Some in Vitro Effects of Adenine Added to Stored Blood

CHARLES BISHOP, PH.D.

From the Department of Medicine, School of Medicine, State University of New York at Buffalo and the Buffalo General Hospital,

Buffalo, New York

When adenine was added to freshly collected ACD blood and the blood incubated at 37C. for one or two days, the concentration of ATP and adenine nucleotides (AMP + ADP + ATP) was higher than in the same blaod without adenine. The addition of inosine with adenine was even more effective. Adenine was used by the red cells and the effective amounts of adenine to be added varied with the experimental conditions, for ex- ample, length of incubation. When adenine or adenine and inmine were incubated with outdated ACD blood, the ATP and adenine nuclcotide con- centrations r e , indicating that synthetic abilities were still present in red cells which had deteriorated energetically as a reault of storage in atrate in the cold. When outdated red cells were returned to a more normal PH, the ATP levels increased. When adenine addition was included along with neu- tralization, the effects were remarkable, the ATP and adenine nucleotide levela going even above normal fresh blood levels. These experiments sug- gest that added adenine enhances ATP synthesis and allows formulation of a acheme for the par- ticipation 0: adenine in the metabolic reactions of stored cells.

THE art of blood storage was advanced considerably when it was recognized that purine nucleosides such as adenosine or inosine were beneficial in maintaining the energetics of the red ce11.6. The impor- tance of the adenine moiety was shown by Nakao ct al.9 who used the combination of adenine and inosine (adenosine is not the equivalent because of the presence in blood of a very active adenosine deaminase). Simon et al.11 then showed that when adenine alone was added to ACD blood, posttransfusion survival was markedly en-

This work was supported in part by Grants A-210, AM-06367-01 and A-5581 of the National Institutes of Health, Bethesda, Maryland.

Received for publication November 14, 1963; ac- cepted January 27. 1964.

hanced. Since the amounts of adenine required were quite small by comparison to the substrate amounts of inosine re- quired, the dangers of provoking hyperuri- cemia from this adjuvant appeared to be remote. Although adenine or adenine plus nucleoside does maintain the ATP levels and glycolytic rate at more normal levelslog 11 the exact mode of beneficial ac- tion of adenine or adenine moiety is not yet clear. T o gain more insight on this point, several experiments were performed in this laboratory, adding adenine alone or in com- bination to fresh or stored blood and fol- lowing in vitm several biochemical param- eters. From the results of these experiments one can infer something of the mode of action of added adenine.

Methods The general approach in all experiments

was essentially the same. Blood was col- lected fresh in ACD solution and used immediately, or outdated ACD blood was obtained from the blood bank. In either case, a pint of blood was usually divided into four aliquots and each aliquot was then manipulated according to the variable to be studied. For example, varying amounts of adenine were added to each aliquot, or the PH’s of the various ali- quots were adjusted in a predetermined fashion. The aliquots were then incubated in spinner flasks at 37 C. as described previ- ously,2 observing aseptic technic through- out. Samples were aseptically withdrawn at intervals. Quantitation of the nucleotides using a previously described chromato-

266 BISHOP

SF- 30 a +AMP + ADP + ATP 0

0 600 SPINNER FLASKS

I. CONTROL

2.AOLNINE 10.8 m N .

400 FINAL CONCN. I B 1. ADENINE 10.1 m N l

IL

w A

AN0 INOSlNE

I*.@ mNl 0 2 0 0

\

z 9 0 24 48

HOURS OF INCUBATION FIG. 1 . ATP and total adenine nucleotide (AMP

+ADP+ATP) concentrationa in the various spinner Basks of Experiment SF-90 after 0, 24 and 48 hours of incubation at Sic. Fresh ACD blood was used.

graphic method4 was the most important determination performed on these samples. Because each experiment evolves from the preceding ones, the particular objective for each is described, and then the results are given. The discussion combines all the experiments.

Results Experiment SF-30. Five hundred ml. of

blood were collected in the usual type of ACD bottle and divided into three aliquots of about 125 ml. each, which were placed in sterile spinner flasks. The first flask had no additions, serving as a control. To the second were added 10 mg. adenine (final concentration 0.6 mM). The third flask contained both adenine (0.6 mM) and ino- sine (9.8 mM) . Because of solubility prob- lems and reluctance to dilute the blood unnecessarily, the adenine and inosine to be added to each flask were weighed as the dry powders and autoclaved as such. They were then stirred into the blood in the spinner flasks. Analytical samples were taken from the spinner flasks after 0, 24, and 48 hours of incubation at 37 C. under anerobic conditions. The complete pattern of blood nucleotides was determined at each time for each sample but to avoid confusion, only the values for A T P and total nucleotide adenine (AMP+ADP+ ATP) are presented. Other values such as

hematocrit, pH, glucose utilization and plasma hemoglobin are not given since they seem to add little useful information.

Figure 1 shows the A1'P and total adenine nucleotides (AMP + ADP + ATP) of the three samples before and after 24 and 48 hours of incubation. In the control flask (1 ) , ATP and adenine nucleotides have decreased markedly over the 48 hours incu- bation period. In contrast, both samples with added adenine (2, 3) maintained their ATP and adenine nucleotide levels much better a t 24 hours but had lost much of this by 48 hours. The combination of ino- sine with adenine appeared to be better than adenine alone. These findings sup- port the notion that adenine moiety is lost in stored blood and give support to the recommendation by Simon et al.11 that adenine be added to ACD solutions. In addition, this experiment shows that ino- sine does furnish an alternate source of energy to the glycolyzing red cells, which again should help to maintain ATP levels higher. In support of this, glucose utiliza- tion for flasks 1 and 2 were 14.4 and 15.5 millimoles of glucose/liter of whole blood in 48 hours while in flask 3 with adenine and inosine, the utilization was only 9.2. The end result was thus better and with some sparing of glucose.

Exfieriment SF-31. This experiment was designed to test various levels of adenine. Simon et ~ 1 . 1 1 varied the amount of added adenine and concluded that 0.75 p moles adenine/ml. of whole blood was optimal for red cell survival and maintenance of ATP concentration and glycolytic rate. In their studies, more adenine appeared to be detrimental. We sought to verify this under slightly different conditions, and selected final adenine concentrations of 0, 0.6, 1.2, and 2.4 mM (i.e.

T h e ATP and adenine nucleotide levels are shown in Figure 2. Without added adenine, the ACD blood behaved much as before. Adenine at the lowest (previous)

moles/ml.) .

EFFECTS OF ADENINE ADDED TO STORED BLOOD 267

level was beneficial as before. However, higher levels of adenine were, under these test conditions, distinctly better than the low level. Since this test system is rather different from that of Simon et d , 1 1 it is perhaps not surprising that the results are not identical with theirs. By chromate graphic analysis4 it was shown that no adenine was left in flask 2 at the end of 24 hours. No interpretation can be made of the results in this flask during the sub- sequent period since at 48 hours it was discovered that the glucose supply had be- come exhausted. In flasks 3 and 4, adenine was present throughout the incubation period but did decrease. This clearly indi- cated that adenine was used by this system and one might infer that in any system, adenine should be added according to the demands of the system involved.

Experiment SF-35. This and the succeed- ing experiments were performed on blood bank ACD blood that had become outdated by a few days. One pint of blood was equally divided among four spinner flasks, giving about the same volume per flask (approximately 125 ml.) as before. Flask 1

ADENINE CON C ENTR AT I ON S

a 0 I. o m m 0 2. 0.6 m m z! 6 0 0 3. 1.2 m Y

4. 2.4 m m W J

400 3 L O 2 0 0 -I \

I 3

0 2 4 48 HOURS OF INCUBATION

QLUCOSE SUPPLY EXHAUSTED

FIG. 2. Results of Experiment SF-31 in which the amount8 of adenine added to freshly collected ACD blood were varied. Clear and hatched ban have same meaning as in Fig. 1 .

a 0 S F - 3 5 2 m 6001

SPINNER FLASKS

w 1

400 3

O 2 0 0 LL

-I \

I ¶

1. CONTROL 2. LIVER EXTRACT

1. ADENINE (1.0 m M )

4. ADENINE (1.0 m Y )

7

AFTER 2 4 HOURS

OF INCUBATION FIL. 3. Results of Experiment SF-35

utilizing outdated ACD blood.

was used with no additions. T o flask 2 was added 2 ml. of crude liver extract (Lilly) with the thought that it might contain needed nutrients not yet identified. T o flask 3 was added 17 mg. of adenine (final concentration about 1.0 mM). T o flask 4 were added the same amount of adenine and 335 mg. inosine (final concentration about 10 mM).

After incubating for 24 hours, samples were analyzed for nucleotides and the re- sults are shown in Figure 3. Again it is apparent that adenine was beneficial with respect to levels of ATP and nucleotide adenine and that the combination of ade- nine and inosine was even better than adenine alone. The liver extract contained no beneficial combination of ingredients. This experiment differs from the previous ones in that the blood used had been stored for more than three weeks and discarded as unusable. Incubation with adenine or adenine and inosine restored the ATP and adenine nucleotide levels to values reminis- cent of those in freshly collected ACD blood. This behavior is already well knowng. 10 and this particular experiment serves only as a basis of comparison for the last two experiments.

268 BISHOP

SF-36 n -

SPINNER FLASKS 0

m 1. CONTROL 2. ADENINE

w -I ’ 6 o o ] 3 . p H 7.1 1 4 0 0 1 4.ADENINE,

p H 7.1 3

0 2 4 HOURS O F INCUBATION

FIG. 4. Results of Experiment SF-36 in which outdated ACD blood was either brought to a more normal PH or had adenine added, or both.

Experiments SF-36, SF-37. These experi- ments explored further the rejuvenation of outdated ACD blood by including another manipulation, namely pH adjustment. In Experiment SF-36, a pint of blood was equally divided among four spinner flasks and the first flask was used as a control. T o the second flask 10 mg. of adenine were added. To the third and fourth flasks 7 ml. of sterile 3.75% NaHC03 solution were added, raising the fJH from 6.7 to 7.1 Nothing further was added to flask 3, but 10 mg. of adenine were added to flask 4. The results are shown in Figure 4.

Experiment SF-37 is similar to Experi- ment SF-36 except that 20 ml. of 3.75% NaHCO, were used for flasks 3 and 4 to raise the fJH of the outdated blood from 6.8 to 7.4. The results are shown in Fig- ure 5.

From the results of both experiments it is apparent that either the addition of adenine or returning the PH to a more physiological range enhances the ATP and nucleotide adenine levels. Together, in SF-36, they appear to act synergistically. Such an effect is not seen in SF-37, perhaps

because the amounts of alkaline sodium bicarbonate solution required were so great as to cause other damage to the red cells. The importance of fJH has been reported not only from this laboratoryz. 3 but also by de Verdier et al.5

Discussion

From the foregoing experiments as well as from the previous literature, it is appar- ent that adenine or adenine moiety is a most important substance for the human red cell. In fact it may be that the avail- ability of adenine is the factor which deter- mines the level of ATP and adenine nu- cleotides in the red cell. It has already been shown that the mature mammalian red cell cannot synthesize purine de novo,1,8 but probably obtains i t ultimately from the liver.’ Blood removed from the body and placed in storage is deprived of any source of new adenine.

Isotopically labeled adenine is incorpo- rated into red cell ATP in vitrol but such a demonstration does not completely eluci- date the role of added adenine in stored blood since what one measures here is what might be termed “steady-state’’ ATP, i.e., the resultant of ATP synthesis and break- down. If added adenine only decreased the rate of disappearance of red cell ATP it would not be clear whether the adenine enhanced the rate of synthesis of ATP or retarded the rate of breakdown of ATP or nucleotide adenine (for example, by inhib- iting the deamination of AMP). The re- sults of the present experiments, however, suggest that ATP synthesis may have been enhanced since more ATP may be present after incubation with adenine than was present initially. These results do not pre- clude the possibility that ATP breakdown may be slowed by added adenine. The results are of practical importance in blood banking-indicating that the ATP level of stored blood can be elevated by adding adenine.

There must be many factors in the suc-

EFFECTS OF ADENINE ADDED T O STORED BLOOD 269 cessful storage of human red cells, all of which are required in concert if successful storage-really long term storage-is to be achieved. Only one facet of the storage problem is examined here-that of ATP maintenance. It has been known for many years that the stored red cell loses its ATP and that posttransfusion survival is more or less correlated with ATP level. Both are probably indicators of fundamental cellular integrity or competence. Insofar as the ATP level is concerned, the following scheme appears to obtain:

ATP

ADP

IMP -AMP

A t .It

c PRPP (phosphoribosyl- T pyrophosphate)

A hypoxanthine

adenine

ATP is the adenine nucleotide present in by far the greatest amount in the red cell, being required for all manner of cellular reactions including ion transport. ADP is readily converted to ATP by active glycolysis in the red cell. As glycolysis be- gins to fail in the stored red cell, ATP continues to be needed and hence ATP concentration falls and ADP concentration rises. ADP can be converted to ATP and AMP by myokinase. By this and other means, more and more ATP and ADP be- come AMP and this compound is very sus- ceptible to deamination. This latter step, moreover, is essentially irreversible in the human red cell1 and hence the amount of adenine moiety (and hence the maximal amount of ATP) in the red cell decreases. This, of course, is only a problem for the stored red cell since if i t were circulating in the body, it could replenish its adenine supply and synthesize more adenine nucle- otides. Hence the red cell must remain energetically active because it must keep most of its adenine nucleotides as ATP. As soon as the glycolytic rate falls a little behind the demand for ATP, the foregoing

I. CONTROL 2. ADENINE

, 3. pH 7.4

0

O i

4. ADENINE, D H 7.4

0 2 4 HOURS OF INCUBATION

FIG. 5. Results of Experiment SF-37 which was of the same design as SF-36 but with more sodium bicarbonate added, to bring the PH to 7.4.

chain of events supervenes. In stored blood this is disastrous if adenine moiety cannot be recouped. Therefore, in retrospect, the addition of adenine to stored blood seems obvious. However, adenine is not just a beneficial additive to be used empirically. It is a metabolite required in this system under these conditions and it may be re- quired in greater amounts or perhaps not even at all if the storage system is changed.

Acknowledgment The author is very happy to acknowledge the

able assistance of Miss Ann Dutton in these experi- ments.

References 1. Bishop, C.: Purine metabolism in human and

chicken blood. in vilro. J. Biol. Chem. 235: 3228, 1960.

2. Bishop, C.: Differences in the effect of lactic acid and neutral lactate on glycolysis and nucleotide pattern in incubated whole human blood. Transfusion 2: 256, 1962.

3. Bishop, C.: Maintenance of ATP level of incu- bated human red cells by controlling the PH. Transfusion 2: 408. 1962.

4. Bishop, C., D. M. Rankine, and J. H. Talbott: The nucleotides in normal human blood. J. Biol. Chem. 234: 1233, 1959.

5. DeVerdier. C., C. Hogman, L. Garby, and J. Killander: Storage of human red blood cells. 11. The effect of PH and the addition of adenine. Acta Physiol. Scand. In press.

BISHOP

6. Ciabrio, B. W.. C. A. Finch, and F. M. Huen- nekens: Erythrocyte preservation: a tmpic in molecular biochemistry. Blood 11: 103, 1956.

7. Henderson, F. J. and G. A. LePage: Transport of adenine-8-CI4 among mouse tissues by blood. J. Biol. Chem. 254: 3219, 1959.

8. Lowy, B. A.. J. L. Cmk, and I. M. London: The biosynthesis of purine nucleotides dc nouo in the rabbit crythrocyte in vitro. J. Biol. Chem. 256: 1442, 1961.

9. Nakao, M., T. Nakao, M. Tatibana, and H. Yoshikawa: Phosphorus metabolism in hu- man erythrocyte. 111. Regeneration of adeno-

sine triphosphate in long-stored erythrocyte by incubation with inosine and adenine. J. Biochem. 47:661, 1960.

10. Shafer, A. W. and G. R. Bartlett: Phos- phorylated intermediates of the human erythrocyte during storage in acid citrate- dextrose (ACD). 111. Effect of incubation at 37" with inosine plus adenine and adenosine after storage for 6, 10, 14, and 18 weeks. J. Clin. Invest. 41:690, 1962.

11. Simon, E. R., R. G. Chapman, and C. A. Finch: Adenine in red cell preservation. 1. Clin. Invest. 41: 351, 1962.