Regulation of oxygen affinity in blood of fetal, newborn and adult mouse

Respiration Physiology (1978) 35, 271-282 © Elsevier/North-Holland Biomedical Press

REGULATION OF OXYGEN AFFINITY IN BLOOD OF FETAL, NEWBORN AND ADULT MOUSE

R. PETSCHOW, D. PETSCHOW, R. BARTELS, R. BAUMANN and H. BARTELS Physiologisches Institut der Medizinischen Hochschule Karl-Wiechert-Allee 9, Hannover, G.F.R.

Abstract. Oxygen half saturation pressure (Ps0) of blood and the role of 2,3 diphosphoglycerate (DPG),

adenosine-triphosphate and red cell pH regulating oxygen affinity were examined in fetuses (16,518,5 days of gestational age), neonatal (1-22 days post partum) and adult mice (Balb/c). The high oxygen affinity of fetal blood (Ps0 = 29 Torr at 37 'C, pH 7.4 and Pco.~ = 40 Torr) decreases to an average adult value of 41 Torr within two weeks after birth, accompanied by an increase of DPG-concentration from 0.2 M/MHb 4 to the average of 1.5 M/MHb 4. At a constant pHe of 7.4 red cell pH decreases from pH 7.3 to 7.18 from 18.5 days of gestational age to ten days post partum. Electrophoretic mobility and functional characteristics of purified fetal and adult hemoglobin were identical. Changes in oxygen affinity occur only due to organic phosphate concentration variations. A rapid replacement of large size fetal red cells by smaller adult cells after birth fairly coincides with the increase of the 2,3-DPG concentration. ATP

2,3-DPG Hematology

Perinatal changes Oxygen affinity Ps0

In most mammals the oxygen affinity of blood decreases rapidly after birth. Different mechanisms responsible for the change in oxygen affinity have been described. In species where fetal hemoglobin is synthesized changes in O2-affinity occur because the adult hemoglobin(s) replacing it have either a lower intrinsic O2-affinity as in ruminants (Blunt et al., 1971; Baumann et al., 1972), or as has been shown for human blood the adult hemoglobin shows a stronger interaction with allosteric effectors that reduce oxygen affinity (Bauer et al., 1968; Tyuma and Shimizu, 1969). In some species (rabbit, dog, rat, pig), where embryonic is replaced directly by adult hemoglobin, changes in O2-affinity during fetal and postnatal Accepted for publication 25 August 1978 I Supported by Deutsche Forschungsgemeinschaft SFB 146 B 8. 271

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R. PETSCHOW et al.

development are caused by alterations in the concentrations of allosteric effectors i.e. organic phosphates (Dhindsa et al., 1972; Baumann et al., 1973: Bunn and Kitchen, 1973; Tweeddale, 1973; Jelkmann and Bauer, 1977). In a previous study (Bauer et al., 1975) the properties of embryonic hemoglobins in the white mouse Balb/c were investigated on a molecular level. This study investigates in the same species hemoglobin properties and regulation by cofactors throughout fetal life until three weeks after birth. By adding the cofactors in physiological concentrations to hemoglobin solutions, whole blood characteristics for oxygen transport could be simulated satisfactorily.

Material and methods

Inbred strain Balb/c of white mouse were chosen for these experiments because the primary structure of embryonic as well as adult hemoglobin in this strain has been investigated recently (Melderis et al., 1974). As the embryonic period in mice ends at the 17th day of gestation (Witschi, 1956), blood was sampled from 16.5 and 18.5 days old fetuses respectively and from newborns up to 22 days of age after decapitation. Blood of adult mice was taken by heart puncture in halothane narcosis. Sodium heparinate powder was added to preve0t clotting. Blood samples were kept at 4 '~C until anal~Csis: The oxygen affinity of whole blood and 30 g ~o hemoglobin solutions was determined using the Lex 02 Con (Lexington Instruments, Waltham, MA). Samples of blood or 30 g~o hemoglobin solutions were equilibrated 20 min at 37 ~C and a Pco~. of 40 Torr with water vapor saturated gas mixtures of known composition, pH was measured with "a Radiometer microglass electrode type G 297/G 2 and pH meter 4 calibrated with Radiometer precision buffers. Ps0-values of Whole blood were corrected with a Bohr factor of -0.47 Alog Po:/ApH to plasma pH 7.4. The relation between red cell pH (pHi) and plasma pH (pile) depends on the concentration of organic phosphates in the red cell as well as on the amount and type of hemoglobin. To determine the relation between intra- and extracellular red cell pH, blood samples were equilibrated at half saturation oxygen pressure and centrifuged anaerobically. The packed cells were hemolysed by freezing and thawing and pH was measured as described above. The red cell 2,3 diphosphoglycerate concentration was determined by the method of Ericson and De Verdier (1972). The ATP-concentration was measured using the test-kit from Boehringer, Mannheim, Germany. To determine the hemoglobin pattern, isoelectric focussing of hemolysates from different developmental stages was performed at 4 'C with ampholine pH 6-8 (LKB Bromma) as described by Drysdale et al. (1971) using the equipment of ORTEC (Oakridge, Tenn.). Hemoglobin solutions were prepared as described by Berman et al. (1971). Packed red cells were hemolysed by adding distilled w~iter. Organic phosphates and inorganic phosphates were removed from the hemolysate chromatographically

BLOOD O2-AFFINITY CHANGES DURING MOUSE DEVELOPMENT

273

on Sephadex G 25 fine, equilibrated with Tris 0.05 M, NaC1 0.1 M, pH 7.6 at 4 °C. No phosphates could be detected after column passage. Two stock solutions of hemoglobin were prepared: (a) Hb 4 g ~ in 0.1 M NaCI and 0.05 M BisTris (Nutritional Biochemics Corporation, Cleveland, OH), pH 7.2 at 37 C ; (b) Hb 4 g ~o in 0.15 M NaC1. Oxygen equilibrium curves of 2 g ~o hemoglobin solutions were determined in a diffusion chamber at 37 C (Niesel and Thews, 1961; Sick and Gersonde, 1969) and in 30 g ~o hemoglobin solutions with the Lex 02 Con. The hemoglobin concentration was measured by the cyanmethemoglobin method (Kleihauer and Betke, 1957), and methemoglobin according to Evelyn and Malloy (1938). Red cell counts were performed by haemocytometer technique. For reticulocyte counts, blood smears were stained with brilliant cresyl blue. Price-Jones curves were constructed from directly measured red cell diameters (Price-Jones, 1933) using a Zeiss ocularmicrometer K 16 X. Results

The oxygen half saturation pressure (P50) in adult mouse blood is 41 Torr at 37 C , 40 Torr CO, and pile 7.4. Gray and Steadman (1964) found 41.5 Torr. The Ps0 value in fetal mice of 16.5 and 18.5 days of gestational age is much lower (29 Torr) increasing to adult levels within 14 days after birth (fig. 1). rorr

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274

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Hemoglobin electrophoresis. From day 16.5 of gestational age up to maturity we found no change in the hemoglobin type. Embryonic hemoglobins were no longer detectable from the 17th day of gestation onwards. Since identical electrophoretic mobility does not decisively prove the hemoglobins to be identical, Pb0 of 'stripped' hemoglobin solutions from fetal mice (18.5 days) was compared to that of adult mice under the same conditions. No difference was seen in Pb0 (fig. 2). Comparison o f Hb f r o m adult and f e t a l m o u s e 3l

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BLOOD O2-AFFINITY CHANGES DURING MOUSE DEVELOPMENT

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Concentration of organic phosphates. The concentration changes of ATP as well as DPG were traced starting day 16.5 of gestation up to the 20th and 26th day post partum. Whereas the concentration of 2,3-DPG remained low during the fetal period until birth, the concentration of ATP decreased continuously from 1 M ATP/M Hb 4 at day 16.5 to 0.35 M ATP/M Hb 4 at birth. After birth the ATPconcentration changed only slightly (fig. 3) whereas the 2,3-DPG concentration increased continuously from 0.2 M/M Hb 4 to the adult value of 1.5 M/M Hb 4 about 20 days after birth. At birth the sum of the intraerythrocytic concentration of ATP and 2,3-DPG as well as the intracellular hydrogen ion concentration reach a minimum which corresponds with the low Ps0 found at this period (figs. 4 and 5). Since the intracellular Bohrfactor of the various development stages could not be determined directly, due to the small size of the blood samples, we used the Bohr factor determined in 2-g ~o hemoglobin solutions to which ATP and 2,3-DPG had Blood ,4

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been added as required. While these values represent only approximations of the intracellular Bohr factor, the actual error introduced in the determination of Ps0 is small, since pH corrections covered only small pH intervals. The relation between intracellular and extracellular pH was derived from plots of pHi against pile in blood of fetal (16.5 days), newborn (4.5 days) and adult mice (table 1) and used to obtain estimates of the extracellular Bohr factor (B Fe). Oxygen affinity in concentrated hemoglobin solutions. Intraerythrocytic conditions were simulated using hemoglobin solutions in the physiological concentration range (30 g ~o) to which allosteric effectors were added in the respective stochiometric ratio characteristic for the developmental stage. Agreement between the Ps0 in hemoglobin solution and the Ps0 values for adult blood and blood obtained at 4.5 days post partum is good (table 1). The larger difference encountered at day 16.5 of development is due to the rapid fall in red cell ATP concentration during equilibration (from 0.9 M ATP/M Hb4 to 0.56 M ATP/M Hb4) whereas in Hb solutions the ATP concentration remained constant throughout the duration of the experiment. The differences in Ps0 between whole blood and dilute hemoglobin solution (2 g ~o) are due to the fact that following the law of mass action at a constant stochiometric ratio less 2,3-DPG or ATP is bound to hemoglobin in dilute than in concentrated solutions.

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RED BLOOD CELL PARAMETERS (figs. 6, 7 and 8) The red blood cell count increases from 0.7 x 106/gl at 16.5 days of gestation to average adult values of 9-10 x 106/~tl within the first 20 days post partum. The reticulocvtefraction at birth up to the 3rd postnatal day is around 250%0, decreasing to about 150%o in 4-6 day old animals. The mean corpuscular volume M C V rapidly decreases from about 280 gm 3 at the 16th day of gestation to about 100 gm 3 at birth, to 50 lain3 at day 12, then remaining fairly constant at that level. Adult values are around 47 gm ~. Price-Jones curves of red cell diameters show a pronounced shift to the left side, that means the appearance of an increasing percentage of smaller red cells towards adult MCD values of around 6.5 gin. Hemoglobin concentrations increase rapidly from 4.5 g/100 ml blood at the 16th day of gestation to 13 g/100 ml around birth. The hematocrit is 15'~Joat the 16th day of gestation going up to 40~'/0 at birth. Adult values are around 43°~,,o. Hemoglobin concentration and hematocrit values show a slight postnatal decrease below adult levels as described for most mammalian species (see Bartels, 1970).

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Discussion

The average weight of the mouse fetus increases from + 0.6 g to 1.5 g (Rugh, 1968) from day 16.5 until birth and fetal oxygen consumption has to rise accordingly. This requires an improvement of oxygen transfer across the placenta. The specific contribution of the fetal blood is twofold. In the first place the hemoglobin concentration nearly trebbles during this period from 4.5 g/100 ml blood to 13 g/100 ml blood with a corresponding increase in oxygen transport capacity from 6.3 to 17 ml O2/100 ml blood. A less pronounced increase in oxygen capacity has been observed in the rabbit (Jelkmann and Bauer, 1977) whereas no such change was observed in sheep (Meschia et al., 1970) and guinea pigs (Bartels et al., 1967). Secondly the oxygen affinity of fetal mice blood is always higher than that of maternal blood, with a difference of about 10 mm Hg in Ps0 at pH 7.4. Mice seem to belong to those mammalian species where embryonic hemoglobin is replaced directly by adult hemoglobin and this transition is completed by the 16th day of development. Although the final proof of the identity of the hemoglobins found in fetal blood with adult hemoglobin rests on primary structure analysis, the identidity of functional and electrophoretic behaviour suggest such a view. Changes in oxygen affinity during fetal and postnatal development are caused by concomitant changes in the concentration of the allosteric effectors ATP and 2,3-DPG (fig. 3). In fetal mice ATP is the main allosteric effector for the regulation of hemoglobin oxygen affinity. Its concentration decreases continuously throughout fetal life whereas the concentration of 2,3-DPG remains constant. Since the ATP concentration was observed to decrease substantially during tonometry one must conclude that the actual Ps0 is about 3 to 4 mm Hg higher at day 16 than at day 20 and fetal blood then has the highest oxygen affinity at birth coinciding with the minimum of the sum of the concentrations of ATP and 2,3-DPG. After birth the concentration of 2,3-DPG increases rapidly to reach the adult values at about 2 weeks post partum. Until now there is no explanation for the rapid increase of red blood cell 2,3-DPG concentration during the first two weeks after birth. It is possible that the erythropoietic changes occurring during fetal and postnatal development are involved in this. During fetal life erythropoiesis switches from the liver and spleen to the bone marrow, the latter site producing the mature adult erythrocytes which are characterized by a much smaller cell size compared to erythrocytes produced in the liver. An indirect reflection of the change in erythrocyte population is probably given by the drastic reduction in mean cellular volume from 250 lxm3 to about 50 lam3. However, these changes do not coincide with the period of rapid accumulation of 2,3-DPG, which makes it unlikely that the postnatal rise in 2,3-DPG is due to simple exchange of two cell populations one (i.e. those ceils coming from the

BLOOD O2-AFFINITY CHANGES D U R I N G MOUSE DEVELOPMENT

281

liver) characterized by a low concentration of 2,3-DPG and the other (i.e. cells produced by the bone marrow) having a high concentration of 2,3-DPG. On the other hand it could be that postnatally the 2,3-DPG synthesis is activated by changes in the intracellular environment which alter the activity of enzymes involved in 2,3-DPG synthesis. In this respect it is noteworthy that Jelkmann and Bauer (1978) found that erythrocytes from fetal rabbit blood had a much larger activity of pyruvate kinase than adult blood and the difference in acitivity was sufficient to account for the low levels of 2,3-DPG in fetal blood. A further resolution of this problem requires an improved characterisation of the different cell populations and their changes during maturation. Female mice had significantly lower 2,3-DPG concentrations compared to males, The Ps0 was, however, not significantly lower compared to Ps0 in male mice. One explanation could be that at the intraerythrocytic 2,3-DPG concentration found in female blood the hemoglobin is already saturated with 2,3-DPG so that a further increase as observed for males can decrease Ps0 only through its reduction of the intracellular pH (Duhm, 1971). However, this should be reflected in a difference of pHi for females and males, when pHi is related to constant pile. This was not observed. Therefore, we assume that the increased 2,3-DPG concentration in male erythrocytes compensates a concomitant increase of the concentration of impermeable extracellular anions.

References Bartels, H., D. E1 Yassin and W. Reinhardt (1967). Comparative studies of placental gas exchange in guinea pigs, rabbits and goats. Respir. Physiol. 2:149 162. Bartels, H. (1970). Prenatal Respiration. Amsterdam, North-Holland Publishing Compagny. Bauer, Ch., I. Ludwig and M. Ludwig (1968). Different effects of 2,3 DPG and adenosine triphosphate on the oxygen affinity of adult and foetal human hemoglobin. L(fe Sci. 7:1339 1343. Bauer, Ch., R. Tamm, D. Petschow, R. Bartels and H. Bartels (1975). Oxygen affinity and allosteric effectors of embryonic mouse hemoglobins. Nature 257:333 334. Baumann, R., Ch. Bauer and A. M. Rathschlag-Schaefer (1972). Causes of the postnatal decrease of blood oxygen affinity in lambs. Respir. Physiol. 15: 151-158. Baumann, R., F. Teischel, R. Zoch and H. Bartels (1973). Changes in red cell 2,3 diphosphoglycerate concentration as cause of the postnatal decrease of pig blood oxygen affinity. Respir. Physiol. 19: 153 161. Berman, M., R. Benesch and R. E. Benesch (1971). The removal of organic phosphates from hemoglobin. Arch. Biochem. Biophys. 145: 236-239. Blunt, M. H., J. L. Kitchen, S. M. Mayson and T. H. J. Huisman (1971). Red cell 2,3 diphosphoglycerate and oxygen affinity in newborn goats and sheep. Proc. Soc. Exptl. Biol. Med. 138: 800-803. Bunn, H.F. and H. Kitchen (1973). Hemoglobin function in the horse: The role of 2,3 diphosphoglycerate in modifying the oxygen affinity df maternal and foetal blood. Blood 42: 471-479. Dhindsa, D.S., A.S. Hoversland and J.W. Templeton (1972). Postnatal changes in oxygen affinity and concentration of 2,3 disphosphoglycerate in dog blood. Biol. Neonate 20 : 226-235. Drysdale, J. W., R. Piergiorgio and H. F. Bunn (1971). The separation of human and animal hemoglobins by isoelectric focussing in polyacrylamide gel. Biochim. Biophys. Acta 229: 42-50.

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Duhm, J. (1971). Effects of 2,3 diphosphoglycerate and other organic phosphate compounds on oxygen affinity and intracellular pH of human erythrocytes. Pfliigers Arch. Ges. Physiol. 326: 341-356. Ericson, A. and C.H. De Verdier (1972). A modified method for the determination of 2,3 diphosphoglycerate in erythrocytes. Scand. J. Clin. Lab. Invest. 29: 85-90. Evelyn, K.A. and H.T. Malloy (1938). Microdetermination of oxyhemoglobin, methemoglobin and sulfhemoglobin in a single sample of blood. J. Biol. Chem. 92:588 597. Gray, L.H. and J.M. Steadman (1964). Determination of oxyhemoglobin dissociation cdrves for mouse and rat blood. J. Physiol. (London) 175:161 171. Jelkmann, W. and C. Bauer (1977). Oxygen affinity and phosphate compounds of red blood cells during intrauterine development of rabbits, plliigers Arch. 372: 149-156. Jelkmann, W. and C. Bauer (1978). The low 2,3-DPG concentration in fetal rabbit erythrocytes is due to a high pyruvate kinase activity, pLTiigers Arch. Suppl. 373 : R 45. Kleihauer, E. and K. Betke (1957). Zur H~imoglobinbestimmung mittels Cyanh/imoglobin nach Betke und Savelsberg. )4rztl. Lab. 3:202 205. Melderis, H., G. Steinheider and W. Ostertag (19743. Evidence for a unique kind of a-type globin chain in early mammalian embryos. Nature 250: 774-776. Meschia, G., E. L. Makowski and F.C. Battaglia (1970). The use of indwelling catheters in the uterine and umbilical veins of sheep for a discription of fetal acid-base balance and oxygenation. Yale J. Biol. Med. 421 : 154. Niesel, W. and G. Thews (1961). Ein neues Verfahren zur schnellen und genauen Aufnahme der Sauerstoffbindungskurve des Blutes und konzentrierter H~moproteinl6sungen. Pfliigers Arch. 273: 380-395. Price-Jones, C. (19333. Red Blood Cell Diameters. London, Oxford Medical Publications. Rugh, R. (1968). The mouse. Minneapolis, MN, Burgess Publishing Company. Sick, M. and K. Gersonde (1969). Method for continuous registration of O2-binding curves of hemoproteins by means of a diffusion chamber. Anal. Biochem. 32:362 476. Tweeddale, P. M. (1973). DPG and the oxygen affinity of maternal and foetal pig blood and hemoglobins. Respir. Physiol. 19:12 18. Tyuma, J. and K. Shimizu (1969). Different response to organic phosphates of human fetal and adult hemoglobins. Arch. Biochem. Biophys. 129: 404-405. Witschi, E. (1956). The Development of Vertebrates. Philadelphia, W. B. Saunders.