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Comparative studies of oxygen equilibria of human adult and cord blood red cell hemolysates and suspensions
1%
CLINICA CHIMICA ACTA
COMPARATIVE OF HUMAN
STUDIES
ADULT
HEMOLYSATES CARL
EQUILIBRIA
CORD BLOOD
AND
M. NECHTMAN
Department
OF OXYGEN
AND
AND
RED
CELL
SUSPENSIONS
TITUS
H. J. HUlSMAN
of Biochemistry, Medical College of Georgia, Augusta, Ga. (U.S.A.) (Received
November
rqth, 1963)
SUMMARY
The oxygen equilibria of suspensions and hemolysates of human adult and cord blood erythrocytes have been compared. Dialyzed hemolysates of adult and cord blood red cells showed small, but distinct, differences in the Bohr effect. Changes in phosphate molarity of the buffer used for the dialysis resulted in identical changes in oxygen affinities. Suspensions of cord blood erythrocytes in 0.9 g y0 NaCl showed a marked increase in oxygen affinity when compared with similar suspensions of adult red blood cells. The same difference in the Bohr effect was observed as seen for dialyzed hemolysates. The oxygen equilibria of concentrated undialyzed hemolysates of cord blood erythrocytes were almost identical to those of cord blood cell suspensions; the Bohr effect, however, was increased. The equilibrium between oxygen and the corresponding adult hemolysates was notably changed; the affinities of these hemolysates were found higher than that of either cord blood cell suspensions or hemolysates. The oxygen affinities of red cell suspensions from a heterozygous carrier of the persistent high Hb-F anomaly with 34% Hb-F were indistinguishable from those of normal adult erythrocyte suspensions. The addition of notable quantities of KCN and of NaF to the 0.9 g% NaCl solution did not affect the oxygen affinities of suspensions of both adult and cord blood erythrocytes. Hypotonic cell suspensions (in 0.6 g% NaCl) demonstrated increased oxygen affinities and a slight decrease in the Bohr effect; the effect of hypotonicity was the same for both cell types. Treatment of cord blood erythrocytes with a hypertonic solution (2.3 g% NaCl) resulted in decrease of the oxygen affinity. A similar treatment of adult erythrocytes increased the oxygen affinity, especially at pH values of 6.9 and higher. The significance of these findings has been discussed. Our studies suggest that differences in cell membrane or cell organization may likely contribute to the observed difference in oxygen affinities of human adult and cord blood erythrocytes. The presence of a specific fetal hemoglobin in the erythrocyte influences the Bohr effect and possibly the pH inside the cell, but not the oxygen affinity.
Clin. Chins. Acta, IO (1964) 165-174
166
C.M. NECHTMAN,T. H. J. HUISMAN
The important functional difference of the fetal and adult hemoglobins of many mammalian species is the increased affinity of the fetal hemoglobin for molecular oxygen. This increased oxygen affinity of the hemoglobin of fetal blood is commonly considered to be advantageous in the transfer of oxygen from the maternal to the fetal circulation. Such differences-as have, for instance, been observed for the goat l-3, the sheep 4fs and the cowB-are demonstrable when comparisons are made either between prepared solutions of the hemoglobins or of cell suspensions. Since these Hb-A and Hb-F types exhibit marked differences in physical and chemical properties (reviewed in’), it is generally assumed that structural differences between the Hb-A and the Hb-F within one species are expressed in variation of oxygen equilibria. In the human the oxygen affinity of cord blood (and also of cord blood suspension) is slightly higher than that of adult blood s- 14. Upon hemolysis of the cells and subsequent dialysis of the hemolysates, the fetal oxygen dissociation curve is changed only slightly, while the adult oxygen dissociation curve is changed so far to the left that no notable difference between the oxygen affinities of these solutions is detectables~14*1s. It seems, therefore, that the observed differences in oxygen equilibria of human cord and adult blood may not result from the many differences in the amino acid sequences of the two proteins. Environmental influences have been suggestedIS. Such an influence is likely to be effective through differences in the environment of the hemoglobin molecule within the cell, since possible effects of fetal plasma on the oxygen equilibria have been excluded by the experiments of ABRAHAMOVAND SMITI-I’~and by the observation that washed fetal red blood cells showed the same relatively high affinity for oxygen as whole cord bloods. In this communication we should like to report additional comparative studies of the oxygen equilibria of human adult and cord blood samples. Our studies were primarily oriented in the following four directions: (a) An investigation was undertaken to determine possible differences in the Bohr effect of fetal and maternal red cell suspensions and dialyzed red cell hemolysates, since contradictory observations have been reported in the literature 13*14. (b) Possible influences of “dialyzable factors” present inside the cell membrane were studied by comparing the oxygen equilibria of undialyzed, concentrated hemolysates of adult and cord blood erythrccytes with those obtained for the corresponding cell suspensions. The effect of increased phosphate levels in the environment was also studied. (c) In view of known differences in metabolic or enzymatic activities I691’ oxygen equilibria of cord blood and adult erythrocytes being suspended in media containing enzyme inhibitors have been investigated. (d) The possibility that differences in membrane’ permeability may be responsible for the observed differences in oxygen equilibria was investigated by comparing the oxygen affinity of cord blood and adult red cell suspensions in hypoand hypertonic salt solutions. MATERIALANDMETHODS Samples of blood from the umbilical cord, taken at birth, were obtained through courtesy of the Departement of Obstetrics and Gynecology of the Medical College of Georgia. Three adult members of a family described before18, being heterozygous for the persistent high Hb-F anomaly (PHF), served as donors of blood samples with 34% Hb-F. Blood samples from healthy normal adults (laboratory workers) Clin. Chim.
A&,
xo (1964)
165-174
0 EQUILIBRIAOF RED CELL HEMOLYSATES
167
were collected simultaneously. In all instances, the blood samples were processed within 12 h after the collection. Samples from either source were centrifuged and the cells washed three times with 0.9 g% NaCl solution. Hemolysis was carried out either by addition of an equal volume of distilled water and 0.4 volume of toluene or by freezing and thawing of cell-water (I : 0.6) mixtures. The stromata were removed by centrifugation at high speed in the cold. The “toluene-treated” hemolysates of cord blood erythrocytes and of normal red blood cells were dialyzed simultaneously against a large quantity (1000 ml VCYSUS IO ml of each hemoglobin solution) of phosphate buffer at 4” for 24 h. The molarities of these buffer solutions varied between 0.01 and 0.2 with pH values ranging from 6.8 to 7.8. The hemoglobin concentration of each sample was adjusted with the appropriate buffer solution to 5 g/roe ml. The hemolysates prepared by freezing and thawing were studied without additional treatment; the final hemoglobin concentration varied between 17 and 20 g/roe ml. Suspensions of cells from different sources in NaCl solutions of three concentrations (0.6, 0.9 and 2.5 g%), in 0.9 g% NaCl containing 300 rng% glucose, and in 0.9 g% NaCl containing IO-~ M KCN and IO-~ M NaF, were prepared simultaneously after two additional washings with the appropriate solution. The packed cell volumes were adjusted to 48 h 2%. Construction of the oxygen dissociation curves at 37” was performed by the technique of BRINKMANAND DIRKENIs. The determinations of the percent oxygen saturation were made with the use of a Zeiss Spectrophotometer (model PMQ-II) following the procedure described by JONXIS AND BOEVE~O.The PH measurements were carried out on a “Radiometer” pH meter, model 4, at a temperature of 25’. The values obtained were corrected to 37’ by means of the factor given by ROSENTHAL~~.All analyses were completed within 48 h after drawing of the samples. From the observed oxygen dissociation curves approximately linear relationships were attained by plotting log [r/(100-y)] ag ainst the log PO,, y being thepercentualoxygen saturation determined. From these linear relationships two constants were calculated, namely, n, a measure of the heme-heme interaction, which is the slope of the curve, and log Pso, the value of the PO, at which 50% saturation is observed. The P,, values have also been plotted as function of PH. The value 4, calculated from 4 = d log P,,/d pH and evaluated at PH 6.6 to 7.6, was used as a measure of the Bohr effect 22. The percentages of Hb-F were determined following the spectrophotometric procedure described beforeZ3. Red cell indices, when required, were measured following standard hematological techniques. RESULTS Fig. I presents the results of the oxygen equilibria of fresh hemoglobin solutions of normal adult and cord blood erythrocytes, after dialysis against 0.1 M phosphate buffers of varying pH values. The final hemoglobin concentration was 5.0 & 0.3 g/IO0 ml. All oxygen measurements were performed after equilibration with oxygen at different pressures (10-60 mm Hg) in 150 ml tonometers, at 40 mm pC0, and at 37”. The results are expressed as the logarithms of the P,, values. Our data indicate a slight but distinct difference in the Bohr effect of the two types of hemolysates. The constant 4, reflecting the magnitude of the Bohr effect, is 0.40 for dialyzed Cl&. Chim.Acta,IO(x964)165-174
168
C. M. NECHTMAN,
T. H. J. HUISMAN
hemolysates of adult erythrocytes and 0.47 for dialyzed hemolysates of cord blood red cells. The oxygen affinities of both types of hemolysates are approximately the same at pH’S between 7.0 and 7.4; below PH 7.0 human cord blood hemoglobin has a distinctly lower affinity for oxygen. The hemc-heme interaction, expressed as the constant n in Hill’s equation, is also significantly diff srent ; the n value obtained for adult hemolysates is 2.90 f 0.05 (rg determinations) and that for cord blood hemolysates 2.65 * 0.1 (14 determinations). Oxygen dissociation curves of five samples of a hemolysate prepared from red cells of a heterozygous carrier of the persistent high Hb-F anomaly (PHF) showed P,, values, being intermediate between those found for the adult and cord blood hemolysates. The mean value of the constant n was 2.75 & 0.1. I
Adult Hb Cord blood Hb 175% Hb-FI Hb 01 PHF csrrler 134’S Hb-FI
n
6.4
b!b
I
6.8
l!O
7!2
7!4
7.‘b
pH l37’CI
Fig. I. The oxygen affinities of dialyzed hemolysates at different pn values.
Possible influences of differences in phosphate concentration were studied by analyzing the oxygen equilibria curves of cord blood and adult erythrocyte hemolysates, which were simultaneously dialyzed versus large quantities of sodium phosphate buffers of varying molarities (0.01, 0.025, 0.05, 0.1 and 0.2 M) and a pH of 7.2-7.6. The log P,, values, which were obtained after equilibriation of the hemolysates with oxygen at various tensions and at 40 mm Hg pCO,, were corrected to pH 7.0 (37’) by means of the 4 values determined in the previously described experiments. Table I summarizes the results. In all experiments, the log P,, values for fetal hemolysates are similar to those found for adult hemolysates. Both types of hemolysates show the same increase in oxygen affinity by decreasing the phosphate molarity, which is in accordance with the findings of ROSSI-FANELLIet aLza~zs. The difference in the constant n of Hill’s equation was also observed in these experiments. Fig. 2 shows data derived from the oxygen dissociation curves of adult erythrocytes and of cord blood red cells, both being suspended in 0.9 g% NaCl. The mean values for total hemoglobin in g %, packed cell volume in o/0 and RBC in IO& per ~1 were 16.6, 4g and 5.15 for the adult cell suspensions, and 15.2, 47 and 4.18 for the Clin.Chim.Acta,
IO (1964)
165-174
0
EQUILIBRIA
OF RED CELL HEMOLYSATES TABLE
THE
OXYGEN
UPTAKE
OF ADULT SODIUM
Molarity of phosphate
AND
CORD
PHOSPHATE
I
BLOOD
BUFFERS
RED
CELL
OF DIFFERENT
0.01
0.1
0.2
* Total hemoglobin of five determinations.
Ps
AGAINST
Covd’ log pa0
constantn
2.5 2.7 2.45 2.55 2.6
1.138 I.196
2.3 2.3 2.25 2.3 2.3
concentration
DIALYZED
MOLARITIES
constant n
I.210
1.231 1.265 5.0 * 0.3 9%;
the values
a 4
HEMOLYSATES
Adult *
0.025 0.05
1%
M M O----e D---m
presented
log pa0 I.143 1.200 I.219
1.240 I.275 are mean values
Cellsof PHF wrriw Adult cells Cord blood cells Cow. undialyxd hemol. Adult cells Cont. unditlped hemol. cord bload cells
pH I3r00
Fig.
2.
The oxygen
affinities of red blood cells suspended in 0.9 9% NaCl and of undialyzed concentrated hemolysates at different PH values.
cord blood cell suspensions. The oxygen dissociation curves were determined after equilibriation of these suspensions with oxygen at various tensions and at a pC0, varying from o to IOO mm Hg in different experiments. The pH values (at 37’) after equilibration with these gas mixtures varied from 7.28 to 6.63 for the adult cell suspensions and from 6.99 to 6.43 for the cord blood cell suspensions. A marked increase in oxygen affinity of the fetal cell suspensions can be noted, the difference being 8.5 mm 0, at a pH of 7.0. A similar difference in the Bohr effect as found for dialyzed hemolysates is demonstrable; the 4 value is 0.405 for adult cell suspensions and 0.47 for cord blood cell suspensions. The oxygen dissociation curves of the suspensions of erythrocytes obtained from a heterozygous PHF carrier, were determined at 20, 40 and 80 mm Hg pC0,. The results do not differ from those found for normal adult cells, despite the presence of 34% of Hb-F. The mean values of the constant 12 are 2.0 f 0.1 for adult cells (17 determinations), 1.7 f 0.15 for cord blood cells (16 determinations) and 2.1, 2.1 and 2.3 for the cells of the PHF carrier indicating a difference between adult and fetal erythrocyte suspensions. Clin. Chim. Acta, IO (1964)
165-174
C. M.
170
NECHTMAN,
T.
H.
J. HUISMAN
The results obtained in studying the oxygen equilibria of undialyzed concentrated hemolysates are also presented in Fig. 2. The P,, values were derived from oxygen dissociation curves, determined as described for cell suspension. The total concentrations of the hemolysates varied between 17 and 20 g %. Several observations may be made upon the data of Fig. 2. The oxygen affinities of the cord blood hemolysates are closely similar to those found for the corresponding cell suspension. The Bohr effect is increased; the 4 value is 0.57 versus 0.47 for the cord blood cell suspension. The constant n in Hill’s equation is not different, namely 1.65 f 0.1 (7 determinations). The affinities of adult hemolysates, on the contrary, are higher than those found for the corresponding cell suspensions. The log P,, values at a pH of 6.8 and lower are significantly lower than those of the concentrated cord
hemoglobin
TABLE IHE OXYGEN
UPTAKE
OF ADULT
AND
CORD
BLOOD
Tl CELLS,
SUSPENDED
IN
DIFFERENT
MEDIA
(expressed as log PC,,)
Type of
hemolysate Adult Adult Adult Adult Cord Cord Cord Cord
Pff 7.20 7.00
6.85 6.75 6.85 6.75 6.60 6.45
0.9 g yO NaCl 1.28 1.38
0.9 g 0h NaCl $ 300 mg yO glucose
0.9 g yO NaCl + IO-~ M KCN $ IO+ M NaF
_
1.32
I.43 1.50 I.25 I.29
I.355 1.42 1.48 _ I.29
I.35 1.42 1.48 1.215 1.24
I.345 I .45
I.35 * .42
I.315 -
blood hemolysates and, probably, also lower than those of cord blood cell suspensions. The Bohr effect and the heme-heme interactions are not markedly altered, the 4 value is 0.38 (0.405 for adult cell suspensions), while the constant n in Hill’s equation is 2.05 f 0.1 (6 determinations). The possibility that differences in metabolic or enzymatic activity within the cell may contribute to the increased oxygen affinity of cord blood cell suspensions was investigated by comparing oxygen equilibria of cells suspended in 0.9 g o/oNaCl, in 0.9 g o/oNaCl to which 10-3 M KCN and IO-~ M NaF was added, and in 0.9 g o/o NaCl with 300 mg glucose per IOO ml. It may be assumed that the addition of notable amounts of KCN and NaF will inhibit several vital reactions active within the cells, such as the carbohydrate metabolism and carbonic anhydrase activity, while alterations in the active transport of Na and K may also occurls. As shown in Table II, no significant differences are detectable. Treatment of erythrocytes with hypotonic NaCl solutions will increase the cell volume due to osmotic swelling. Such influences probably will also produce alterations in the hydration of the membrane 17. Hypertonic NaCl solutions affect the erythrocytes’ membrane in an opposite way. The influence of such treatment of adult and cord blood red cells on the oxygen equilibria has been studied by determining the log P,, values of adult and cord blood cells, being suspended either in 0.6 g y. NaCl or 2.5 g o/o NaCl solutions. The oxygen equilibria experiments were performed at pC0, pressures varying from o to IOO mm Hg. Table III represents the mean values
of the indices of the cell suspensions used. The indices for the 0.9 g o/oNaCl suspenClin.Chim.Acta,
IO (1964) 165-174
0
EQUILIBRIA
OF RED CELL HEMOLYSATES TABLE
THE MEAN
III
CELL INDICES OFSUSPENSI~NS OFADULT AND CORDBLOOD SOLUTIONS OFVARIOUS CONCENTRATIONS
Type of
NaCl
cells
g %
Total Hb g %
Adult
0.9 0.6 2.5
16.6 12.4 24.2
Fetal
0.9 0.6 2.5
15.2 13.3 21.0
171
RED
CELLS
IN
NaCl
RBC x IO’/,ul
MCV* rug
MCH*
49 48 50
5.15 4.06 6.99
95 118 71.5
32.2 30.5 34.5
33.9 25.9 48.3
47 49 46
4.18 3.67 5.89
112.5 133.5 78
36.4 36.0 35.6
32.4 26.9 45.7
pcv* %
Ps
MCHC * %
* PCV = packed cell volume; MCV = mean cell vohrme; MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration.
Cellsin 0.6 + NaCl -
Adult cells Cord bloal celllr
Cells -
in 2.5 % NaCl Adult cells Cord bloal cells
Fig. 3. The oxygen affinities of adult and cord blood erythrocytes, suspended in hypo- and hypertonic NaCl solutions, at different PH values. The dotted lines represent the values found for erythrocytes suspended in 0.9 g % NaCl.
sions of cord bIood cell show that the increased cell volume and mean cellular hemoglobin content of the cells * are not changed during the preparation of the suspensions. Treatment of both types of cells with 0.6 g yO NaCl increased the cellular volume with approximately ZOO/~, while the MCHC decreased correspondingly. The opposite effect is seen for the 2.5 g y. NaCl suspensions; the MCV is decreased for 25 to 30%, while the concentration of the hemoglobin in the cells is increased as much as 40%. The results of the oxygen equilibrium experiments are presented in Fig. 3. Treatment of both adult and cord blood erythrocytes with hypotonic NaCl solution results in a slight increase of the oxygen affinities; the differences in log P,, between the two types of suspensions are approximately the same as those found for 0.9 g yO NaCI suspensions. The Bohr effects are slightly decreased and almost identical (d, for A cells 0.36 and for F cells 0.37). The effect of a hypertonic NaCl solution on cord blood red cells is demonstrated by a significant decrease in oxygen affinity, while the CEin.Chim. Acta,
IO (x964)
165-174
172
c. hf. NECHTMAN,T. H. J. HUISMAN
Bohr effect is only slightly altered (4 value 0.42). The changes observed for adult red blood cells are quite different ; the oxygen affinities at pH values of 6.9 and higher are markedly increased, while the Bohr effect is considerably elevated (4 value 0.54). Hemolysis of red cells was not observed in any of these experiments. DISCUSSION The data derived from the experiments with dialyzed hemolysates of human adult and cord blood erythrocytes suggest the presence of a slight, but distinct, difference in the Bohr effect resulting in a decreased oxygen affinity of cord blood hemolysates at PH values below 7.0, while the oxygen affinities of both types of hemolysates are similar at pH values between 7.0 and 7.4. These results are in agreement with observations by MANWELLl3 who reported that the oxygen affinities of “ion-equalized” cord blood hemolysates at pH values below 7.0 are lower than those of adult hemolysates. A similar small difference in the Bohr effect is observed between suspensions of both cell types in 0.9 g o/0NaCl; the oxygen affinity of cord blood cell suspensions, however, was found markedly higher than that of adult red cell suspensions. These observations agree with the work of MCCARTHY*, who also studied oxygen equilibrium curves of cell suspensions. It seems, therefore. that suspensions of corpuscles obtained from human adult and fetal blood show similar differences as found for whole blood samples 8- 14. Our studies also indicate the value of n, a measurement of the hemeheme interaction, to be significantly different; the n values for cord blood cell suspensions and dialyzed hemoglobin solutions are slightly less than those found for the adult samples. In this respect our results confirm those of MCCARTHY~obtained for hemoglobin solutions, and of HELLECERS et al. 26 for whole blood samples, and are opposite to the findings on concentrated hemolysates according to the work of ALLEN et a1.16.
There seems little doubt from this work and from that of other93 13m15 that, upon hemolysis, the oxygen equilibrium of cord blood hemoglobin is almost unchanged whereas the oxygen affinity of adult hemolysates has increased to such an extent that its oxygen dissociation curves are to the left of those of cord blood hemolysates. The difference, however, is strongly dependent of the PH due to the increased Bohr effect of the fetal hemolysates. As to the factor, or factors, which determine the considerable change in the position of the oxygen equilibrium curves of adult red blood cells upon hemolysis in contrast to cord blood erythrocytes, different possibilities may be considered: (I) the absence of fetal hemoglobin inside the adult cell; (2) the presence of dialyzable component(s) present in the evironment of the hemoglobin molecule within the cell; (3) differences in metabolic or enzyme activity within adult and cord blood erythrocytes; (4) differences in the adult and cord blood cell membranes or in the organization of the red cell. The first possibility has become unlikely through the studies of SCHRUEFER~~, who demonstrated that whole blood samples from an adult individual, containing 69% Hb-F and 31% adult hemoglobin, yielded oxygen equilibrium curves, indistinguishable from those of whole adult blood with no notable quantities of Hb-F. This observation is supported by our data obtained with suspensions of erythrocytes from a persistent high Hb-F carrier. The oxygen affinities of those cells are identical C&n.Chim.
Ada,
10 (1964)
165-174
0 EQUILIBRIA OF RED CELL HEMOLYSATES
I73
with those of cell suspensions of normal adult erythrocytes, despite the presence of 34% Hb-F which is equally distributed over all cellsls. Fetal hemoglobin, however, seems to exert another effect. During our experiments it was noted that the PH values (37’) of cord blood cells suspended in 0.9 g o/oNaCl and of undialyzed cord blood cell hemolysates were considerably lower than the pH values of the corresponding adult cells or hemolysates. The linear relations between the pC0, and the logarithms of the PH values are replotted in Fig. 4. The difference in pH between adult and cord blood cell suspensions, which was approximately 0.15 units, is not dependent of the pC0,. Since a similar but smaller decrease in PH was also found for suspensions of adult erythrocytes containing 34% of Hb-F it seems likely that the fetal hemoglobin itself is responsible for this phenomenon,
Suspended Cells
0
M
40
60 80 IW
I:oncenlrated Hemolyrales
0
20 40
60 80
100
PC02 Fig. 4. The relation between the pC0, and the PH of erythrocytes, being suspended in 0.9 g y0 m ,.,...A Ll^^A. w0r1 _LIU _“A ,& I.n...-I..,.^A-^ A.OUI “I rn~.w,,.,Crr+~A L”II&GXILI(LL.zU ur;uL”lyxzLoJ. ^ u-v^ --..-..I ‘,“l,,lal ^-l..IC. &L&L&IL, u- _u L”‘U “I”“U, G _ t heteiozygons PHF carrier.
The second possibility, namely the effect of a dialyzable substance present within the red cell, has become less likely, since undialyzed hemolysates of adult and cord blood erythrocytes demonstrated only those differences in oxygen affinities, which could be expected from differences in Bohr effects. The third possible explanation is based on possible differences in metabolic or enzymatic activities within both cell types which could interfere with the oxygen uptake. A study of the oxygen affinities of both types of cells, being suspended in 0.9 g o/oNaCl to which excessive amounts of NaF and KCN had been added, admittedly do not exclude this possibility. Nevertheless, there is no evidence that the presence of these inhibitors changes significantly the oxygen equilibria of both adult and cord blood erythrocytes, despite the fact that many important enzymatic reactions were almost completely abolished. The fourth possibility, namely a difference in cell membranes, should be considered as a cause for increased oxygen affinity of cord blood erythrocytes. The data obtained in the study of the oxygen equilibria of adult and cord blood erythrocytes being suspended in hypo- and hypertonic NaCl solutions indicate a different response to such a treatment. Both types of cells show a similar increase in oxygen affinity when suspended in hypotonic solutions. When cells are dehydrated by hypertonic solutions so that the mean hemoglobin concentration per cell increases considerably a significant decrease in the oxygen affinity may be expected. Such an effect has been demonstrated for cord blood cells. For suspensions of adult erythrocytes, however, an increase in oxygen affinity was found which was most marked at low CO, Clin.Chim.
Acta,
IO (1964)
165-174
I74
C. M.
NECHTMAN,
T.
H. J. HUISMAN
pressures. There is, therefore, some reason to believe that the differences in the overall kinetics of oxygen uptake by the hemoglobin inside the red cell of fetal and of adult origin are due to unknown differences in cell membranes or cell organization rather than to either specific differences in the oxygen equilibria of the hemoglobin types or the presence of factors in the environment of the hemoglobin molecules. ACKNOWLEDGEMENT
This study was supported by the U.S.P.H.S.
Research grant No. H-6982.
REFERENCES 1 A. ST. G. HUGGETT, J. Physiol. (London), 62 (1927) 373. 2 T. BARCROFT, L. B. FLEXNER, W. HERKEL, E. F. MCCARTHY AND T. MCCLURKIN, J. Physiol.
(London), 83 (‘934) 192. 3 E. F. MCCARTHY, J. Physiol.
(London), 102 (1943) 55. 4 V. K. RIEGEL, P. HILPERT AND H. BARTELS, Acta Haematol., 25 (1961) 164. 5 T. H. J. HUISMAN, in F. LINNEWEH, (Ed.), Die physiologische Entwicklung des Kindes, Springer, Berlin, 1959. p. 296. 8 T. Roos AND C. ROMIJN, J. Physiol. (London), g2 (1938) 249. 7 C. J. MULLER, Doctoral Thesis, Groningen, Van Gorcum & Co., Publ., Assen, The Netherlands, 1961. * E. F. MCCARTHY, J. Physiol. (London), 102 (1943) 55. 0 H. BARTELS, H. HARMS, V. PROBST, K. RIEGEL AND J. SCHNEIDER, Klin. Wochschr., 37 (1959) 664. 10 K. RIEGEL, H. BARTELS AND J. SCHNEIDER, Z. Kinderheilk., 83 (1959) 2og. 11 H. BARTELS, P. HILPERT AND K. RIEGEL, P@gers Arch. Ges. Physiol., 271 (1960) 169. 12 A. ABRAHAMOV AND C. A. SMITH, A. M. A. J. Diseases Children, g7 (1959) 375. la C. MANWELL, Ann. Rev. Physiol., 22 (1960) Igr. 14 J. J. P. SCHRUEFER, C. J. HELLER, F. C. BATTAGLIA AND A. E. HELLEGERS, Nature, 196 (1962) 550. 16 D. W. ALLEN, J. WYMAN JR. AND C. i\. SMITH, J. Biol. Chem., 203 (1953) 81. I6 T. A. 1. PRANKERD. The Red Cell. Charles C. Thomas. Publ.. SDrinnfieId, Ill., 1961. I7 J. W. *HARRIS, The ‘Red Cell, Harvard University Press Publ., Cambridge, M&s., 1963. I* R. B. THOMPSON, J. W. MITCHENER AND T. H. J. HUISMAN, Blood, 18 (1961) 267. I8 R. BRINKMAN AND M. N. J. DIRKEN, Acta Brev. Neevl., IO (1940) 228. 20 J. H. P. JONXIS AND H. W. BOEVE, Acta Med. Stand., 115 (1956) 157. 2* T. B. ROSENTHAL, J. Biol. Chem., 187 (1948) 25. 22 J. WYMAN JR., Advances Protein Chem., 4 (1948) 407. 23 J. H. P. JONXIS AND T. H. J. HUISMAN, Blood, II (1956) Ioog. 24 A. ROSSI-FANELLI, E. ANTONINI~AND A. CAPUTO, J. Biol. Chem., 236 (1961) 397. 25 A. ROSSI-FANELLI, E. ANTONINI-AND A. CAPUTO, J. Biol. Chem., 236 (1961) 391. z6 A. E. HELLEGERS, G.‘MESCHIA. H. PRYSTOWSKY, S. WOLKOFF AND D. H. BARRON, Quart. J. Exptl. Physiol., 44 (1959) 215. Clin. Chim. Acta, IO (x964) 165-174
CLINICA CHIMICA ACTA
COMPARATIVE OF HUMAN
STUDIES
ADULT
HEMOLYSATES CARL
EQUILIBRIA
CORD BLOOD
AND
M. NECHTMAN
Department
OF OXYGEN
AND
AND
RED
CELL
SUSPENSIONS
TITUS
H. J. HUlSMAN
of Biochemistry, Medical College of Georgia, Augusta, Ga. (U.S.A.) (Received
November
rqth, 1963)
SUMMARY
The oxygen equilibria of suspensions and hemolysates of human adult and cord blood erythrocytes have been compared. Dialyzed hemolysates of adult and cord blood red cells showed small, but distinct, differences in the Bohr effect. Changes in phosphate molarity of the buffer used for the dialysis resulted in identical changes in oxygen affinities. Suspensions of cord blood erythrocytes in 0.9 g y0 NaCl showed a marked increase in oxygen affinity when compared with similar suspensions of adult red blood cells. The same difference in the Bohr effect was observed as seen for dialyzed hemolysates. The oxygen equilibria of concentrated undialyzed hemolysates of cord blood erythrocytes were almost identical to those of cord blood cell suspensions; the Bohr effect, however, was increased. The equilibrium between oxygen and the corresponding adult hemolysates was notably changed; the affinities of these hemolysates were found higher than that of either cord blood cell suspensions or hemolysates. The oxygen affinities of red cell suspensions from a heterozygous carrier of the persistent high Hb-F anomaly with 34% Hb-F were indistinguishable from those of normal adult erythrocyte suspensions. The addition of notable quantities of KCN and of NaF to the 0.9 g% NaCl solution did not affect the oxygen affinities of suspensions of both adult and cord blood erythrocytes. Hypotonic cell suspensions (in 0.6 g% NaCl) demonstrated increased oxygen affinities and a slight decrease in the Bohr effect; the effect of hypotonicity was the same for both cell types. Treatment of cord blood erythrocytes with a hypertonic solution (2.3 g% NaCl) resulted in decrease of the oxygen affinity. A similar treatment of adult erythrocytes increased the oxygen affinity, especially at pH values of 6.9 and higher. The significance of these findings has been discussed. Our studies suggest that differences in cell membrane or cell organization may likely contribute to the observed difference in oxygen affinities of human adult and cord blood erythrocytes. The presence of a specific fetal hemoglobin in the erythrocyte influences the Bohr effect and possibly the pH inside the cell, but not the oxygen affinity.
Clin. Chins. Acta, IO (1964) 165-174
166
C.M. NECHTMAN,T. H. J. HUISMAN
The important functional difference of the fetal and adult hemoglobins of many mammalian species is the increased affinity of the fetal hemoglobin for molecular oxygen. This increased oxygen affinity of the hemoglobin of fetal blood is commonly considered to be advantageous in the transfer of oxygen from the maternal to the fetal circulation. Such differences-as have, for instance, been observed for the goat l-3, the sheep 4fs and the cowB-are demonstrable when comparisons are made either between prepared solutions of the hemoglobins or of cell suspensions. Since these Hb-A and Hb-F types exhibit marked differences in physical and chemical properties (reviewed in’), it is generally assumed that structural differences between the Hb-A and the Hb-F within one species are expressed in variation of oxygen equilibria. In the human the oxygen affinity of cord blood (and also of cord blood suspension) is slightly higher than that of adult blood s- 14. Upon hemolysis of the cells and subsequent dialysis of the hemolysates, the fetal oxygen dissociation curve is changed only slightly, while the adult oxygen dissociation curve is changed so far to the left that no notable difference between the oxygen affinities of these solutions is detectables~14*1s. It seems, therefore, that the observed differences in oxygen equilibria of human cord and adult blood may not result from the many differences in the amino acid sequences of the two proteins. Environmental influences have been suggestedIS. Such an influence is likely to be effective through differences in the environment of the hemoglobin molecule within the cell, since possible effects of fetal plasma on the oxygen equilibria have been excluded by the experiments of ABRAHAMOVAND SMITI-I’~and by the observation that washed fetal red blood cells showed the same relatively high affinity for oxygen as whole cord bloods. In this communication we should like to report additional comparative studies of the oxygen equilibria of human adult and cord blood samples. Our studies were primarily oriented in the following four directions: (a) An investigation was undertaken to determine possible differences in the Bohr effect of fetal and maternal red cell suspensions and dialyzed red cell hemolysates, since contradictory observations have been reported in the literature 13*14. (b) Possible influences of “dialyzable factors” present inside the cell membrane were studied by comparing the oxygen equilibria of undialyzed, concentrated hemolysates of adult and cord blood erythrccytes with those obtained for the corresponding cell suspensions. The effect of increased phosphate levels in the environment was also studied. (c) In view of known differences in metabolic or enzymatic activities I691’ oxygen equilibria of cord blood and adult erythrocytes being suspended in media containing enzyme inhibitors have been investigated. (d) The possibility that differences in membrane’ permeability may be responsible for the observed differences in oxygen equilibria was investigated by comparing the oxygen affinity of cord blood and adult red cell suspensions in hypoand hypertonic salt solutions. MATERIALANDMETHODS Samples of blood from the umbilical cord, taken at birth, were obtained through courtesy of the Departement of Obstetrics and Gynecology of the Medical College of Georgia. Three adult members of a family described before18, being heterozygous for the persistent high Hb-F anomaly (PHF), served as donors of blood samples with 34% Hb-F. Blood samples from healthy normal adults (laboratory workers) Clin. Chim.
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xo (1964)
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0 EQUILIBRIAOF RED CELL HEMOLYSATES
167
were collected simultaneously. In all instances, the blood samples were processed within 12 h after the collection. Samples from either source were centrifuged and the cells washed three times with 0.9 g% NaCl solution. Hemolysis was carried out either by addition of an equal volume of distilled water and 0.4 volume of toluene or by freezing and thawing of cell-water (I : 0.6) mixtures. The stromata were removed by centrifugation at high speed in the cold. The “toluene-treated” hemolysates of cord blood erythrocytes and of normal red blood cells were dialyzed simultaneously against a large quantity (1000 ml VCYSUS IO ml of each hemoglobin solution) of phosphate buffer at 4” for 24 h. The molarities of these buffer solutions varied between 0.01 and 0.2 with pH values ranging from 6.8 to 7.8. The hemoglobin concentration of each sample was adjusted with the appropriate buffer solution to 5 g/roe ml. The hemolysates prepared by freezing and thawing were studied without additional treatment; the final hemoglobin concentration varied between 17 and 20 g/roe ml. Suspensions of cells from different sources in NaCl solutions of three concentrations (0.6, 0.9 and 2.5 g%), in 0.9 g% NaCl containing 300 rng% glucose, and in 0.9 g% NaCl containing IO-~ M KCN and IO-~ M NaF, were prepared simultaneously after two additional washings with the appropriate solution. The packed cell volumes were adjusted to 48 h 2%. Construction of the oxygen dissociation curves at 37” was performed by the technique of BRINKMANAND DIRKENIs. The determinations of the percent oxygen saturation were made with the use of a Zeiss Spectrophotometer (model PMQ-II) following the procedure described by JONXIS AND BOEVE~O.The PH measurements were carried out on a “Radiometer” pH meter, model 4, at a temperature of 25’. The values obtained were corrected to 37’ by means of the factor given by ROSENTHAL~~.All analyses were completed within 48 h after drawing of the samples. From the observed oxygen dissociation curves approximately linear relationships were attained by plotting log [r/(100-y)] ag ainst the log PO,, y being thepercentualoxygen saturation determined. From these linear relationships two constants were calculated, namely, n, a measure of the heme-heme interaction, which is the slope of the curve, and log Pso, the value of the PO, at which 50% saturation is observed. The P,, values have also been plotted as function of PH. The value 4, calculated from 4 = d log P,,/d pH and evaluated at PH 6.6 to 7.6, was used as a measure of the Bohr effect 22. The percentages of Hb-F were determined following the spectrophotometric procedure described beforeZ3. Red cell indices, when required, were measured following standard hematological techniques. RESULTS Fig. I presents the results of the oxygen equilibria of fresh hemoglobin solutions of normal adult and cord blood erythrocytes, after dialysis against 0.1 M phosphate buffers of varying pH values. The final hemoglobin concentration was 5.0 & 0.3 g/IO0 ml. All oxygen measurements were performed after equilibration with oxygen at different pressures (10-60 mm Hg) in 150 ml tonometers, at 40 mm pC0, and at 37”. The results are expressed as the logarithms of the P,, values. Our data indicate a slight but distinct difference in the Bohr effect of the two types of hemolysates. The constant 4, reflecting the magnitude of the Bohr effect, is 0.40 for dialyzed Cl&. Chim.Acta,IO(x964)165-174
168
C. M. NECHTMAN,
T. H. J. HUISMAN
hemolysates of adult erythrocytes and 0.47 for dialyzed hemolysates of cord blood red cells. The oxygen affinities of both types of hemolysates are approximately the same at pH’S between 7.0 and 7.4; below PH 7.0 human cord blood hemoglobin has a distinctly lower affinity for oxygen. The hemc-heme interaction, expressed as the constant n in Hill’s equation, is also significantly diff srent ; the n value obtained for adult hemolysates is 2.90 f 0.05 (rg determinations) and that for cord blood hemolysates 2.65 * 0.1 (14 determinations). Oxygen dissociation curves of five samples of a hemolysate prepared from red cells of a heterozygous carrier of the persistent high Hb-F anomaly (PHF) showed P,, values, being intermediate between those found for the adult and cord blood hemolysates. The mean value of the constant n was 2.75 & 0.1. I
Adult Hb Cord blood Hb 175% Hb-FI Hb 01 PHF csrrler 134’S Hb-FI
n
6.4
b!b
I
6.8
l!O
7!2
7!4
7.‘b
pH l37’CI
Fig. I. The oxygen affinities of dialyzed hemolysates at different pn values.
Possible influences of differences in phosphate concentration were studied by analyzing the oxygen equilibria curves of cord blood and adult erythrocyte hemolysates, which were simultaneously dialyzed versus large quantities of sodium phosphate buffers of varying molarities (0.01, 0.025, 0.05, 0.1 and 0.2 M) and a pH of 7.2-7.6. The log P,, values, which were obtained after equilibriation of the hemolysates with oxygen at various tensions and at 40 mm Hg pCO,, were corrected to pH 7.0 (37’) by means of the 4 values determined in the previously described experiments. Table I summarizes the results. In all experiments, the log P,, values for fetal hemolysates are similar to those found for adult hemolysates. Both types of hemolysates show the same increase in oxygen affinity by decreasing the phosphate molarity, which is in accordance with the findings of ROSSI-FANELLIet aLza~zs. The difference in the constant n of Hill’s equation was also observed in these experiments. Fig. 2 shows data derived from the oxygen dissociation curves of adult erythrocytes and of cord blood red cells, both being suspended in 0.9 g% NaCl. The mean values for total hemoglobin in g %, packed cell volume in o/0 and RBC in IO& per ~1 were 16.6, 4g and 5.15 for the adult cell suspensions, and 15.2, 47 and 4.18 for the Clin.Chim.Acta,
IO (1964)
165-174
0
EQUILIBRIA
OF RED CELL HEMOLYSATES TABLE
THE
OXYGEN
UPTAKE
OF ADULT SODIUM
Molarity of phosphate
AND
CORD
PHOSPHATE
I
BLOOD
BUFFERS
RED
CELL
OF DIFFERENT
0.01
0.1
0.2
* Total hemoglobin of five determinations.
Ps
AGAINST
Covd’ log pa0
constantn
2.5 2.7 2.45 2.55 2.6
1.138 I.196
2.3 2.3 2.25 2.3 2.3
concentration
DIALYZED
MOLARITIES
constant n
I.210
1.231 1.265 5.0 * 0.3 9%;
the values
a 4
HEMOLYSATES
Adult *
0.025 0.05
1%
M M O----e D---m
presented
log pa0 I.143 1.200 I.219
1.240 I.275 are mean values
Cellsof PHF wrriw Adult cells Cord blood cells Cow. undialyxd hemol. Adult cells Cont. unditlped hemol. cord bload cells
pH I3r00
Fig.
2.
The oxygen
affinities of red blood cells suspended in 0.9 9% NaCl and of undialyzed concentrated hemolysates at different PH values.
cord blood cell suspensions. The oxygen dissociation curves were determined after equilibriation of these suspensions with oxygen at various tensions and at a pC0, varying from o to IOO mm Hg in different experiments. The pH values (at 37’) after equilibration with these gas mixtures varied from 7.28 to 6.63 for the adult cell suspensions and from 6.99 to 6.43 for the cord blood cell suspensions. A marked increase in oxygen affinity of the fetal cell suspensions can be noted, the difference being 8.5 mm 0, at a pH of 7.0. A similar difference in the Bohr effect as found for dialyzed hemolysates is demonstrable; the 4 value is 0.405 for adult cell suspensions and 0.47 for cord blood cell suspensions. The oxygen dissociation curves of the suspensions of erythrocytes obtained from a heterozygous PHF carrier, were determined at 20, 40 and 80 mm Hg pC0,. The results do not differ from those found for normal adult cells, despite the presence of 34% of Hb-F. The mean values of the constant 12 are 2.0 f 0.1 for adult cells (17 determinations), 1.7 f 0.15 for cord blood cells (16 determinations) and 2.1, 2.1 and 2.3 for the cells of the PHF carrier indicating a difference between adult and fetal erythrocyte suspensions. Clin. Chim. Acta, IO (1964)
165-174
C. M.
170
NECHTMAN,
T.
H.
J. HUISMAN
The results obtained in studying the oxygen equilibria of undialyzed concentrated hemolysates are also presented in Fig. 2. The P,, values were derived from oxygen dissociation curves, determined as described for cell suspension. The total concentrations of the hemolysates varied between 17 and 20 g %. Several observations may be made upon the data of Fig. 2. The oxygen affinities of the cord blood hemolysates are closely similar to those found for the corresponding cell suspension. The Bohr effect is increased; the 4 value is 0.57 versus 0.47 for the cord blood cell suspension. The constant n in Hill’s equation is not different, namely 1.65 f 0.1 (7 determinations). The affinities of adult hemolysates, on the contrary, are higher than those found for the corresponding cell suspensions. The log P,, values at a pH of 6.8 and lower are significantly lower than those of the concentrated cord
hemoglobin
TABLE IHE OXYGEN
UPTAKE
OF ADULT
AND
CORD
BLOOD
Tl CELLS,
SUSPENDED
IN
DIFFERENT
MEDIA
(expressed as log PC,,)
Type of
hemolysate Adult Adult Adult Adult Cord Cord Cord Cord
Pff 7.20 7.00
6.85 6.75 6.85 6.75 6.60 6.45
0.9 g yO NaCl 1.28 1.38
0.9 g 0h NaCl $ 300 mg yO glucose
0.9 g yO NaCl + IO-~ M KCN $ IO+ M NaF
_
1.32
I.43 1.50 I.25 I.29
I.355 1.42 1.48 _ I.29
I.35 1.42 1.48 1.215 1.24
I.345 I .45
I.35 * .42
I.315 -
blood hemolysates and, probably, also lower than those of cord blood cell suspensions. The Bohr effect and the heme-heme interactions are not markedly altered, the 4 value is 0.38 (0.405 for adult cell suspensions), while the constant n in Hill’s equation is 2.05 f 0.1 (6 determinations). The possibility that differences in metabolic or enzymatic activity within the cell may contribute to the increased oxygen affinity of cord blood cell suspensions was investigated by comparing oxygen equilibria of cells suspended in 0.9 g o/oNaCl, in 0.9 g o/oNaCl to which 10-3 M KCN and IO-~ M NaF was added, and in 0.9 g o/o NaCl with 300 mg glucose per IOO ml. It may be assumed that the addition of notable amounts of KCN and NaF will inhibit several vital reactions active within the cells, such as the carbohydrate metabolism and carbonic anhydrase activity, while alterations in the active transport of Na and K may also occurls. As shown in Table II, no significant differences are detectable. Treatment of erythrocytes with hypotonic NaCl solutions will increase the cell volume due to osmotic swelling. Such influences probably will also produce alterations in the hydration of the membrane 17. Hypertonic NaCl solutions affect the erythrocytes’ membrane in an opposite way. The influence of such treatment of adult and cord blood red cells on the oxygen equilibria has been studied by determining the log P,, values of adult and cord blood cells, being suspended either in 0.6 g y. NaCl or 2.5 g o/o NaCl solutions. The oxygen equilibria experiments were performed at pC0, pressures varying from o to IOO mm Hg. Table III represents the mean values
of the indices of the cell suspensions used. The indices for the 0.9 g o/oNaCl suspenClin.Chim.Acta,
IO (1964) 165-174
0
EQUILIBRIA
OF RED CELL HEMOLYSATES TABLE
THE MEAN
III
CELL INDICES OFSUSPENSI~NS OFADULT AND CORDBLOOD SOLUTIONS OFVARIOUS CONCENTRATIONS
Type of
NaCl
cells
g %
Total Hb g %
Adult
0.9 0.6 2.5
16.6 12.4 24.2
Fetal
0.9 0.6 2.5
15.2 13.3 21.0
171
RED
CELLS
IN
NaCl
RBC x IO’/,ul
MCV* rug
MCH*
49 48 50
5.15 4.06 6.99
95 118 71.5
32.2 30.5 34.5
33.9 25.9 48.3
47 49 46
4.18 3.67 5.89
112.5 133.5 78
36.4 36.0 35.6
32.4 26.9 45.7
pcv* %
Ps
MCHC * %
* PCV = packed cell volume; MCV = mean cell vohrme; MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration.
Cellsin 0.6 + NaCl -
Adult cells Cord bloal celllr
Cells -
in 2.5 % NaCl Adult cells Cord bloal cells
Fig. 3. The oxygen affinities of adult and cord blood erythrocytes, suspended in hypo- and hypertonic NaCl solutions, at different PH values. The dotted lines represent the values found for erythrocytes suspended in 0.9 g % NaCl.
sions of cord bIood cell show that the increased cell volume and mean cellular hemoglobin content of the cells * are not changed during the preparation of the suspensions. Treatment of both types of cells with 0.6 g yO NaCl increased the cellular volume with approximately ZOO/~, while the MCHC decreased correspondingly. The opposite effect is seen for the 2.5 g y. NaCl suspensions; the MCV is decreased for 25 to 30%, while the concentration of the hemoglobin in the cells is increased as much as 40%. The results of the oxygen equilibrium experiments are presented in Fig. 3. Treatment of both adult and cord blood erythrocytes with hypotonic NaCl solution results in a slight increase of the oxygen affinities; the differences in log P,, between the two types of suspensions are approximately the same as those found for 0.9 g yO NaCI suspensions. The Bohr effects are slightly decreased and almost identical (d, for A cells 0.36 and for F cells 0.37). The effect of a hypertonic NaCl solution on cord blood red cells is demonstrated by a significant decrease in oxygen affinity, while the CEin.Chim. Acta,
IO (x964)
165-174
172
c. hf. NECHTMAN,T. H. J. HUISMAN
Bohr effect is only slightly altered (4 value 0.42). The changes observed for adult red blood cells are quite different ; the oxygen affinities at pH values of 6.9 and higher are markedly increased, while the Bohr effect is considerably elevated (4 value 0.54). Hemolysis of red cells was not observed in any of these experiments. DISCUSSION The data derived from the experiments with dialyzed hemolysates of human adult and cord blood erythrocytes suggest the presence of a slight, but distinct, difference in the Bohr effect resulting in a decreased oxygen affinity of cord blood hemolysates at PH values below 7.0, while the oxygen affinities of both types of hemolysates are similar at pH values between 7.0 and 7.4. These results are in agreement with observations by MANWELLl3 who reported that the oxygen affinities of “ion-equalized” cord blood hemolysates at pH values below 7.0 are lower than those of adult hemolysates. A similar small difference in the Bohr effect is observed between suspensions of both cell types in 0.9 g o/0NaCl; the oxygen affinity of cord blood cell suspensions, however, was found markedly higher than that of adult red cell suspensions. These observations agree with the work of MCCARTHY*, who also studied oxygen equilibrium curves of cell suspensions. It seems, therefore. that suspensions of corpuscles obtained from human adult and fetal blood show similar differences as found for whole blood samples 8- 14. Our studies also indicate the value of n, a measurement of the hemeheme interaction, to be significantly different; the n values for cord blood cell suspensions and dialyzed hemoglobin solutions are slightly less than those found for the adult samples. In this respect our results confirm those of MCCARTHY~obtained for hemoglobin solutions, and of HELLECERS et al. 26 for whole blood samples, and are opposite to the findings on concentrated hemolysates according to the work of ALLEN et a1.16.
There seems little doubt from this work and from that of other93 13m15 that, upon hemolysis, the oxygen equilibrium of cord blood hemoglobin is almost unchanged whereas the oxygen affinity of adult hemolysates has increased to such an extent that its oxygen dissociation curves are to the left of those of cord blood hemolysates. The difference, however, is strongly dependent of the PH due to the increased Bohr effect of the fetal hemolysates. As to the factor, or factors, which determine the considerable change in the position of the oxygen equilibrium curves of adult red blood cells upon hemolysis in contrast to cord blood erythrocytes, different possibilities may be considered: (I) the absence of fetal hemoglobin inside the adult cell; (2) the presence of dialyzable component(s) present in the evironment of the hemoglobin molecule within the cell; (3) differences in metabolic or enzyme activity within adult and cord blood erythrocytes; (4) differences in the adult and cord blood cell membranes or in the organization of the red cell. The first possibility has become unlikely through the studies of SCHRUEFER~~, who demonstrated that whole blood samples from an adult individual, containing 69% Hb-F and 31% adult hemoglobin, yielded oxygen equilibrium curves, indistinguishable from those of whole adult blood with no notable quantities of Hb-F. This observation is supported by our data obtained with suspensions of erythrocytes from a persistent high Hb-F carrier. The oxygen affinities of those cells are identical C&n.Chim.
Ada,
10 (1964)
165-174
0 EQUILIBRIA OF RED CELL HEMOLYSATES
I73
with those of cell suspensions of normal adult erythrocytes, despite the presence of 34% Hb-F which is equally distributed over all cellsls. Fetal hemoglobin, however, seems to exert another effect. During our experiments it was noted that the PH values (37’) of cord blood cells suspended in 0.9 g o/oNaCl and of undialyzed cord blood cell hemolysates were considerably lower than the pH values of the corresponding adult cells or hemolysates. The linear relations between the pC0, and the logarithms of the PH values are replotted in Fig. 4. The difference in pH between adult and cord blood cell suspensions, which was approximately 0.15 units, is not dependent of the pC0,. Since a similar but smaller decrease in PH was also found for suspensions of adult erythrocytes containing 34% of Hb-F it seems likely that the fetal hemoglobin itself is responsible for this phenomenon,
Suspended Cells
0
M
40
60 80 IW
I:oncenlrated Hemolyrales
0
20 40
60 80
100
PC02 Fig. 4. The relation between the pC0, and the PH of erythrocytes, being suspended in 0.9 g y0 m ,.,...A Ll^^A. w0r1 _LIU _“A ,& I.n...-I..,.^A-^ A.OUI “I rn~.w,,.,Crr+~A L”II&GXILI(LL.zU ur;uL”lyxzLoJ. ^ u-v^ --..-..I ‘,“l,,lal ^-l..IC. &L&L&IL, u- _u L”‘U “I”“U, G _ t heteiozygons PHF carrier.
The second possibility, namely the effect of a dialyzable substance present within the red cell, has become less likely, since undialyzed hemolysates of adult and cord blood erythrocytes demonstrated only those differences in oxygen affinities, which could be expected from differences in Bohr effects. The third possible explanation is based on possible differences in metabolic or enzymatic activities within both cell types which could interfere with the oxygen uptake. A study of the oxygen affinities of both types of cells, being suspended in 0.9 g o/oNaCl to which excessive amounts of NaF and KCN had been added, admittedly do not exclude this possibility. Nevertheless, there is no evidence that the presence of these inhibitors changes significantly the oxygen equilibria of both adult and cord blood erythrocytes, despite the fact that many important enzymatic reactions were almost completely abolished. The fourth possibility, namely a difference in cell membranes, should be considered as a cause for increased oxygen affinity of cord blood erythrocytes. The data obtained in the study of the oxygen equilibria of adult and cord blood erythrocytes being suspended in hypo- and hypertonic NaCl solutions indicate a different response to such a treatment. Both types of cells show a similar increase in oxygen affinity when suspended in hypotonic solutions. When cells are dehydrated by hypertonic solutions so that the mean hemoglobin concentration per cell increases considerably a significant decrease in the oxygen affinity may be expected. Such an effect has been demonstrated for cord blood cells. For suspensions of adult erythrocytes, however, an increase in oxygen affinity was found which was most marked at low CO, Clin.Chim.
Acta,
IO (1964)
165-174
I74
C. M.
NECHTMAN,
T.
H. J. HUISMAN
pressures. There is, therefore, some reason to believe that the differences in the overall kinetics of oxygen uptake by the hemoglobin inside the red cell of fetal and of adult origin are due to unknown differences in cell membranes or cell organization rather than to either specific differences in the oxygen equilibria of the hemoglobin types or the presence of factors in the environment of the hemoglobin molecules. ACKNOWLEDGEMENT
This study was supported by the U.S.P.H.S.
Research grant No. H-6982.
REFERENCES 1 A. ST. G. HUGGETT, J. Physiol. (London), 62 (1927) 373. 2 T. BARCROFT, L. B. FLEXNER, W. HERKEL, E. F. MCCARTHY AND T. MCCLURKIN, J. Physiol.
(London), 83 (‘934) 192. 3 E. F. MCCARTHY, J. Physiol.
(London), 102 (1943) 55. 4 V. K. RIEGEL, P. HILPERT AND H. BARTELS, Acta Haematol., 25 (1961) 164. 5 T. H. J. HUISMAN, in F. LINNEWEH, (Ed.), Die physiologische Entwicklung des Kindes, Springer, Berlin, 1959. p. 296. 8 T. Roos AND C. ROMIJN, J. Physiol. (London), g2 (1938) 249. 7 C. J. MULLER, Doctoral Thesis, Groningen, Van Gorcum & Co., Publ., Assen, The Netherlands, 1961. * E. F. MCCARTHY, J. Physiol. (London), 102 (1943) 55. 0 H. BARTELS, H. HARMS, V. PROBST, K. RIEGEL AND J. SCHNEIDER, Klin. Wochschr., 37 (1959) 664. 10 K. RIEGEL, H. BARTELS AND J. SCHNEIDER, Z. Kinderheilk., 83 (1959) 2og. 11 H. BARTELS, P. HILPERT AND K. RIEGEL, P@gers Arch. Ges. Physiol., 271 (1960) 169. 12 A. ABRAHAMOV AND C. A. SMITH, A. M. A. J. Diseases Children, g7 (1959) 375. la C. MANWELL, Ann. Rev. Physiol., 22 (1960) Igr. 14 J. J. P. SCHRUEFER, C. J. HELLER, F. C. BATTAGLIA AND A. E. HELLEGERS, Nature, 196 (1962) 550. 16 D. W. ALLEN, J. WYMAN JR. AND C. i\. SMITH, J. Biol. Chem., 203 (1953) 81. I6 T. A. 1. PRANKERD. The Red Cell. Charles C. Thomas. Publ.. SDrinnfieId, Ill., 1961. I7 J. W. *HARRIS, The ‘Red Cell, Harvard University Press Publ., Cambridge, M&s., 1963. I* R. B. THOMPSON, J. W. MITCHENER AND T. H. J. HUISMAN, Blood, 18 (1961) 267. I8 R. BRINKMAN AND M. N. J. DIRKEN, Acta Brev. Neevl., IO (1940) 228. 20 J. H. P. JONXIS AND H. W. BOEVE, Acta Med. Stand., 115 (1956) 157. 2* T. B. ROSENTHAL, J. Biol. Chem., 187 (1948) 25. 22 J. WYMAN JR., Advances Protein Chem., 4 (1948) 407. 23 J. H. P. JONXIS AND T. H. J. HUISMAN, Blood, II (1956) Ioog. 24 A. ROSSI-FANELLI, E. ANTONINI~AND A. CAPUTO, J. Biol. Chem., 236 (1961) 397. 25 A. ROSSI-FANELLI, E. ANTONINI-AND A. CAPUTO, J. Biol. Chem., 236 (1961) 391. z6 A. E. HELLEGERS, G.‘MESCHIA. H. PRYSTOWSKY, S. WOLKOFF AND D. H. BARRON, Quart. J. Exptl. Physiol., 44 (1959) 215. Clin. Chim. Acta, IO (x964) 165-174