British loirrnalo! Haernatology. 1992, 81. 585-590
Amino acid metabolism during platelet storage for transfusion SCOTTMURPHY,SANTIAGO MUNOZ,MARKPARRY-BILLINGS* A N D 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. Leucodepletion studies excluded a significant contribution of contaminating leucocytes to these measurements. Thrombin stimulation 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 quantitatively 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 stoichiometricallyinto lactate which suggests that it does not provide a substrate for oxidative metabolism. From the incorporation of I4C from I4C-labelled free fatty
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 (Windmueller & Spaeth, 1978). Therefore, since amino acids are both important sources for ammonia generation and potential 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 previously described (Edenbrandt & Murphy, 1990; Holme et al, 1978) 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
Correspondence:Dr Scott Murphy, Cardeza Foundation for Hematologic Research, 101 5 Walnut Street, Philadelphia, PA 19107. U.S.A.
585
586
Scott Murphy et a1 Table I. Amino acid concentrations(PM)during PC and PFP storage. Amino acid levels
PFP
AIanine Glycine Valine Threonine Lysine Leucine Serine Histidme Arginine Isoleucine Tryptophan Ornithine Phenylalanine Tryosine Hydroxyproline
PC
Day 0
Day 1
Day 5
Day 1
Day 5
576f150 330593 320f55 248 f 30 237f35 172f42 157f38 134f21 128f22 8 6 f 19 82f14 79f18 79f5 78f15 36f13
618f170 317f94 344f56 259f30 235f38 175f53 153f41 142f25 127f19 90f23 87f5 82f21 82f6 80f16 40f 13
635f180 320f96 339f42 257jz31 234jz40 179152 157h40 143f27 134f23 93f23 84f8 81f22 85jz7 81jz17 38f12'
565f66 398f101 317f33 250f27 253f37 197f46 158f28 138f19 141 f 2 3 94f22 72f14 82f16 83f6 80f 16 32f12
795f89* 521f131* 332f37 289 f 30' 305f48' 234f41' 168f31 145f24 163f 30' 114f19' 70f 16 103f20' 105f8' 97f17' 32fll
Values given as mean f 1 SD. * P
Amino acid metabolism during platelet storage for transfusion SCOTTMURPHY,SANTIAGO MUNOZ,MARKPARRY-BILLINGS* A N D 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. Leucodepletion studies excluded a significant contribution of contaminating leucocytes to these measurements. Thrombin stimulation 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 quantitatively 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 stoichiometricallyinto lactate which suggests that it does not provide a substrate for oxidative metabolism. From the incorporation of I4C from I4C-labelled free fatty
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 (Windmueller & Spaeth, 1978). Therefore, since amino acids are both important sources for ammonia generation and potential 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 previously described (Edenbrandt & Murphy, 1990; Holme et al, 1978) 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
Correspondence:Dr Scott Murphy, Cardeza Foundation for Hematologic Research, 101 5 Walnut Street, Philadelphia, PA 19107. U.S.A.
585
586
Scott Murphy et a1 Table I. Amino acid concentrations(PM)during PC and PFP storage. Amino acid levels
PFP
AIanine Glycine Valine Threonine Lysine Leucine Serine Histidme Arginine Isoleucine Tryptophan Ornithine Phenylalanine Tryosine Hydroxyproline
PC
Day 0
Day 1
Day 5
Day 1
Day 5
576f150 330593 320f55 248 f 30 237f35 172f42 157f38 134f21 128f22 8 6 f 19 82f14 79f18 79f5 78f15 36f13
618f170 317f94 344f56 259f30 235f38 175f53 153f41 142f25 127f19 90f23 87f5 82f21 82f6 80f16 40f 13
635f180 320f96 339f42 257jz31 234jz40 179152 157h40 143f27 134f23 93f23 84f8 81f22 85jz7 81jz17 38f12'
565f66 398f101 317f33 250f27 253f37 197f46 158f28 138f19 141 f 2 3 94f22 72f14 82f16 83f6 80f 16 32f12
795f89* 521f131* 332f37 289 f 30' 305f48' 234f41' 168f31 145f24 163f 30' 114f19' 70f 16 103f20' 105f8' 97f17' 32fll
Values given as mean f 1 SD. * P
