Altered tissue polyamine levels due to ornithine-α-ketoglutarate in traumatized growing rats

Altered Tissue Polyamine Levels Due to Ornithine-a-Ketoglutarate Traumatized Growing Rats Malayappa

Jeevanandam,

Nancy J. Holaday,

and Mohammad

in

R. Ali

All cells contain significant amounts of polyamines (PA), and their concentrations are highly regulated. Metabolic activity within a tissue may be reflected in the amount of intracellular PA. Since trauma involves accelerated death and regeneration of tissues, the related levels of PA in extracellular and intracellular fluids may reflect altered protein metabolism. Trauma induces an increased excretion of urinary PA, and the tissues responsible for this whole-body activity are not known. During posttraumatic nutritional management, supplementation with ornithine-ol-ketoglutarate (OKG) seems to improve nitrogen economy. The present study evaluates the significance of muscle, liver, and intestine PA responses in traumatized (bilateral femur fractures) rats to the feeding of an isonitrogenous liquid diet supplemented with or without OKG. Uninjured control rats were pair-fed with respective traumatized rats. After 2 days of starvation and 4 days of feeding, the traumatized and control rats were killed and the tissues were excised and analyzed. Starvation decreases and refeeding increases urinary PA excretion. Trauma-induced PA response is predominantly seen in muscle tissues, and this may be responsible for parallel increases in PA excretion. Liver PA responses show a varying tendency confirming the increased protein synthetic activity due to trauma. Intestine has the highest intracellular PA levels, and there is a general smaller (statistically insignificant) increase in all the individual PA contents due to trauma. OKG supplementation augments tissue and urine PA responses in control rats; however, in trauma rats muscle PA levels show very little change, although nitrogen retention is significantly better (88% to 77%). Mechanistic studies are needed to evaluate the significances of the time-dependent, injury-induced, individual intracellular PA levels. Copyright

0 1992 by W.B.-Saunders

Company

P

OLYAMINES (PA), the low-molecular-weight polycationic amines, putrescine (PU), spermidine (SD), and spermine (SM) play an essential role in cellular proliferation, cell growth, and synthesis of protein. The specific functions of PA are still not well understood, but their ubiquitous distribution, high concentration in cells, and increase in concentration seen in rapidly growing tissues have stimulated many investigations of these compounds. The factors that influence urinary patterns of PA include the rate of PA formation in various organs, metabolic activity within the tissue, rate of transfer from the cells into the circulation, rate of degradation within circulation, and rate of cell death in various organs.’ Very little seems to be known about the major source of excreted PA. Cells and tissues do not ordinarily use extracellular PA, and the fate of synthesized PA is urinary excretion in the free, acetylated, or degraded forms. Thus extracellular PA levels are excellent markers of various cellular events, and urinary excretion seems to reflect the rate of PA formation due to whole-body activities. The early posttraumatic period is characterized by enhanced proteolysis, lipolysis, and gluconeogenesis. Both anabolic and catabolic processes accelerate following severe injury, with catabolism predominating. Since trauma involves accelerated degeneration/death and regeneration of tissues, associated PA levels in plasma and urine could reflect altered whole-body protein metabolism. One of the basic characteristics of severe trauma is an increased

From the Trauma Center, St Joseph’s Hospital and Medical Center, Phoenix, AZ. Presented in part at the 13th Congress of ESPEN, Anhverp, Belgium September l-4, 1991, Clin Nutr lO:lOS, 1991 (suppl2). Address reprint requests to Malayappa Jeevanandam, PhD, Trauma Center, St Joseph’s Hospital and Medical Center, 350 W Thomas Rd, Phoenk, AZ 85013. Copyright 0 1992 by W.B. Saunders Company 00260495l9214111-0010$03.00/0

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excretion of PA,2 which is further exaggerated during nutritional support in the “flow” phase of injury.3 Provision of conventional nutrition alone may not be adequate at this stage of injury to meet dietary demands, and attention has been focused recently on the search for novel substrates as dietary supplements. The addition of an anabolic stimulus during intensive nutritional therapy seems to be a reasonable adjunct for minimizing injury-induced erosion of muscle mass.4,5 Among several adjuvant therapies, supplementation with ornithine-cu-ketoglutarate (OKG) seems to be most promising. This treatment enhances protein kinetics,6-9promotes wound healing, lo improves cellular immunity,” stimulates secretion of anabolic hormones such as insulin12 and growth hormone, ‘3and involves biotransformation into metabolites like glutamine, arginine, proline, ketoacids, and PA.6J4-17 The objectives of the present study were to evaluate the significance of the altered tissue (muscle, liver, and intestine) PA responses in traumatized rats fed a liquid diet supplemented with or without OKG. We were able to examine the effects of OKG supplementation on tissue and urine PA levels in starved-refed, uninjured rats, and to investigate the effects of trauma on tissue and urine PA levels in rats fed a diet with or without OKG supplementation. MATERIALS

AND METHODS

A group of 32 young, male, Sprague-Dawley rats (ACE Animals, Boyertown, PA) weighing between 155 and 165 g were housed in individual metabolic cages (Plas-Labs, Lansing, MI) and kept in our well-ventilated, temperatureand humidity-regulated, vivarium equipped with controlled 12-hour dark-light cycles. They were adapted to ad-libitum liquid-diet 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. This research protocol was reviewed and approved by the Institutional Animal Care and Use Committee, and we adhered to the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health publication no. 85-23,

Metabotism, Vol41, No 11 (November), 1992: pp 1204-l 209

TISSUE POLYAMINE LEVELS IN TRAUMATIZED

1205

RATS

revised). Four sets of eight rats each were studied. The rats were weight-matched, and one half of the animals acted as controls and the rest were traumatized. Food intake was determined daily by weighing the liquid-diet feeding tubes at the beginning and end of each 24-hour period, and urine output was collected from 800 AM to 800 AM for the determination of 24-hour urine excretion of PA and creatinine. Each day between 8:00 and 9:30 AM, animals were weighed, urine output was measured and collected, and feeding tubes were changed. On day 0. ail rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar. 50 mg/mL, ParkeDavis, Morris Plains, NJ: 10 mg/lOO g body weight), and the animals were divided into four weight-matched groups. One group of 16 rats received a closed bilateral femoral fracture involving a standard-force fracture at the midshaft of the femur by twice releasing the arm of a common, spring-loaded rat-trap onto the femur. This ensured a uniform closed fracture and soft-tissue damage. Another group of 16 rats acted as pair-fed controls for the traumatized animals. The animals were then returned to individual cages and had free access to water. but were deprived of food for 2 days. This condition was imposed to simulate the conditions of human subjects treated in the trauma intensive care unit. On the third day, ad-libitum feeding was started and continued for 4 days. Half of the control and traumatized animals were given a cdsein-based, control oral liquid diet (no. F1259, BioServ, Frenchtown. NJ) that had 6.5 mg nitrogen and 1 kcal energy/ml. The amino acid composition of this diet is given in Table 1, and the calorie sources are 18% protein 34% fat, and 47% carbohydrate. The other half of the animals were fed a test oral liquid diet. This isonitrogenous test diet contained the above control diet, from which 146 mg nitrogen/L was replaced by nitrogen from OKG (Cetornan. Laboratories J. Logeais, Paris, France). Each gram of OKG, which is a neutral, monohydrate salt containing 2 mol basic amino acid r.-ornithine (ORN) and 1 mol alphaketoglutarate (AI(G). has 131 mg nitrogen. The test diet therefore had 2.3% nitrogen replaced by OKG-N and was isonitrogenous but slightly (1.8%) hypocaloric to the control diet. Considering the higher Table 1. Amino Acid Composition of the Two lsonitrogenous Diets (g/L) Control Diet (BioServ)

Test Diet (OKG)

Alanine

0.95

0.93

Glycine

0.87

0.85

Serine

2.00

1.96

Proline

3.57

3.49

-

Arginine

1.31

1.28

Histidine

0.98

0.96 6.94

Glutamic acid

7.10

Tyrosine

2.00

1.96

Aspartic acid

2.26

2.21

Cysteine

0.11

0.11

Ornithine

0

1.38

Valine

2.30

2.25

Leucine

2.91

2.85

lsoleucine

1.93

1.89

Phenylalanine

1.60

1.56

Threonine

1.57

1.53

Tryptophan

0.55

0.54

Methionine

0.87

0.85

Lysine

2.59

2.53

NOTE. Each liter of BioServ no. F1259 diet contains 6.5 g nitrogen and 1,000

kcal; calorie

carbohydrate.

sources

are 18%

protein,

34%

fat, and 47%

metabolic rate of rats, on the basis of body weight, this dose of OKG for the rat (79.7 mg OKG-N/kg/d) corresponds to twice the normal 20-g OKG dose (37.4 mg OKG-N/kg/d) recommended daily for the enteral route in a 70-kg man.‘j Each rat would have received roughly a daily supplementary dose of 429 umol ORN and 215 umol AKG. In each weight-matched group, control rats were pair-fed with traumatized animals and all animals had free access to water. On day 6, at the end of 4 days of oral feeding, food was withdrawn for 2 hours and the rats were then anesthetized. Blood was collected by cardiac puncture, and within 5 minutes the tissues (uninjured forelimb muscle. injured hindlimb muscle, intestine (ileum), and liver) were excised, cleaned, and frozen by plunging into liquid nitrogen. Urine concentrations of creatinine were determined using standard procedures with a Micro Centrifugal Analyzer (Multistat Plus, Instrumentations Laboratory, Lexington, MA). Daily urinary nitrogen levels were determined using a chemiluminescence Digital Analyzer (Antek Instruments, Houston, TX). Fatty tissues from muscle were carefully removed. A portion of the intestine was cut longitudinally, washed with buffered saline to remove any food, and blotted. Tissue water contents were determined by freezedrying to constant weight. PA in various dry tissues were determined by the automated ion-exchange method using an amino acid analyzer (Model 7300, Beckman Instruments, Palo Alto, CA), as described previously in other reportsZ,3,1band modified. Briefy, 500 ILL4% sulphosalicilic acid (SSA) containing the internal standard (Benzylamine, 400 pmol, Sigma no. B6625, Sigma. St Louis, MO) was added to 5 to 7 mg freeze-dried. powdered tissue, vortexed slowly, and kept in ice for 1 hour with occasional shaking. It was then centrifuged at 10,000 x g for 10 minutes in a refrigerated centrifuge. The supernatant was filtered through 0.22~urn membrane filter (Millipore, Bedford, MA), and a 20-FL aliquot was injected into an automated amino acid analyzer fitted with a IO-cm ion-exchange column and a fluorescence detector. Post-column derivatization was accomplished with O-phthaldehyde (Fluor-R reagent, Beckman). The total run time for each sample was 73 minutes. including 62 minutes of column sample separation run, 1 minute of column regeneration by buffer B6 (NaOH. 3 mol/L), and 10 minutes of column equilibration by buffer Bl (67.2 g/L NaCl and 34.3 g/L sodium citrate dihydrate, pH 5.43) at 70°C. The flow rate was 20 mL buffer and 10 mL Fluor-R reagent/h. Ten minutes after sample injection, the temperature of the column was changed from 70” to 80°C and the buffer was changed to B2 (137.4 g/L NaCl and 34.3 g/L sodium citrate dihydrate, pH 3.50). The changes in temperature and buffer were found to be necessary for a better separation of PA including the isomers of acetylated (AC) SD. N’AcSD and NsAcSD. AcPU was distinctly separated from amino acids and ammonia with buffer Bl at 70°C. The retention times (min) were as follows: AcPU, 6.1; N’AcSD, 16.9; NsAcSD, 17.2; PU, 18.9; AcSM, 26.3; SD, 30.8; Benzylamine (internal standard), 38.9; and SM, 55.3. When present in equal molar quantities, N’AcSD and N8AcSD were clearly separated. However. if one AcSD was present predominantly, it masked the other peak. In all rat samples, total AcSD levels were very low (2% in urine and 0.04% in muscle tissue), NXAcSD was the major component, and hence the sum of N’AcSD and N*AcSD was reported as AcSD. A calibration standard was analyzed at the beginning of every run and after every six samples. The coefficient of variation of multiple analyses was within 4%. The urine sample was deproteinized with SSA, mixed with the internal standard, and analyzed as described for tissues. The results were grouped as control-OKG (C-O), trauma-OKG (T-O), control-Non-OKG (C-N). and trauma-Non-OKG (T-N). Effects of trauma with relation to diet supplementation were analyzed between corresponding groups. The values listed are

1206

JEEVANANDAM,

means ? SEM. The variables of control and trauma rats with or without OKG supplementation were compared by means of Student’s t test18 for significant differences. A P value of .05 or less was considered statistically significant. RESULTS

The rats weighed 160 ? 3 g initially, and in 2 days of starvation they lost 24 g. Control rats were pair-fed with respective traumatized animals. When the feeding was resumed after 2 days of food deprivation, all rats were able to consume almost the full (93%) ration and regained the lost weight in 2 days of feeding. The 4-day mean nitrogen intakes were 439 ? 6,443 ? 9,496 ? 11, and 511 2 17 mg nitrogen/d; the 4-day cumulative nitrogen balances were 988 + 50, 757 + 35, 1,136 f 103, and 1,000 2 88 mg nitrogen; and the 4-day weight gains were 29.5, 23.5, 36.5, and 33.0 g for C-N, T-N, C-O, and T-O groups, respectively. C-O and T-O groups consumed more food, retained more nitrogen, and gained more weight than the respective non-OKG groups. The mean creatinine excretions were 3.6 + 0.1 and 4.1 + 0.1 mg/d for OKG-supplemented and nonsupplemented rats, respectively. The nutritional efficiency indexes (g nitrogen retained/g nitrogen intake) were 56% and 57% in C-N and C-O rats, and they were considerably lower in trauma rats (42% in T-N and 49% in T-O). T-O rats were able to retain nitrogen more efficiently than the corresponding non-OKG rats. The gain in weight/ gram nitrogen retained was 30.0 g and 33.2 g in rats fed control and test diets, respectively. Daily urinary excretion of PU and SD are illustrated in Figs 1 and 2, respectively. In rat urine, both free SM and ACSM levels are undetectable, although they were more abundant in tissues. AcPU is the major PA in rat urine, and its excretion decreases by approximately 60% in 2 days of starvation in control rats; however, the decrease is not significant in T-N rats, and this may be due to the countereffects of injury and starvation. Refeeding enhances AcPU excretion in all groups. In contrast to AcPU, there is an increase in the excretion of PU, AcSD, and SD on the first day of starvation, followed by a subsequent decrease on the second day of starvation and a further decrease on the first day of feeding in both injured and uninjured animals. In general, traumatized animals excrete more PA than the respective controls, and C-O and T-O rats excrete 22% and 36% less PA than respective nonsupplemented animals. Individual tissue PA levels are given in Table 2. The trauma-induced PA response is seen predominantly in muscle tissues (Fig 3). Total PA levels significantly (P = .OOl) increased (71%) in muscle tissue due to trauma in C-N rats. Parallel increases in individual PA levels due to trauma are seen in urine and muscle tissue. In normal rats, OKG supplementation alone significantly (P = .OOl) increased (51%) muscle PA levels (Fig 3, C-O v C-N); however, this increase in muscle PA levels is not seen in trauma rats (T-O v T-N). Although total PA levels in liver and intestine are 3 to 10 times higher than in muscle tissue, they are relatively unresponsive to injury or dietary change. Muscle seems to be the major contributor to the increased PA excretion in injury. In muscle tissue, unconjugated free

Acetylated

4500 r

g

4000

-

‘E ‘= 5I

3500

-

c $

3000

-

2500

-

2000

-

,500

-

HOLADAY, AND ALlI

Putrescine

E ; p w” : a z

-Starved

-

-FedDays t‘ Control

- No-OKG

B Trauma

- No-OKG

0 Control

- OKG

m Trauma

- OKG

Mean t SEM loxlo

”

r

= 8

each group

Putrescine

0 -Starved

1 -

2 IF,“.,” Days

Fig 1. Daily excretion of AcPU and free PU in control and traumatized growing rats. Animals were starved for 2 days and then fed for 4 days with or without OKG supplementation.

SM levels constitute 54% of total PA. The high levels of SM in muscle (54%), liver (41%), and intestine (21%) and its complete absence in urine, along with the absence of AcSM in any tissue and urine, indicate a slow turnover of SM and/or the rapid utilization of SM to SD or AcPU. In control diet-fed rats, muscle AcPU, PU, and SM levels are significantly increased due to trauma (C-N v T-N), whereas in liver tissue they are significantly decreased and in intestine they showed an increasing trend. OKG supplemen-

tation has no significant effects due to trauma in any of the tissues. However, in uninjured rats, OKG supplementation significantly increases AcPU and SM levels in muscle, with a decreasing trend in the majority of PA present in liver and intestine. In this rat-trauma model, femur bones of both hindlimbs were fractured and there was also extensive muscle tissue damage. The traumatized rats were not immobilized at any time, and displayed little hindrance to motion from bilateral femoral fractures except in the ability to exert force from the hind legs. To assess the systemic effect of injury in the remote part, PA contents of muscle tissues from both hindlimbs and forelimbs were measured, and the results are reported in Table 3. Hindlimb (injured) muscle PA contents in both OKG-supplemented and nonsupplemented

IISSUE POLYAMINE LEVELS IN TRAUMATIZED

250 r

Fylated

’

0

” 0

1207

RATS

Spermidine

T

”

I+

” 1

”

”

”

-Starved

@ Trauma - No-OKG 0 Control - OKG q Trauma - OKG

Spermidine (326+40)

0

1

-Slarved

Fig 2. Daily excretion growing rats.

of AcSD and free SD in four groups of

groups show a small increase due to trauma. However, as previously noted, the forelimb muscle PA contents increased significantly due to trauma in rats fed the diet without OKG. AcPU levels decreased significantly in damaged hindlimb muscle compared with uninjured forelimb muscle in both control and traumatized rats fed diet with or without OKG. There were no significant changes between forelimbs and hindlimbs in other muscle PA levels. DISCUSSION

Significantly elevated urinary PA levels seen in polytrauma patients’ and their increase due to feeding3 are confirmed in this rat-trauma model. Even a short-term fasting period in the rat corresponds to a large catabolic drain and large metabolic adaptations, since the energy turnover in such a small animal is extremely high. Food deprivation for 2 days in control rats results in the loss of 15% of body weight, nitrogen loss corresponding to 43% of the later daily nitrogen intake, and 60% less excretion of AcPU, which is the major (89%) PA component of urine. Starved and traumatized rats lost 14% of their body weight, and also lost nitrogen corresponding to 50% of the later daily nitrogen intake. Since starvation decreases and trauma increases AcPU excretion, it shows a 16% decrease on the first day (starvation effect) and a 17% increase on the

1208

Fig 3. Muscle (forelimb) tissue total PA levels in growing rats. P = ,001 for control rats with or without OKG; P = .OOl for control and trauma rats without OKG; NS for rats with OKG.

second day (trauma effect), thus resulting in no change in AcPU excretion at the end of 2 days of injury and fasting. Excretion of AcPU is increased during refeeding in all groups of rats; however, excretion of PU, A&D, and SD are decreased on the first day of feeding and then slowly increase to normal on the fourth day. The significance of this delay is not apparent. OKG-supplemented rats excrete less PA than nonsupplemented rats, and this is more pronounced in trauma rats. Injury-induced PA effect seems to be partially modulated by OKG. Intracellular PA metabolism can be formulated as a cyclic process, which explains the transformation of one PA into another. This kind of cyclic process is necessary for the precise control of cellular PA concentrations, as it allows relatively rapid SM to SD concentration changes, despite a slow basal turnover rate.19 The acetylation of PA is the limiting factor in the interconversion of PA. The conspicuous absence of AcSM or SM in urine and plasma (unpublished data) of rats and higher intracellular SM levels may be due to a slow turnover of tissue SM or rapid interconversion of SM to SD or PU, or to a block in intracellular transport of SM into the plasma compartment. Higher intracellular SM levels seem to act as reservoirs of PA for rapid conversion to other PA in case of urgent physiological demand. Muscle seems to be the major organ contributing to the increased PA excretion due to trauma, since the injury response is only mild in other organs. Although intestine has 12 times more PA than muscle on a dry-weight basis, the increase in PA due to trauma is 70% in muscle and only 11% in intestine. Trauma induces all individual muscle PA levels, and it becomes significant in AcPU, PU, and SM in control diet-fed rats; this may be related to accelerated protein metabolism. OKG supplementation induces total muscle PA levels in control rats (C-N v C-O), and it becomes significant in AcPU and SM. Trauma has no significant effect on any tissue PA levels in OKGsupplemented rats (C-O v T-O). However, the cumulative nitrogen retention is improved by 32% (from 757 to 1,000 mg), and the nutritional efficiency index is enhanced to 49% from 42% in T-O rats compared with T-N rats. The mechanism for the lack of PA responses in T-O rats in the face of improved nitrogen economy is not apparent. The

TISSUE POLYAMINE LEVELS IN TRAUMATIZED

RATS

possibility that the trauma effects on PA due to OKG supplementation may be so rapid and transient that we missed them cannot be ruled out. Muscle PA accumulation patterns in response to denervation were strikingly different from those in response to tenotomy. 2o There was an earlier significant elevation of PU, SD, and SM in tenotomy, reflecting an increased cell-proliferation component, in line with the early marked central fiber necrosis. Normal PA concentrations were restored by 6 weeks posttenotomy with normal histological appearance. Denervation, on the other hand, resulted in alterations in PA patterns in muscle that did not return to normal, but continued to reflect an increased proliferation component. Significant elevations in skeletal muscle PA concentrations were seen in patients with different muscular diseases compared with normal men.*’ In our rattrauma model hindlimb muscles of rats were damaged, and this experimental model allowed us to compare injury and dietary effects between uninjured forelimb and injured hindlimb muscles of the same rat. There is a significant increase in the AcPU level in forelimb muscle compared with hindlimb muscle of the same control rat, and this effect is not altered by diet supplementation. However, total PA levels are similar in both limbs of control rats fed control diet, and are increased in both limbs when supplemented with OKG. Trauma significantly enhances muscle PA levels in both limbs in control diet-fed rats, and this is more pronounced in uninjured forelimb muscle. This demonstrates the predominance of the systemic trauma effect over local muscular damage. OKG supplementation in control rats significantly increases muscular PA levels, particularly AcPU, in both limbs, and this effect is somewhat moderated in trauma rats, as also shown in urinary PA data and nitrogen-retention data. Table 3. Comparison of Hindlimb and Forelimb Muscle Tissue PA Levels ControlRats Hindlimb

Trauma Rats

Forelimb

Hindlimb

Forelimb

AC PU Non-OKG

238 + 8’

269 k IOt

285 + 31*

504 k 88

OKG

294 -t 4f

379 2 20

316 c 22

320 + I2

PU Non-OKG

17 t- I

13 2 2

62 2 24

27 + 6

OKG

37 + 9

47 2 18

31 2 5

432

I3

AC SD Non-OKG

4 2 0.2

5+1

3 -t 0.4

5~2

OKG

3 + 0.6

3 + 0.3

211

4 2 0.5

SD Non-OKG

320 + I8

350 -c 17

644 2 178

599 + 123

OKG

433 c 69

562 k 106

490 zk 54

617 + 126

SM Non-OKG OKG

877 + 28 1,038 e 55

791 2

91

1,017 2 80

1,310 + 176

1,168 2 73

1,089 k 61

1,134 + 39

Sum PA Non-OKG

1,456 I 23t

1,428 + 91t

2,010 -t 96,

2,437 k 129

OKG

1,802 + 62”

2,162 + 100

1,928 2 46

2,188 + 59

NOTE. Results are nmol/g dry tissue, means + SEM (n = 8). *P I

.05,

hindlimb versus forelimb of the same group.

tP 5 .05, control versus trauma (forelimb or hindlimb).

TISSUE

POLYAMINE

LEVELS

IN TRAUMATIZED

1209

RATS

Enhanced activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of PU. is an early feature of liver growth after partial hepatectomy,‘* and the elevation of PU synthesis coincides with changes in RNA synthesis in tissue.‘” Fasting decreases liver ODC activity,24 and feeding hypertonic glucosez2 or specific amino acidz5 increases PA synthesis. When hepatic synthetic activity is depressed, the concentration of SD in the liver is markedly reduced.2h SD and SM are the major components of rat liver PA, and the trauma effect in animals fed control or OKG-supplemented diets is a decrease in AcPU, AcSD, and SM levels and an increase in SD levels, with an overall decrease of 8% total PA. The results indicate that the liver protein synthetic activity in trauma may be moderately increased. This same effect is seen in control rats when the diet is supplemented with OKG.

Increased ODC activity, with the resultant increase in PA content, plays an essential role in intestinal mucosal maturation and regeneration from injury in adult rats.27 Injury is also accompanied by a marked decrease in the activity of diamine oxidase of the mature mucosa.27 The interplay between PA synthesis and oxidative degradation may seem to be responsible for the observed tissue level. Intestine (ileum) has the highest intracellular PA levels, and SD forms the major component (46%). There is a general increase in all intestinal PA contents due to trauma and a general decrease due to OKG supplementation. More studies are needed to evaluate the significances of individual intracellular PA levels, since there seems to be a time-dependent stress response counteracting the selfregulation of various individual PA.

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administered ornithine alphaketoglutarate on plasma and urinary amino acid levels after burn injury. J Trauma 24:590-596, 1984 15. Hammarqvist F, Wernerman J, Ah R, et al: Effects of an amino acid solution enriched with either BCAA or OKG on the post-operative intracellular amino acid concentrations on skeletal muscle. Br J Surg 77:214-218, 1990 16. Jeevanandam M, Ali MR, Ramias L, et al: Efficacy of OKGA as a dietary supplement. Clin Nutr 10:155-161.1991 17. Ah MR. Jeevanandam M: Altered tissue polyamine levels in malnourished growing rats fed supplemented oral diet. FASEB J 5:A596, 1991 (abstr) 18. Snedecor GW. Cochran WG: Statistical Methods (ed 7). Ames, IA, Iowa State University Press, 1971 19. Seiler N: Functions of polyamine acetylation. Can J Physiol Pharmacol65:2024-2035.1987 20. Kaminska AM, Stern LZ, Russell DH: Polyamine metabolism in muscle: Differential response to tenotomy and denervation in the soleus and gastrocnemius muscle of adult rats. Exp Neural 78:331-339. 1982 21. Rudman D, Kutner MH. Chawla RK, et al: Abnormal polyamine metabolism in hereditary muscular dystrophies: Effect of human growth hormone. J Clin Invest 65:95-102. 1980 22. Schrock TR, Oakman NJ, Bucher NLR: Ornithine decarboxylase activity in relation to growth of rat liver: Effects of partial hepatectomy, hypertonic infusion, celite injection or other stressful procedures. Biochim Biophys Acta 204:564-577, 1970 23. Faust0 N: Studies on ornithine decarboxylase activity in normal and regenerating livers. Biochim Biophys Acta 190:193201, 1969 24. Domschke S. Soling HD: Polyamine metabolism in rat liver: Effect of starvation and refeeding. Horm Metab Res 5:97-101, 1973 25. Sens DA, Levine JH, Buse MG: Stimulation of hepatic and renal ornithine decarboxylase activity by selected amino acids. Metabolism 32:787-792. 1983 26. Kostyo JL: Changes in polyamine content of rat liver following hypophysectomy and treatment with growth hormone. Biochem Biophys Res Commun 23:150-155. 1966 27. Luk GD, Marton LJ. Baylin SB: Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats. Nature 210:195-198, 1980