/ . Biochem., 78, 469-474 (1975)

Dextrose Medium Effects of Temperature and Citrate Anions1 Sachiko TSUDA,2 Akio TOMODA, and Shigeki MINAKAMI Department of Biochemistry, Kyushu University School of Medicine, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, March 31, 1975

The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5, 5'-dimethyloxazolidine-2,4-dione (DMO) method. The initial pHi at 4° was about 7.6 and was higher than the extracellular pH (pHe) at 4°. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4°) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2, 3-diphosphogIycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4° were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood.

The importance of pH for red cell preservation has been known since the introduction of the acid citrate dextrose (ACD) medium during World War II (2). Better preservation of ATP in blood stored in ACD compared with that in neutral citrate glucose medium was shown (3). Although pH measurements of ACD blood during storage were carried out by

many investigators (e.g. Ref. 4), most of the pH measurements were done at 37° and not at the storage temperature. Beutler and Duron (5) have shown that the initial pH of ACD blood at the storage temperature is approximately 0.5 unit higher than that at 37°. They have shown that the optimal storage pH at 4° is approximately 7.5, if pH measurement is done at the storage temperature. Their results suggest that pH measurements of bloodanticoagulant mixtures should be done at the storage temperature and not at 37°. Little attention has so far been paid to the pHi of stored blood, though it seems indisputable that the contents of ATP and 2,3-diphosphoglycerate (DPG) as well as the stability of red cell enzymes during storage are mainly

1 A part of this investigation has already been reported in a preliminary form ( / ) . 2 Present address: Higashi Tsukushi Junior College, Kokurakita-ku, Kitakyushu 803. Abbreviations: ACD, acid citrate dextrose; DMO, 5,5'-dimethyloxazolidine-2,4-dione; DPG, 2,3-diphospho-D-glycerate ; pHi, intracellular pH ; pHe, extracellular pH.

Vol. 78, No. 3, 1975

469

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

Intracellular pH (pHi) of Red Cells Stored in Acid Citrate

S. TSUDA, A. TOMODA, and S. MINAKAMI

470

METHODS ACD blood was taken by venipuncture into an evacuated glass bottle which contained ACD solution (NIH formula A). After addition of 4 //Ci of [HC]5,5'-dimethyloxazolidine-2,4-dione (DMO) per 100 ml of blood, the bottle was kept in a refrigerator. Samples (5 ml) were taken out at intervals with a sterile syringe. The procedure for the determination of the pHi was essentially according to Calvey (6). The pHe, the radioactivity in both whole blood and plasma, hematocrit and the water content of the sample were measured. The pHe was measured with a Hitachi-Horiba Expanded Scale F-5 pH meter at 4° with a 6028-10T combination electrode. It was standardized with primary standards (NBS phosphate D and phthalate) at 4°. The hematocrit value was determined with a 1.5 mm diameter tube at 12,000 rpm for 5 min. The extracellular water content was calculated from the hematocrit value with correction for intercellularly trapped water in packed cells, which amounted 2%. The extracellular water in packed cells was determined for ACD blood by the use of [14C]inulin. The intracellular water was determined as the difference between the total water and the extracellular water. Total water was obtained by weighing 100 ft] samples before and after dessication at 110°±10° for 24 hr. The sample was centrifuged in a refrigerated centrifuge at 5,000 rpm for 10 min. Before and after centrifugation, 100 ftl3 aliquots were taken into 1 ml of 5 M NaH2PO4 solution and extracted with 5 ml of water-saturated 1 : 1 (v/v) ethyl acetate-toluene mixture. The upper layer (2 ml) was added to 5 ml of 3

scintillator (4.0 g 2,5-diphenyloxazole, 0.1 g 1,4bis-2-(5-phenyloxazolyl)benzene and 40 ml of methanol in 1 liter of toluene). The radioactivity was determined with a Hitachi-Horiba LS-10 liquid scintillation spectrometer with correction for quenching by the external standard channel ratio method. The intracellular pH was calculated using the equation of Irvine et at. (7) from the pHe, extra- and intracellular water content (Ve, Vi), radioactivities in the plasma and whole blood (Cp, Cw) and pK of DMO at 4° (6.52, as determined by titration), as follows;



'



Determination of pHi in heparinized blood at 4° and at 37° was carried out similarly using [14C]DMO. Blood was kept for at least 10 min at 4° and at 37° with [UC]DMO (0.04 pCi per ml) and then centrifuged. For studies at 37°, blood was centrifuged with a prewarmed rotor: the temperature change was not more than 2°. The measurement of pHe was done with standardization at 37° as 6.13 (8). ATP and 2,3-diphosphoglycerate (DPG) measurements in ACD and heparinized blood stored in a refrigerator were carried out as follows: 50 ml aliquots of blood were withdrawn into evacuated bottles containing 7.5 ml of ACD solution or iso-osmotic saline solution containing 10 mg of heparin sodium (1,700 USA units) and 165 mg of glucose. For heparinized blood, heparin solution (100 units in 0.1 ml) was added every day before collection of the sample. Samples were taken evey day with sterile syringes and used for the determination of pHe, pHi and the intracellular concentrations of ATP and DPG. The determinations of pHe and pHi were as described above. ATP and DPG were determined after deproteinization with perchloric acid: ATP with glyceraldehyde3-phosphate dehydrogenase [EC 1. 2.1.12] and 3-phosphoglycerate kinase [EC 2.7.2.3] ( 9 ) and DPG by the method of Rose and Liebowitz (.10).

The same calibrated Levy-type pipette was used for both whole blood and plasma. J.

Biochem.

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

influenced by the intracellular pH (pHi) rather than by the extracellular pH (pHe) and that the difference between pHe and pHi is affected by several factors such as temperature and the concentrations of impermeable ions. This paper deals with the pHi of ACD blood as measured at 4°, and the effects of temperature and citrate anions on the pHi.

471

pHi OF ACD BLOOD

pH of Whole Blood and Plasma at 4° and at 37"—Table I shows the pH of ACD blood and ACD plasma as measured at 4 and 37°. The pH of ACD blood at 4° was 0.5 unit higher than that at 37°, in good agreement with the result reported by Beutler and Duron (5), but the difference for ACD plasma was 0.3 unit. This can be readily explained in terms of the large negative temperature coefficient (4pH/Jf) of protein solutions as a buffer system and the high concentration of hemoglobin. This result also suggests that the effects of temperature on pHe and pHi may be different. Similar results were obtained for heparinized blood: the pH of heparinized blood changed from pH 7.37 to pH 8.00 when the blood was cooled from 37° to 4°. pK of 5,5'-Dimethyloxazolidine-2, 4-dione(DM0) at 4°—Since the pK of DM0 at 4° must be known for the calculation of pHi at 4° and is not available from the literature ( / / ) , DM0 solution was titrated at 4° and 37" (Fig. 1). The pK of DM0 at 4° was determined to be 6.52, which was used for the calculation at 4°. The pK at 37° was 6.13, in good agreement with the value given by Waddell and Butler

90

Fig. 1. Titration curves of 5,5'-dimethyloxazolidine2,4-dione (DM0) at 4° and 37°. DM0 solution (100 mitf) was titrated with 0.1 N NaOH. 4° ( • ) ; 37° (O).

Extracellular pH and Intracellular pH of ACD Blood— Figure 2 shows the changes of pHe and pHi of ACD blood during storage. The pHe was obtained by measuring the pH of ACD blood at 4° and the pHi was calculated TABLE I. Effect of temperature on the pH of ACD blood and ACD plasma (number of samples: 9). Sample

Measurement

Mean±S.E.

ACD blooda

pH at 4° pH at 37° JpH"

7.43+0.043 6.93+0.028 0.50±0.022

pH at 4° pH at 37° JpHt>

7.40+0.047

ACD plasma

a

7.10+0.038

0.30 ±0.015

Hematocrit values of ACD blood range from 4044%. " The difference of pH measured at 4° and 37°. Vol. 78, No. 3, 1975

68 28

Fig. 2. Changes of extracellular pH (pHe) and intracellular pH (pHi) of acid citrate dextrose (ACD) blood during storage at 4°. Samples were taken from the storage bottle at intervals, the pHe was measured with a pH-meter and the pHi was calculated by the [l4C]DM0 method as described in "METHODS." Plots were drawn by the least-squares method.

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

RESULTS

from the pHe and the distribution of [UC]DMO between ACD plasma and cells at 4°. The pHe was about 0.5 unit higher than reported by previous investigators, who measured the value at 37°. This is due to the temperature effect described above. Unexpectedly, the pHi was higher than the pHe, in apparent contradiction of the usual concept that the pHi of red cells is lower than the pHe. This can be explained as an effect of the temperature at which the pHi was obtained and the presence of citrate anions in ACD medium, as shown

S. TSUDA, A. TOMODA, and S. MINAKAMI

472

(Fig. 3) shows that the pHe-pHi line for ACD blood is shifted upward compared with the line for heparinized blood. This can be explained in terms of the Donnan-Gibbs equilibrium, since citrate anions are known to be unable to permeate the red cell membrane (13). ATP and 2,3-Diphosphoglycerate Levels in Stored Blood—Changes of ATP and DPG were studied for both ACD and heparinized blood

s i •3

Days

pHe

7.4

78

Fig. 3. Extracellular pH (pHe) vs. intracellular pH (pHi) of heparinized blood at 37 and 4°, and of acid citrate dextrose (ACD) blood at 4°. Heparinized and ACD blood were stored at 4° and used for the determination of pHe and pHi as described in Fig. 2. Samples of heparinized blood at 37° were also measured as above, except that the plasma was replaced by isotonic saline solution, containing 1 mM MgCl2, 5 mM KC1, 1 mM NaH2PO4, and 10 mM glucose. The extracellular pH was adjusted with 0.2 N NaOH. Heparinized blood at 4° (O) and at 37° ( • ) ; ACD blood at 4° (A).

Fig. 4. A: Changes of extracellular pH (pHe) and intracellular pH (pHi) in acid citrate dextrose (ACD) blood and in heparinized blood. Fifty milliliter aliquots of blood were withdrawn into evacuated bottles with 7.5 ml of ACD solution or isotonic saline containing heparin and 165 rag of glucose. After addition of 2 fid [14C]DM0 per bottle, they were kept at 4° for a week. Changes of pHe and pHi were measured as described in Fig. 2. pHe ( • ) and pHi (O) of heparinized blood; pHe ( A ) and pHi (A) of ACD blood. B: Changes of ATP and 2,3-diphosphoglycerate (DPG) in acid citrate dextrose (ACD) blood and in heparinized blood. The samples described in Fig. 4A were used for the determination of ATP and DPG. ATP in heparinized ( • ) and ACD ( A ) blood; DPG in heparinized (C) and ACD (A) blood. / . Biochem.

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

in the following experiments. Another point of interest is that the decrease of pHi was slower than that of pHe. Effect of Temperature and Extracellular Citrate on pHi—When the pHi of heparinized blood was measured at 37° by the DMO method, the result was essentially in agreement with those reported by previous investigators {12), as shown in Fig. 3. When both the pHe and the pHi were measured at 4°, a line was obtained approximately parallel to that of the blood at 37°, but with an upward shift of about 0.5 unit. This shows that the pHi of blood shifts to the alkaline side on cooling, and the extent of the shift is more than that of the pHe. The intracellular and extracellular pH's of heparinized blood at pH 7.4 were about the same at 4°. Another factor which produces a high intracellular pH of ACD blood is the presence of citrate in the medium. The relation of pHe and pHi in ACD and heparinized blood at 4°

pHi OF ACD BLOOD

during storage in a refrigerator for a week, with simultaneous measurement of the pHi and pHe at 4°. Figure 4A shows the changes of pHe and pHi, and Fig. 4B the changes of ATP and DPG. The most significant observation is the rapid breakdown of ATP in heparinized blood, reflected in the cessation of the pH fall shown in Fig. 4A, probably due to less of glycolytic activity. If this rapid decrease of ATP is due to the high pH of the heparinized blood, adjustment of the pH may delay ATP breakdown at low temperature. This was demonstrated by the addition of lactic acid to make the pHi around pH 7.4 at 4°: essentially no decrease of ATP or DPG was observed for a week (Fig. 5). DISCUSSION It has been usual practice to measure the pH of ACD blood at 37°. This is probably for the practical reason that instruments to measure blood pH at 37° are available in clinical laboratories. However, since the extracellular milieu of the red cells will be at physiological pH as soon as ACD blood is transfused into the body and since we are interested in the stability of red cells during storage, the pH as measured at the temperature of storage, i.e. Vol. 78, No. 3, 1975

at 4°, will be more useful than the pH at 37°. This is clearly shown by Beutler and Duron (5), who found little difference in the optimum pH for the preservation of red cell ATP at 37° and 4°, when the pH was measured at the actual temperature of storage. A remarkable effect of temperature on the pH of whole blood and plasma has been found since the introduction of blood pH measurements (14, 15). This can be readily explained in terms of the increase in the pK of protein solutions with increase in temperature. The observation that the difference of pH at 37 and 4° for whole blood is larger than that for plasma, indicates that the increase of proton concentration inside the cells is greater than that in the plasma. The difference of pH on both sides of the membrane follows the Donnan-Gibbs equilibrium (16, 17). If the increase in the dissociation of protein anions inside the cells is greater than that outside the cells, which happens when blood is warmed, the Donnan ratio: r=[Cl-]i/[Cl-] 0 = [H+]0/[H+]l (suffixes i and o indicate concentrations inside and outside the cells) decreases: this means that the difference of pH on both sides of the membrane (pHe-pHi) decreases when the temperature of the blood increases. When the blood is cooled, the difference increases, so that the pHi at a given pHe shifts to the alkaline side with decrease in temperature. Similar considerations can be applied to the effect of citrate on pHi. When citrate is added to the blood, the concentration of impermeable anions outside the cells increases so that the Donnan ratio: r=[Cl-]i/[Cl-] 0 = [H + ] 0 / [H+]i increases: this results in a pHi increase. The increase of pHi on addition of citrate has been demonstrated by Funder and Wieth (18). Another point to be noted is the change of the pHi due to changes of intracellular organic phosphate compounds as shown by Duhm (17). We confirmed his results by the DMO method in red cells incubated without glucose: the pHi at a constant level of pHe increased when DPG and ATP were exhausted (unpublished data), which also explains the high pHi of blood stored for a long period. Although these changes of the pHi can be anticipated by sim-

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

Days Fig. 5. Changes of A T P and 2,3-diphosphoglycerate (DPG), as well as those of extracellular pH (pHe) and intracellular pH (pHi), in acidified heparinized blood. Heparinized blood was acidified by addition of lactic acid to make the pHi of the blood around pH 7.4 at 4° and stored for a week. Other conditions were the same as in Fig. 4, A and B. pHe ( • ) and pHi ( O ) ; A T P (H) and DPG ( • ) in acidified heparinized blood.

473

474

S. TSUDA, A. TOMODA, and S. MINAKAMI

REFERENCES 1. Tsuda, S., Kakinuma, K., & Minakami, S. (1972) Experientia 28, 1481-1482 2. Loutit, J.F., Mollison, P.L., & Young, L.M. (1943) Quart. J. Exp. Physiol. 32, 183-202 3. Rapoport, S. (1947) / . Clin. Invest. 26, 591-615

4. Shafer, A.W., Tague, L.L., Welch, M.H., & Guenter, C.A. (1971) / . Lab. Clin. Med. 11, 430437 5. Beutler, E. & Duron, O. (1965) Transfusion 5, 17-24 6. Calvey, T.N. (1970) Quart. J. Exp. Physiol. 55, 238-252 7. Irvine, R.O.H., Saunders, S.J., Milne, M.D., & Crawford, M.A. (1960) Clin. Sci. 20, 1-18 8. Waddell, W J . & Butler, T.C. (1959) / . Clin. Invest. 38, 720-729 9. Minakami, S., Suzuki, C , Saito, T., & Yoshikawa, H. (1965) / . Biochem. 58, 543-550 10. Rose, Z.W. & Liebowitz, J. (1970) Anal. Biochem. 35, 117-180 11. Albers, C , Usinger, W., & Spaich, P. (1971) Resp. Physiol. 11, 211-222 12. Waddell, W J . & Bates, R.G. (1969) Physiol. Rev. 49, 285-329 13. Garby, L. (1961/62) Folia. Haemal. 78, 295-297 14. Yoshiraura, H. & Fujimoto, T. (1937) / . Biochem. 25, 493-518 15. Rosenthal, T.B. (1948) / . Biol. Chem. 173, 25-30 16. VanSlyke, D.D., Wu, H., & McLean, F.C. (1923) / . Biol. Chem. 56, 765-849 17. Duhm, J. (1971) Pflugers. Arch. Ges. 326, 341356 18. Funder, J. & Wieth, J.O. (1966) Ada Physiol. Scand. 68, 234-245 19. Minakami, S. & Yoshikawa, H. (1966) / . Biochem. 59, 145-150 20. Asakura, T., Sato, Y., Minakami, S., & Yoshikawa, H. (1966) / . Biochem. 59, 524-526 21. Duhm, J. (1973) in Erythrocytes Thrombocytes Leucocytes: Recent Advances in Membrane and Metabolic Research (Gerlach, E., Moser, K., Deutsch, E., & Wilmanns, W., eds.) pp. 149-157, Georg, Thieme, Stuttgart 22. Tsuboi, K.K. & Fukunaga, K. (1970) Biochim. Biophys. Ada 196, 215-220 23. Jacobs, M.H. & Parpart, A.K. (1933) Biol. Bull. 65, 512-528 24. Wilbrandt, W. (1940) Pflugers. Arch. Ges. Physiol. 243, 537-556 25. Tomoda, A., Tsuda, S., & Minakami, S. (1973) Experientia 29, 539-540

/ . Biochem.

Downloaded from https://academic.oup.com/jb/article-abstract/78/3/469/777708 by Western Sydney University Library user on 12 January 2019

pie application of thermodynamic principles, they have not been sufficiently considered in relation to red cell glycolysis and blood preservation. The role of pH as one of the most important factors regulating red cell glycolysis has been noted (19). This has been further extended to show the implications of the intracellular pH for the level of 2,3-diphosphoglycerate, which is important for red cell oxygen transport: the increase at low oxygen tension is suggested to be due to the intracellular increase on pH (20, 21). As another example, acceleration of red cell glycolysis has been observed on incubation of red cells either with addition of impermeable anions or on replacing the saline solution with a non-electrolyte (22). The latter condition is known to cause an efflux of Cl" and influx of OH~ and thereby causes an increase of the pHi (23, 24). Changes of glycolytic rate as well as in the levels of glycolytic intermediates on the addition of citrate were as expected from the change of the pHi (25). In conclusion, we would like to stress the importance of pHi determination for the study of red cell preservation. This does not mean, however, that the pHe does not influence the stability of red cells, since the pHe may also affect the cells in various ways either directly on the cell membrane or on cellular metabolism through membrane transport. Further improvement of blood preservation in fluid media may be possible by a systematic study of storage conditions with a consideration of both pHe and pHi.

Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions.

/ . Biochem., 78, 469-474 (1975) Dextrose Medium Effects of Temperature and Citrate Anions1 Sachiko TSUDA,2 Akio TOMODA, and Shigeki MINAKAMI Departm...
431KB Sizes 0 Downloads 0 Views