1987 70: 1389-1393
E Beutler, L Forman and C West
Effect of oxalate and malonate on red cell metabolism
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Effect of Oxalate and Malonate on Red Cell Metabolism
B/cod. Vol 70. No 5 (November), 1987: 1389-1393 1389
By Ernest Beutler, Linda Forman, and Carol West
The addition of oxalate to blood stored in Citrate-
phosphate-dextrose (CPD) produces a marked improve-
ment in 2,3-diphosphoglycerate (2.3-DPG) preservation; an
increase in 2.3-DPG levels can also be documented in
short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase. monophospho- glycerate mutase, and pyruvate kinase (PK). In the pres- ence of fructose 1 .6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP).
Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step. indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde
O
VER A DECADE AGO it was demonstrated thatascorbic acid exerted a favorable effect on the 2,3- diphosphoglycerate (2,3-DPG) level oferythrocytes in stored blood.”2 Subsequent studies revealed that ascorbic phos- phate, a more stable derivative of ascorbic acid had a similar
effect.3 Although this phenomenon has been confirmed
repeatedly,6 our investigation of the mechanism of the
ascorbate effect failed to provide an explanation for the increase in 2,3-DPG that occurred.”78 Specifically, we could not find significant inhibition of glycolytic enzymes by
ascorbate,’ no evidence of oxidation of either NADH or
NADPH, and no evidence that the ascorbate was metabo-
lized or that it exerted a significant effect on intracellular pH.7
Recently, Kandler et al9 found that in contrast to the usual preparations, highly purified ascorbic acid and ascorbic phosphate had no effect on the 2,3-DPG of stored red cells.
Searching for a contaminant that might explain the effect of
less purified batches, they discovered that oxalate found in
the ascorbate preparations could account for the effect on the 2,3-DPG levels of red cells in stored blood.
We now report on the effects of oxalate on the metabolism of human erythrocytes, identifying some of the inhibitory
actions that may explain the effect of this compound on
2,3-DPG levels. We have also explored the effect of malonic acid, a natural metabolic intermediate and the 3-carbon analogue of the 2-carbon dicarboxylic acid, oxalic acid, on 2,3-DPG levels.
MATERIALS AND METHODS
After obtaining informed consent, blood samples from normal
blood donors were drawn into citrate-phosphate-dextrose (CPD), for
storage studies at 4#{176}Cor into I mg/mL ethylenediamine tetracetic acid (EDTA) for short-term incubations at 37#{176}C.Potassium oxalate
and sodium malonate were purchased from Sigma Chemical Com-
pany, St. Louis.
Unless otherwise indicated red cell enzyme activities were mea- sured using standard methods,’#{176} adding the stated amount of oxalate to the assay system. Pyruvate kinase (PK) activity was estimated using a system in which the generation of adenosine triphosphate (ATP) is measured in order to circumvent the inhibitory effect of oxalate on the lactate dehydrogenase reaction used as an indicator in the standard method. In this technique, the reaction mixture con-
phosphate dehydrogenase step is apparently due to an
increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media. but appre- ciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives.
Malonate. the 3-carbon dicarboxylic acid analogue of oxa- late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.
S 1987 by Grune & Stratton. Inc.
tamed the following: 100 mmol/L Tris-HCI, pH 8; 0.5 mmol/L
EDTA; 10 mmol/L MgC12, 100 mmol/L KCI; 0.2 mmol/L NADP;
2 mmol/L glucose; 1.5 mmol/L adenosine diphosphate (ADP); 0.1
U each of glucose-6-phosphate (G-6-P) dehydrogenase and hexoki- nase. The reaction was started by the addition of phospho(enol)pyru- vate (PEP) to provide a concentration of 5 mmol/L. The effect of oxalate on the kinetics of the PK reaction were studied using
partially purified PK.” Diphosphoglycerate phosphatase (DPGP)
activity was measured by estimating inorganic phosphate (P1)
release from labeled 2,3-DPG.’2
ATP, 2,3-DPG, and pyruvate and lactate levels were measured spectrophotometrically as previously described.’0 G-6-P, fructose- 6-phosphate (F-6-P), fructose- I ,6-diphosphate (FDP), dihydroxy- acetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 3-phosphoglyceric acid (3-PGA), 2-phosphoglyceric acid (2-PGA), and PEP were all measured fluorometrically.’#{176} In some instances, the levels of DHAP, GAP, and FDP were sufficiently elevated in the
erythrocytes so that their levels could also be measured by a
spectrophotometric modification of the standard fluorometric tech- nique. The internal pH ofstored red cells was measured at 4#{176}Cusing a Corning model I 50 with a glass electrode, packing the erythrocytes by centrifugation, freezing, and thawing. Red cell viability was studied in New Zealand white rabbits using the combined “Tc-5’Cr method.’3
RESULTS
Cold storage of CPD anticoagulated blood with and
without oxalate. Four hundred fifty milliliters of blood
From the Department of Basic and Clinical Research. Scripps Clinic and Research Foundation. La Jolla. CA.
Submitted March 9. 1987; accepted June 29. 1987.
Supported in part by National institutes of Health Grant No.
HL25552.
This is publication number 4723BCR from the Research insti- lute ofScripps Clinic.
Address reprint requests to Ernest Beutler. MD. Department of Basic and Clinical Research, Scripps Clinic and Research Founda- lion. 10666 N. Torrey Pines Rd. La Jolla. CA 92037.
The publication costs ofihis article were defrayed in part by page charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
© I 987 by Grune & Stratton. inc.
0006-4971/87/7005-0018$3.00/0.
DAYS OF STORAGE
1,
E
0 0
a
(3
G/yco/y/: Et7lyfl7es
Enol
GP, Aid GM3D PGK
lix PFK TP1 DPCM DPGP
ecu
CSHPx CR
6PCD COT
DAYS OF STORAGE
Fig 1. 2.3-DPG and ATP levels of blood stored in CPD (0. #{149}) and CPD plus sufficient oxalate to provide a final concentration of 500 smoI/L (0. U). Four 450 ml units of blood were studied. Each point represents the mean of two observations. The 0 time points were obtained after a one to two hour holding period at room temperature. Following this the samples were stored at 4’C and mixed only at the time of sampling.
1390 BEUTLER. FORMAN. AND WEST
from each of four normal donors was collected into 63 mL of CPD and into CPD containing sufficient potassium oxalate to provide a final concentration of 500 mol/L. The blood was stored at 4#{176}C,mixed at intervals, and the levels of ATP and 2,3-DPG were determined. The results of these investi-
gations are shown in Fig I. There was no difference in
internal red cell pH for the first 2 weeks of storage. At 3 weeks the internal pH of the samples with oxalate averaged
0.061 U higher and at 4 weeks 0.11 U higher than the
controls. The initial determination of red cell ATP and
2,3-DPG levels was made after the anticoagulated blood had been held at room temperature for approximately two hours.
It was apparent that ATP levels in blood collected into
oxalate-containing anticoagulants had already declined sub- stantially in this brief time. This immediate effect of oxalate on red cell ATP levels was confirmed in other studies (data not shown). Over the 4-week storage period oxalate greatly
improved preservation of 2,3-DPG but had a markedly
deleterious effect on ATP levels. The same experiment was carried out with a final oxalate concentration of only 50 Mmol/L. The results of this study are shown in Fig. 2.
The effect of oxalate on red cell enzyme activities. The effect of 500 smol/L potassium oxalate on red cell enzyme
activities is summarized in Fig 3. The enzyme most sensitive
to inhibition by oxalate was found to be lactate dehydrogen- ase (LDH), followed closely by monophosphoglyceromutase
(MPGM) and then by PK. In contrast to the effect of
oxalate, 5 mmol/L malonate failed to inhibit erythrocyte
MPGM, PK, or LDH activity. The effect of oxalate on PK
kinetics is shown in Fig 4. The inhibition of PK by oxalate
was competitive with PEP in the presence of FDP, the
allosteric effector of PK, but noncompetitive in the absence
of FDP.
The effect of oxalate on red cell metabolism at
37C. Estimation of the levels of metabolic intermediates
other than ATP and 2,3-DPG at the end of the 28-day
storage period proved to be unreliable because of the very large amounts of pyruvate and lactate that had accumulated.
B
U
S
0 0
Fig 2. The same as Fig 1.except that the oxalate concentra- tion was only 50 ,mol/L.
However, studies of the effect of oxalate on red cell interme- diates could readily be carried out on red cells incubated in autologous plasma for several hours at 37#{176}C,since a rise in the red cell 2,3-DPG level was demonstrable under these conditions. It was necessary to use an anticoagulant other than CPD for such studies, because of the very substantial effect that temperature exerts on the pH of blood. It is -0.7 pH units higher at 4#{176}Cthan at 37#{176}C’4and the citrate
preservatives provide an unsuitable medium for study of
metabolism at the latter temperature. A suspension of red
cells in EDTA anticoagulated plasma incubated under a
suitable C02-air atmosphere is very suitable for such investi- gations. Preliminary studies showed that maximum elevation of red cell 2,3-DPG levels could be observed after four to six hours incubation at 37#{176}Cand that the amount of phos- phate in plasma was insufficient to support an optimal effect of oxalate on 2,3-DPG concentration.
Erythrocytes from blood drawn into EDTA anticoagulant
were resuspended at a hematocrit of 30% in their own
plasma. Sufficient KH2PO4/K2HPO4 pH 7.4 was added to
provide a 2.5 mmol/L increase in P04 concentration. Ali- quots of the cell suspension were incubated at 37#{176}Cin a shaking incubator in an atmosphere of 5% CO2/95% air with
Fig 3. The effect of 500 .tmol/L oxalate on the activity of some red cell enzymes. Abbreviations: Hx. hexokinase; GPI. glu- cose phosphate isomerase; PFK. phosphofructokinase. AID. aldo- lase, TPI. triose phosphate isomerase; GAPD. glyceraldehyde phosphate dehydrogenase; DPGM, diphosphoglycerate mutase;
PGK. phosphoglycerate kinase; MPGM. monophosphoglycerate mutase; Enol. enolase; LDH. lactate dehydrogenase; G6PD. glu- cose-6-phosphate dehydrogenase; 6PGD. 6-phosphogluconate dehydrogenase; GSHPx. glutathione peroxidase; GOT. glutamate oxalocetate transaminase; GR. glutatione reductase.
0
B
8
> -1.0
.J
-l.0
Log PEP (mM) Log PEP (mM)
OXALATE AND MALONATE IN RBC STORAGE 1391
Fig 4. The kinetics of the inhibition of PK by 500 Mmol/I oxalate. Hill plots are presented (A) in the presence of 500 smol/L FDP. and (B) in the absence of FDP. PK was purified using standard methods.” The inhibition by oxalate was competitive with respect to PEP in the presence of FDP and noncompetitive in its absence.
and without the addition of 2 mmol/L oxalate. The possible limiting effect of phosphate and of NAD was investigated
in incubation mixtures to which excess phosphate or pyru-
vate had been added. The pH and the levels of metabolic intermediates are presented in Table I. Figure 5 represents a
“cross-over” plot’5 based on these data. In a similar short-
term incubation experiment malonate failed to exert any
effect on the level of 2,3-DPG.
The effect of oxalate on red cell survival. Twenty
milliliters of rabbit blood was collected into 2.8 mL of CPD solution and divided into bags to which no oxalate was added and those to which sufficient potassium oxalate was added to provide a final concentration of 500 imol/L. At the end of 21 days storage the survival of the red cells of one of the samples was measured in a normal recipient rabbit, and at the end of 22 days the other sample was labeled with a larger amount of 51Cr and viability again determined in a rabbit. The order of presentation was varied so that two of the rabbits received red cells that had been stored with oxalate on day 21 and the other two those that had been stored with oxalate on day 22.
The results of these studies are presented in Table 2. Shown also is the effect of storage of rabbit blood on red cell ATP and 2,3-DPG levels. Oxalate exerted the same favorable effect on 2,3-DPG concentrations and unfavorable effect on
ATP concentration of rabbit erythrocytes as were observed with human cells.
DISCUSSION
Although oxalate has long been used as an anticoagulant for the collection of blood for diagnostic studies, no thought has apparently been given to its use for blood storage. This has probably been due to the presumed toxicity of oxalate.
However, the concentration of oxalate required to aid in the maintenance of red cell 2,3-DPG levels is much lower than that required as an anticoagulant. Even a concentration of only 50 gzmol/L exerted a substantial favorable affect on red cell 2,3-DPG: after 21 days’ storage the levels had declined by only about one-third. Assuming uniform distribution throughout body water, the instant administration of450 mL of blood containing this concentration of oxalate would result in a rise of only -0.6 micromolar in the oxalate level, an increase of the order of 20%. One must take into account, of course, the effect of the administration of multiple units of blood, the rate of clearance of oxalate in normal and diseased individuals, and the existence in the population of many individuals who may have hereditary abnormalities in oxa- late transport’6 potentially leading to calcium oxalate stone formation.
Table 1. Levels of Metabolic Intermediat es in Red Cell Suspensions Incu bated With and Without Oxala te and the Indicated Additives
2.5 mmol/L Phosphate 1 0 mmol/L Phosphate
2.5 mmol/L Phosphate 10 mmol/L Piuvate
Control Oxalate Control Oxalate Control Oxalate
Glucose-6-phosphate (nmol/mL) Fructose-6-phosphate (nmol/mL) Fructose-1-6-diphosphate (nmol/mL) Dihydroxyacetone-phosphate (nmol/mL) Glyceraldehyde-3-phosphate
2,3 Diphosphoglycerate (jmoI/gHb) 3-Phosphoglyceric acid (nmol/mL) 2-Phosphoglyceric acid (nmol/mL) Phosphoenolpyruvate (nmol/mL) Pyruvate (moI/L)
Lactate(imol/L)
63.78 24.84
8.68 9.80
2.55 14.56
3 1 .38 779.83
ND. N.D.
1 3.82 1 8.27
98.65 560.84
8.46 36.24
1 5.86 16 1 .66
328.9 46.50
7320.65 8532.15
65.93 23.60
29.67 16.85
1 1 .5 325.4
36 1084
ND. ND.
1 1 .45 1 3.26
81.1 556.38
7.99 33.23
7.53 205.6
254.4 20.03
7460.9 8522.5
52.84 29.65
25.36 15.63
2.25 3.79
1 1 .7 1 46.56
ND. ND.
1 4.35 18.72
94.70 448.55
N.D. ND.
1 9.9 154.9
7984.2 14055.0
7147.0 6788.1
pHatt-6hr 7.506 7.498 7.668 7.614 7.543 7.696
E
a
C)
0. 4 4 “ V
a. Q. Q. 0. nn a. -
ID ID 0 : d Q. Q. Li > 0
(3 U. u 0 (3 ,() ,,
c’J 0
The same animals served as controls in a concurrent investigation of
12
1392 BEUTLER. FORMAN, AND WEST
2.5mMP, 0-c
I0mMP #{149}-#{149}
(2.5mMPi
\ 0mM
Fig 5. A crossover plot demonstrating the effect of oxalate after six hours incubation in autologous plasma at 37’C under 95%
air-5% CO2. The effect of added P1 and pyruvate are shown:
concentrations represent the amount added to those already present in plasma.
The discovery that oxalate increases the levels of 2,3-DPG in stored blood provides a solution to the problem of why ascorbate appeared to have this effect. We are unable to explain why ascorbate preparations failed, in our earlier studies,’ to result in enzyme inhibition. It is possible that the preparation used in the enzyme assays was of higher purity than those used in the storage studies. Since these investiga-
tions were carried out some I 5 years ago, the purity of the
various lots used can no longer be determined. Similar differences in purity probably account for the discrepancy between our finding that dehydroascorbate was ineffective7 while Dawson et a14 subsequently reported that it was more effective than was ascorbate itself.
Efforts to maintain 2,3-DPG during prolonged blood
storage are generally directed at creating appropriate modi- fications of the metabolic flow within the erythrocyte. Such a modification may consist of providing an additional substrate for red cell metabolism. Dihydroxyacetone and inosine are
Table 2. T he Effect of Ox alate on the Stora ge of Rabbit Blood
Al? 2.3-DPG
(mol/9 Hb) (imol/g Hb)
Viability (%) Storage Medium Day 0 Day 2 1 Day 0 Day 2 1 Days 21-22
CPD 7.34 1.76 28.65 13.12 71.2. 68.8. 65.6. 64.4
CPD + oxalate 7.34 0.72 28.65 28.12 72.6. 78.5, 66.8, 69.6
such substances. Dihydroxyacetone is converted to DHAP by red cell triokinase.’T !nosine undergoes phosphorolysis to ribose-!-phosphate and hypoxanthine, and the ribose-!- phosphate is converted in the hexose monophosphate path- way to F-6-P and to GAP.’8 In each of these cases use of the new substrate is less sensitive to a fall in the pH of stored blood than is glucose, providing a distinct metabolic advan- tage to the stored erythrocytes. An alternative to providing a more efficient substrate is modifying red cell preservatives so as to inhibit or to stimulate reactions that influence 2,3-DPG synthesis or degradation. In the past, manipulations of hydrogen ion, pyruvate, and phosphate concentrations have been used for this purpose.’9 Increasing the pH stimulates the
diphosphoglycerate mutase (DPGM) and inhibits the DPGP
reactions.2#{176} Pyruvate serves to oxidize NADH to NAD, thereby increasing the level of one of the required co-factors
for the glyceraldehyde phosphate dehydrogenase (GAPD)
reaction.21’22 The product of this reaction, I,3-DPG is the immediate substrate for the formation of 2,3-DPG. Phos- phate stimulates glycolysis, probably largely by relieving the inhibition of hexokinase by G-6-P.23
It is the DPGM and DPGP reactions that directly control the level of 2,3-DPG in erythrocytes, but these reactions, in
turn, are influenced by other metabolic changes in the
erythrocyte. PK deficiency is associated classically with increased 2,3-DPG levels, sometimes even in the heterozy- gous state.24 The effect of PK deficiency is presumably due to the accumulation of substrates proximal to the PK reaction, ie, PEP, 2-PGA, and 3-PGA. The latter compound inhibits
DPGP and stimulates DPGM. Vora recently suggested that
inhibitors of this reaction might be useful in maintaining 2,3-DPG levels during blood storage.25
In the present investigation we extended the observations of Kandler et al.9 Red cell membranes are readily permeable to the oxalate anion,26 even at 0#{176}C.’6Oxalate increases 2,3-DPG levels not only in blood stored in an artificial additive solution, but also in CPD anticoagulant. Although ATP depletion accompanied the improved 2,3-DPG mainte- nance, red cell viability was not impaired, at least in rabbits.
Oxalate did not exert its effect by altering intracellular pH.
It is a known inhibitor of both plasma LDH2T and of L type
(red cell) PK.28 We found that not only these enzymes but also red cell monophosphoglycerate mutase is inhibited by oxalate. Our kinetic studies indicated that the inhibition of PK is competitive with respect to PEP when the allosteric effector, FDP is present, a finding that is consistent with data obtained earlier in the study of L type PK.28
At 37#{176}Coxalate increases the 2,3-DPG levels of red cells incubated in their own plasma even for a few hours. This provided us with the opportunity of addressing the question of how oxalate accomplishes its effect on 2,3-DPG levels.
The studies in whole red cells pin-point the major functional lesion induced by oxalate to the PK reaction: the cross-over plot (Fig 5) shows that the concentration of intermediate compounds proximal to the PK reaction is increased and that those distal to the reaction are decreased. However, a second cross-over was also present at the GAPD step. A deficiency of either phosphate or NAD could produce such an effect.
The cross-over before GAPD is not eliminated by increasing
REFERENCES
OXALATE AND MALONATE IN ABC STORAGE 1393
the phosphate concentration by 7.5 mmol/L. However,
increasing the available NAD by the addition of pyruvate21’22 greatly decreased the accumulation of substrates proximal to the GAPD reaction. Thus, it appears that in addition to its effect on the PK reaction, oxalate produces a change in the NADH/NAD ratio of red cells, possibly merely by inhibit- ing the LDH reaction.
Although the potential toxicity of oxalate might preclude its clinical use, its effect suggests that other PK inhibitors
1. Wood LA, Beutler E: The effect of ascorbate on the mainte- nance of 2,3-diphosphoglycerate (2,3-DPG) in stored red cells. Br J Haematol 25:61 1, 1973
2. Wood L, Beutler E: The effect of ascorbate and dihydroxyace- tone on the 2,3-diphosphoglycerate and ATP levels of stored human red cells. Transfusion 14:272, 1974
3. Bensinger TA, Zuck TF: Trisodium ascorbate phosphate, a
stabilized form of ascorbic acid, promotes 2,3-diphosphoglycerate maintenance during blood storage. Transfusion 16:518, 1976
4. Dawson RB, Hershey RT, Myers CS, Eaton JW: Blood
preservation. XLIV. 2,3-DPG maintenance by dehydroascorbate
better than D-ascorbic acid. Transfusion 20:321, 1980
5. Dawson RB, Dabezies M, Hershey RT, Myers CS, Holmes 5,
Sisk LD, Miller RM: Blood preservatives and hemoglobin oxygen
affinity. XXX. 2,3-DPG and its maintenance by ascorbate. Prog
Clin Biol Res 21:627, 1978
6. Moore GL, Ledford ME, Brummell MR: Improved red cell storage using optional additive systems (OAS) containing adenine, glucose and ascorbate-2-phosphate. Transfusion 21 :723, 1981
7. Beutler E: Viability, function, and rejuvenation of liquid- stored red cells, in Chaplin H Jr, Jaffe ER, Lenfant C, Valeri CR (eds): Preservation of Red Blood Cells. Washington, DC, National Academy ofSciences, 1973, p 195
8. Beutler E: The maintenance of red cell function during liquid storage, in Schmidt PJ (ed): Progress in Transfusion and Transplan- tation. Washington, DC, 1972 p 285
9. Kandler R, Grode G, Symbol R, Hickey G: Oxalate is the
active component that produces increased 2,3-DPG in ascorbate
stored red cells. Transfusion 26:563, 1986 (abstr)
10. Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods, Orlando, FL, Grune & Stratton, 1984
1 1. Miwa 5, Boivin P. Blume KG, Arnold H, Black JA, Kahn A, Staal GE, Nakashima K, Tanaka KR, Paglia DE, Valentine WN, Yoshida A, Beutler E: Recommended methods for the characteriza- tion of red cell pyruvate kinase variants. Br J Haematol 43:275, 1979
1 2. Beutler E, Forman L, West C, Gelbart T: The mechanism of
improved maintenance of 2,3-diphosphoglycerate in stored blood by the xanthone compound BW A440C. Biochem Pharmacol (in press, 1987)
I 3. Beutler E, West C: Measurement ofthe viability ofstored red cells by the single-isotope technique using 51-Cr: Analysis of valid- ity. Transfusion 24:100, 1984
I 4. Bensinger TA, Metro J, Beutler E: Redesigned apparatus for
anaerobic measurement of blood pH at low temperature. Am J Clin
Pathol 63:264, 1975
might be useful, ifone that was sufficiently nontoxic could be found. It is of interest, in this respect that malonate, the
structure of which superficially resembles PEP more closely
than does that of oxalate, has no effect on either the PK reaction or on 2,3-DPG levels in incubated red cell suspen- sion. Oxalate is a normal plasma constituent, circulating at a level of -4 imol/L.29’#{176} It may be that this compound may exert some in vivo regulatory effect, and further studies are underway to explore this possibility.
15. Minakami 5: Regulation of glycolysis in human red cells- application of “cross over” theorem, in Deutsch E, Gerlach E, Moser K (eds): Metabolism and Membrane Permeability of Erythrocytes and Thrombocytes. Verlag, Stuttgart, Georg Thieme, 1968, plO
16. Baggio B, Gambaro G, Marchini F, Cicerello E, Tenconi R, Clementi M, Borsatti A: An inheritable anomaly of red-cell oxalate
transport in “primary” calcium nephrolithiasis correctable with
diuretics. N Engl J Med 314:599, 1986
I 7. Beutler E, Guinto E: Dihydroxyacetone metabolism by human erythrocytes: Demonstration of triokinase activity and its characterization. Blood 41:559, 1973
18. Gabrio BW, Finch CA, Huennekens FM: Erythrocyte preser- vation: A topic in molecular biochemistry. Blood 11:103, 1956
I 9. Beutler E: Experimental blood preservatives for liquid stor- age, in Greenwalt Ti, Jamieson GA (eds): The Human Red Cell In Vitro. New York, 1974, p 189
20. Rose ZB: The enzymology of 2,3-bisphosphoglycerate. Adv Enzymol 51:211, 1980
21. McManus Ti, Borgese TA: Effect of pyruvate on metabolism of inosine by erythrocytes. Fed Proc 21:68, 1961
22. Paniker NV, Beutler E: Pyruvate effect in maintenance of
ATP and 2,3-DPG of stored blood. i Lab Clin Med 78:472, 1971
23. Gerber G, Kloppick E, Rapoport 5: Ueber den Einfluss des
Anorganisschen Phosphats auf die Glykolyse; seine Unwirksamkeit auf die Hexokinase des Menschenerythrozyten. Acta Biol Med Ger 18:305, 1967
24. Tanaka KR, Paglia DE: Pyruvate kinase deficiency. Semin
Hematol 8:367, 1971
25. Vora 5: Red cell preservation: A novel proposal for the
maintenance of 2,3-DPG level. Blood 68:303a, 1986 (suppl) 26. Hubbard AR, Sprandel U, Chalmers RA: Transport of oxalic acid, glycollic acid, glyoxylic acid and benzoic acid by resealed erythrocyte ‘ghosts’ prepared by a dialysis technique. Biochem Soc Trans7:958, 1979
27. Emerson PM, Wilkinson iH: Urea and oxalate inhibition of
the serum lactate dehydrogenase. i Clin Pathol 18:803, 1965 28. Buc H, Demaugre F, Leroux JP: The kinetic effects of oxalate on liver and erythrocyte pyruvate kinases. Biochem Biophys Res Commun 85:774,1978
29. Cole FE, Gladden KM. Bennett Di, Erwin DT: Human plasma oxalate concentration re-examined. Clin Chim Acta 139:137, 1984
30. Kasidas GP, Rose GA: Measurement of plasma oxalate in healthy subjects and in patients with chronic renal failure using immobilised oxalate oxidase. Clin Chim Acta 154:49, 1986
