Europ. J. appl. Physiol. 34, tt--17 (1975) 9 by Springer-Verlag 1975

Diurnal Changes of the 2,3-Diphosphoglycerate Concentration in Human Red Cells and the Influence of Posture* D. B6ning, U. Meier, U. Schweigart, and M. Kunze Physiologisches Institut der Deutschen Sporthochschule K61n :Received September tt, 1974

Abstract. The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (Ps0), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I an almost synchronous diurnal behavior of Pc0,2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to ~_3.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; the Pc0 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a. posture dependent aldosterone secretion either directly on the 2,3-DPG metabolism or indirectly via mediating the red cell p g and thus ruling the formation of this organic phosphoris compouud is discussed. Key words: Blood - - Red Cell pH - - 2,3-Diphosphoglycerate - - Circadian Rhythm - Oxygen Dissociation Curve. Previous investigations carried out on rabbits revealed diurnal variations of the hemoglobin affinity for oxygen as indicated by a slight increase of the half saturation pressure for pH 7.4 (Ps0) during day-time (Bauer and RathschlagSchaefer, 1968; Schultz and Rathschlag-Schaefer, i969). The question arises whether such Pro changes are induced by corresponding changes of the intraerythrocytie 2,3-diphosphoglycerate concentration (2,3-DPG). So far results of preliminary experiments give evidence of diurnal variations of this organic phosphorus compound in man, but with the corresponding Ps0 to follow this r h y t h m only between 6.00 and 9.00 hrs (Schweigart et al., 1973). Some of the factors which are known to influence the 2,3-DPG metabolism, such as p H (f.i. Duhm and Gerlach, ~971) or inorganic phosphate concentration (Astrup et al., 1970), could already be shown to underlie a 24-hr periodicity (B6ning et al., ~[974). The purpose of the present study was to confirm possible diurnal alterations of both the [2,3-DPG] and Ps0 in man under conditions of unrestricted daily activity. In order to trace back the influence of postural effects additional experiments were undertaken during recumbency alone.

~Iethods Experimental Procedure. All subjects were clinically healthy male non-smokers (aged 20 to 30 years). 9 of them took part in experiments lasting 24 hrs (series I), which started either at 12.00, 06.00, or 23.00 hr. :Bed rest time in a dark room covered from 23.00 to 07.00 hr. * Supported by Deutsche Forsehungsgemeinschaft BO 360/2.

t2

D. BSning el al.

During day-time the subjects pursued their normal activity. Severe physical engagements or sports were not allowed. Issues of meals followed a strict time schedule (09.t5, t2.15, and !8.t5 hr). All subjects could drink ad libitum; alcohol was prohibited. Blood samples were collected 9 times in 3 hrs intervals with the first and last sample being averaged. In addition, some results of 9 identical experiments performed earlier (BSning et al., 1974) are included. In a second set of experiments (series II) 6 subjects lay down just after their arrival at 23.00 hrs and remained recumbent until the end of the investigation at 12.00 hrs next day; otherwise the environmental conditions did not differ from those of series I. Unless the subjects had assumed a recumbent position they were required to sit down about 15 rain before venepuncture. 6 rnl of blood were drawn into heparinized (0.01 ml Liquemin "Roche", 2500 I.U./ml) syringes, taking care to avoid any stasis. Thereafter, portions of 2 ml have been equilibrated in spheric tonometers at 37~ C for about 25 min. Gas mixtures contained as much oxygen as to provide an oxygen saturation of approximately 60 % at carbon dioxide tensions of 35 to 40 mm Hg. By use of Severinghaus' blood gas calculator (Severinghaus, t966) we obtained half saturation pressures (Ps0) corrected to pH 7.4 and 37~ C. Immediately after equilibration following quantities were determined: oxygen saturation (So2), plasma pH, base excess (BE), hematocrit value, blood hemoglobin, and mean corpuscular hemoglobin concentration (MCHC). Analytical procedures had been the same as reported in a pt;ior publication (BSning et al., 1974). In the 9 earlier described 24-hr experiments and those of series II venous oxygen saturation and venous pt{ in plasma as well as in red cells were instantly measured after blood sampling. For the determination of the 2,3-diphosphoglyeerate concentration 3 ml of whole blood were centrifuged, 0.5 ml of the resulting red cell sediment deproteinized by 1.5 ml 0.6 n perchlorie acid, and thereafter neutralized by potassium hydroxide. Aliquots were determined according to the method of Krimsky (1970). Analysis of variance was applied for statistical evaluation.

Results Series I . The 2 , 3 - D P G c o n c e n t r a t i o n exhibits d i u r n a l oscillations ( P < 0.0t) with lowest values i n the early m o r n i n g a n d highest i n the afternoon (Fig. l). The greatest alterations (between 06.00 a n d t8.00 hrs) a m o u n t to 0.6 mmol/1 red cells or t 3 . 5 % of the daily mean, correspondingly ~t2.6% when calculated on the basis

"]

PSO(rnm Hg)

[DPG] (rnmoIII red ceils) 5

3

....

o

~

;

,'2

,'6

2'0

A h~

~'ig. 1. Diurnal behavior of P~o ('~ - 9) and 2,3-DPG concentration (?~= 8). Solid lille: unrestricted daily activity. Dotted line: recumbency. 3Ieans and standard errors

00.00

33A • 0.3

7.354 _+ 0.008

7.183 • 0.007

57.4 • 3.1

Day time

M C t t C (g %)

pile1

priory

So2 (%)

54.5 + 4.0

7.t58 • 0.009

7.329 • 0.008

32.8 • 0.2

03.00

58.6 • 2.9

7A68 • 0.006

7.337 • 0.006

32.7 • 0.4

06.00

46.2 • 2.4

7.t76 • 0.006

7.338 _+ 0.008

32.3 • 0.3

09.00

48.5 +_ 3.8

7A98 _+ 0.007

7.35i • 0.009

32.6 • 0.3

~12.00

52.8 • 3.6

7.185 • 0.010

7.358 • 0.010

33.2 • 0.3

15.00

52.0 • 3.8

7.189 _+ 0.008

7.358 _+ 0.007

33.2 • 0.4

:18.00

57.2 + 4.0

7.179 _+ 0.006

7.355 • 0.007

33.6 • 0.2

2t.00

T a b l e t . M C H C , p H a n d So~. in v e n o u s b l o o d d u r i n g n o r m a l d a i l y a c t i v i t y . M e a n s al]d s t a n d a r d errors

P < 0.05

P < 0.05

P < 0.05

P < 0.05

Significance

g-

c~ 9

c~

9

o

'14

D. BSning et al.

Table 2. MCHC, pH, SoD and BE in venous blood during recumbency. Means and standard errors Day time

06.00

09.00

~2.00

~ICHC (g %)

33.9 • 0.4 7.341 ! 0.009 7.175 • 0.013 73.9 • 3.4 --0A • 0.6

34.7 _+0.3 7.373 +_0.004 7.i99 • 0.006 65.9 +_5.3 +1.9 • 0.3

35.4 • 0.5 7.368 _+0.009 7.t89 • 0.012 6'1.3 • 6.7 +IA

pHrl priory So2 (%) BE(meq/1)

• 0.i

Significance n.s. P < 0.05 n.s. P < 0.05 P

< 0.01

of ~mol/g hemoglobin. The time course of/)5o shows an almost parallel behavior (Fig. i). The maximal diurnal change reaches i m m Hg (P < 0.00t) or about 4% of the average day value. The 5ICHC (Table i) also underlies a 24 hrs rhythmicity with highest values in the evening (maximM change t.1 g% or 3.3% of the daily mean ; P < 0.05). For BE no diurnal variation could statistically be detected. I n addition, p H and SO 2 values of venous cubital blood abtained previously in nine identical experiments (B6ning et al., 1974) are presented for comparison (Table 1). For both quantities the changes throughout the daytime - - which are similar to those of [2,3-DPG] - - were proved significant (P < 0.05). Series I I . Here the subjects did not get up in the morning but remained recumbent until noon. In contrast to series I the [2,3-DPG] exhibits a tendency to decline between 06.00 and i2.00 hrs which is with the _Y-value of 3.9 close to 4.t for a significance level P < 0.05 (Fig. t). P~0 does not change significantly (Fig. 1), also the increments of MCHC cannot statistically be verified (Table 2). B E shows a morning increase (P < 0.0i). The venous pI-I in plasma increases (P < 0.05), but does not change significantly in the red cells. The venous oxygen saturation decreases from 73.9 % to 61.3 % (P < 0.05).

Discussion The maximal daily variation of the [2,3-DPG] in series I which tallies 13.5% of the 24 hrs mean value is relatively pronounced when compared with other blood constituents. As shown in a previous investigation the diurnal changes of most of the electrolyte concentrations, except inorganic phosphate, never exceed 10 % of the average day value (B6ning et al., t974). The concomitant Ps0 alterations can satisfactorily be explained when assuming t h a t an increase of l mmol DPG/1 erythrocytes results in a/)5o rise of about 1.5 to 2.0 m m I.ig (Duhm, i971). Changes of the base excess and MCI.IC - - to the extent as occuring in our experiments =- are negligible due to their minor influence on the hemoglobin affinity for oxygen (Arturson and Robert, t97i). Astrup et al. (t970) have demonstrated t h a t elevated concentrations of inorganic phosphate (Pal in serum lead to higher 2,3-DPG concentrations.

Diurnal Changes of the 2,3-DiphosphoglycerateConcentration

15

,',pH 0.26 0.22 0.16 9 0.14. 0.1

-' ~12 1'6 '0 2Z, hr Fig. 2. An illustration of pit differencebetween whole blood and red ceils during day time. Solid line: unrestricted daily activity. Dotted line: recumbency 0

i

-'

However, inorganic phosphate as a possible cause for regulating the [2,3-DPG] does not agree upon our findings. While the circadian course of P~ was shown to have a minimum at noon (B6ning et al., t974), in the present study the corresponding [2,3-DPG] at the same time exhibits almost maximal values during unrestricted daily activity. The predominant role of pH for the 2,3-DPG metabolism was repeatedly confirmed, such as, at high altitude (Lenfant et al., i968) or during artificially provoked hypoxia (Duhm and Gerlach, 197i). In both cases a respiratory alkalosis caused by hyperventilation led to increased intraerythrocytic 2,3-DPG contents. Even p H alterations in erythrocytes solely due to different states of oxygenation of hemoglobin are sufficient to influence the content of this phosphorus compound, whereas the regulation via product inhibition of DPG-mutase is less important (Duhm and Gerlach, t97t). According to the data of Astrup et al. (1970) sustained pH changes of about 0.0i will change the 2,3-DPG concentration as much as 0.04 moles 2,3-DPG/mole Hb tetramer. During unrestricted daily activity the diurnal oscillations of the [2,3-DPG] and venous red cell pH show good similarity; thus supporting the assumption that under these circumstances altered concentrations of this organic phosphate might be due overwhelmingly to corresponding alteration of the proton activity in blood. When the subjects remained recumbent throughout the morning hours a consistently elevated plasma pH could be observed, which was higher than the corresponding pH during unrestricted daily activity. At the same time recumbency did not result in a continously raised red cell pH. Thereby the difference between plasma and red cell pH tends to become greater during recumbency which is demonstrated in Fig. 2. As shown in Tables I and 2 this cannot numerically be explained solely by the small difference of the saturation decrease; taking into account that the state of oxygenation exerts an influence on the Donnan distribution for H+-ions across the red cell membrane (f.i. Duhm, t971). Previously published results (Sehweigart et al., 1972) accounting for a more pronounced alteration of saturation while changing body position are based on computational errors. A possible explanation is given by results showing that the aldosterone excretion (Wolfe et al., t966; Mtiller et al., 1958) parallels the 2,3-DPG concentration. Both substances could be proved to reach circadian day maxima, which were

16

D. B6ning et al.

missing when the subjects remained recumbent. A direct influence of aldosterone on the 2,3-DPG metabolism is not yet known. I t is, however, generally accepted t h a t aldosterone somehow mediates the Na +- and K+-ion transport across cell membrane either by direct interaction with the ion pumps or by enzyme induction accompanied with an enhanced ATP formation (Pelletier et al., t972). A change of the Donnan ratio for tt+-ions in red cells m a y occur according to following relationship (BSning, 1970, modified): [~+] P1 r =

[H+]E,,,

[Cat+-- g b - - - x-]~,~ --

[ C l - + ~CO,,-],,,

(1)

(Cat+ = cations, H b - = ions of H b and Oxy-tIb, X - other nondiffusible anions which mainly consist of organic phosphorus compounds, such as 2,3-DPG, A T P ; unit : meq/1 water). The logarithm of r can be set equal to the difference between plasma and red cell p H and is consequently dependent upon the erybhroeytic concentration of cations. The regulation of latter by mineralocorticoids is, according to contradictory results (Losert et al., i964; Streeten and Salomon, 1954; Bauer et al., t968), not completely elucidated. On the other hand, the Donnan ratio can be influenced as well by organic phosphates, such as ATP. However, it remains questionable whether the results showing an enhanced A T P production in the presence of aldosterone in tissue cells are also applicable to erythrocytes. Proof for a possible, either direct or indirect, influence of aldosterone on the 2,3-DPG metabolism still awaits further investigation. The present findings lead to the conclusion t h a t the practical importance of the changed oxygen affinity for work capacity is not pronounced, despite of the rather marked alterations of [2,3-DPG]. Due to the sigmoid shape of the oxygen dissociation curve the resulting Ps0 increase of t m m Hg lowers the oxygen saturation b y 2.5% or t % at 50% or t 0 % saturation, respectively. This means a by i to 5% improved oxygen extraction from blood in the vessels of the working muscles at constant P c , which cannot usually be detected when determining the maximal 0 2 uptake, but m a y play a role for utmost athletieal performance. Acknowledgement. We are indebted to Miss Vosdellen for her skillful technical assistance.

References Arturson, G., Robert, ~. : Oxygen affinity of whole blood in human subjects. Acts anaesth. scand. 45 (Suppl.), 22--25 (1971) Astrup, P., l~6rth, i~., Thorshauge, C.: Dependency on acid-base status of oxyhemoglobin dissociation and 2,3-diphosphoglycerate level in human erythrocytes. II. In vivo studies. Scan& J. olin. Lab. Invest. 26, 4~7--52 (1970) Bauer, Ch., Rathschlag-Schaefer, A. 1~. : The influence of aldosterone and cortisol on oxygen affinity and cation concentrations of the blood, l~esp. Physiol. ~, 360-370 (1968) B6ning, D. : Wirkungen eines akuten Sauerstoffmangels auf die Blutelektrolytkonzentrationen bei h6henangepa6ten und nichthShenangepagten ~enschen. Pfliigers Arch. 814, 217--230 (1970) B6ning, D., Schweigart, U., Kunze, M. : Diurnal variations of protein and eleetrolySe concentrations and acid-base status in plasma and red cells of normal man. Europ. J. appl. Physiol. 8~, 239--250 (t974) Duhm, J.: Effects of 2,3-diphosphoglycerate and other organic phosphate compounds on oxygen affinity and intraeellular plcI of human erythroeytes. Pfliigers Arch. 826, 341 --356 (197'1)

Diurnal Changes of the 2,3-Diphosphoglycerate Concentration

17

Duhm, J., Gerlach, E. : On the mechanisms of the hypoxia-induced increase of 2,3-diphosphoglycerate in erythrocytes. Pfliigers Arch. 326, 254--269 (1971) Krimsky, J.: D-glycerate-2,3.diphosphate. In: }Iethoden der Enzymatischen Analyse, Vol. II, I-I. V. Bergmeyer, Hrsg., S. t397--1399. Weinheim (Bergstr.): Verlag Chemie 1970 Lenfant, C., Torrance, J., English, E., Finch, C. A., Reynafarje, C., Ramos, J., Faura, J.: Effect of altitude on oxygen binding by hemoglobin and on organic phosphate levels. J. elin. Invest. 47, 2652--2656 (1968) Losert, W., Senft, C., Senft, G. : Extrarenale Wirkungen des Aldosterons und der Spirolaetone. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 248, 450--463 (t964) 5Iuller, A. F., Manning, E. L., Riondel., A M. : Influence of position and activity on the secretion of aldosterone. Lancet 1988 I, 7tf--713 Pelletier, N., Ludens, J. H., Fanestil, D. D. : The role of aldosterone in active sodium transport. Arch. intern, lV[ed. 129, 248--257 (1972) Schultz, D., Rathschlag-Sehaefer, A. M. : Changes of oxygen affinity and cation concentration in the blood during 24 hrs. Pfltigers Arch. 307, R t l (1969) Schweigart, U., BSning, D., Tibes, U., Hemmer, B. : The influence of physical activity on the 2,3-diphosphoglycerate concentration of erythroeytes and the oxygen dissociation curve in man. In: Erythrocytes, thromhoeytes, leucocytes. IInd. Symp. Vienna 1972, E. Gerlaeh, K. ?,loser, E. Deutseh, W. Wilmans, eds., pp. 168--170. Stuttgart: Thieme 1973 Severinghaus, J. W.: Blood gas calculator. J. appl. Physiol. 21, 1108--1116 (1966) Streeten, D. H. P., Solomon, A. X. : The effect of ACTH and adrenal steroids on K transport in human erythrocytes. J. gen. Physiol. 37, 643--661 (1954) Wolfe, L. K., Gordon, R. D., Island, D. P., Liddle, S. W. : An analysis of factors determining the circadian pattern of aldosterone excretion. J. olin. Endocr. 26, 1261--1266 (1966) Priv.-Doz. Dr. D. BSning Physiologisehes Institut der Deutschen Sporthochschule KS!n D-5000 K61n 41 Carl-Diem-Weg Pederal l~epublic of Germany