British loirrnalo! Haernatology. 1992, 81. 585-590

Amino acid metabolism during platelet storage for transfusion

SCOTT MURPHY, SANTIAGO MUNOZ, MARK PARRY-BILLINGS* AND ERIC NEWSHOLME* Cardeza Foundation for Hematologic Research, Division of Gastroenterology, and Department of Medicine of Jefferson Medical College of the Thomas lefferson University, Philadelphia, Pa., U.S.A., and *The Cellular Nutrition Research Group, Department of Biochemistry, Oxford University, Oxford, U.K.

Received 2 January 1992; acceptedfor publication 17 March 1992

Summary. Previous studies indicated that the concentration of ammonia rises during storage of platelet concentrates (PC) at 22OC for transfusion and that fuels other than glucose are important for metabolism. Therefore, in the current study, we measured the concentrations of 17 plasma amino acids during PC storage: 16 of these either rose or were unchanged while the concentration of glutamine fell to zero by day 4. As the concentration of glutamine fell, the concentration of glutamate rose with a relationship suggesting that 65-75% of the glutamine was metabolized no further than glutamate. Phosphate-dependent glutaminase activity was present in platelets at 22.3 + 6 . 3 nmol/min/mg protein, a level similar

to that seen in lymphocytes and macrophages. Leucodeple- tion studies excluded a significant contribution of contami- nating leucocytes to these measurements. Thrombin stimula- tion did not increase the rate of glutamine metabolism. Analysis of the rates of glutamine metabolism suggests that it accounts for most of the ammonia produced during PC storage. However, it appears to be relatively insignificant as a metabolic fuel. The role of glutamine metabolism for platelets is uncertain. It may be a vestige of a pathway in the megakaryocyte. The ammonia which it produces may be deleterious for platelets and for patients with liver disease who receive PC infusions.

In 1981, Ukrainski et al first demonstrated that ammonia accumulated in platelet concentrates (PC) during their storage at 22°C for transfusion (Ukrainski et al, 1981). We recently confirmed and extended these observations by showing that, relative to cell-free plasma stored as a control, ammonia levels rose by 0.1 5 mM per day during the first 2 d of storage and by 0 .5 mM after 7 d of storage (Edenbrandt & Murphy, 1990). One source of ammonia is the deamination of AMP to IMP with the eventual further metabolism of the latter to hypoxanthine. In that study (Fdenbrandt & Murphy, 1990). we showed that adenine nucleotide levels dodecrease to approximately two-thirds of control values by day 7 of storage and that this decrease can be accounted for quantita- tively by a rise in concentration of hypoxanthine to 0.08 mM. Thus, this pathway, although present, could not account for the large amounts of ammonia which accumulate.

In another study (Kilkson et al. 1984), we measured the rate of oxygen consumption during PC storage and could calculate that approximately 8 5% of ATP regeneration could be accounted for by oxidative metabolism. However, glucose was converted stoichiometrically into lactate which suggests that it does not provide a substrate for oxidative metabolism. From the incorporation of I4C from I4C-labelled free fatty

Correspondence: Dr Scott Murphy, Cardeza Foundation for Hemato- logic Research, 101 5 Walnut Street, Philadelphia, PA 19107. U.S.A.

acids into C02 it was calculated that fatty acid oxidation could account for only approximately one half of the oxygen consumption (Cesar et al, 1987). It was therefore considered important to identify the additional fuel or fuels which are oxidized. One possibility is glutamine. This amino acid is a major respiratory substrate for the small intestine (Wind- mueller & Spaeth, 1978). Therefore, since amino acids are both important sources for ammonia generation and poten- tial substrates for oxidative metabolism, we have measured changes in the concentrations of amino acids during PC storage. In addition, we have measured in platelets the activity of glutaminase, a key enzyme in the metabolism of glutamine. Finally, we have determined the response of platelet glutamine metabolism to stimulation by thrombin.

MATERIALS AND METHODS PC and platelet-free plasma (PFP) were prepared as pre- viously described (Edenbrandt & Murphy, 1990; Holme et al, 19 78) from blood drawn from normal volunteers into citrate-phosphate-dextrose anticoagulant. 5 5-60 ml PC or PFP were stored in plastic containers (PL-732, Fenwal, Deerfield, Ill.) at 22 f2'C on a rotator (Heher Labs Inc., St Paul, Minn.) at 6 cycles/min. For comparison with an unprocessed, control PC, leucodepleted PC were prepared by passage through a filter (Kickler et al, 1989) (PL-SOS, Pall

585

586 Scott Murphy et a1 Table I. Amino acid concentrations (PM) during PC and PFP storage.

Amino acid levels

PFP PC

Day 0 Day 1 Day 5 Day 1 Day 5

AIanine Glycine Valine Threonine Lysine Leucine Serine Histidme Arginine Isoleucine Tryptophan Ornithine Phenylalanine Tryosine Hydroxyproline

576f150 330593 3 2 0 f 5 5 248 f 30 2 3 7 f 3 5 1 7 2 f 4 2 157f38 1 3 4 f 2 1 1 2 8 f 2 2 8 6 f 19 8 2 f 1 4 7 9 f 1 8 7 9 f 5 78f15 36f13

618f170 3 1 7 f 9 4 3 4 4 f 5 6 2 5 9 f 3 0 235f38 1 7 5 f 5 3 153f41 1 4 2 f 2 5 1 2 7 f 1 9 9 0 f 2 3 8 7 f 5 8 2 f 2 1 8 2 f 6 80 f16 40f 13

635f180 320f96 3 3 9 f 4 2 257jz31 234jz40 179152 157h40 1 4 3 f 2 7 1 3 4 f 2 3 9 3 f 2 3 8 4 f 8 81 f22 85jz7 81jz17 38f12'

565f66 398f101 3 1 7 f 3 3 2 5 0 f 2 7 2 5 3 f 3 7 1 9 7 f 4 6 158f28 1 3 8 f 1 9 141 f 2 3

9 4 f 2 2 72 f14 82 f16 8 3 f 6 80f 16 32f12

795f89* 521f131* 3 3 2 f 3 7 289 f 30' 305f48' 234f41' 1 6 8 f 3 1 1 4 5 f 2 4 163f 30' 114f19'

70f 16 103f20' 105f8' 97 f17 ' 3 2 f l l

Values given as mean f 1 SD. * P<0.05 v. day 1. n=5.

Corp., Glen Cove, N.Y.) according to the manufacturer's instructions. Platelet counts of control and leucodepleted PC were equalized by addition of PFP to the PC with the highest platelet count. Routine platelet and leucocyte counting was done as previously described (Edenbrandt & Murphy, 1990). Leucocyte counts of leucodepleted PC were done with the Nageotte chamber (Poly-Labo, Paul Block Co., Strasbourg, France) (Andreu et al, 1989).

Amino acid levels in PC supernatants and PFP were determined after separation by high-pressure ion exchange chromatography and identification with ninhydrin (Spach- man et al, 1958) using a 6300 Beckman Amino Acid Analyser (Beckman, Palo Alto, Calif.). In addition, enzymatic methods and a DU-50 spectrophotometer (Beckman, Palo Alto, Calif.) were used to measure the levels of lactate (Kikson et al. 1984), and of glutamine, glutamate and aspartate (Bergmeyer, 1985). Bovine thrombin (Sigma Co., St Louis, Mo.) was used for stimulation of glutamine metabolism by platelets. To determine the activities of phosphatedependent glutaminase and citrate synthase, platelets from PC on day 1 of storage or from fresh blood drawn into EDTA were washed as previously described (Bills et al, 1976), and homogenized in appropriate extraction media (Ardawi & Newsholme, 1982: Alp et al, 1976). The glutaminase assay involved determination of the rate of conversion of glutamine to glutamate at 37OC in a solution containing 20 m~ gluta- mine, 150 mM phosphate, 0.2 m~ EDTA, and 50 mM Tris (pH 8.6) (Ardawi & Neosholme, 1982). The glutamate formed was measured spectrophotometrically (Bergmeyer, 1985). Citrate synthase activity was determined by the method of Alp et a1 (1976) in a solution containing 50 m~

Tris, 1 mM EDTA, 0 - 2 mM 5.51dithiobis-(2-nitrobemic

acid) (DTNB). 0.1 mM acetyl CoA, 0.5 mM oxaloacetate and 0.05% (v/v) triton XlOO using a Model 240 spectrophoto- meter (Gilford. Oberlin. Ohio). In the assay, CoA formed from acetyl CoA reacts with DTNB leading to an increase in extinction at 412 nm. The results of enzyme assays were expressed per lo9 platelets or per mg protein (Bradford, 1976).

Informed consent and statistics. Volunteers gave written, informed consent to protocols approved by the Institutional Review Board of Thomas Jefferson University. Student's paired and unpaired t tests were used for statistical compari- sons. Unless otherwise indicated, results are given as mean f 1 standard deviation (SD).

RESULTS

Amino acid levels during storage of PC and PFP Using the products obtained from five individual donations, the levels of 17 amino acids were measured by ion exchange chromatography in fresh PFP and on days 1 and 5 of storage of PC and PFP. The levels of glutamine and glutamate could not be measured accurately by this method because of overlap between the adjacent peaks. Although quantification was not possible, it was clear that the glutamine peak disappeared during storage while the glutamate peak increased. The levels of the other 15 amino acids are given in Table I. Of the 15, there was an increase in 10. The percentage increase ranged from 15% to 30% except for glycine. 31% and alanine, 41%.

The levels of glutamine and glutamate were measured daily by the enzymatic technique: the results are given in Fig 1. At the start of storage, the glutamine concentration was

Amino Acid Metabolism in Stored Platelets 5 8 7

0.4

I MEAN flSD 0'51

. - ;

0.4

0.3

mM

0.2

0. I

0 I 2 3 4

D A Y S OF STORAGE

Fig 1. Fall in glutamine concentration (solid symbols and line) and rise in glutamate concentration (open circles and interrupted line) during PC storage. Measurements were by the enzymatic technique (Bergmeyer. 1985).

= E 1 . *: **

0 . f z L < 0'3t * * **

O o.2 t . 0. I 1 ; ,*; 0; * ,

0 0.1 0.2 0.3 0.4

GLUTAMATE mM Fig 2. Glutamine and glutamate concentrations of individual PC samples during the first 2 d of storage. The two concentrations were highly correlated (r= -0.86). The regression line was described by the equation: [glutamate] = -0.65 x [glutamine] +0.29.

rn 301

(0 0

m v- \

10

0

I , 30 6 0 90 MINUTES THROMBIN

OR SALINE

Fig 3. Stimulation by thrombin of lactate production by platelets. Aliquots of PC on day 1 of storage were incubated at 3 7°C for 90 min with either thrombin (1 unit/&). closed circles, or saline, open circles. The rate of lactate production was approximately doubled by thrombin relative to saline controls.

0.363 f 0 . 0 6 5 mM. and it decreased to a non-detectable level by day 4. At the same time, the glutamate concentration increased. A plot of the concentration of glutamine against that of glutamate during the first 2 d of storage is given in Fig 2. The two were highly correlated ( r= -0.86) and the slope of the regression line was -0.65 suggesting that two molecules of glutamate were produced for each three mol- ecules of glutamine consumed. The data from these same 12 studies was also analysed in a different way. The mean ratio of glutamate appearance to glutamine disappearance between day 1 and day 2 was 0 . 7 4 f 0 . 1 9 , suggesting that three molecules of glutamate were produced for each four molecules of glutamine utilized. Thus, we conclude that, under the conditions of PC storage, only one-third to one- quarter of the glutamine utilized is metabolized to products other that glutamate. In studies of the donations from five individuals, aspartate was not detectable in fresh PFP or in PC on day 7 of storage (data not shown).

Role of plasma and contaminating leucocytes Table I shows that there was essentially no change in the levels of the 15 amino acids listed when PFP was stored as a control under the same conditions as those which were used for storing PC. Furthermore. when PFP samples from 11 donations were stored for 3 d, glutamine levels were 3 5 3 f 8 5 and 320 f 66 mM on days 1 and 3 respectively and glutamate levels were 49 f 28 and 64 f 26 on days 1 and 3 respectively. Thus, cells were required for the changes in amino acid concentrations that occurred when PC was stored.

The mean leucocyte count of the PC was 2.9 f 1.9 x 109/1. Filtration through the Pall filter reduced this level to less than

588 Scott Murphy et al

mM 0.3E 0.2

O . ' t u A 30 60 90

THROMBIN ( I unitlml)

0.3 t mM 0.2

0. I

I I I I Fig 4. Glutamine and glutamate A 3 0 6 0 9 0 concentrations of PC do not change after

THROMBIN ( 1 unitlml)

stimulation with thrombin. These are measurements from the same four aliquots

MINUTES AT 37'C

2 x 106/1, the detection limit of the counting method used. In three paired studies, the decreases in glutamine concentra- tion between days 1 and 2 of storage were 0.18 f 0 . 0 2 mM and 0.18 f 0.04 mM for control and filtered PC respectively while the increases in glutamate concentration during the same time interval were 0.14f0.04 and 0.15f0.02. Therefore, we conclude that platelets can carry out the conversion of glutamine to glutamate in the virtual absence of leucocytes (i.e. greater than 3 log depletion) and that platelets, not contaminating leucocytes, are responsible for the metabolism of glutamine during PC storage.

Platelet enyme activities Enzyme activities were measured in platelets freshly prepared from blood drawn into EDTA from three normal volunteers. Mean glutaminase and citrate synthetase activities were 22.3 f 6 . 3 and 32.8f4.3 nmol/min/mg protein respect- ively. In five studies of platelets from PC on day 1 of storage, mean glutaminase activity was 46.8 f25 .6 nmol/min/lOY platelets. Assuming that lo9 platelets contain 1.8 mg protein (Holmsen. 1990), there is no significant difference between the two values for glutaminase. Glutaminase activity was also measured in these PC after leucodepletion using the Pall filter. There was no statistically significant difference in glutaminase activity before and after leucodepletion. Thus the glutaminase activity measured in PC appears to be due to platelets and not to contaminating cells.

Lack of eflect of thrombin on glutamine utilization Aliquots of PC on day 1 of storage were incubated at 3 7°C for 90 min with either thrombin (1 unit/ml) or saliie. Fig 3 shows that these experimental conditions resulted in the well-known stimulatory effect of thrombin on glycolysis (Akkerman, 19 78). However, Fig 4 indicates that glutamino- lysis was not stimulated by thrombin in these same aliquots.

DISCUSSION

These studies show that the glutamine present in the plasma of PC is utilized during PC storage so that its concentration is nondetectable by day 4. The rate of fall in glutamine concentration during the 6rst 2 d was approximately 0.15 mM per day (Fig 1) which was similar to the rate of increase in ammonia concentration in our previous study (Edenbrandt &

used in Fig 3.

Murphy, 1990). Thus, the deamination of glutamine appears to account for the increase in ammonia concentration during PC storage. Our leucodepletion studies suggest that platelets are responsible for most of the utilization of glutamine since the rate of utilization was not decreased by removal of leucocytes. This is the first description of the glutaminase activity of platelets.

Two-thirds to three-quarters of the glutamine metabolized could be accounted for as glutamate produced. This is similar to glutamine metabolism in lymphocytes and rat macro- phages in which 59% and 74% respectively of the glutamine utilized is converted to glutamate with no further metabolism of the latter (Newsholme et al, 1988). The potential fates for glutamate are shown in Fig 5. It can be deaminated directly by glutamate dehydrogenase or its amino group can be transaminated with keto acids such as oxaloacetate or pyruvate to form aspartate or alanine respectively. Gluta- mate dehydrogenase and glutamate-oxaloacetate transami- nase have been previously demonstrated to be present in platelets (Waller et al, 1959). We found (Table I) that the alanine concentration increased by approximately 0.2 mM from day 1 to day 5 (i.e. 0.05 mM/d). but there was no increase in the concentration of aspartate. However, it is possible that any aspartate formed might be used to provide the amino group required for the salvage of hypoxanthine to AMP (Kovacevic et al, 1988). In a previous study (Eden- brandt & Murphy, 1990). we found a rate of hypoxanthine salvage equivalent to 0.003 mM/d during PC storage. Thus these two transamination pathways could account for the difference between the rate of glutamine utilization and glutamate production.

Either transamination or deamination of glutamate forms 2-oxoglutarate which can enter the tricarboxylic acid (TCA) cycle at the level of 2-oxoglutarate dehydrogenase. One of the aims of the present study was to determine if one or more amino acids could serve as substrate for oxidative metab- olism. The fall in glutamine concentration of 0.1 5 mM/d is equivalent to a rate of utilization of 0.1 mmol/d/l0l2 platelets, assuming PC platelet concentration to be 1500 x 109/l. Our data show that at most one third of the glutamine, i.e. 0.033 mmol/d/lO'* platelets. is metabolized from glutamate to 2-oxoglutarate. In order for a 2-oxoglutar- ate molecule to be completely oxidized. four molecules of

Amino Acid Metabolism in Stored Platelets 589 Clutamine

G 1 u t ama t e Oxaloacetate Pyruva te

A s p a r t a t e A lan ine

$ x \

2-Oxoqlu tara te N H 3

Isocitrate /

S u c c i n a t e

f Fumarate

I( Malate

TCA

CYCLE Citrate

tc

oxygen are required. Even making the unlikely assumption that all of the 2-oxoglutarate was fully oxidized, this would require only 0- 132 mmol oxygen per day per l0l2 platelets, one tenth the known oxygen consumption rate (Kilkson et a / , 1984). Thus. it is unlikely that glutamine is a major oxidative substrate as it is in other cells (Windmueller & Spaeth, 1978). Although not tested in the current study, previous work (Cesar et al, 198 7) suggested that free fatty acids may provide an important fuel during PC storage. Guppy et al(1990) have recently suggested that platelets may utilize internal fuel. It will be a subject for further study to determine why more glutamine carbon cannot pass through the TCA cycle.

As demonstrated in Table I, no other plasma amino acid declined in concentration during PC storage. In fact 10 of the 15 amino acids examined showed a statistically significant, albeit relatively small, increase. Although, as previously mentioned, alanine might accumulate due to transamination ofpyruvate, it seems likely that some proteolytic activity may be present during PC storage thus releasing amino acids from plasma or platelet proteins. We (Cesar e t al, 1987) and others (Bode 81 Miller, 1988) have provided data suggesting that this might be the case. If proteolysis is occurring, platelets are required since the amino acid levels were stable when cell- free plasma was stored as a control. We point out that we have not yet excluded utilization of one or more of these amino acids by simply measuring concentrations before and after storage. In our studies of plasma free fatty acids, fatty acid levels in PC increased in spite of the fact that they were being oxidized. We suggested that this might be due to the presence of lipoprotein lipase in plasma during storage of PC (Cesar et a / . 1987).

In spite of these reservations, glutamine seems to be uniquely utilized by platelets in comparison to other amino acids. What then is the role for glutaminolysis in platelets?

Acety lCoA

Fig 5. Potential fates for glutamate formed from glutamine by glutaminase (1) activity. Glutamate may be deaminated by glutamate dehydrogenase (2) or transaminated (3) with oxaloacetate, pyruvate or other keto acids. The 2-oxoglutarate formed can then enter the TCA cycle. Citrate synthetase (4) is another mitochondria1 enzyme which we have measured for comparison with glutaminase. It is responsible for the condensation of acetyl CoA with oxaloacetate to form citrate.

Using the same technique for its measurement+ our figure for the level of phosphate-dependent glutaminase is essentially the same as has been observed in human and rat lympho- cytes and about one quarter of that seen in rat peritoneal macrophages (Newsholme et al, 1988). We have also made the first measurement in platelets of the activity of another mitochondria1 enzyme, citrate synthetase (Fig 5). Its levels are similar to those of glutaminase. This is also the case for lymphocytes and macrophages. These results suggest that glutamine metabolism is important in platelets. However, in this study, we found no stimulation of glutamine metabolism by the platelet’s most potent agonist, thrombin (Figs 3 and 4). On this basis, it is tempting to speculate that glutaminolysis in platelets is vestigial, simply reflecting a capacity to use glutamine which was important in the platelet’s precursor cell, the megakaryocyte. Its role in the latter cell would be to provide precursors for purine and pyrimidine nucleotides from which RNA and DNA would be synthesized (News- holme el al, 1988). Similar reasoning can be applied to the high rate of glycolysis displayed by platelets (Kilkson et al, 1984). Glucose-6-phosphate, an intermediate in glycolysis, is used for the synthesis of ribose and deoxyribose which, in turn. are necessary for RNA and DNA synthesis. Nucleic acid synthesis occurs at a high rate in megakaryocytes but does not occur in platelets.

The impact of glutamine metabolism on the platelet storage lesion and platelet transfusion therapy is not yet clear. Glutamine’s potential entry into the TCA cycle and its role as a source of amino groups in the salvage pathway for hypoxanthine may be viewed as probably beneficial. On the other hand, glutamine’s metabolism is responsible for the very high ammonia levels which are reached during PC storage. Ammonia levels higher than 0.2 mM are toxic to many cells (Newsholme & Leach, 1984). Furthermore,

590 Scott Murphy et a1 glutamate at the concentrations reached during storage ofPC is toxic to many cells particularly those of the central nervous system (Rothman, 1973: Choi, 1990). Thus glutamine metabolism might be viewed as deleterious for the platelets. In at least one study (Holme et al, 1987) PC stored in a synthetic medium which lacked glutamine had better in vitro and in vivo characteristics after 7 d than did PC stored in plasma.

The ammonia content of stored platelets may be clinically important in the case of patients with disorders characterized by ammonia accumulation and toxicity. For instance, plate- lets are frequently transfused daily to patients with liver cirrhosis and thrombocytopenia who are actively bleeding from oesophagogastric varices. In this setting, the amount of ammonia administered as a passenger with the transfused platelets is not far from that which is capable of elevating blood ammonia levels above the normal range and inducing hepatic encephalopathy in some cases (Rudman et al, 1973). Finally, a syndrome of idiopathic hyperammonaemia has been reported in patients who have received high-dose chemotherapy for haematologic malignancy (Mitchell et al, 1988). The potential role of ammonia infused during the transfusion of blood products was not considered in this report. Further studies will be required to clarify these points.

ACKNOWLEDGMENTS Supported in part by research grant HL 20818 from the National Institutes of Health and grant TWO 1526 from the Fogarty International Center of the National Institutes of Health.

REFERENCES Akkerman. J.W.N. (1 978) Regulation ofcarbohydrate metabolism in

platelets. A review. Thrombosis and Haemostasis, 39, 712-724. Alp. P.R., Newsholme. E.A. & Zammit. V.A. (1976) Activities of

citrate synthetase and NAD-linked and NADP-linked Isocitrate dehydrogenase in muscle from vertebrates and invertebrates. Biochemical Journal. 154, 689-700.

Andreu. G.. Masse. M. & Lecrubier, C. (1989) Evaluation of a new leukocyte removal filter for platelet concentrates: Sepacell PL 10 N. Transfusion. 29, 40s.

Ardawi. M.S.M. & Newsholme. E.A. (1982) Maximum activities of some enzymes of glycolysis. the tricarboxylic acid cycle and ketone-body and glutamine utilization pathways in lymphocytes of the rat. Biochemical Journal. 208, 743-748.

Bergmeyer. H.U. (1985) Methods of Emumatic Analusis. Vol. 111. pp. 350-363. Academic Press, London.

Bills, T.K., Smith, J.B. & Silver. M. (1976) Metabolism of ['*C] arachidonic acid by human platelets. Biochirnica et Biophysica Acta,

Bode, A.P. & Miller. D.T. (1988) Preservation of in vitro function of platelets stored in the presence of inhibitors of platelet activation and a speciec inhibitor of thrombin. Journal of hboratoru and Clinical Medicine, 1 11, 1 18-1 24.

Bradford, M. (1 976) A rapid, sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Analutical Biochernistru. 72, 248-254.

424, 303-314.

Cesar. J.. DiMinno, G., Alam, I.. Silver, M. & Murphy, S. (1987) Plasma free fatty acid metabolism during storage of platelet concentrates for transfusion. Transfusion. 27,434437.

Choi, D.W. (1990) Cerebral hypoxia: some new approaches and unanswered questions. Journal of Neuroscience, 10, 2497-2 501.

Edenbrandt. C.M. & Murphy, S. (1990) Adenine and guanine neucleotide metabolism during platelet storage at 22OC. Blood. 76,

Guppy, M.. Whisson. M.E., Sabaratnam, R.. Withers, P. & Brand. K. (1990) Alternative fuels for platelet storage: a metabolic study. Voz Sanguinis. 59, 146-1 52.

Holme. S.. Heaton, W.A. & Cartwright. M. (1987) Improved in viw and in vitro viability of platelet concentrates stored for 7 days in platelet additive solution. British Journal of Haematologu. 66,23 3- 238.

Holme. S., Vaidya. K. & Murphy, S. (1978) Platelet storage at 22OC Effect of type of agitation on morphology, viability and function in vitro. Blood. 52, 425435.

Holmsen, H. (1990) Composition of platelets. In: Hematologu (ed. by W. J. Williams et al). McGraw Hill, New York.

Kickler, T.S.. BUI. W., Neu. P.M.. Drew, H. & Pall, D. (1989) Depletion of white cells from platelet concentrates with a new absorption filter. Transfusion, 29, 41 1-414.

Kilkson, H., Holme. S. & Murphy, S. (1984) Platelet metabolism during storage of platelet concentrates. Blood, 64, 406414.

Kovacevic, A.. Jerance. D. & Brkljac, 0. (1988) The role of glutamine oxidation and the purine nucleotide cycle for adaptation of tumor energetics to the transition from the anaerobic to the aerobic state. Biochemical Journal, 252, 381-386.

Mitchell, R.B.. Wagner, J.E.. Karp. J.E., Watson, A.J.. Brusilow, W.. Pmpiorka. D.. Storb, R.. Santos, G.W., Burke, P.J. & Saral, R. (1 988) Syndrome of idiopathic hyperammonemia after high-dose chemotherapy: review of nine cases. American Journal of Medicine.

Newsholme, E.A. & Leach, A.R. (1 984) Biochernistru for the Medical Sciences. John Wiley and Sons, London.

Newsholme, E.A.. Newsholme, P.. Curi, R., Challoner. M.A. & Ardawi, M.S.H. (1988) A role for muscle in the immune system and its importance in surgery, trauma, sepsis, and bums. Nutri-

Rothman, S. (1973) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. Journal of Neurosciences, 5 , 1884-1 89 1.

Rudman. D., Smith. R.. Salam. A., Warren, D.. Galambos. J. & Wenger. J. (1973) Ammonia content of food. American Journal of Clinical Nutrition, 26, 487490.

Spachman. D.H., Stein, W.H. & Moore. S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analutical Chemistru, 30, 1190-1206.

Ukrainski. C.T.. GoldEnger. D.. Pomerance. J.J.. Lee. H.C., Parber. S. & Sanchez, R. (1981) Ammonia accumulation in platelet concen- trates during storage. Transfusion. 21, 11 3-1 17.

Waller, H.D., Lohr. G.W., Grignani. F. & Gross, R. (1959) Uber den energienstoffwechsel normaler menschlicher Thrombozyten. Thrombosis et Diathesis Haemorrhugica. 3, 520-547.

Windmueller. H.G. & Spaeth, A.E. (1978) Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestlne. Journal of Biological Chemistru.

1884-1892.

85,662-667.

tion. 4, 261-268.

253,69-76.