Pfl Jgers Archiv

Pfltigers Arch. 363, 61-67 (1976)

EuropeanJournal of Physiology 9 by Springer-Verlag 1976

Influence of 2,3-Diphosphoglycerate on the Buffering Properties of Human Blood R o l e o f the R e d Cell M e m b r a n e * JOCHEN DUHM PhyiologischesInstitut, UniversitfitMtinchen, Pettenkoferstrage 12, D-8000 M/inchen 2, Federal Republic of Germany

Summary. The effect of the concentration of red cell 2,3-diphosphoglycerate (2,3-DPG, 0 . 5 - 2 1 gmoles/g cells) on the buffering properties and on the slope of the relation between the extracellular and intracellular pH (ApHi/ApHe) of human blood was studied. The results were evaluated in connection with previous findings concerning the effect of 2,3-DPG on the Donnan ratio rn+ = He+/H + . ApHi/ApHe decreases with rising red cell 2,3-DPG content as well as with rising extracellular pH. ApHi/ApHe and rn§ can be related fo each other by the empirical equation dpHi/ApHe = 1 + log rn+ = 1 + p H i - pHi. The validity of this equation appears to be restricted to conditions where the Donnan ratio rt+ is altered between 0.3 and 1 either by changes of the red cell concentration of buffering anions such as 2,3-DPG or by changes of the extracellular pH. As determined in suspensions of red cells with intact membranes, the 2,3-DPG- and pH-induced changes of ApHi/ApH~ lead to proportional changes in the buffering power of the non-bicarbonate buffers of erythrocytes. Due to this effect the buffering power of suspensions of cells containing 5 times the normal concentration of the buffer 2,3-DPG is lower than that of cells with normal 2,3-DPG content (at extracellular pH values above 7). These findings demonstrate that the action of intracellular nonbicarbonate buffers in blood is effectively modulated by the physico-chemical properties of the red cell membrane.

Key words." Erythrocytes - 2,3-Diphosphoglycerate - Buffers in blood - Red cell membrane. * Part of the resutts was presented on the 3rd InternationalConferenceon Red Cell Metabolismand Function,Ann Arbor, October 16- 19, 1974 (cf. Ref. [131).

INTRODUCTION Elevation of red cell 2,3-DPG 1 content alters the oxygen affinity [4, 9, 11 - 13,17] and the Bohr effect of human blood [2, 3, 5, 7, 8, 20, 24] and causes an inhibition of red cell glycolysis [6,14]. These effects have been shown to result from two separate actions of 2,3-DPG. On the one hand, 2,3-DPG can bind to hemoglobin [1,4, 9, 13] and probably also to some enzyme proteins [6,14], thereby altering the functional properties of the proteins. On the other hand, the nonpenetrating 2,3-DPG anion alters the Donnan distribution of penetrating ions across the red cell membrane [10-12,25]. Consequently, elevation of the red cell 2,3-DPG content induces a decrease of the red cell pH. The red cell pH, however, is another factor influencing the function of hemoglobin and enzyme proteins as well as the strength of the interaction of 2,3-DPG and hemoglobin [5,9, 13]. It appears then, that the direct interaction of 2,3-DPG with red cell proteins is modulated in whole blood by the physicochemical properties of the red cell membrane. In studies on the effect of red cell 2,3-DPG content on the oxygen affinity [11[, on the Bohr effect [16] and on the glycolytic metabolism of human blood [14] it has been shown that such membrane-mediated effects can be of considerable importance. It therefore seemed of interest to examine whether a property of 2,3-DPG which is not mediated by binding to proteins might also be modulated by the red cell membrane. Such a property is the buffering capacity of 2,3-DPG. Since the pK values of the second dissociation of the two phosphate groups of 2,3-DPG are close to 7.0 [5, 7], one would expect that rising 2,3-DPG concentrations should be associated with a rising buffering capacity of erythrocytes [27,28]. It will be shown, however, that elevation of the red cell 2,31 2,3-DPG: 2,3-diphosphoglycerate, subscripts i and e: intracellularand extracellular.

62

Pfltigers Arch. 363 (1976)

D P G c o n t e n t c a n b e a s s o c i a t e d w i t h a decrease o f the b u f f e r i n g p o w e r o f r e d cell s u s p e n s i o n s .

METHODS Human blood was obtained from non-smoking volunteers, heparin (0.1 mg/ml blood) being used as anticoagulant. After centrifugation the buffy coat was removed. The red cell 2,3-DPG content was elevated by incubating the cells for t00 rain in isotonic media containing 10 mM inosine, 10 mM pyruvate, 50 mM inorganic phosphate and NaC1 (37~C, pH 7.4, hematocrit 10~) D0]. The fresh or incubated cells were then washed 3 times in a solution containing (raM): NaC1 (150), KCI (5), NaHCO3 (6), glucose (5.5), CaCIz (2.2), Na2HPO4 (0.9), and NaH2PO4 (0.1) (pH 7.4). Subsequently the cells were resuspended in the same medium to yield a hemoglobin content of 2.2 _+ 0.05 mmoles hemoglobin tetramer/1 suspension (hematocrit about 40 ~). The pH was adjusted to a value of about 8 by addition of 0.3 M NaHCO3. Aliquots of the suspensions (5 ml) were stirred thoroughly by means of a magnetic stirrer at 37~C in free contact with room air for more than 10 rain to equilibrate the Pco2 of the suspensions with that of the room air. Subsequently, portions of 50 to 100 btl of 0.3 n HC1 were added stepwise within 30 s using the Autoburette ABU 12 (Radiometer, Copenhagen). The resulting changes of the extracellular pH were recorded (glass electrode G 298A, calomel electrode K 4112, pH meter PHM 64, Radiometer, Copenhagen). The extracellular pH reached a new stable value within 1 - 2 rain after each addition of titrant (pH change per titration step about 0.1 pH units). Another sample of the suspensions was hemolyzed by freezing at - 2 0 ~ and thawing (3 times) and immediately titrated as described above. The intracellular pH of the erythrocytes was measured by the freezethaw technique as previously described [11,19]. Hemoglobin was quantitated as cyanmethemoglobin at 540 nm using a millimolar absorption coefficient of 11. Methemoglobin was measured by the method of Evelyn and Malloy [18] and was found to be less than 4 in the cell suspensions and less than 9 ~ in the hemolysates at the end of the experiments. The red cell 2,3-DPG concentration was determined enzymatically on neutralized perchloric acid extracts [21].

RESULTS

AND

DISCUSSION

T o s t u d y the effect o f t h e red cell 2 , 3 - D P G c o n t e n t o n t h e b u f f e r i n g p r o p e r t i e s o f red cell n o n - b i c a r b o n a t e buffers, s a l i n e s u s p e n s i o n s o f e r y t h r o c y t e s c o n t a i n i n g n o r m a l o r e l e v a t e d c o n c e n t r a t i o n s o f 2 , 3 - D P G were t i t r a t e d w i t h HC1 w h i l s t i n c o n t a c t w i t h r o o m air as described in Methods. The molar buffer values (added acid per h e m o g l o b i n t e t r a m e r p e r p H c h a n g e = flHbH +) (cf. [29]) o b t a i n e d w i t h s u s p e n s i o n s o f i n t a c t cells a n d their h e m o l y s a t e s are s u m m a r i z e d in F i g u r e 1. In interpreting the buffer values given in Figure 1 it has to be considered that due to metabolic processes H + is generated by intact erythrocytes as well as by fresh hemolysates. H + generation by intact erythrocytes may be attributed entirely to glycolysis. From lactate production rates of erythrocytes with normal or elevated 2,3-DPG content [14] it can be estimated that H + was formed by the two types of erythrocytes at a rate of 0.012 (0.005) and 0.004 (0.003) moles/mole hemoglobin tetramer per rain at extracellular pH values of 8.0 and 7.0, respectively. Values for the cells with high 2,3-DPG

content are given in parentheses. The time interval between two titration steps was about 2 rain. Accordingly the underestimation of the buffer values resulting from glycolytic H + generation can be estimated not to exceed 0.4 ~. It seems reasonable to assume that metabolic H + generation in hemolysates (e.g. due to degradation of ATP) did not exceed this value. The cells were suspended in saline media to avoid participation of plasma proteins in the measurements of buffer values. However, the saline suspension media contained 1 mM inorganic phosphate since this phosphate concentration is necessary for the metabolic integrity of human erythrocytes. The molar ratio of extracellular phosphate to hemoglobin tetramer was about 0.3. Hence, extracellular non-bicarbonate buffers contributed only little to the buffer values given in Figure t (at most about 0.17 buffer units at the pK of inorganic phosphate). Finally it has to be taken into account that NaHCO3 was used to adjust the pH of the suspension media, of the cell suspensions and of the hemolysates (see Methods). Since the cell suspensions and hemolysates were stirred vigorously for more than 10 rain in broad contact with room air before starting the titration experiments, it seems reasonable to assume that the Pco2 in the solutions did not exceed 1 mm Hg 2. Thus the bicarbonate concentrations should not have exceeded 2.38 and 0.024 mM at pH 8 and pH 6, for instance, as can be calculated using the Henderson-Hasselbalch equation (pK' of carbonic acid = 6.1 ; ~co2 = 0.03 mmoles x t -I x mmHg-1). These bicarbonate concentrations are one to three orders of magnitude lower than those in normal blood. According to all these considerations it appears that only a small error is introduced by the assumption that the buffer values given in Figure 1 reflect almost exclusively the properties of red cell non-bicarbonate buffers. T h e d a t a s h o w n in F i g u r e 1 A d e m o n s t r a t e t h a t the buffering capacity of intact human erythrocytes d e p e n d s o n t h e e x t r a c e l l u l a r p H . F o r n o r m a l cells the b u f f e r i n g c a p a c i t y is m a x i m a l at a n e x t r a c e l l u l a r p H o f a b o u t 6.8. F o r r e d cells w i t h elevated 2 , 3 - D P G c o n t e n t this m a x i m u m is d i s p l a c e d to a l o w e r p H v a l u e o f 6.4. T h e m o s t s t r i k i n g r e s u l t is t h a t t h e b u f f e r i n g c a p a c i t y o f r e d cells c o n t a i n i n g 5 t i m e s the n o r m a l c o n c e n t r a t i o n o f t h e b u f f e r 2 , 3 - D P G is lower t h a n i n n o r m a l cells at e x t r a c e l l u l a r p H v a l u e s a b o v e 7.0. O n l y b e l o w this p H v a l u e is a n i n c r e a s e o f the b u f f e r i n g c a p a c i t y to b e o b s e r v e d in t h e s u s p e n s i o n s w i t h e l e v a t e d red cell 2 , 3 - D P G c o n t e n t . A different p i c t u r e w a s o b t a i n e d w h e n the cell s u s p e n s i o n s were first h e m o l y z e d a n d t h e n t i t r a t e d (Fig. 1 B). T h e b u f f e r v a l u e s i n the h e m o l y s a t e s w i t h h i g h 2 , 3 - D P G c o n t e n t were e l e v a t e d relative to t h o s e o f n o r m a l cells at all p H values. T h e t w o c u r v e s s h o w n i n F i g u r e 1 B agree closely w i t h d a t a p u b l i s h e d b y S i g g a a r d - A n d e r s e n [27,28]. By c o m p a r i n g F i g u r e s i A a n d 1 B it b e c o m e s e v i d e n t , t h a t , a l t h o u g h t h e buffer c o n c e n t r a t i o n s ( m o l e s h e m o g l o b i n + m o l e s o r g a n i c p h o s p h a t e s / 1 s u s p e n s i o n ) were i d e n t i c a l u n d e r b o t h c o n d i t i o n s , the b u f f e r v a l u e s are c o n s i d e r a b l y 2 TO examine this assumption the Pco2 in the red cell suspensions and hemolysates to be titrated was measured with a Pco2 electrode. The results obtained before and after equilibration of the solutions with a Pco2 of 3 mm Hg demonstrated that the Pco2 of the solutions was with certainty below 3 mm Hg.

J. Duhm: 3pHdztpHe, Red Cell 2,3-DPG and Non-Bicarbonate Buffers in Blood

63

Fig. 1 A and B. Effect of 2,3-DPG and pH on 18 the buffer value of human erythrocytes (37~ C, soA moles H+ lutions in contact with room air). The cells were mole Hbl.. A pH suspended in saline media at a hemoglobin concentration of 2.2 mmoles/1 (by tetramer) and titrated 14 T T TTTTTT T with HC1 (A), or first hemolyzed and then titrated (B) (see Methods). Mean values _+ 1 S.D. from 6 - i 0 experiments on blood of 5 different donors. i~ (O~) 4 gmoles 2,3-DPG/g erythrocytes, 10 (0 ,,~) 21 ~moles 2,3-DPG/g erythrocytes

TTTT

TT,~#.I> = N

i

Ig

K,

.T ~T

~

~:r

%

INTACT C E L L S

I

I&TI~

HEMOLYZED CELLS

:| I

6

higher in hemolysates than in suspensions of intact erythrocytes. It may be noted, that an increase of the buffer value of blood upon hemolysis has been observed more than 50 years ago by Van Slyke et al. [30] and Peters et al. [22]. The entire system of non-bicarbonate buffers in red cell suspensions can be described by Equation (1) fiH +(S) = / ? U + (E). A p r i l . r ApH~ + flH + (M)-toM(S),

fiUbH + (S)

zlpH i

ApH~

I

I

7 EXTRACELLULAR pH

1.o

I

I

I

I

8

6

I

I

I

7 HEMOLYSATE pH

i

i

i

&pHi ~i~..~, pile ApHe -'~.~--~'~'~~.~ 6.6--.

06-

(1)

where #H + = buffer value (A H+/1 . ApH), S = cell suspension, E = erythrocytes, M = medium, q0 = volume fraction, pHi and pile = intracellular and extracellular pH. fiH+ (S) and #H + (E) refer to the extracellular and intracellular pH, respectively [28]. As outlined above, the buffer value of the medium [fill + (M) - qoM(S)] was kept very small in the experiments and, therefore, may be omitted in the following calculations without introducing an error exceeding the experimental error. By converting the buffer values in Equation (1) to molar buffer values of hemoglobin (fi.bH +) and rearranging Equation (1) one obtains

fiHbH + ( E )

I

(2)

According to this equation a deviation of ApHi/ ApH~ from one should be responsible for differences in the buffering capacities of intact and hemolyzed cells. ApHi/ApH~ is a function of both the red cell 2,3DPG content and of the extracellular pH (Fig. 2). At an extracellular pH of 7.4, for instance, ApHi/ApHe decreased from 0.8 at normal 2,3-DPG levels to about 0.5 at a 2,3-DPG concentration of 21 l.tmoles/g cells. ApH]ApHe decreases with rising extracellular pH. This effect is more pronounced at high red cell 2,3DPG concentrations.

I

0

I

I

I

I

/

8 16 24 2,3 -DPG [# moLes/g ERYTROCYTES]

Fig. 2. Dependence of ApHi/ApHe on red cell 2,3-DPG content and on the extracellular pH (37 ~C, cells suspended in plasma). Closed symbols: Values taken from Figure 3, Ref. [11] and from Figure 10, Ref. [13] (100% 02 saturation, Pco2 < 1 mmHg). Open symbols: Values taken from Ref. [16] ( 3 0 - 7 0 % 02 saturation, Pco2= 38 mmHg)

From all these results it follows that in quantitative estimations of the role of changes of ApH]ApHe in altering the buffering properties of the red cell contents it has to be considered that 1. the buffering capacity of the red cell contents depends on the actual red cell pH (see Fig. 1), 2. the red cell pH is a function of the extracellular pH as well as of the red cell 2,3-DPG content [11], and 3. ApHjApHe depends both on the extracellular pH and on the red cell 2,3-DPG concentration (see Fig. 2). At an extracellular pH of 7.4, for instance, the buffer values of red cell suspensions [fl~bH+ (S)] were 10.2 for normal cells and 9.0 for cells containing 21 gmoles 2,3-DPG/g, whereas the buffer values of the erythrolysates at the erythrolysate pH which corresponds to the intracellular pH of the suspended cells

Pfl/igers Arch. 363 (1976)

64 Table 1. Valuesfor the parameters of Equations (2) and (3) 2,3-DPG [pmoles/g cells]

21

" b ~ a ~

pHe~

pHia

flitba + (s)b

/~nbH+ (E) b

/~HbH+ (S) flnbH+ (E)

April c ApHe

rn +d

1 + log rn+~

6.6 7.0 7.4 7.8

6.53 6.89 7.23 7.53

12.0 11.9 10.2 8.1

13.4 13.4 11.8 9.9

0.90 0.89 0.86 0.82

0.91 0.75 0.80 0.73

0.86 0.75 0.63 0.52

0.98 0.87 0.80 0.72

6.6 7.0 7.4 7.8

6.34 6.61 6.82 7.00

13.1 11.8 9.0 6.2

17.1 17.5 17.6 17.2

0.77 0.67 0.51 0.36

0.76 0.64 0.48 0.34

0.55 0.42 0.29 0.16

0.74 0.62 0.45 0.19

Valuestaken from Figure 3 of Ref. [11]. Valuestaken from Figure 1. Valuestaken from Figure 2. Valuestaken from Figure 3. See Equation (3).

[flHbH + (E)] were 11.8 a n d 17.6, respectively (see T a b l e 1). The c o r r e s p o n d i n g ratios of the buffer values o f red cell suspensions a n d of erythrolysates [i.e. left side of E q u a t i o n (2)] were 0.86 a n d 0.51 a n d the values of ApH~/ApH~ [right side o f E q u a t i o n (2)] were 0.80 a n d 0.48, respectively. Similar a g r e e m e n t between the ratio of the buffer values a n d ApH~/ApH~is observed at p H values differing c o n s i d e r a b l y f r o m n o r m a l (Table 1). The deviations f r o m ideal b e h a v i o u r m a y be the result of experimental error a n d / o r of inaccuracies due to the a s s u m p t i o n that the m e a s u r e d buffer values reflect almost exclusively the properties of red cell n o n b i c a r b o n a t e buffers (see above). T h u s is c o n c l u d e d that the deviations of ApHi/ApHe from one a n d the 2,3D P G - a n d p H - i n d u c e d alterations of April~April, respectively, are responsible for the differences in the buffering capacities of intact a n d hemolyzed erythrocytes. The increase of the buffer value of hemolysates occurring upon elevation of 2,3-DPG concentration corresponds fairly well to that to be expected theoretically. The following considerations may serve as an example. One should expect that at the pK2 of the two phosphate groups of 2,3-DPG (pH 7,0) the buffer value of hemolysates increases by about 3.7 buffer units upon elevation of 2,3DPG concentration from 4 to 21 gmoles/g ceils (i.e. from a molar ratio of 2,3-DPG to hemoglobin of about 0.8 to 4). This increase of the buffer value is obtained by multiplying the molar buffer value of a buffering group at its pK value (0.576) [29] with the increase of the molar ratio of 2,3-DPG to hemoglobin (3.2), taking into account that two buffering groups of 2,3-DPG are involved (0.576 x 3.2 x 2 = 3.69). At an erythrolysate pH of 7.0 an increase of the buffer value by 4.1 units was observed (see Fig. 1B). The differencebetween the calculated and the measured values (4.1-3.7 = 0.4) may be attributed mainly to the increase in the concentrations of buffering organic phosphates other than 2,3-DPG during the preincubation (cf. [10,11,31]) and to a minor extent to changes of pK values of interacting groups of 2,3-DPG and hemoglobin which may occur with rising 2,3-DPG concentrations [7, 8, 20].

The contribution of 2,3-DPG to the red cell non-bicarbonate buffers may be estimated as follows: At the normal plasma pH of 7.4 the red cell pH of erythrocytes with a 2,3-DPG content of 0.8 and 4 moles/mole hemoglobin is about 7.2 and 6.8, respectively (see Table 1), i.e. the red cell pH deviates from the pK2 values of 2,3-DPG by 0.2 pH units in both types of cells. Thus the molar buffer value of 2,3-DPG should be about 0.576 x 0.9 x 2 = 1.04 [29]. Accordingly the contribution of 2,3-DPG to the total buffer value of the red cell contents should be about 7 % at the normal 2,3-DPG concentration (1.04 x 0.8 = 0.83 of the total of 11.8) and 24 % at the high 2,3-DPG concentration (1.04x4 = 4.16 of the total of 17.6). The questions n o w arise why ApHi/ApHe is n o t equal to one in n o r m a l b l o o d a n d why ApHi/ApHe changes with the red cell 2 , 3 - D P G c o n t e n t a n d with the extracellular p H (see Fig. 2). It is t e m p t i n g to assume that changes of ApHi/ApHe m i g h t be causally related to changes of the D o n n a n ratio r H+ = H +e /iH ,.+ 9 As s h o w n in F i g u r e 3 ru+ decreases b o t h with rising red cell 2 , 3 - D P G c o n t e n t a n d with rising extracellular pH. ApHi/ApHe increases in a curvilinear m a n n e r with rising values of rn+ (Fig. 4). W h e n plotting ApHi/ApHe against I + log r~+ the n u m e r i c a l values are close to the line o f identity of b o t h p a r a m e t e r s (Fig. 5). T h u s the following e q u a t i o n c a n serve as a n a p p r o x i m a t i o n describing the r e l a t i o n s h i p between ApHi/ApHe a n d rH+ : ApHi ApH~

-- 1 + log rn+ = 1 + p H i - p i l e .

(3)

It m a y be p o i n t e d o u t that E q u a t i o n (3) could n o t be o b t a i n e d from the G i b b s - D o n n a n law a n d that alterations o f the D o n n a n ratio i n d u c e d by a d d i t i o n of small a m o u n t s of extracellular citrate are n o t acc o m p a n i e d by changes of ApHI/ApHo [23]. I n o w n experiments (not shown) it was observed, that a n ele-

J. Duhm: 1.0

--

ApHjApH~, Red Cell 2,3-DPG and Non-Bicarbonate Buffers in Blood I

I

I

I

I

"~

FH+ :"J~o~O 0,

,.,

". -

l

07

""-~.

"---

~ .

7.8-----------" 0.2

L I

0

7.0'

I

I

P

I

8 16 24 2,3-DPG [tJ moleslg ERYTROCYTES]

Fig. 3. Dependence of rn+ on red cell 2,3-DPG content and on the extracellular pH (37 ~C, cells suspended in plasma). Symbols as in Figure 2

vation of rn+ to about 1.3 by replacement of 80 % of extracellular NaC1 by sucrose, citrate or 2,3-DPG was not accompanied by the increase of ApH~/ApH~ to be expected from Equation (3), but rather by a small decrease (extracellular pH = 7.4). Furthermore it is obvious that Equation (3) cannot be valid at rn+ values below 0.1, since under this condition ApH(ApHe would become negative, a situation which does not occur. Estimated from Table 1, the minimum value of rt~+ for which Equation (3) holds would appear to be about 0.3. Consequently the empirical Equation (3) can only be applied to conditions where the Donnan ratio r.+ is altered between 0.3 and I by changes of the extracellular pH or of the red cell concentration of buffering anions such as 2,3-DPG.

CONCLUSION

1.0

I

I

I

I

/m

ApHi ApHe

-1

9

o/,o,

0.6

/

/

0.? I

0

I

07

I

I

1.0 FH+ Fig. 4. Dependenceof ApHUApHeon r.+ in human blood (37~C). Values taken from Figures 2 and 3. The red cell 2,3-DPG content was 0.8, 3.8, 11.3 and 20.7 lxmoles/g (closed symbols) and 0.5, 4 and 16 Ixmoles/g(open symbols). (U) pile = 6.6; (O, O) pile = 7.4; (i) pile = 8.8

1.0

06

I

i

I

ApHe

ll~

0.6

0.2-/

o I

ApH~

pHe-pH

" 0.80.6 0

65

0.4

0.2 I

I

I

I

02

04

06

08 10 1 + log rH+ Fig. 5. Relationship between ApHi/ApHe and J + log r.+ and pH~- pHi, respectively(37~C, cells suspended in plasma). The line represents the line of identity of ApH~/ApHoand of I + log rn+. Symbols as in Figure 4

The buffer value of red cell non-bicarbonate buffers as determined in suspensions of intact cells is directly proportional to ApHi/ApHe, and, of course, to the intracellular buffer concentration. ApHi/ApHe decreases with rising red cell 2,3-DPG content and with rising extracellular pH. The effect of 2,3-DPG is of such a magnitude that an increase of the intracellular concentration of the buffer 2,3-DPG can lead to a decrease of the buffering capacity of suspensions of human erythrocytes. This phenomenon which is of theoretical interest probably bears no practical implications, since the absolute decrease of the buffer value of intact erythrocytes occurring upon elevation of red cell 2,3-DPG content is only small at an extracellular pH of 7.4. Furthermore, it has been stated that changes of the amount of non-bicarbonate buffers in blood are of little clinical significance [26]. Finally, it has been shown that the total buffering capacity of the bicarbonate + non-bicarbonate buffers of whole blood (as quantitated by the slope ApH/A log Pco2 of CO2 titration curves) is not significantly different for blood with normal or elevated red cell 2,3-DPG content [13]. ApHjApHe varies as a function of the D o n n a n ratio rH+ and of the pH-difference across the red cell membrane, respectively (Figs. 4 and 5). ApH]ApHe and rH+ can be related to each other by Equation (3). The validity of this empirical equation appears to be restricted to conditions where rH+ is altered between 0.3 and 1 by changes of the intraerythrocytic concentration of buffering anions such as 2,3-DPG or by changes of the extracellular pH. The studies conducted so far concerning the effects of 2,3-DPG-induced changes of the Donnan equilibrium demonstrate that a number of physiological properties of human erythrocytes are affected [11, 12,

66

Pflfigers Arch. 363 (1976)

Table 2.

Effects of 2,3-DPG on human erythrocytes mediated by changes of the Donnan equilibrium a

Primary effect: Alteration of the Donnan ratio r due to the charge and the osmotic activity of 2,3-DPG [11 ] Secondary effects:

Tertiary effects:

Decrease of the red cell pH [11,25]

Change of the activities of pH-dependent metabolic pathways (inhibition of glycolysis [14]) Decrease in the oxygen affinity of hemoglobin [11]

Decrease of red cell CI- and HCO3 concentrations

Changes in the activities of enzymes dependent on the concentrations of these anions (?)

Decrease of red cell water content [10- 12,14]

Increase of red cell hemoglobin concentration [12,15] Increase of osmotic resistance [15]

Decrease of

a

ApHi/ApHe ([16] this paper)

Decrease of the Bohr effects of blood [16] Decrease of the buffering power of non-bicarbonate buffers within the red cell ([13] this paper)

The numbers in Square brackets refer to References.

14-16,25]. According to Table 2 the effects can be classified as primary, secondary and tertiary ones. Changes of the Donnan equilibrium alter the most important function of the red cell, namely its function in gas transport, as well as its glycolytic metabolism, which is essential not only for a normal survival of erythrocytes but also for the normal functioning of hemoglobin. Thus is appears that the physico-chemical characteristics of the red cell membrane and 2,3-DPGinduced changes of the Donnan equilibrium in human blood, respectively, play a substantial role in determining some of the most important physiological properties of the erythrocyte.

REFERENCES 1. Arnone, A. : Mechanism of action of hemoglobin. Ann. Rev. Med. 25, 123-164 (1974) 2. Bauer, Ch. : Antagonistic influence of CO2 and 2,3-diphosphoglycerate on the Bohr effect of human hemoglobin. Life Sci. 8, 1041 (1969) 3. Bauer, C. : On the respiratory function of hemoglobin. Rev. Physiol. Biochem. Pharmacol. 70, 1 - 31 (1974) 4. Benesch, R., Benesch, R. E. : The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem. biophys. Res. Commnn. 26, 162-167 (1967) 5. Benesch, R. E., Benesch, R., Yu, C. I.: The oxygenation o f hemoglobin in the presence of 2,3-diphosphoglycerate. Effect of temperature, pH, ionic strength and hemoglobin concentration. Biochemistry 8, 2567-2571 (1969) 6. Beutler, E., Matsumoto, F., Guinto, E. : The effect of 2,3-DPG on red cell enzymes. Experientia (Basel) 30, 190-192 (1974) 7. de Bruin, S. H., Janssen, L. H. M.: The interaction of 2,3-diphosphoglycerate with human hemoglobin. Effects on the alkaline and acid Bohr effect. J. biol. Chem. 248, 2774-2777 (1973) 8. de Bruin, S. H., Rollema, H. S., Janssen, L. H. M., van Os, A. J.: The interaction of 2,3-diphosphoglycerate with human deoxy- and oxyhemoglobin. Biochem. biophys. Res. Commun. 58, 204--209 (1.974)

9. Chanutin, A., Curnish, R. R. : Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biphys. 121, 9 6 - 1 0 2 (1967) 10. Deuticke, B., Duhm, J., Dierkesmann, R. : Maximal elevation of 2,3-diphosphoglycerate concentrations in human erythrocytes : Influence on glycolytic metabolism and intracellular pH. Pfltigers Arch. 326, 1 5 - 34 (1971) 11. Duhm, J.: Effects of 2,3-diphosphoglycerate and other organic phosphate compounds on the oxygen affinity and intracellular pH of human erythrocytes. Pfltigers Arch. 326, 341 - 356 (1971) 12, Duhm, J. : The effect of 2,3-DPG and other organic phosphates on the Donnan equilibrium and the oxygen affinity of human blood. In: Oxygen affinity of hemoglobin and red cell acid base status (M. R~brth and P. Astrup, eds.), pp. 583-594. Copenhagen: Munksgaard 1972, and New York: Academic Press 1972 13. Duhm, J. : Studies on 2,3-diphosphoglycerate: Effects on hemoglobin, glycolysis and on buffering properties of human erythrocytes. In: Erythrocyte structure and function, vol. 1, Progress in clinical and biological research (G. Brewer, ed.) pp. 167-197. New York: Alan R. Liss 1975 14. Duhm, J. : Glycolysis in human erythrocytes containing elevated concentrations of 2,3-P2-glycerate. Biochem. biophys. Acta (Amst.) 385, 6 8 - 8 0 (1975) 15. Duhm, J.: Interactions of 2,3-diphosphoglycerate with hemoglobin and intact erythrocytes. International Society of Haematology, European and African Division, Third Meeting, London, Aug. 2 4 - 2 8 , 1975, Abstracts 1, 1 : 16 16. Duhm, J. : Dual effect of 2,3-diphosphoglycerate on the Bohr effects of human blood. Pftiigers Arch. 363, 5 5 - 6 0 (1976) 17. Duhm, J., Gerlach, E.: Metabolism and function of 2,3-diphosphoglycerate in red blood cells. In: The human red cell in vitro. (T. Greenwalt and G. A. Jamieson, eds.) pp. 111-148. New York-London: Grune and Stratton 1974 18. Evelyn, K. A., Malloy, H. T.: Micro determination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J. biol. Chem. 126, 655-662 (t938) 19. Funder, J., Wieth, J. O.: Chloride and hydrogen ion distribution between human red cells and plasma. Acta physiol, scan& 68, 234-245 (1966) 20. Kilmartin, J. V. : Influence of DPG on the Bohr effect of human hemoglobin. FEBS Letters 38, 147-148 (1974) 21. Michal, G.: D-Glycerat-2,3-diphosphat. In: Methoden der enzymatischen Analyse (H. U. Bergmeyer, ed.), pp. 1478-1483. Weinheim: Verlag Chemie 1974 22. Peters, J. P., Bulger, H. A., Eisenmann, A. J.: Studies of the carbon dioxide absorption curve of human blood. IV. The

J. Duhm: ApH~/ApHo, Red Cell 2,3-DPG and Non-Bicarbonate Buffers in Blood

23.

24.

25.

26.

27.

relation of the hemoglobin content of blood to the form of the carbon dioxide absorption curve. J. biol. Chem. 58, 747-768 (1924) Poyart, C. F., Bursaux, E., Freminet, A. : Citrate and the Donnan equilibrium in human blood: PsoT.4 or Psow2. Biomedicine Express 19, 5 2 - 55 (1973) Riggs, A. : Mechanism of the enhancement of the Bohr effect in mammalian hemoglobin by diphosphoglycerate. Proc. nat. Acad. Sci. (Wash.) 68, 2062-2065 (1971) Salhany, J. M., Keitt, A. S., Eliot, R. S.: The rate of deoxygenation of red blood cells: Effect of intracellular 2,3-diphosphoglycerate and pH. FEBS Letters 16, 257-261 (1971) Siggaard-Andersen, O.: An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scand. J. clin. Lab. Invest. 27, 239-245 (1971 a) Siggaard-Andersen, O. : Oxygen-linked hydrogen ion binding of human hemoglobin. Effects of carbon dioxide and 2,3-di-

28. 29.

30.

31.

67

phosphoglycerate. I. Studies on erythrolysate. Scand. J. clin. Lab. Invest. 27, 351-360 (1971b) Siggaard-Andersen, O.: The acid-base status of the blood. Copenhagen : Munksgaard 1974 Van Slyke, D. D. : On the me~tsurement of buffer value and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution. J. biol. Chem. 52, 525-570 (1922) Van Slyke, D. D., Wu, H., McLean, F.: Studies of gas and electrolyte equilibria in the blood. V. Factors controlling the electrolyte and water distribution in the blood. J. biol. Chem. 56, 765-849 (1923) Zachara, B., Lewandowski, J.: Isolation and identification of inosine triphosphate from human erythrocytes. Biochim. biophys. Acta (Amst.) 353, 253-259 (1974)

Received December 16, 1975