Hemoglobin-ligand interaction in fetal and maternal sheep blood

Respiration Physiology (1978) 34, 185 194 © Elsevier/North-Holland Biomedical Press

HEMOGLOBIN-LIGAND INTERACTION IN FETAL AND MATERNAL SHEEP BLOOD l

M.P. HLASTALA, T.A. STANDAERT, R. L. F R A N A D A and H. P. McKENNA D~Tartmenls q[' Medicine, q[' Physiolo~,:v and Biophysic.s, and qf Pediatrics University 01' Washington, Seattle, Washington 98195, U.S.A.

Abstract. Hemoglobin ligand interaction was studied in maternal and fetal sheep blood as a function of oxygen saturation, pH was changed by varying CO, concentration (CO 2 Bohr effect) or by addition of N a O H or HCI at constant Pco~ (fixed acid Bohr effect). For maternal blood, CO~ Bohr factor was -0.41 at 50!~, oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. For fetal blood, CO, Bohr factor was - 0 . 4 5 at 50!~,i oxygen saturation, unchanging at lower saturation and decreasing in magnitude at higher saturation. Fixed acid Bohr lhctor was relatively saturation independent with a value of - 0 . 3 6 for fetal blood and - 0 . 2 7 lbr maternal blood. The pH-independent effect of molecular CO, on oxygen affinity was markedly saturation dependent being greatest at low oxygen saturation. The CO 2 effect was greater in maternal blood than fetal blood. However, the magnitude of the saturation dependency of Bohr factor is not great enough to have major physiological significance in oxygen transfer across the sheep placenta. Bohr effect Fetus Hemoglobin oxygen affinity

Placental oxygen exchange Sheep

Detailed analysis of gas exchange in the placenta has involved two major avenues in recent years: theoretical modelling and experimental animal preparations. Using the modelling approach, it has been shown by Hill et al. (1973) that oxygen transfer across the placenta is facilitated by carbon dioxide transfer and resultant hemoglobinligand interaction. As the maternal blood delivers oxygen, it also takes up CO~, thus shifting the maternal oxygen dissociation curve to the right (decreased oxygen affinity) and facilitating oxygen unloading. Fetal blood is simultaneously giving off Acc~Tted.fbr publication 20 March 1978 I This study was supported in part by U.S. Public Health Service Grants HL-12174, HL-14152, HD00095, and Research Career Development Award HL-00182. 185

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M.P. H L A S T A L A et al,

CO~ in the placenta as it is taking up oxygen facilitating that oxygen exchange. Hill et al. (1973) calculated that approximately 8%~ of the oxygen exchange is a result of this double Bohr effect. On the other hand, these authors calculated that 46"; of the CO, exchange is a result of the double Haldane effect in an analogous manner. Recently, there have been many studies into the details of the interaction of hemoglobin with its ligands in a great many animal species. It has been shown in studies with whole blood in our laboratory (Hlastala and Woodson, 1975) and by others (Garby et al., 1972; Arturson et al., 1974: Meier et al., 1974; Teisseire el al.. 1975) that the complex interactions between human hemoglobin and hydrogen ion, carbon dioxide and 2,3-diphosphoglycerate are strongly dependent on oxygen saturation. However, this saturation dependency has been studied only in man. Previous studies in sheep blood have documented the magnitude of the Bohr effect in both maternal and fetal blood but these data are limited to either 50'% saturation or calculated as an average over the entire saturation range masking any saturation dependency. Since the magnitude of the Bohr effect might significantly alter the O~ and CO2 exchange dynamics across the placenta and since most animal placental experimental preparations involve the sheep, this study was carried out to determine the saturation dependence of hemoglobin ligand interaction in both maternal and fetal sheep blood.

Methods

Heparinized samples of both fetal and maternal sheep blood were obtained from Suffolk ewes and fetuses (table 1) between two and six days before term. Fetal blood was obtained from umbilical catheters placed for another study. Blood was prepared by spinning down at 800 x g for 30 minutes. Plasma was drawn off and the buffy coat discarded. Red cells were then re-suspended in plasma. The procedure was then repeated again after spinning at 1,000 x g for 10 minutes. This effort to eliminate platelets helped to prevent accumulations on the electrode membrane. Oxygen dissociation curves (ODC) wei'e determined using the dissociation curve TABLE 1 Fetal N (sheep) Hb (g~;) Hct D P G (mol/mol Hb) N (samples) Ps0 ( m m Hg) n (Hill coefficient)

8 14.3 _+ 1.2" 0.43 + 0.03 1.08 +_0.09 53 16.1 2.35

Maternal 8 11.6_+1.1 0.29 Jr 0.04 0.07 _+0.03 101 35.5 2.68

* Mean + standard deviation.

BOHR EFFECT

IN S H E E P B L O O D

187

analyzer described by Duvelleroy et al. (1970). In this method blood is deoxygenated by tonometry with a nitrogen-CO2 gas mixture. A continuous curve is then inscribed by polarographic measurements as oxygen is taken up by the deoxygenated blood from an oxygen atmosphere. All experiments were performed at 37 "C. Blood pH was varied by two methods: (1) addition of isotonic (0.15 N) HC1 or NaOH (fixed acid titration). Prior to pH alteration plasma was separated from whole blood by centrifugation. HCl or NaOH was then added to plasma and the cells resuspended. ODC were determined at three pH values for each sample at a constant Pco: of 42 torr. (2) Varying CO, concentrations of the equilibrating gas (CO_, titration). 0 Three CO, concentrations were used for each sample: 2.5 O/~ .... 5.7~, and 8.5~o. Procedures (1) or (2) were usually performed on aliquots of the same blood sample. Pco: was measured before and after each run and found to change by less than _+2 torr. pH was measured before and after each run. pH at intermediate saturations was obtained by straight-line interpolation (Hlastala and Woodson, 1975; Hahn et al., 1976). Oxygen pressure at a given saturation for each experimental curve was then plotted semilogarithmically against pH, and the Bohr effect expressed as AlogPo~/ApH was calculated by least squares regression. Reproducibility of the method was assessed by determining P50 on 9 separate aliquots of the same blood sample with resulting standard deviation in Ps0 of 0.92 tort. Red cell organic phosphates were measured by the enzymatic method as modified by Detter et al. (1975). Hemoglobin concentration was determined as cyanmethemoglobin and hematocrit by the microhematocrit method.

Results

The linear relationship between log Poe and pH is shown in fig. 1 for all experimental ODC at 50% saturation. Bohr factor is the slope of the indicated regression lines and the 'standard Po,'* at 50% saturation is the Po: at pH = 7.40. For CO2 titration the Bohr factors for maternal (-0.41) and fetal (-0.44) blood are nearly identical. There is a considerable difference in Ps0; 34.8 torr for maternal and 15.3 torr for fetal. With fixed acid titration there is a difference of both Ps0 and Bohr factor. The fetal Bohr factor at 50% saturation of -0.36 is significantly greater (P < 0.001) than the maternal Bohr factor of -0.27. Ps0 was 36.1 torr for maternal and 16.9 torr for fetal blood. Since the 'standard Po~' obtained by regression of fixed acid data is slightly different from that obtained from CO, data, the two are averaged to obtain the actual 'standard Po_~'which is listed in tables 1 and 2. Bohr factors for each 10°/0 oxygen saturation are also in table 2. The relationship between CO: Bohr factor and oxygen saturation for both maternal and fetal sheep blood is shown in fig. 2. The maternal CO, Bohr factor is independent * S l a n d a r d Poe is the P % at a p a r t i c u l a r s a t u r a t i o n f o r p H = 7.a, b a s e excess = 0.0 meq/1, t e m p e r a t u r e = 37 C.

80

CO2 Titration

40"

~

~6

MOternal •

Po 2

°

•

(torr) o

20"

I0 80-

H + Titration

40-

~

%

*

Molerrl MolerrlQI

(torr) 8

20-

t0 68

o°

70

72

•

74

76

78

pH

Fig. 1. Po2 at 50?.; saturation (Ps0) is plotted on a log scale as a function of pH. Bohr l:actor (A log Po2/ApH) is indicated by slope of regression line.

TABLE 2 Saturation

A log Po~ BE ApH

A log Po2

A log Po_~1 A pH[

Pco,

'standard Pc)-'

A log P(o:

Maternal sheep blood 10 20 30 40 50 60 70 80 90

-0.48 -0.44 -0.42 -0.42 -0.4t -0.41 -0.40 -0.38 -0.34

± 0.05* ± 0.04 ± 0.04 ± 0.04 ±0.04 ±_ 0.04 ± 0.04 ± 0.03 ± 0.03

-0.25 -0.27 -0.27 -0.27 -0.27 -0.26 -0.26 -0.25 -0.24

± 0.03* ± 0.03 i 0.02 ± 0.02 ±0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.02

0.13 0.10 0.09 0.09 0.09 0.09 0.09 0.07 0.06

14.3 20.5 25.6 30.5 35.5 41.2 48.1 57.6 74.5

-0.45 -0.45 -0.43 -0.44 -0.44 -0.43 -0.40 -0.39 -0.37

4- 0.07 ±_ 0.05 ± 0.05 ± 0.04 ± 0.04 ± 0.04 ± 0.04 ± 0.04 ± 0.05

-0.34 -0.36 -0.36 -0.37 -0.36 -0.36 -0.36 -0.36 -0.35

+ 0.04 _+0.03 +_0.03 ± 0.02 _+0.02 ± 0.03 ± 0.03 ± 0.03 ± 0.04

0.06 0.05 0.04 0.04 0.04 0.04 0.02 0.02 0.01

6.2 8.9 11.2 13.5 16.2 19.0 23.0 28.7 40.3

Fetal sheep blood 10 20 30 40 50 60 70 80 90

* Mean ± standard error.

189

B O H R E F F E C T IN S H E E P B L O O D

COz Bohr Factor -.8-.6zxlog Po2

z~pH _I4. Maternal

-.20

2'o 4b

0

16o

Saturation (%) Fig. 2. C O 2 Bohr factor v s . oxygen saturation for maternal and fetal blood. Bars indicate + 1 SE of slope of the regression line. Range of pH for maternal and fetal data, respectively, was 7.14 to 7.66 and 6.89 to 7.52.

of oxygen saturation over most of the range. However, there is a slight increase at 10~ saturation and a decrease at greater than 75~o saturation. The difference between CO2 Bohr factor at 10~ and 90~o saturation is significant at the P < 0.001 level (Student t-test). This is very similar to the relationship found in normal adult human blood (Hlastala and Woodson, 1975). The fetal blood CO2 Bohr factor is nearly constant over the entire range with a slight but insignificant decrease at oxygen saturations above 65,°/o. The fixed acid Bohr factor for both maternal and fetal sheep blood is shown in fig. 3. In this case the Bohr factor is relatively saturation independent for both maternal and fetal blood. There is a slight, but statistically insignificant, decrease in Bohr factor at both 10~o and 9 0 ~ for both bloods. The maternal H + Bohr factor is less (P < 0.001) than the fetal H + Bohr factor at all saturations. Again, these curves resemble but are not precisely the same as the analogous measurements in whole adult human blood (Hlastala and Woodson, 1975).

H+

8ohr Factor

-.8-

-.6-

A 10g P02 ~pH

Fetal

--.4-.20 0

Maternal

2'o 4'o ob

ob

Saturation (%)

Fig. 3. Fixed acid Bohr factor vs. oxygen saturation maternal and fetal blood. Bars indicate _+ 1 SE of slope of the regression line. Range of pH for maternal and fetal data, respectively, was 7.07 to 7.84 and 7.07 to 7.66.

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M.P. HLASTALA el al,

14-

C02

Effecl

12IOA log Poz A log Pco2.08 .0604020

0

~b

4b

do ~b 1do

Soturation (%) Fig. 4. Effect of CO~ on oxygen affinity at constant pH(A log Po./A log Pco~) vs. oxygen saturation lbr maternal and fetal blood.

The CO2 Bohr effect is a sum of effects on oxygen affinity of the carbamino reaction and of pH change mediated through the hydration of CO2, whereas the fixed acid Bohr effect is due to the change in hydrogen activity alone. The two Bohr factors can then be used to calculate the influence of molecular CO~ via carbamino formation on oxygen affinity (Hlastala and Woodson, 1975) as shown in fig. 4. The molecular CO2 effect is much greater at very low saturations than at high saturations. And this effect is much more pronounced in maternal blood than in fetal blood.

Discussion The oxygen dissociation curve for sheep blood has been studied by numerous authors (Battaglia et al., 1970; Bauer and Jung, 1975; Baumann et al., 1972: Blunt et al., 1971 ; Hilpert et al., 1967; Huisman and Kitchens, 1968; Meschia et al., 1961 ; Naughton et al., 1963; Parer et al,, 1967). A Ps, varying between 27.7 torr and 44.7 torr has been reported. Some of this variability is because of experimental inaccuracy, some because of temperature differences, and some is caused by difference in hemoglobin type. Electrophoretically distinct forms of hemoglobin occur in adult sheep. In adult Dorset sheep, type A hemoglobin has a Ps0 of 26.8* tort and type B hemoglobin has a Ps, of 36.7* torr (Naughton et al., 1963). Heterozygotes 15ave mixtures of the two with resultant intermediate Ps0. In addition, Huisman and Kitchens (1968) identified a type C which is produced in severe anemia and has O, dissociation properties similar to type A. In our study, the mean Ps0 of individual maternal blood samples was 36.3 _+ 1.1 torr (mean + SD) with a range of 34.2 to

B O H R E F F E C T IN SHEEP B L O O D

191

37.8 torr. There may have been some mixture of hemoglobin types but we were unable to analyze this. No data is available in the literature concerning variability of hemoglobin types in Suffolk sheep which were used in this study. We have found only four papers which have measured the P50 of fetal sheep blood which varies between 15.5" torr (Meschia et al., 1961) and 15.7" torr (Naughton etal., 1963) to 18.0"* torr (Blunt et al., 1971) and 18.8" torr (Battaglia et al., 1970). Our value of 16.0 (table 1) is within the range reported. DPG concentrations reported in table 1 are similar to previous reports. Our value of 0.07 mol/mol Hb compares with 0.07** mol/mol Hb (Battaglia et al., 1970), 0.02** mol/mol Hb (Baumann et al., 1972) and < 0.02** mol/mol Hb (Bunn, 1971) in adult sheep blood. In fetal sheep blood, our value of 1.08 mol/mol Hb compares with 2.0** mol/mol Hb (Baumann et al., 1972) and 0.53** mol/mol Hb (Battaglia et al., 1970). The greater variation in reported fetal DPG levels may be related to large changes in DPG immediately after birth (Battaglia et al., 1970; Baumann et al., 1972). Several authors have measured the Bohr factor at 50~ saturation for both adult and fetal sheep blood. Values for CO, Bohr factor in adult sheep blood vary between -0.34 (Meschia et al., 1969) to -0.49 (Huisman and Kitchens, 1968). Our value of -0.42 (at 50~ saturation) falls well within that range. The adult H + Bohr factor has been determined as -0.25 by Baumann et al. (1975) and between -0.21 and -0.34 by Huisman and Kitchens (1968). Our value of -0.27 falls in the middle of that range. For fetal blood, CO2 Bohr factors have been determined by three authors: -0.32 (Meschia et al., 1969), -0.49 (Meschia et al., 1961) and -0.71 (Huisman and Kitchens, 1968). This is a wide range but our value of -0.44 agrees more closely with the earlier value of Meschia. Only one value for fetal hydrogen ion Bohr factor appears in the literature and that is the value of -0.38 measured by Huisman and Kitchens (1968). There is a good deal of variability in the results reported in the literature, which is partly related to methodologic differences. In addition, the measurement of Bohr factor is subject to a certain degree of measurement error. The standard error of estimate of our regression slopes (table 2) varied between 0.02 and 0.07 which was probably typical of the other studies although little information as to the regression errors is available in that literature. These results are similar in many ways to the results obtained in human blood. Typically, it has been shown that the CO2 Bohr effect is dependent on oxygen saturation. This is less evident in sheep blood (fig. 2). The saturation dependency of the Bohr effect is much smaller. There is very little change in Bohr factor from 10°/oto 60~o but there is a statistically significant decrease at the very high saturations. As in human blood the fixed acid Bohr factor (fig. 3) is essentially independent of oxygen saturation. Maternal sheep blood fixed acid Bohr factor is approximately 75°/, of the fetal sheep blood Bohr factor. This might be expected from human blood * Corrected to 3 7 C using the Van "t H o f f p l o t for sheep hemoglobin from Bauer and Jung (1975). ** Calculated from the form of presentation in paper.

192

M . P . H L A S T A L A ~'I al.

data because of the extremely low concentration of DPG in maternal sheep blood (0.07 mol/mol Hb). However, it has been shown that there is only minimal interaction between DPG and sheep hemoglobin (Baumann et al., 1972; Blunt et al., 1971; Bunn, 1971) implying that the difference in fixed acid Bohr l:actor between maternal and fetal blood is related to molecular differences or intracellular pH differences rather than DPG concentration. The interaction of CO~ with hemoglobin is also different between maternal and fetal blood. In maternal blood, the effect of COt on oxygen affinity is saturation dependent. These data are like that in adult human blood depleted of DPG (Hlastala and Woodson, 1975). In fetal sheep blood, the COt effect on oxygen affinity is similar but of reduced magnitude. This relationship has also been seen by Arturson et al. (1974) in human blood but is in contrast to the results of Baumann el al. (1975) in sheep blood. Baumann found that oxygen linked carbamate formation was constant as a function of oxygen saturation. They were unable to demonstrate a change of shape of the oxygen dissociation curve when plotted as a Hill plot, which is consistent with our finding of a fairly flat saturation dependency ot" the CO, Bohr factor (fig. 2). However, Baumann et al. carried out their experiments with very dilute hemoglobin solutions rather than whole blood. It is interesting that our findings show a significant oxygen saturation dependence of the direct COt effect. In human blood this has been thought to be related to DPG binding on the/~ chain and subsequent interaction with oxygen affinity at higher oxygen saturation (Hlastala and Woodson, 1975). Another interesting finding is the lowered CO, effect in fetal blood which might be consistent with the high DPG levels ( 1.08 tool/tool Hb) in fetal blood. However, it has been shown by Bauer and Jung (1975) and Blunt et al. (1971) that there is very little interaction between sheep hemoglobin and DPG presumably because of a deletion of the N-terminal valine on fl chain of ruminant hemoglobins (Bunn, 1971 ). There is. however, a high concentration of DPG in the fetal erythrocytes and it may be that sufficient binding is taking place to alter the CO~ binding properties. However, the reduced COt effect in fetal blood may be due to inherent molecular differences in fetal hemoglobin. The oxygen transfer across the placenta of the sheep can be represented with the fetal and maternal oxygen dissociation curves in fig. 5. For both cases an arterial and venous curve is represented (an entire oxygen dissociation curve at the specific pH and Pco: of either arterial or venous blood). The fetal dissociation curve plateaus at a higher content because of its higher hemoglobin concentration than the maternal blood. Also shown are typical arterial and venous points lbr both the maternal uterine circulation and the fetal umbilical circulation taken from the data of Comline and Silver (1975) (table 3). Using these curves, the contribution of the Bohr effect to oxygen exchange can be calculated to be 6.70o in the maternal blood and 15.5'!,, in the fetal blood. These results indicate a weaker Bohr mechanism in fetal sheep blood than in human fetal blood. Our previous studies (Hlastala and Woodson, 1974) indicate a greater COn Bohr effect in human fetal blood averaging -0.58 at 50"41 saturation.

BOHR EFFECT IN SHEEP BLOOD

20]

193

Fetal

u~bi,i~ol

] Vein ///T

O,

o

Maternal

2'o 4b

& PO2

sb ldo (torr)

Fig. 5. Oxygen dissociation curves for fetal and maternal sheep blood. Using data from table 3. Arterial and venous points are shown for both umbilical and uterine circulations.

TABLE 3 Placental blood gases Poe (torr)

P(o~ (torr)

pH

CO, * (ml/100 ml)

Maternal circulation uterine artery uterine vein

98.9 53.1

33.6 37.9

7.478 7.440

15.2 12.1

Fetal circulation umbilical vein umbilical artery

34.8 22.8

41.5 45.7

7.399 7.346

17.4 13.0

Data taken from Comline and Silver (1975). * Calculated using hemoglobin concentrations measured in our experiments of 11.6 gi!,, for maternal blood and 14.3 g!l,, for fetal blood.

T h i s is largely due to a greater m o l e c u l a r CO2 effect at 50~o s a t u r a t i o n o f 0.077 c o m p a r e d with 0.042 in fetal sheep blood. This m a y be a result o f the low intera c t i o n o f h u m a n fetal h e m o g l o b i n with D P G in spite o f the high fetal D P G conc e n t r a t i o n ( a p p r o x i m a t e l y 1.0 m o l / m o l Hb).

Acknowledgement T h e a u t h o r s wish to t h a n k Dr. J i m D e t t e r for m e a s u r e m e n t o f D P G c o n c e n t r a t i o n s .

194

M.P. H L A S T A L A et al.

References Arturson, G., L. Garby, B. Wranne and B. Zaar (1974). Effect of 2,3-diphosphoglycerate on the oxygen affinity and on the proton- and carbamino-linked oxygen affinity of hemoglobin in h u m a n whole blood. Acta Physiol. Stand. 9 2 : 3 3 4 340. Battaglia, F.C., H. McGaughey, E. L. Makowski and G. Meschia (1970). Postnatal changes in oxygen affinity of sheep red cells: a dual role of diphosphoglyceric acid. Am. J. Physiol. 219 : 217 22 I. Bauer, C. and H. D. Jung (1975). A comparison of respiratory properties of sheep haemoglobin A and B. J. Comp. Physiol. 102:167 172. Baumann. R., C. Bauer and A . M . Rathsclag-Schaefer (1972). Causes of the postnatal decrease of blood oxygen affinity in lambs. Respir. Physiol. 15:151 158. Baumann, R.. C. Bauer and E.H. Hailer (1975). Oxygen-linked CO 2 transport in sheep blood. Am. J. Plo'siol. 229: 334~339. Blunt, M. H.. J. L, Kitchens, S. M. Mayson and T. H.J. Huisman (1971). Red cell 2.3-DPG and oxygen affinity in newborn goats and sheep. Proc. Soc. Exp. Biol. Med. 138 : 800 803. Bunn, H . F . (1971). Differences in the interaction of 2,3-diphosphoglycerate with certain m a m m a l i a n hemoglobins. Science 172:1049 1050. Comline, R. S. and M. Silver (1975). Placental transfer of blood gases. Br. Med. Bull. 31 : 25 31. Detter, J.C., D . F . Gibson. S.F. MacMillan and T . H . Oas (1975). Manual determination of intraerythrocytic 2,3-diphosphoglycerate in whole blood by enzymatic analysis, and comparison with ion-exchange chromatography. Clm. Chem. 2l: 376 380. Duvelleroy, M. A., R . G . Buckles, S. Rosenkaimer, C. T u n g and M. B. Laver (1970). An oxyhemoglobin dissociation analyzer. J. Appl. Physiol. 28:227 233. Garby. L.. M. Robert and B. Zaar (1972). Proton- and carbamino-linked oxygen affinity of normal h u m a n blood. Acre Physiol. Stand. 84: 482. Hahn, C. E. W.. P. Focx and C. M. Raynor (1976). A development of the oxyhemoglobin dissociation curve analyzer. J. Appl. Physiol. 41: 259-267. Hill, E. P., G. G. Power and L.D. Longo (1973). A mathematical model of carbon dioxide transfer in the placenta and its interaction with oxygen. Am. J. Physiol. 224:283 299. Hilpert, P., R. G. Fleischmann, D. Kempe and H. Bartels (1963). The Bohr effect related to blood and erythrocyte pH. Am. J. Pto'siol. 205 : 337 340. Hlastala, M. P. and R. D. Woodson (1974). Oxygen delivery in the fetus : influence of CO, and the Bohr effect. Proc. X X V I hTt. Congr. Physiol. Sci. 11 : 237. Hlastala, M.P. and R . D . Woodson (1975). Saturation dependency of the Bohr effect: interactions a m o n g H + , CO~ and DPG. J. Appl. Physiol. 38:1126 1131, Huisman. T. H. J. and J. Kitchens (1968). Oxygen equilibrium studies of the hemoglobin from normal and anemic sheeps and goats. Am. J. Physiol. 215:140 146. Meier. U.. D. Boning and H.J. Rubenstein (1974). Oxygenation dependent variations of the Bohr coefficient related to whole blood and erythrocyte pH. p/liigers Arch. 349:203 213. Meschia, G., A. Hellegers. J.N. Blechner, A.S. Wolkoff and D . H . Barron (1961). A comparison ot" the oxygen dissociation curves of the bloods of maternal, fetal and newborn sheep at various pHs. Q. J. Exp. Physiol. 4 6 : 9 5 100. Meschia, G.. F. C. Battaglia, E. L. Makowski and W. Droegemueller (1969). Effect of varying umbilical blood 02 affinity on umbilical vein PO,. J. ,4ppl. Physiol. 26:410 416. Naughton, M . A . , G. Meschia, F.C. Battaglia, A. Hellegers, H. Hagopian and D . H . Barron (1963). Hemoglobin characteristics and the oxygen affinity of the bloods of dorset sheep. Q. J. Exp. Physiol. 48:313 323. Parer, J.T., W . D . Jones and J. Metcalfe (1967). A quantitative comparison of oxygen transport in sheep and h u m a n subjects. Re~])ir. Physiol. 2 : 19(~206. Teisseire, B., L. Teisseire, R. Herigault and C. Soulard (1975). Methode d'enregistrement en continu sur micro-6chantillons de la courbe d'association Hb-O,. 11. Etude de l'effet Bohr et de la carbaminoforrnation. Btdl. Physiol.-Pathol. Re.v~ir. 11:853 862.