Hb Potomac (101 Glu -k Asp): Speculations on Placental Transport in Carriers of High-Affinity

By S. Charache, R. Jacobson, B. Brimhall, E. A. Murphy, P. Hathaway, R. Winslow, R. Jones, C. Rath, and Joan Simkovich

Blood from a woman with unexplained of the proband’s four surviving siblings erythrocytosis had increased oxygen af- and both of her children were carriers. Dif-

finity,butnoabnormality could be detected ferences in the ratio of carrier:normal chil- Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 by electrophoresis or chromatography of dren born to male or female carriers of 23 her hemolysate. Separation of the tryptic other high-affinity hemoglobins were not peptides of her f chains disclosed two significant. The high proportion of carriers half-sized peaks in the region of 13 1-1 1 . in this kindred was probably due to chance The faster of these was abnormal, with alone, and not because high maternal oxy- the structure (. 101 Glu Asp. The new gen affinity interfered with oxygen trans- was called “Potomac.” Three port to fetuses with normal hemoglobin.

I NHERITED’ or acquired4 abnormalities of oxygen affinity do not play a critical role in oxygen transport in healthy, resting subjects at sea level, for a variety ofcompensatory mechanisms are used to restore homeostasis.5 When reserves are exhausted or stressed, oxygen affinity becomes much more impor- tant. Such situations include heart failure,6 severe anemia,7 high altitude,8 severe exercise,9 and pregnancy.’#{176} Textbooks of obstetrics point out that transport of oxygen from mother to fetus is aided by the difference in their oxygen affinities.” Most mammals show such a difference, often greater than that in man’2----although the domestic cat’3 and (in early pregnancy) the elephant’4 do not. In 1967 Moore et al.’5 pointed out that the usual difference in affinity between adult and is not necessary for delivery of a healthy human fetus. A female carrier of a high- affinity hemoglobin (Hb Zurich, P50 21.6 mm Hg) had a hematologically nor- mal child, although the difference in P,0” across the placenta had probably only been 1.1 mm Hg rather than the usual 6+ mm Hg (normal maternal P50 26.6, fetal 20.5 mm Hg). Parer made the same point in 1970, this time in a carrier of Hb Rainier(P50 12.5 mm Hg).’#{176}

*: the oxygen pressure in mm Hg necessary for 50 saturation of hemoglobin, corrected to pH 7.4 and 37’C unless specified to the contrary. From the Department of Medicine. Johns Hopkins Universitt School of Medicine. Baltimore. Md.. the Department of Medicine. Georgetown University School of Medicine. Washington, D. C. . the

Department of Biochemistry. Universitt of Oregon School of Medicine, Portland. Ore. . and the Chemical Hematology Branch, National Heart. Lung and Blood Institute. Bethesda. Md. Submitted June 29. 1977. accepted September 12, 1977. Supported b.v N/H Research Grants HL-02799 and A M-17850 and Genetics Center Grant 5P50- GM-I 9489. Address for reprint requests: Dr. Samuel Charache. 1030 Blalock Building. Johns Hopkins Hospital. Baltimore. Md. 21205.

1978 by Grune & Stratton, Inc. ISSNOIYi6-4971/78/5I02-YiI6$0/.00/0

Blood, Vol. 51, No. 2 (February). 1978 331 332 CHARACHE El AL.

Since then a number of hemoglobins with much higher affinity than Hb Zurich have been described: in some [for instance, a family with hemoglobin Osler (P50 11.0 mm Hg)’6] more than the expected 50% ofthe children of female probands were also carriers, suggesting that when maternal oxygen affinity is very high, reversal ofthe usual fetomaternal difference in 02 affinity diminishes the chance for successful completion of pregnancy. Against that hypothesis were the observations that (1) most hemoglobins with very high oxygen af- finity have been f3-chain variants, and the affinity of carrier fetuses would be expected to be abnormally high only late in pregnancy when fl-chain synthesis increases, and (2) spontaneous abortions have been infrequently reported Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 among carriers of high-affinity hemoglobins. We have recently identified a new high-affinity hemoglobin, which we have called “Potomac” because the proband lives near that river. Both of her chil- dren (and three of her four siblings) are also carriers, a segregation ratio which suggests that the high affinity of her blood may have had a selective influence on fetal survival. We report here data on the structure of the new hemoglobin. We have been unable to separate it from Hb A, and data on the purified component are lacking. We also present a review of pedigrees of carriers of other high- affinity hemoglobins to explore the question as to whether the unusually high proportion of carriers of Hb Potomac is due to chance alone.

MATERIALS AND METHODS

Methods

Hematologic, electrophoretic, and chromatographic procedures, and measurement of 02 af- finity of whole blood were carried out as described previously.’7 Additionally, continuous whole blood oxygen equilibrium curves were measured at constant pH and P2 by the slow addition of H202 to deoxygenated blood samples.18 Blood samples were anticoagulated with EDTA, and measurements of whole blood #{176}2affinity were performed within 2 hr of blood collection. The oxygen affinity of hemolysates was measured by the method of Imai et al.t9 using 0.05 M bis- Tris-0.IOM NaCI buffers. The affinity of samples of whole blood, collected in EDTA-containing Vacutainers and mailed to Baltimore, Md., from other cities without refrigeration, was estimated from measurements on unstripped dilute hemolysates (1.5 x l0 M Hb) to which 2,3-diphosphoglycerate (DPG) was added to give a final concentration of 1.4 x l0 M (if no DPG was present in the original sample). Inositol hexaphosphate (IHP) was added to other samples to yield the same final con- centration. A -reducing system2#{176}was added in every case. When measurements were made in this fashion, the oxygen affinities of hemolysates of erythrocytes from carriers who lived in the Baltimore-Washington area (111-2, for instance) did not differ from those of carriers living in New England (111-3), although the former samples were studied within a few hours of collection and the latter were mailed. Heat stability and denaturation by 17% isopropanol were measured by previously described methods21.22 chains were separated by the method ofClegg et al. and then aminoethylated,23 dialyzed, lyophilized, and sent to Portland, Ore. There, the chains were digested with trypsin and the peptides were separated by the method of Jones.24 Further details of peptide analyses are given in Results in the discussion of structural studies.

Case Report

A 27-yr-old woman was found to have a hematocrit of55% in 1949 at the time of a spontaneous abortion. Three years later her hemoglobin concentration was 18.6 g/dl. Her physician recognized that this value was abnormally high, but she was neither treated nor evaluated for the cause of her erythrocytosis. In 1966, she sought medical advice because of vertigo. The hematocrit was Hb POTOMAC (101 Glu ‘ Asp) 333

“ ‘ c#{243}c / 2F 4 r:L, 6 7

“I ,v ::b ,()PROBAND FEMALE I 2 ENORMAL MALE

Fig. 1. Pedigreeoftheproband. DECEASED

57.5#{176}/a, platelet and white cell counts were normal, and red cell mass was 34 mI/kg. One 500-mI Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 phlebotomy was performed. She did not return until 2 yr later, when she developed increasing fatigue. Her hematocrit was 58.5#{176}/aand her red cell mass was 39 mI/kg. White cell and platelet counts and an intravenous pyelogram were normal. She was treated with phlebotomies every 2-4 wk. In 1969, when she was 47 yr old, Dr. J. Simkovich demonstrated that the oxygen affinity ofher blood was increased. She returned in 1976, at the age of 55, because of recurrent vertigo; the hematocrit was 57.8%. The leukocyte alkaline phosphatase score was normal, arterial P02 was 85 mm Hg, and red cell mass was 36.4 mI/kg. Removal of 500 ml of blood did not ameliorate her symptoms. Several members of her family knew that they had erythrocytosis. The pedigree is shown in

Fig. 1 and hematologic data are given in Table 1. Patients whose hemoglobin concentrations equaled or exceeded 17 g/dl had low P when dilute hemolysates were studied. None of the af- fected family members had received treatment for a high hematocrit and none had an elevated reticulocyte count.

RESULTS

Oxygen Affinity of Blood

Continuous oxygen dissociation curves of whole blood were biphasic (Fig. 2), suggesting that the sample contained more than one type of hemoglobin; the estimated P50 was 12.5 mm Hg at 37#{176}C,Pc02 40 mm Hg, and pH 7.4 (normal 27-29 mm Hg). Independent measurements, using analyzed gas mixtures and a

Table 1 . Hematologic Data From the Proband’s Family

Age Sex Hb (g/dI) Retic. (%) P’ (mm Hg)

Il-i 56 F 19 1.4 3.63 2 60 F 19 0.7 2.3

3 57 F 19 - 2.02

4 51 F 13.7 - -

5 53 F 20.9 - 0.09

Ill-i 21 M 19 1.90 2 23 F 17 2.81 3 31 F 17.9 2.62 4 25 F 14.8 6.5 5 14 M 17.8 2.64 6 26 M 16.4 7.23 7 16 M 14.8 6.24 8 27 M 14.5 5.87 9 19 F 14.9 6.98

IV-1 9 M 17.1 1.09 2 12 F 14.2 8.38

* Hemolysate 0.1 g/dI at 25C, pH 7.4, 0.05 bis#{149}tris-0.1M NaCI, in the presence of 1 .4 x 10 M DPG. 334 CHARACHE El AL.

100 ___..J.__._-.--.

75 I, ,; SAT

(%) 50 ,‘ ; ------PROBAND

‘I : #{149}#{149}NORMAL

25 :

0 #{149}‘ I’OO I ) Fig. 2. Continuous oxygen dissociation curve of P02 the proband’s blood at 37’C, pH 7.40, P2 40 mm C tort) Hg. Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021

Co-Oximeter, gave a P of 1 1 .7 mm Hg under the same conditions, with a normal value of 26.3. Dilute hemolysates from all members of the proband’s family with erythro- cytosis also yielded biphasic dissociation curves. The P of red cell hemolysate of Il-S was remarkably low on duplicate determinations; her hemoglobin con- centration was the highest in the kindred. The Bohr effect of hemolysates and reactivity with DPG and IHP, measured at pH 6.5-7.5, were approximately normal (Fig. 3). No difference in Bohr effect was found when the P25 of carrier hemolysates (which reflected predominantly the properties of Hb Potomac) were compared with P75 (which reflected the properties of HbA).

Stability Fresh hemolysates containing Hb Potomac did not show more precipitation than normal samples when incubated in 17% isopropanol at 37#{176}Cor phosphate buffer at 6OC. Hemolysates prepared from mailed blood samples of both carriers and normal family members yielded positive isopropanol tests, al- though they did not contain methemoglobin.25

Structural Studies

An abnormal component could not be separated by electrophoresis at pH 8.6 on cellulose acetate or at pH 6.0 on agar. No abnormalities were found after chromatography of hemoglobin on DEAE-Sephadex (Pharmacia Fine Chemi- cals, Piscataway, N.J.) or Biorex-70 (BioRad Laboratories, Richmond, Calif.)

NO ADDITIVE OPG HP 24

22 QNORMAL ) . 20

18 1 0

LOG 6 . 0 0 p50 4 2 0 0 0 ‘: #{149} . 0 Fig. 3. Variation of P50 . of dilute (1O M) hemo- lysates, “stripped” and in the presence of 1.4 x iO M 6% 8 07’2 74 6’6 d8 107’2 74 6’6687’O 72 74

DPGorO.64x 1O4MIHP. pH Hb POTOMAC (101 Glu ‘ Asp) 335

0 00 200 300 400 500 600 ML. EFFLUENT Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021

Fig. 4. Tryptic peptides from the proband’s /3 chain after chromatography on Aminex A-5. or chromatography of globin on CM-52 (Whatman, Clifton, N.J.). Amino- ethylated /3 chains from the A-Potomac hemolysate were digested with trypsin. The peptides from 40 mg of tryptic hydrolysate were separated on a 0.9 x 30 cm column of Aminex A-5 (BioRad)) cation-exchange resin. A linear gra- dient oftwo buffers, 375 ml each, was used at a flow rate of 30 mI/hr. The first buffer was 0.05 M pyridine-acetate, pH 3.1; the second was 0.39 M pyridine- acetate, pH 4.5. The remaining peaks were eluted with 0.5 M pH 5.0 pyridine- acetate. This procedure gave a peptide pattern which looked like that of a normal j chain from HbA, except that two half-sized peaks were eluted near the position usually occupied by peptide fi T-l 1 (Fig. 4). On amino acid analysis, the faster ofthe two peaks proved to have the composition of normal fi T-l I, except that it had one less glutamyl residue and one extra aspartyl residue, suggesting that the replacement had occurred at residue 101 (Table 2). The slower of the two peaks had the same amino acid analysis as a normal f3 T-l 1 peptide. The aberrant /3 T- I 1 peptide was then treated overnight with 0.5 M acetic acid in an evacuated ampoule at 1 10#{176}Cin order to cleave it at its aspartyl residues. The resulting solution was chromatographed on a 0.9 x 60 cm col- umn of Aminex 50W-X2 resin with pyridine-acetate buffers. On amino acid analysis, the products of cleavage in order of their elution from the column were found to be: free aspartic acid; free proline; a tripeptide containing Leu, His, Val; and another containing Asx, Phe, Arg. The sequence of normal /3 T-l 1 and the probable sequence of the abnormal /3 T-ll peptide from Hb

Table 2. Amino Acid Composition of Normal and Abnormal /1 1-1 1 Peptides From Hb Potomac (Moles/Mole Peptide)

Abnormal fi 1. 1 1 Normal l T 1 1

Histidine (His) 1 . 1 1 .1 Arginine (Arg) 0.9 0.9 Aspartic acid and asparagine (Asx) 3.1 2.0 Glutamic acid (Glu) 0 1.0 Proline(Pro) 0.9 1.0 Valine (Val) 1.0 1.0 Leucine(Leu) 1.1 1.1 Phenylalanine (Phe) 1 .0 1.0 336 CHARACHE El AL.

Potomac are compared below. (Lines represent cleavage points with 0.5 M acetic acid; see Table 2 for definitions of all abbreviations.)

Residue in Chain

Normal /3 T-ll Leu-His-Val-Asp- Pro-Glu-Asn-Phe-Arg

fiT- 1 1 from H b Potomac Leu-H is-Val-Asp- Pro- Aspj -Asn-Phe-Arg

Amino acid analyses were run on the other peptides from the original tryptic hydrolysate and all had normal compositions. Residue 101 was inferred to be aspartic acid (Asp) and not asparagine (Asn) for three reasons: it was cleaved Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 by 0.5 M acetic acid; no charge difference was observed between HbA and Hb Potomac on electrophoresis on cellulose acetate; and the codon for Glu could change to that for Asp with only one nucleotide change, while two would be necessary for the change ofGlu to Asn.

Review ofOther High-Affinity Hemoglobins

Pedigrees of families with high-affinity hemoglobins were compiled, using published data and details provided by generous personal communications. Twenty-three families were studied; copies of their pedigrees and a detailed analysis were published elsewhere.26 The hypothesis to be tested was that high maternal oxygen affinity decreases the probability that children with normal hemoglobins will be born. The ratio of affected to normal children was compared between carrier females and car- rier males (whose wives had normal oxygen affinity). After correction for bias ofascertainment, the probabilities that differences in ratios were due to chance alone were 0.95 for all families and 0.96 for families with P less than 16 mm Hg, based on analysis of I 1 1 children born to male carriers and 96 born to females.

DISCUSSION The site of the amino acid substitution (residue G3f3 in helical notation) in Hb Potomac is at an interface between the a and /3 chains of hemoglobin.27 Dr. Max Perutz suggests that the high oxygen affinity of Hb Potomac is pro- duced by destabilization of its deoxy structure as follows: The side chain of the normal glutamic acid residue is only 3.5 A from aspartic acid G 1 of the neigh- boring a chain in the deoxy conformation. If aspartic acid is substituted at G3fl, its carboxylated come within 2.5 A, close enough to produce strong repulsive forces, distort the deoxy structure, and render it unstable. If the side chain slips into an alternative conformation, which avoids contact with aspartic Gla, it contacts aspartic acid G1f3, with similar results. Equilibrium between the oxy and deoxy conformations is shifted toward the more stable oxy struc- ture, which has intrinsically high affinity, and patients develop erythrocytosis as a result. Electrophoretic mobility ofthe abnormal hemoglobin is not altered, for glutamic and aspartic acids have like charges at pH 8.6. Three other substitutions are known at position G3/3 (Table 3). They produce combinations of increased oxygen affinity, decreased stability, ability to form stable hybrids (af3Af3X) and electrophoretic mobility different from that ex- Hb POTOMAC (101 GIu ‘ Asp) 337

Table 3. Properties of Other Hemoglobins With Substitutions at $ 101 (G3)

Charge Mobility* Whole Blood Stable Hemoglobin Substitution Change pH 8.6 P (mm Hg) Stability Hybrid Ref

Alberta Gly +2 +1.5 15.5 Normal + 28 British Columbia Lys +4 + 1 23.2 Normal 0 29 Rush Glu + 2 + 1 .5 Normal Low + 30 acetate electrophoresis: HbS, + 1 ; H bC, +2.

pected solely on the basis ofthe change in charge of the amino acid side chain. In Hb Alberta and Hb British Columbia, destabilization of the deoxy con- formation may result from introduction of water or excess positive charge into Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 the interior of the molecule. The latter hypothesis has also been advanced to ex- plain the instability of Hb Rush.3#{176} It has recently been suggested that HbA,, found in increased concentration

in diabetics,3’ raises maternal oxygen affinity32 and hence might pose a threat to the fetus.33 Our data suggest that if diabetes does increase oxygen affinity of the mother,34 the hazard to the fetus is not significant. Review of pedigrees of other families with high-affinity hemoglobins has pro- vided no evidence that being a carrier has survival value for the fetus of an af- fected mother. Even if placental blood flow, P02, P02, and hemoglobin con- centration in the fetus35 remain normal, the high hemoglobin concentration of the carrier mother would tend to maintain oxygenation. Since there are very few high-affinity a-chain mutants, we can conclude only that reversal of the usual gradient in oxygen affinity is not lethal in late pregnancy, when /3-chain synthesis begins. No statement can be made as to whether reversal produces less obvious damage, such as low birth weight or retarded development, for data are insufficient.

ACKNOWLEDGMENT

We thank Dr. Max Perutz for his interpretation of the molecular basis for increased oxygen affinity in Hb Potomac, and Dr. Andr#{233}Hellegers and Dr. Joan Simkovich for their advice.

REFERENCES

I . Bromberg PA, Padilla F, Guy JT, 5. Metcalfe J, Dhindsa DS: The physiologi- Balcerzak SP: Effect of hemoglobin Little Rock cal effects of displacements of the oxygen on the physiology of oxygen delivery. Chest dissociation curve, in Astrup P, Rorth M (ed): 6l(Suppl]: 145, 1972 (Abstr) Oxygen affinity of Hemoglobin and Red Cell 2. Woodson RD. Heywood JD, Lenfant C: Acid Base Status. New York, Academic, 1972, Oxygen transport in hemoglobin K#{246}ln. Effect pp 6 13-628 of increased oxygen affinity in absence of corn- 6. Metcalfe 1, Dhindsa D, Edwards Mi, pensatory erythrocytosis. Arch Intern Med 134: Mourdjinis A: Decreased affinity of blood for 711-715, 1974 oxygen in patients with low-output heart fail- 3. Gillies IDS, White YS, White JM: Corn- ure. Circ Res 25:47-51, 1969 pensatory mechanisms for the severe anaemia 7. Torrance J, Jacobs P. Restrepo A, Esch- caused by haemoglobin Hammersmith. Eur J bach J, Lenfant C, Finch CA: Intraerythro- Clin Invest 6:213-220, 1976 cytic adaptation to anemia. N EngI J Med 283: 4. Wranne B, Nordgren L, Woodson 165-169, 1970 Increased blood oxygen affinity and physical 8. Pace N: Respiration at high altitude. Fed work capacity in man. Scand J Clin Lab Invest Proc 33:2126-2132, 1974 33:347-352, 1974 9. Thomson JM, Dernpsy JA, Chosy LW, 338 CHARACHE El AL.

Shahidi NT, Reddan WG: Oxygen transport Hemoglobin Hasharon (a247 his (CD5) p2); A and oxyhemoglobin dissociation during pro- hemoglobin found in low concentration. J Clin longed muscular work. J AppI Physiol 37:658- Invest 48:834-847, 1969 664, 1974 22. Carrell RW, Kay R: A simple method 10. Parer iT: Reversed relationship of oxy- for the detection of unstable hemoglobins. Br gen affinity in maternal and fetal blood. Am J J Haematol 23:615-619, 1972 Obstet Gynecol 108:323-325, 1970 23. Clegg JB, Naughton MA, Weatherall Di: I I Pritchard JA, MacDonald PC: Williams An improved method for the characterization Obstetrics (ed 15). New York, Appleton- of human haemoglobin mutants: Identification Century-Crofts, 1976, p 156 ofa2fi29t Glu haemoglobin N (Baltimore). Na- 12. Metcalfe J, Dhindsa DS, Novy Mi: Gen- ture 207:945-947, 1965 eral aspects of oxygen transport in maternal 24. Jones RI: Automatic peptide chromatog- Downloaded from http://ashpublications.org/blood/article-pdf/51/2/331/581130/331.pdf by guest on 28 September 2021 and fetal blood, in Longo LD, Bartels H (ed): raphy, in Glick D (ed): Methods of Biochemical Respiratory Gas Exchange and Blood Flow in Analysis. New York, Interscience, 18:205, 1970 the Placenta. DHEW PubI NIH 73-361, 1973, 25. Bensinger TA, Beutler E: Instability of pp 63-74 the oxy form of sickle hemoglobin and of 13. Novy MI, Parer iT: Absence of high methemoglobin in isopropanol. Am i Clin blood oxygen affinity in the fetal cat. Resp Pathol 67:180-183, 1977 Physiol 6:144-ISO, 1969 26. Charache S. Murphy EA: Is placental 14. Riegel K, Bartels H, Buss 10, Wright oxygen transport normal in carriers of high PG. Kleinhauer E, Luck CP, Parer iT, Met- affinity hemoglobins? Proceedings, INSERM calfe J: Comparative studies of the respiratory Conference on Abnormal Hemoglobins, 1977 functions of mammalian blood. IV. Fetal and 27. Perutz MF: Structure and mechanism adult African elephant blood. Resp Physiol ofhaemoglobin. Br Med Bull 32:195-208, 1976 2:182-195, 1967 28. Mant Mi, Salkie ML, Cope N, Appling 15. Moore WMO, Battaglia FC, Hellegers F, Bolch K, iayalakshmi M, Gravely M, AE: Whole blood oxygen affinities of women Wilson iB, Huisman THi: Hb-Alberta or

with various hemoglobinopathies. Am I Obstet a2$2 lOl(G3) Glu - Gly) a new high-oxygen- Gynecol 97:63-66, 1967 affinity hemoglobin variant causing erythro- 16. Charache S. Brimhall B, Jones RT: cytosis. Hemoglobin I: 183-194, 1977 Polycythemia produced by hemoglobin Osler 29. Jones RI, Brimhall B, Gray G: Hemo- (fil45(HC2)Tyr-’ Asp). Johns Hopkins Med I globin British Columbia (a2$2 lOl(G3) Glu 136:132-136, 1975 Lys) a new variant with high oxygen affinity. 17. Charache 5, Fox I, McCurdy P. Hemoglobin 1:171-182, 1977 Kazazian H Jr, Winslow RM: Postsynthetic 30. Adams iG III, Winter WP, Tausk K, deamidation of hemoglobin Providence (f182 Heller P: Hemoglobin Rush (8101(G3) gluta- Lys-” Asn, Asp) and its effect on oxygen mine): A new unstable hemoglobin causing transport. I Clin Invest 59:652-658, 1977 mild hemolytic anemia. Blood 43:263-296, 1974 18. Winslow RM, Swenberg M-l, Berger 31. Trivelli LA, Ranney HM, Lai H-I: RL, Shrager RI, Luzzanna M, Sarnaja Hemoglobin components in patients with dia- Rossi-Bernardi L: The oxygen equilibrium betes mellitus. N EngI J Med 284:353-357, curve of normal human blood and its evalua- 1971 tion in terms of Adair’s equation. J Biol Chem 32. Ditzel i: Oxygen transport impairment 252:2331-2337, 1977 in diabetes. Diabetes 25[Suppl 2]:832-838, 1976 19. Imai K, Morirnoto H, Kotani M, 33. Schwartz HC, King KC, Schwartz AL, Watari H, Hirata W, Kuroda M: Studies on Edmunds D, Schwartz R: Effects of pregnancy the function of abnormal hemoglobins. 1. An on , in normal, gestational dia- improved method for automatic measurement betic, and diabetic women. Diabetes 25: 1 1 18- of the oxygen equilibrium curve of hemo- 1122, 1976 globin. Biochirn Biophys Acta 200:189-196, 34. Arturson G, Garby L, Robert M, Zaar B: 1970 Oxygen affinity of whole blood in vivo and 20. Hayashi A, Suzuki T, Shin M: An under standard conditions in subjects with enzymatic reduction system for metmyoglobin diabetes mellitus. Scand J Clin Lab Invest and methernoglobin, and its application to 34:19-22, 1974 functional studies of oxygen carriers. Biochim 35. Metcalfe I, Bartels H, Moll W: Gas cx- Biophys Acta 310:309-316, 1973 change in the pregnant uterus. Physiol Rev 21. Charache 5, Mondzac AM, Gessner U: 47:782-838, 1967