Maintenance of ATP Level of Incubated Human Red Cells by Controlling the pH

CHARLES BISHOP, PH.D. #row llrr Depnrtment of Medicine, University of Buflnlo Srhool of i\fecliritic mrtl

the Rufalo General Hospital, Buffalo, A’eui l’orh

F m h human blood waa incubated at 37C. in ACD solution, heparin-glucoae solution, and h e p aringlucose solution with #€I controlled by a u t e matic titration with rterilc NaHCOs solution. In ACD solution glycolysh of the red cella slowed down with time and concomitantly the ATP level de- c r e d iharply. In heparin%lucose solution, glycol- yds continued more vigorously and the ATP levels were maintained longer. In the laaka in which the lactic acid was neutralized continuously, glycolyds praceeded almost unabated, and the ATP levels were correspondingly well maintained. Red cell viability and iurvival are presently assumed to be related to glycolytic maintenance of adequate 1weL of ATP. Thae d e s demonstrate that itorage of blood in heparinglucose may prolong the red cell viability over that in ACD, and that neutralization of the lactic acid p r o d d in glycolyaia gives iuch dramatic improvemmt that a whole new system of blood itorage may k predicated on this principle

THE human red cell has a life span of about 120 days when circulating in the body at 37 C. If in uivo conditions could be du- plicated in uitro, one would expect that in an average sample of human blood stored at 37C.. 50 per cent of the cells would re- main viable for at least 60 days or 75 per cent for at least 30 days. Cooling the blood to 4 C. should multiply this by some factor which at present can only be guessed. At any rate, blood stored at 4C. under simu- lated in uivo conditions should have a red cell survival of 75 per cent for well over 30

Received for publication February 14, 1962; ac- cepted May 5, 1962.

Preliminary results of these experiments were presented at the annual meeting of the American A d a t i o n of Blood Banks, Chicago, Illinois, Octo- ber 26-28, 1961.

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

days. In attempting to reconcile the dis- parity between this and the presently ac- cepted figure of 21 days, this laboratory has been systematically examining various as- pects of blood storage to ascertain the principal factors involved in the present need for a limitation on the storage period of 21 days. The present studies demonstrate that the collection system and the continued maintenance of neutral fiH are two very important factors in prolonging red cell metabolic activity.

Experhen ts

Three separate experiments were per- formed. In each, two consecutive aliquots of blood from a single donor were collected, one (125 ml.) in the usual ACD solution and the other (250 ml.) in heparin and glucose.* The ACD blood was transferred aseptically to a water-jacketed spinner flask** and two aliquots (125 ml. each) of the heparin-glucose blood were transferred to identical spinner flasks. One of the latter samples was not controlled for pH but into the other flask was inserted aseptically a combined electrode (Radiometer #GK- 2021C) which was connected to a Ratliom- eter TTT 1 Titrator. The Titrator con- trolled a Radiometer #MNVIc magnetic valve which controlled the flow of sterile 5 per cent NaCHO, solution into the spin- ner flask. In the subsequent discussion the

‘ACD solution. NIH formula A. Heparin and glucoae, heparin 2 ml. (2.OOO units) and 3 ml. 10 per cent sterile dextrose per 100 ml. of blood.

++ Bellco Class lnc., Vineland, N. J.

408

MAINTENANCE OF RED CELL ATP 409

three experimental conditions will be desig- nated:

A - ACD blood H - Heparinglucose blood P - Heparinglucose blood with

pH controlled

In Experiment SF-13 the PH in Flask P was allowed to fall to 7.2 and was then controlled at this PH. Incubation of all samples was continued for 72 hours. Be- cause glycolysis was faster in Flasks H and P, several ml. of sterile 10 per cent dextrose solution was added every 24 hours to these flasks. This insured that the blood glucose level did not fall below 100 mg./100 ml. Glycolysis was slower in Flask A as demon- strated by excess glucose at the conclusion of the 72 hour incubation. No extra dex- trose was added to this flask. This finding is consistent with the common observation that outdated ACD blood contains ade- quate glucose concentration for continuing glycolysis, even though glycolysis has vir- tually ceased.

In Experiment SF-14, the same experi- mental design was followed except that the PH of Flask P was controlled at 7.4.

In Experiment SF-15, the same three flasks were set up except that to Flask P was initially added sterile isotonic lactic acid solution to bring the pH to 6.8. The titrator was set to maintain this pH for the rest of the incubation period.

Aliquots of blood (25 ml.) from each flask were removed periodically by means of sterile seven inch 17 gauge needles. The pH of the aliquots of blood was measured using the Radiometer Astrup apparatus.t Hematocrits were read on the centrifuged blood. Blood glucose was determined by the Glucostat (glucose oxidase) method,H lactate by a modification of the lactic de- hydrogenase method of Horn and Bruns,6 and nucleotides and purines by absorption

?The London Company, Cleveland 11, Ohio. j-t Worthington Company, Freehold, N. J.

mr-n

(Cmelmnlllkr whmh bind) 0 - A fIa#k

400 I m-P fI.mk

ATP CONCENTRATION

0- H flank

20

GLUCOSE UTILIZATION - LACTATE /2 PRODUCTION - 0 A

fdl l lwlarlfyl

0 24 40 72 HOURS OF INCUBATION (37%)

FIG. 1. Experiment SF-13. Concentration of ATP in human blood samples incubated in ACD (A Flask), Heparin-Glucose (H Flask) or Heparin- Glucose with PH controlled at 7.4 (P Flask) by continuous titration with sterile 5 per cent NaHCOS. In the bottom graph is shown in solid symbols the glucose utilization in each flask. The data for lactate production, depicted in open symbols, are also shown. The lactate production valuea have been divided by 2 in order to demonstrate agree- ment with the theoretical production of two moles of lactate for one mole of glucose utilized via the Embden-Meyerhof pathway. The PH values at each interval are shown for each flask, the initial ones being grouped together above their three curves but in the proper respective order.

and elution using Dowex-1 formate ancl Dowex-50-H+ columns according to our own method.4

Results

The complete nucleotide pattern (ATP, ADP, AMP, IMP, inosine and hypox- anthine and also DPN, TPN, uric acid ancl GTP) was determined for each blood but the individual data are not shown because of the complexity of such a presentation. In every instance, the breakdown of ATP is accompanied by the same sort of in-

410 BISHOP

W - W 1 ATP CONCENTRATION 600

0- A fm 0- n fI.IL

40 0- c f l n b

20

GLUCOSE UTILIZATION - 0 A

LACTATE/2 PRODUCTION- o O A

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HOURS OF INCUBATION (37'C) FIG. 2. Experiment SF-14. Same plots as in Figure 1. The PH in Flask P wan controlled 7 2 .

creases and decreases of breakdown prod- ucts as has already been described from this laboratory.' Therefore, in the interest of simplicity only ATP concentrations are plotted in Figures 1 to 3. These concentra- tions are expressed as micromoles of ATP per liter of whole blood. Underneath, in each figure, are curves for glucose utiliza- tion and lactate production. Glucose utiliza- tion is often expressed by plotting against time the amount of glucose remaining. In these experiments such a plot would have been difficult to follow inasmuch as extra glucose was added at intervals to Flasks H and P. The acutal glucose utilized was therefore plotted against time, the decre- ments in glucose concentration being suc- cessively added together to give a contin- ually rising curve. By plotting the glucose utilization this way and by superimposing the rising lactate concentration divided by two, the reader can compare on a molar basis the actual lactate production with that

predicted on the assumption of two moles of lactate produced for each mole of glucose utilized. The units are concentration, i.e., millimolarity of the whole blood. The PH of each blood sample is noted on the glu- cose utilization curves.

The red cell derives its energy by gly- colysis and hence it is not surprising that in Figures 1, 2 and 3 the ability to maintain the ATP level goes hand in hand with the ability to utilize glucose. In Figure 1, for example, the ACD blood (A) lost its ATP level fairly rapidly and the rate of glycol- ysis (measured both by glucose utilization and lactate production) was comparatively slow. Heparinized blood with glucose (H) maintained its ATP level much better and concomitantly the rate of glycolysis was greater. The flask with heparin and glu- cose, maintained at a PH of 7.2 (P), showed the highest ATP levels and also the great- est rate of glycolysis. The converse of this situation has been well demonstrated,z namely, that when glycolysis is stopped by inhibitors such as iodoacetate or fluoride, the blood ATP level falls promptly.

We can now compare ACD blood with heparin-glucose blood. In every experi- ment there is no question that heparin- glucose blood carried on glycolysis much better and maintained its ATP levels much longer. One unresolved question is whether the poorer showing in the ACD solution was due to the presence of the citrate or only because of the differences in PH of the solutions in which the blood was col- lected. Studies to resolve this problem are now in progress.

The marked superiority of the heparin- glucose system with PH control over either the ACD or the simple heparinglucose sys- tem is readily apparent in Experiments SF-13 and SF-15. In SF-14 a technical fault led to an interesting result. Through an oversight, glucose was not added to Flasks H and P at 24 hours. Because glycolysis was proceeding at such a rapid rate in

MAINTENANCE 01; RED CE1.L ATP 41 1

30

Flask P, the glucose supply to the red cells was exhausted and the cells perforce ceased glycolyzing. The drop in ATP in this sample a t 24 hours is therefore readily ex- plained. The interesting aspect, however, is that when glucose was added back sev- eral hours later the ATP level rose and maintained itself so that by 72 hours it was a t a higher level than in H.

A small error crept into SF-15 also, in that on the night before the 48 hours sam- ple was taken, a fuse blew in the titrator, allowing the PH of the blood in Flask P to drop to 6.6. Replacement of the fuse was followed by restoration of the proper PH. No serious difficulty seems to have resulted.

Although the rates of glycolysis in Flasks M and P in Experiment SF-15 appear almost equal. in marked contrast to comparable flasks in SF-I3 and SF-14, this appearance is deceptive. In Flask P of SF-15, the initial glycolytic rate was reduced because the blood was acidified with lactic acid to pH 6.8. Glycolysis in Flask H thus proceeded laster initially, but later slowed down as its pH fell progressively below that in Flask P. Meanwhile, glycolysis in Flask P proceeded steadily and hence the two curves crossed between 48 and 72 hours. The ATP in Flask P was therefore much higher than in Flask H at 72 hours. The glucose utiliza- tion curves would probably have diverged even more if the study had been extended another 24 hours. It is interesting to com- pare the glucose utilization in Flask P of SF-13 with the same flask in SF-15. In the first experiment the PH was held at 7.2 and glycolysis continued at a good rate for the 72 hours. When the blood was acidified to PH 6.8 in the last experiment, glycolysis went only about half as fast, right from the start of incubation.

GLUCOSE UTILIZATION - . A LACTATE/L PRODUCTION - 0 0 A

(millimolmrityl

Discussion

A previous communication from this laboratory3 demonstrated quite unequivo-

HOURS OF INCUBATION (3PC) FIG. 3. Experiment SF-15. The H in Flask P

was controlled at 6.8 except at 48 ku;. See text for explanation of this disparity.

cally that increasing acidity, and not lac- tate accumulation, per se, was indeed the factor slowing glycolysis in the incubated red cell. That demonstration led logically to the present experiments in which glycol- ysis was maintained by continuously neu- tralizing the lactic acid formed by glycolysis and thus maintaining the PH above the point at which the glycolytic rate would be jeopardized. As expected, the ATP levels of the incubated blood were maintained much longer than under the usual circum- stances. Furthermore, i t was shown that ACD solution with its initial blood storage PH of about 6.9 was markedly inferior to neutral heparin and glucose as a collection medium in terms of its ability to sustain both glycolysis and ATP levels.

Many earlier systems of blood storage were predicated on the assumption that glycolysis must be suppressed because of its harmful consequences due to the lactic acid accumulation. Realization that red cell metabolic activity is dependent on gly- colysis posed a dilemma. Encouragement of glycolysis would lead to loss of metabolic

412 BISHOP

activity. Suppression of glycolysis would lead to the same result. The present studies resolve this dilemma by demonstrating that red cell metabolic activity is maintained by allowing glycolysis to proceed, but prevent- ing the drop in PH by neutralization of the lactic acid formed. This concept gives rise to a whole new approach to the blood storage problem inasmuch as the neutraliza- tion of lactic acid in a blood storage con- tainer is a technological problem admitting of several relatively simple solutions.

It may be assumed that red cell viability, as measured in studies such as this, and the ability of cells to survive after transfusion, go hand in hand. Exact correlation under rigorous experimental conditions has not as yet been reported although there have been suggestions to this effect.5 It is our intention to correlate in uitro changes in blood stored in these heparinglucose sys- tems with in vivo red cell survival. At the moment, we are hampered by anomalous CrS1 labeling in heparinglucose blood, even in freshly drawn blood. This appears to be due to a competition between Clal and Ca++, the latter being free in the heparin system instead of complexed as in the ACD

system.’ Resolution of this problem will speed our attack on correlation studies.

Acknowledgments The author expresses his appreciation to Miss

Ann Dutton for her skilled technical assistance and to Misa Jeanette LaDuca for preparation of the figures.

References 1. Bishop, C.: Changes in the nucleotidea of stored

or incubated human blood. Transfusion. 1: 349, 1961.

2. Bishop. C.: Factors in the in vitro maintenance of the nucleotide pattern of whole human blood. Transfusion. 1:355, 1961.

3. Bishop, C.: Differences in the effect of lactic acid and neutral lactate on glycosis and nu- cleotide pattern in incubated whole human blood. Transfusion, in presa.

4. Biahop, C., D. M. Rankine and J. H. Talbott: The nucleotides of normal human blood. J. Biol. Chem. W: 1233, 1959.

5. Gabrio, B. W., C. W. Finch and F. M. Huen- nekens: Erythrocyte preservation: A topic in molecular biochemistry. Blood. 11: 103, 1956.

6. Horn. H. and F. H. Bruns: Quantitativ Bestim- mung von L (+)-Milchtaiure mit Milch- aiure-dehydrogenase. Biochim. Biophys. Acta. 21: 379, 1956.

7. Zachariaa, B.: The binding of chromate to red corpusclea, hemoglobin and globin. Acta Chem. Scand. 13:456, 1959.

Note Added in Proof

Additional proof for the relationship between ATP and red cell survival is given by Nakao, K., T . Wada, T. Kamiyama, M. Nakao, and K. Nagano: A direct relationship between adenosine triphosphate-level and in uiuo viability of eryth- rocytes. Nature. 194: 877, 1962.