Short Communications Differences in the Effect of Lactic Acid and Neutral Lactate on Glycolysis and Nucleotide Pattern in Incubated Whole Human Blood

CHARLES BISHOP From the Department of Medicine, University of Bufjalo School of Medicine

and the Buflalo General Hospital, Buflalo, N e w York

Whole human blood collected in heparin and glucoM was incubated for up to 48 hours. Cly- colysb proceeded, giving rise to lactic acid forma- tion, and in consequence the PH decreased con- tinuously. Blwd ATP levels also decreased as glycolysia slowed since glycolysis is the only energy source in the red cells of whole human blood. T h e decrease in glycolytic rate was shown to be entirely dependent upon the increasing acidity and not on the lactate pc’ se, since added neu!ral lactate had no effect on the rate of glycolyis or the ATP level whereas added lactic acid had a profound effect on both glycolysia and ATP level.

THE main metabolic activity of mam- malian blood resides in the non-nucleated red cells1 and in these, anerobir glycolysis is the only apparent source of energy. Re- gardless of whether glucose is catabolized via the Embden-Meyerhof pathway only, or partially via the pentose shunt, lactic acid is the end product of the energy-producing system. As glycolysis proceeds in blood in- cubated in vitro, lactic acid accumulates. Concomitantly, the pH gradually decreases and glycolysis likewise decreases. The ob- ject of this study was to determine if gly- colysis decreased because of product inhibi- tion by the accumulating lactate or because the pH had become unfavorably low. The latter was shown to be the case since added lactate acid slowed glycolysis while added neutral lactate did not. It has been re-

Received for publication November 24, 1961; ac- cepted January 15, 1962.

Supported in part by Grant A-210 of the Na- tional Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, Bethesda, Maryland, and the Western New York Section of the Arthritis and Rheumatism Foundation.

Preliminary report presented before the Amer- ican Society of Biological Chemists, Atlantic City, April 10-1.5, 1961.

peatedly noted2.3.4 that glycolysis declines as the pH of incubated blood falls, but the mechanism of this effect has not been eluci- dated. Denstedt’s groups. 6 felt that hexo- kinase became less active as the PH de- creased and that this could be responsible for the gradual failure to utilize glucose. The stimulation of lactate production by the addition of nucleosides to blood which has ceased to utilize glucose,7 inter a h , sug- gests that some step in glucose utilization above glyceraldehyde-%phosphate is more acid-sensitive than the rest of the glycolytic pathway.

Methods In the first experiment (SF-9) approxi-

mately 250 ml. of human blood was col- lected in a standard vacuum blood bottle containing approximately 6,000 units of sodium heparin solution (6 ml. of sterile solution containing 1,000 unitslml.) and 1,000 mg. of sterile glucose solution (10 ml. of sterile 10% dextrose solution). Aliquots of approximately 100 ml. were transferred to two water-jacketed spinner flasks’ and to one flask were added 20 ml. of sterile physiologic saline. To the other were added 20 ml. of a solution of neutral racemic sodium lactate containing five millimoles of lactate. Samples were drawn aseptically from the two flasks at several time intervals for the measurement of glucose, PH, and for preparation of trichloracetic acid ex- tracts for subsequent nucleotide fractiona- tion on Dowex-l-formate columns.1 Three

+ Bellco Glass Co., Vineland, New Jeisey.

256

257 EFFECT OF LACTATE O N Gl.YCOLYSIS AND NUCI.EOTIDES OF BLOOD

additional experiments were performed in essentially the same way except that in SF- 10 one flask received neutral sodium lac- tate while the other received the same amount of lactic acid. In SF-1 1, one flask received neutral lactate while the other received lactate plus lactic acid, the mix- ture being used to give a smaller PH drop than in SF-10. In SF-12, the control flask received saline while the other flask re- ceived a mixture of lactic acid and neutral lactate. In all experiments, the total amounts of lactate added to any flask as neutral lactate, lactic acid, or both, was approximately the same, i e . , 5 millimoles.

Results

The glucose utilization and changes in ATP level in all experiments are shown in Figures 1 to 4. The pH values after 24 and 48 hours of incubation are indicated. Al- though complete nucleotide patterns were run at all time intervals on all samples,' the changes in ATP level were most easily seen. In each instance, however, the changes in the other nucleotides were con- sistent with the degree of ATP breakdown noted.

Discussion

The reported number of studies per- formed on whole blood stored or incubated

5 S f - 9

PM 3 I

mM

UTILIZATION

pH7.0 I

0 4 8 24 48 HOURS

FIG. 1. Glucose utilization and ATP levels in Experiment SF-9. T h e control values for ATP are shown as plain bars while the ATP level for the sodium lactate flask are hatched.

in all sorts of solutions is prodigious. Ke- gardless of the system, however, i t is the common observation that glycolysis pro- ceeds for a while and the pH continues to

(rM 300 I00

GLUCOSE

LACTIC ACID

PH~. I - . - - - _ _ - _ _ - - - -. _ - - - 0 4 8 24 48

HOURS FIG. 2. Same as Figure 1, data of Experiment SF-10.

T h e plain bars for ATP level are from the sodium lactate flask while the hatched bars relate to the more acid combination of sodium lactate and lactic acid.

SF-ll A T P

(rM 3 I

rnM - - - A- - LACTIC ACID

t No LACTATE

0 4 8 24 48 HOURS

FIG. 3. Same as Figure 2, data of Experiment SF-1 I .

mM

GLUCOSE UTILIZATION

ma9 / pH6.I

CONTROL A . 5

L A C T I C A C I D

N O L A C T A T E

-*- -- - - - 'pH 6.3

24 48 0 4 8 HOURS

FIG. 4. Same as Figure 1. data of Experiment SF-12.

258 BISHOP

drop (unless the PH is initially completely unphysiological or i f there are glycolytic inhibitors present). The present four ex- periments are typical. It can be clearly seen from Figure 1, however, that lactate per se has no detrimental effect on glucose utilization and that as long as glycolysis proceeds, the ATP level is maintained. From FIgure 4, however, it can be seen that a mixture of neutral lactate and lactic acid cause a marked lowering in pH which is decidedly detrimental to glucose utiliza- tion and likewise to ATP maintenance. Since the total lactate (neutral lactate plus lactic acid) was approximately the same in Experiment SF-9 (F:g. 1) and SF-12 (Fig. 4), i t must be concluded that acidity is the fac- tor leading to glycolytic slow-down and the decline in blood ATP level.

Experiments SF-I0 (Fig. 2) and SF-11 (Fig. 3) indicate the same results. To vary the experimental design, each Fair of flasks, in these experiments, contained the same total lactate concentration. In SF-10, the final pH in the lactic acid flask was very low and Lhe effec:s on glycolysis and ATP level were dramatic. In SF-1 1, on the other hand, the pH values were not greatly dif- ferent at 24 houis and not at all different at 48 hours. The maintenance of ATP mirrored this similarity, although glucose utilization was somewhat different in the two flasks.

I t has thus been clearly demonstrated that glycolysis in whole blood will proceed in the face of accumulating lactate provided that the pH does not fall. Increasing acid- ity, on the other hand, will act as a brake

on glycolysis, and since glycolysis supplies the sole energy for ATP synthesis in the red cell, the ATP level of the blood will fall. T h e mechanism for the inhibition of gly- colysis by rising acidity is at present conjectural .

Acknowledgment The author is happy to acknowledge the expert

assistance of Miss Ann Dutton in all aspects of this study.

References I . Bishop, C., D. M. Rankine and J. H. Talbott:

The nucleotides in normal human blood. J. Biol. Chem. 234: 1233. 1959.

2. Dische, 2. and C. Rand: Zur Frage der Differenz zwischen Zucker Schwund und Milschdure- biltlung bei der Blutglykolyae. Biochem. 1.. 276: 132, 1935.

3. Rapoport, S. and M. Wing: Dimensional, os- motic and chemical changes of erythrocytes in stored blood. (I) Blood preserved in so- dium citrate, neutral and acid citrate-dex- trosc (ACD) mixtures. J . Clin. Invest. 26: 591. 1947.

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5. Kashket, S., D. Rubinstein. 0. F. Denstedt and S. M. Cosselin: Preservation of blood. V. T h e influence of the hydrogen-ion concentration on certain changes in blood during storage. Canad. J . Biochem. 35:827. 1957.

6. Rubinstein, D.. S. Kashket and 0. F. Denstedt: Studies on the preservation of blood. \'I. T h e influence of adenosine and inosine on the metabolism of the erythrocyte. Canad. J. Biochem. 3 6 1269, 1958.

7. Whittam, R.: Active potassium transport in human red cells by nucleosides and deoxy- nucleosides. J . Physiol. 154: 6 0 R , 1960.