fmole) Urine volume (ml)

Mean + SEM; (n = 8)

These data show that rats fed ad libitum with an isonitrogenous diet supplemented with OKGA consumed relatively more food and retain more N resulting in the promotion of growth compared to controls. Their enhanced protein synthetic activity can also be infer&d from the increased urinary excretion of polyamines. The reduced excretion of erotic acid by OKGA fed rats may be due to a reduction in the availability of ammonia due to

diet in growing rats 2

3

4

5

6

7

168 t 4 69 + 2

179 + 4 74 f 2

190 + 4 78 f 3

193 f 4 80 + 6

203 f 5 81 +4

209 + 3 79 2 7

212 + 3 76 + 3

65 + 5 56 + 8 1.9 * 0.2 2.6 ? 0.4 0.06 +0.03 39 + 3

75 Ir 12 60t 11 2.7 + 0.4 3.7 t 0.7 0.10 50.03 48 It 1

69 59 2.5 2.2

85 f 13 68+ 11 2.8 f 0.4 3.7 f 0.6 0.16 +0.04 42 ? 3

105 f 5 83 + 4 3.6 f 0.3 4.2 f 0.3 0.08 20.03 44 + 2

109 f 6 89 + 6 3.6 t 0.4 4.6 t 0.1 0.12 50.03 43 + 4

115 + 19 83 + 14 3.2 * 0.4 4.2 f 0.9 0.09 f0.04 43 + 6

154 + 2 82 rt 7

163 It 2 88 + 3

172 + 3 84 f 4

182 + 3 87 + 8

191 f 2 89 f 5

196 t 2 88 + 7

203 + 4 80 + 8

153 t 14 106 + 14 3.2 + 0.2 3.5 + 0.7 0.26 to.04 48 * 3

143 rt 16 109 + 12 3.0 * 0.2 4.3 + 0.7 0.23 kO.02 59 rt 3

113 ? 19 96 * 19 3.3 + 0.5 4.9 + 0.7 0.20 kO.03 55 f 3

106 * 13 102 f 28 2.6 f 0.3 4.3 f 0.6 0.15 +0.02 57 f 3

121 f 10 109 5 13 3.0 f 0.7 5.8 f 0.9 0.46 +0.19 51 + 5

137 t 21 99 + 16 3.2 t 0.5 5.3 +_0.7 0.38 20.18 55 f 3

122 t 21 107 f 17 3.6 + 0.4 4.9 + 0.8 0.32 +0.06 53 + 3

+ 9 t 8 + 0.2 f 0.7 0.05 +0.01 46 f 2

CLINICAL Table 2 Nutritional

parameters

during

7 days of free feeding

in growing

Without Weight gain, g/d food intake, mgN/d Nitrogen retention, mgN/d Excretion: Urine volume. ml/d Nitrogen, mgN/d Urea. mgN/d Creatinine. mg/d 3 Methylhistidine, FmoUmg Orotic acid, l&d Putrescine, PmoVd Spermidine, PmoUd Mean

OKGA

creatinine

t + + f * + +

t-test (unpaired);

supplementation

in plasma

Plasma Without OKGA Essential VAL LEU ILE PHE TRP MET THR LYS

amino-acids

(EAA): 176 + 7 107 2 6

70 52 74 57 2‘l5 434

+ & * I ii-

.l 3 5 4 Xl 29

Non-essrntial amino-acids (NEAA): ALA 339 i 37 GLY 289 + 22 SER 273 * 17 GLN 927 + 38 I23 f 7 PRO ARG 123 i 16 5‘t + 4 HIS TAU 109 * 24 GLU 1.39 + 8 TYR 57 + 3 ORN 53 + 9 CIT 80 f 6 s-1 t 3 ASN CYS 1.2 i 0.76 2 BCAA (3 1 302 2 16 Z EAA (8) 1158 + 56 X NEAA (14) 2780 f 129 3938 ? 173 2: TAA (22,

135 117 73 55 67 45 339 5o‘l 380 209 227 Y42 123 123 53

f f * + f + + *

9 9 5 3 6 3 37

i‘l

& 32 f 13 + 16 t 65 i 8 t 18 i- 8 172 2 21 149 * 13 62 & 3 46 ? 8 81 f5 1-1 * 3 3.7 + 0.1 322 + 33 1334 + 118 2750 + 245 4085 f 327

8.2 + 0.2

0.001 u.005 0.025

399 +_ 9

2 8 5 0.2 0.15 5 o.‘t

55 128 108 3.4 0.54 38 1.7

+ 1 i + + t?

3 6 5 0.2 0.10 6 0.3

u.010 0.005 0.001 0.025 0.050 0.050

0.29 * 0.04

0.0&i

levels of urea cycle amino-acids. Molimard et al (15) demonstrated, in cirrhotic patients, an OKGA-induced decrease in plasma ammonia which was not found when either alphaketoglutarate or ornithine were administered alone. It has been shown that OKGA not only attenuates the

and urine free amino-acid

levels 01 growing

(kmol/l)

With OKGA

P

n = 8 each group

efficient removal in the urea biosynthetic pathway and also due to the amination of alphaketoglutarate to glutamate. These factors are also evident from the ratio of urea N to the total N excreted in the urine. which is increased by 9% in the OKGA fed rats, and also from the increase in the plasma Table 3 Effect of OKGA

With OKGA 527 t 16

0.09 L 0.02

t SEM: P values by Student’s

159

rats

7.1 f 0.2 456 * II 369 t 8 44 89 69 2.7 0.55 54 3.6

NUTRITION

rats Urine (l.tmoUdJ

“/0 change +7

Without OKGA

With OKGA

+4 +6 - 10 -1.5 f3Y +14

I .63 * 1.30 * O.XY F 0.74, t 0.7x * 1.1x i 1.71 t 1.36 t

0.41 II.43 0.24 0.22 (I.16 Cl.16 0.19 rr42

7.06 i- 0.50 “6 ? o.s4 -,_ I .3h ” 0.3 I .‘6 a 0.31

-13 -28 -17 _?

6.81 k 1.1‘) 14.1 t 3.Y 1.03 * 0.74 _

J.02 r ‘.JX u.34 * 2.45 I I1 k 0.37 _

0 0 0 +5X +7 f9 -13 0 -19 +208 +7 +15 -1 +4

0.85 i 0.27 l.US + 0.25 0.28 * 0.05 55.0 + 7.6 6.46 * 0.80 0.s-l t U.10 0.13 * 0.11 0.42 * 0.11 0.37 F 0.10 0.14 f 0.11 3.82 Y.11

3.41 i_ i.li 7.06 t 0.39 0.69 + 0.75 38.0 + Y.Y Y.90 i 3.41 1.63 + 0.1x 0.94 2 0.41 (I.67 + (I.15 1.07 + 0.75 (I.64 t O.OY 5.68 15.14 3X.48’ j3.62”

+Y

Mean ? SEM; n = 8 in each category. Plasma levels are the averages 7th dav of feeding; * Taurine level not included

32.78*

‘l1.92’

I .osk

o.?:

3.19 t 0.4Y I .83 * 0.34 I .x3 i- 0.5i

on the 7th day and the urine levels are the averages

?O change

t7h +51 + 52 +54 +33 tlYX +51 +35

-z ix 1

_

+301 +Yh +1-M - 31

+13 +x11 +119 +ho +1X’) +%I

of 4th and

160

ORNITHINE-ALPHA-KETOGLUTARATE

AS A DIETARY

rise in blood ammonia but also favourably affects these changes in brain metabolism (16) and hence is widely used in Europe to reduce blood ammonia levels in patients with hepatic encephalopathy. Ornithine is a key metabolite not only in the urea cycle but also in proline synthesis and the first precursor of polyamine pathway. Ornithine and alphaketoglutarate possess certain common metabolic pathways that lead to the production of glutamate. A possible interaction between these two components of OKGA therefore appears to be feasible. Unlabelled alphaketoglutarate caused a decrease in the rate of intestinal absorption of 14C-ornithine, whereas the inverse was not true (8). It is possible that this alphaketoglutarateinduced decrease in ornithine metabolism diminishes the ornithine gradient between the intestinal lumen and intracellular medium which in turn may influence its different metabolic pathways. The efficient utilisation of ornithine when given as OKGA is evident from its unchanged plasma level and not a large urinary excretion of the ingested ornithine in growing rats. The increased production of proline in OKGA fed rats is indicated in part from its increased excretion with no change in plasma level. Only OKGA, but not the components separately, was able to increase by 35% the plasma levels of proline at 60 min after administration in healthy man (7). The simultaneous increase in polyamine, proline and urea metabolism in OKGA fed rats illustrates the stimulation of the diverse pathways of ornithine metabolism in the presence of alphaketoglutarate. The effect of polyamines on growth and protein synthesis may be regulated by the rapid influx of amino-acids into cells rather than by the influence of hormones secreted in response to dietary stimulation (17). OKGA increased directly the hepatocyte albumin synthesis by the way of its component L-ornithine and this effect is primarily mediated by poiyamine synthesis (18). The increased synthesis of polyamines from ornithine of the supplemented OKGA may permissively stimulate anabolic processes in tissues and subsequent growth. It is note-worthy that labelled ornithine is able to give rise to labelled polyamines in several organs of the rat after enteral administration (8). The simultaneous increases in the activities of PU and SD indicates the enhanced stimulation of both anabolit and catabolic processes in OKGA treated rats. The reduced excretory ratio of PU to SD due to OKGA is mainly due to a relatively large increase in SD excretion. signals are probably metabolic Multiple

SUPPLEMENT

involved in the initiation of a meal in animals. However, the control of food intake is extremely complex and involves central as well as peripheral mechanisms comprising effects of nutrients, metabolites, endocrine factors and neural mechanisms. The onset of feeding is also associated with a rapid change in the profile of circulating hormones and substrates (19). It has been hypothesised that the sensitivity to amino-acid intake in animals is due to the essential nature of these nutrients to its health (20). This led to the proposal that fluctuations in plasma amino-acid concentrations give rise to appropriately regulated food choice as well as influencing the total amount of food consumed (21). Examination of the plasma amino-acid ratios in relation to self-selected food consumption in normal healthy man (22) and in growing rats (20) has shown that ratio of the plasma TRP to the sum of competing iarge neutral amino-acids (PHE + TYR + VAL + LEU + ILE) correlates negatively with both long-term and immediate protein consumption. This ratio appears to comprise the elements of food intake control mechanisms, which are based on serotoninergic and catecholaminergic pathways in the brain (23). In OKGA fed rats, this ratio (0.14) is smaller than in the control rats (0.18), in conformity with the above mechanism for an increased voluntary food intake. The taste produced by the L-amino-acids released from foods by chewing may be a detection signal for foods (24)) and the increased glutamate formation might have enhanced the flavour and umami taste. The favourable change of the palatability by altering the amino-acid component of the diet needs further investigation (24, 25). In these freely-fed healthy rats the protein efficiency ratio (PER), which is the gain in body weight per gm of nitrogen consumed, is 15.6 and is identical in both groups. The fraction of N intake which is retained in the body is also identical (0.76) in both of the groups. It appears that the increased voluntary dietary intake by the OKGA supplemented healthy rats mainly accounts for the metabolic benefits. It remains to be seen how efficacious the adjuvant use of OKGA is in the malnourished state (26) and in depleted injured conditions. In the malnourished state there may be an enhancement in the overall metabolism if supplementary OKGA adjuvant therapy is provided. Acknowledgements The generous supply of OKGA (‘Cetronan’) by Laboratories Jaques Logeais. Issy les Moulineaux 92130. France is

CLINICAL gratefully acknowledged. We thank Dr. Guy Dorf for his helpful guidance and fruitful discussions. 14.

References 15. I. Krassowski J, Rousselle J, Maeder E, Felber J P 1981 The effect of ornithine alpha-ketoglutarate on insulin and glucagon secretion in normal subjects. Acta Endocrinologica 98: 252-255 2. Gay G. Villaume C. Beaufrand M J, Felber J P, Debry G 1979 Effects of ornithine alpha-ketoglutarate on blood insulin, glucagon and amino acids in alcoholic cirrhosis. Biomedicine 30: 173-177 J. Grimble G, Cahill E. Silk D B A 1989 3. Payne-James Enteral administration of OKGA in man: effects on hormone profiles and nitrogen metabolism. Journal of Parenteral and Enteral Nutrition 13 (Suppl), 108 (Abst 36) 3. Hammarqvist F, Wernerman J, Ah R, Vinnars E 1990 Effects of an amino acid solution enriched with either branched-chain amino acids or ornithinealphaketoglutarate on the post-operative intracellular amino acid concentration of skeletal muscle. British Journal of Surgery 77: 214-218 5. Wernerman J, Hammarqvist F, von der Decken A, Vinnars E 1987 Omithine-alpha-ketoglutarate improves skeletal muscle protein synthesis as assessed by ribosomal analysis and nitrogen use after surgery. Annals of Surgery 206: 674-678 6. Smith T K, Lindqvist L, Alakuijala L, Eloranta 1989 Effects of dietary polyamine precursors on the metabolism and tissue concentrations of amino acids in the rat. Annals of Nutrition and Metabolism 33: 14%152 7. Cynober L, Coudray-Lucas C, de Brandt J, Guchot J, Aussel C, Salvucci M, Giboudeau J 1990 Action of ornithine alpha-ketoglutarate, ornithine hydrochloride and calcium alpha-ketoglutarate on plasma and hormonal patterns in healthy subjects. Journal of the American College of Nutrition 9: 2-12 8. Vaubourdolle M, Jardel A, Coudray-Lucas C. Ekindjian 0 G. Agneray J. Cynober L 1989 Fate of enterally administered ornithine in healthy animals: Interactions with alpha-ketoglutarate. Nutrition 5: 183-187 9. Cynober L. Saizy R, Dinh F N, Lioret N, Giboudeau J 1984 Effect of enterally administered ornithinealphaketoglutarate on plasma and urinary amino acid levels after burn injury. Journal of Trauma 24: 59&596 M, Young D H, Ramias L. Schiller W R 10. Jeevanandam 1989 Aminoaciduria of severe trauma. American Journal of Clinical Nutrition 49: 814-822 11. Jeevanandam M. Ali M R, Young D H, Schiller W R 1989 Polyamine levels as biomarkers of injury response in polytrauma victims. Metabolism 38: 625-630 12. Jeevanandam M, Shoemaker J D, Horowitz G D, Lowry S F. Brennan M F 1985 Orotic acid excretion during starvation and refeeding in normal man. Metabolism 34: 325-329 13. Pui Y M L, Fisher H 1979 Factorial supplementation

Submission date: 13 August

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

1990; Accepted after revision: 15 February

NCTRITION

I61

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1991