Blood oxygen affinity and hemoglobin type in adult, newborn, and fetal pigs

Respirafion

BLOOD

Physiology

( 1973) 19, l--l 1: North-Holland

OXYGEN

Dicision

Medicine

and the Heart

Research

School, Portland,

Abstract.

The

newborn

and

oxygen-binding

and the concentrations pigs within

24 hr of birth

The red cell 2,3-DPG from

the newborn

pig blood

concentration

within

miniature

responded

related

visible

peptide

patterns

after tryptic

during

late fetal

is maintained

and

and

hemoglobin

against

The P,,

at a plasma

phosphate

by a lower concentration

or bicarbonate

spectra.

resistance

suggests

that

drawn

adult,

newborn

dialysates

stripped

The hemoglobin

of the

on the basis of starch

to alkali

oxygen

from

39.5 ‘C.

as in blood from

denaturation.

that the same hemoglobin

the higher

adult.

of blood

of 7.20 and

buffers;

of 2.3-DPG.

from

pH of 7.40 and 38 “C

The P,,

solutions

from adult pig hemoglobin

Our evidence

life in the pig and

in fresh maternal

of hemoglobin

of excess amounts

absorption

hydrolysis.

Medical

of the hemoglobins

blood

as high

The P,,

to the addition

Oregon

solutions

characteristics

was twice

and ultraviolet adult

blood

to biochemical

(2,3-DPG).

pig in late fetal life is indistinguishable

electrophoresis,

of

pH

birth.

S. DHINDSA

University

was 25.0 mm Hg at an intracellular

24 hr after

identically

Laboratory.

and 22.0 mm Hg for fetal pig blood.

and fetal pigs was the same after dialysis of 2.3-DPG

DHARAM

Oregon 97201. U.S.A.

of whole

and

of 2,3_diphosphoglycerate

was 32.5 mm Hg for adult newborn

properties

fetal pigs were studied

TYPE IN ADULT,

AND FETAL PIGS’

J. NOVY, ARTHUR S. HOVERSLAND2, and JAMES METCALFE of Perinatal

Company, Amsterdam

AFFINITY AND HEMOGLOBIN NEWBORN,

MILES

Publishing

affinity

and

gel the

is synthesized

of fetal

pig blood

of red cell 2,3-DPG.

Fetal blood

Oxygen

Hemoglobin

P 50

Miniature

pig

Newborn

blood

equilibrium

curve

2,3_diphosphoglycerate

A high blood oxygen affinity is a nearly universal characteristic of mammalian fetuses (Bartels and Bauer, 1969) and suggests an adaptation to encourage placental gas exchange. Comparative studies of the oxygen binding properties of maternal and fetal blood from a variety of species indicate that different mechanisms maintain Accepted for publication

1 This work

16 April 1973.

was supported

HL-06042, HL-14121 * Present address: 93710. U.S.A.

in part

by USPHS,

National

Institute

of Health

grants

HL-05499,

and HD-06159, the Oregon Heart Association, and the Cammack Fund. Department of Animal Science, California State University, Fresno, California

2

M. J. NOVY, A. S. HOVERSLAND, D. S. DHINDSA AND J. METCALFE

oxygen affinity higher in fetal blood than in maternal blood at the same pH and temperature (Metcalfe, Dhindsa and Novy, 1973). These mechanisms include a structurally distinct fetal hemoglobin with an intrinsically higher oxygen affinity, fetal hemoglobin which is less responsive to 2,3-diphosphoglycerate (2,3-DPG) than the corresponding adult hemoglobin, and differences in the intraerythrocytic concentration of 2,3-DPG. The oxygen equilibrium characteristics of whole blood and hemoglobin solutions in the fetal pig have not previously been reported in detail. Furthermore, there is disagreement regarding the existence of a unique hemoglobin in the pig during fetal life (Novy et al., 1967; Glauser and Glauser, 1971; Kleihauer and Tautz, 1972). The relationship of the hemoglobin type in the adult and fetal pig to differences between their blood oxygen binding properties and red cell metabolism is the subject of this report. Materials and methods The animals used for the study of adult and fetal blood were “miniature swine” of the Pitman-Moore strain. Eleven pregnant and three nonpregnant sows were anesthetized with 15 trig/kg of sodium pentobarbital and blood samples were obtained from the femoral artery. After opening the abdomen and uterus of the pregnant animals, samples of fetal blood were obtained from the umbilical vessels. All of the gravid sows were in the last weeks of pregnancy (fetal crown-rump length 120 to 250 mm). Pigs of Duroc and Yorkshire breeds were used for the comparison of adult and newborn blood, and samples were drawn from these animals without general anesthesia. Each blood sample was drawn into a syringe whose dead space was tilled with heparin and sodium fluoride solution and placed in ice water. The “mixing technique” of Edwards and Martin (1966) was used to construct blood oxygen equilibrium curves and to determine the half saturation 0, tension (PJ and the Bohr effect at 38 “C. Oxygen tension and pH were measured in duplicate with Radiometer electrodes and a Radiometer model 27 meter. Measurements for comparison of fetal with adult blood were made at 38 “C, but measurements comparing newborn and adult blood and their hemolysates were made at. 39.5 “C. The oxygen electrode was calibrated before and after each determination with water-saturated gases of known O,, CO,, and N, composition. The-oxygen saturation of maternal and fetal blood after tonometry at known gas tensions was also measured by the manometric technique (Van Slyke and Neill, 1924) and the results of the two methods of measuring blood 0, affinity were compared. Studies were usually completed within 3 hr of blood withdrawal. The observed relationships of P,, and pH to the percentage saturation of hemoglobin with oxygen in the range of 20 to 80% saturation were processed by a computer program based on the method of least squares to yield the K values in a modification of Hill’s equation (Hellegers et al., 1959): log Po,=K,-K,pH+K,

S

log 1oo_s.

HEMOGLOBIN

TYPE AND OXYGEN

AFFINITY

3

OF PIG BLOOD

In this equation K, is a constant, K, is a measure of the Bohr effect in whole blood, and K, is the reciprocal of the constant n in Hill’s equation. Hemoglobin solutions were prepared from lysed red cells of fetal and adult pig blood (anticoagulated with heparin alone) after extraction of stroma and lipid with toluene. Aliquots ofthese solutions were dialyzed for a minimum of 24 hr against 1 liter of 0.05 M phosphate buffer at pH 6.5 to 7.0. Blood obtained from newborn pigs (within 24 hr of delivery) was incubated for 24 hr at 37 “C, then hemolyzed by freezing and dialyzed against 10 liters of a bicarbonate buffer in Visking bags for 48 hr according to a method previously described by Bauer et al. (1969). In these studies (results are shown in table 3) the plasma pH and the intracellular pH of blood were determined. One ml of fully oxygenated blood was mixed with one ml of deoxygenated blood in a plastic syringe, capped and centrifuged for 10 min. Plasma pH measurements were made from this plasma. The red cells from the spun samples were separated anaerobically and hemolyzed by freezing in liquid nitrogen and thawing. The pH of the red cell hemolysate was measured as an approximation of intracellular pH. The data were corrected to an intracellular pH of 7.2 for comparison with data from hemoglobin solhtions at the same pH. Methemoglobin concentrations were measured before and after tonometry by a modification of the method of Evelyn and Malloy (Paul and Kemp, 1944). Hemoglobin concentrations were determined spectrophotometrically as cyanmethemoglobin. Starch gel electrophoresis of the hemoglobin solutions was carried out in phosphate buffer at pH 6.9 and in Tris EDTA-borate buffer at pH 8.3 and the gels were stained with benzidine (Jones et al., 1967)..Hydrolysates of unfractionated hemoglobin from adult and fetal blood were prepared with trypsin in a solution buffered with triethylamine at pH 8 to 9 for 4 hr at room temperature. The peptides were then separated in the laboratory of Dr. Richard D. Jones3 on a column of Spinco 15 A resin using a linear gradient of pyridine acetic acid developer from pH 3.1 to pH 5.0 with continuous recording of ninhydrin color of the eluate at 570 rnp (Jones, 1964). Alkali denaturation of adult and fetal pig hemoglobin solutions was carried out by exposure to 0.1 to 1.0 M NaOH. The change with time in optical density of oxyhemoglobin at 540 rnp was recorded continuously in a Gilford recording spectrophotometer at 35 “C. The absorption spectra of adult and fetal oxyhemoglobins at ultraviolet and visible wave lengths were determined on a Cary recording spectrophotometer. The concentration of 2,3-DPG in adult and newborn pig blood was determined by the method of Grisolia et al. (1969). Purified 2,3_diphosphoglycerate was obtained from the Sigma Chemical Co. and added to the dialysates of adult and newborn blood to compare the effects of 2,3-DPG on the oxygen affinity of the hemoglobin solutinns measured as P,,. ’ Professor

and Chairman,

Department

of Biochemistry,

University

of Oregon

Medical

School.

4

M. J. NOVY, A. S. HOVERSLAND,

D. S. DHINDSA

AND J. METCALFE

Results

The oxygen equilibrium curves of whole blood from adult and fetal pigs (in late gestation) are shown in fig. 1. The individual data points, expressed at a plasma pH of 7.40, are shown with the curves of best fit calculated from the respective K values computed from the pooled data. The in uitro points obtained by the mixing technique are in good agreement with those obtained by the manometric method. The oxygen dissociation curves for adult and fetal pig blood are significantly different from one another. The mean P,, values at a plasma pH of 7.40 and 38 “C are 32.5 kO.9 mm Hg (S.D.) for adult pig blood and 22.0*1 .O mm Hg (S.D.) for fetal pig blood. The average deviation of the experimental points about the curve of best tit was 1.5-J 1.1 mm Hg for the adult pig dissociation curve and 0.9f 1.4 mm Hg for that of fetal pig blood. We found no difference in P,, between pregnant and nonpregnant adult pigs. IOQ-

90 -

gN40

-

MINIATURE PIG 30-

38“C,

20 -

l

/ IO // //’

//

1’

I

I

I

I

20

30

40

50

PO,

best fit derived

Van Slyke

0 Mixing

//

IO

Fig. 1. Oxygen-hemoglobin plasma pH of 7.40 and

pH 7.40

CO, Partial

60

Pressure)-mm

I

I

I

70

60

90

Hg

equilibrium curves for adult and fetal miniature pig blood expressed at a 38 “C. The individually measured points are shown about the curves of

from pooled

in

citro

data obtained

by manometric

and mixing methods.

The values for n in Hill’s equation (between 20 and 80% oxygen saturation) and the Bohr-effect factors are similar in adult and fetal pig blood (table 1). Following equilibration of blood with a carbon dioxide tension of approximately 41 mm Hg and oxygen tensions resulting in saturations close to 50x, the mean

HEMOGLOBIN

TYPE AND OXYGEN

TABLE Empirical

equations

describing

the oxygen equilibrium

AFFINITY

OF PIG BLOOD

1 curves of blood from adult and fetal miniature

pigs

Adult pig:

log Paz = 4.732-0.434

pH +0.322

log &

Fetal pig: log PI& =*4.656 -0.448

pH + 0.363 log &

TABLE Oxygen-hemoglobin

equilibrium dialysis Saturation

data

of maternal

against

2

and fetal pig hemoglobin

0.05 M potassium

phosphate

solutions

at 38 “C after

buffer

Fetal

Maternal

(““)

47.0

55.0

Hemoglobin

concentration

P 02

Paz (mm Hg)

PH

6.87

25.8

6.87

25.5

6.87

25.0

6.87

25.0

6.87

27.0

6.87

26.5

6.87

27.4

6.87

26.5

PH

6.0 g per

(mm Hg)

100 ml. Methemoglobin

concentration

was

less than

l”,

of

total hemoglobin.

TABLE Comparison and

of P,,

newborn

pig

(mm

Hg) and 2,3-DPG

blood,

after

3

concentration

incubation,

after

(nmoles/g

dialysis,

and

after

hemoglobin) addition

in fresh maternal of 2.3-DPG

to

the

dialysates _ Fresh blood

Incubated

blood

Dialysateb

Dialysate

+

2.3-DPG P 50

2,3-DPG

P,,

.2.3-DPG

P,,

2,3-DPG

~ P 50

2,3-DPG

Mother

32

30.93

21

9.47

21

0.43

31

54.09

Newborn

25

15.75

24

15.78

21

0.43

31

47.43

’ Incubation b Dialysis Data

for 24 hr at 37 “C. against

for P,,

solutions

10 liters of bicarbonate

buffer for 48 hr.

are expressed at an intracellular pH of 7.2 for comparison at the same pH. Hemoglobin solutions contained approximately

with data from 12.0 g hemoglobin

hemoglobin per 100 ml.

6

M. J. NOVY, A. S. HOVERSLAND, D. S. DHINDSA AND J. MBTCALFE

fetal blood pH was 7.415 (,range 7.290 to 7.465) and the mean maternal blood pH was 7.410 (range 7.335 to 7.470). After equilibration of blood with gases of high and low carbon dioxide concentrations for measurement of the Bohr effect, the pH ranged from 7.000 to 7.795. The mean hemoglobin concentration in 11 maternal blood samples was 10.1 f 1.57 gm/lOO ml (S.D.) with a MCHC of 35.7 + 2.69 (SD.). The mean hemoglobin concentration in fetal umbilical cord blood samples was 8.9 kO.86 gm/lOO ml (SD.) with a MCHC of 31.8*3.05 (S.D.). The oxyhemoglobin equilibrium data of dialyzed hemoglobin solutions are shown in table 2. Table 3 gives a comparison of oxygen affinity data from (1) fresh blood from sows and newborn pigs, (2) the same samples after 24 hr of incubation at 37 “C, (3) after hemolysis by freezing and dialysis against a bicarbonate buffer, and finally (4) after addition of 2,3-DPG to the dialysates. All oxygen affinity data in table 3 were obtained at 39.5 “C and expressed at an intracellular (for blood) or solution (for hemoglobin) pH of 7.20. Data for the pH gradient across the red cell membrane are shown in table 4. TABLE 4 Plasma and erythrocyte

(ERY) pH in blood from adult pigs and newborn pigs within 24 hr of birth* PHUW-

Adult

7.460 7.399 7.471

7.102 7.110 7.180

0.358 0.289 0.291

Newborn

7.354 7.387 7.403

7.103 7.129 7.143

0.251 0.258 0.287

PHW

*At 39.5 ‘C, HbO, approximately 50°j0and Pc,, 4@42 mm Hg.

It appears from these data that dialysis of adult and fetal hemoglobin solutions against either a phosphate or a bicarbonate buffer eliminates the differences in P,, observed in whole blood and the oxygen binding properties of the hemoglobin solutions become identical. Moreover, dialysates of adult and fetal pig hemoglobin stripped of 2,3-DPG respond identically to the addition of excess amounts of 2,3-DPG in vitro. A finding of some interest was the stability of the 2,3-DPG concentration in the red cells from newborn pigs after incubation of blood for 24 hr at 37 “C without additives. There was no change in oxygen affmity or in red cell 2,3-DPG concentration in the blood from the newborn pig while the P,, and the 2,3-DPG concentration of adult blood had fallen sharply. The 2,3-DPG concentration (expressed in pmoles/g Hb) is approximately twice as high in fresh maternal blood as in newborn pig blood drawn within 24 hr after birth (table 3).

HEMOGLOBIN

TYPE AND OXYGEN

AFFINITY

ORIGIN

+

Adult

Fig.

2. Starch

Tris-EDTA-borate

gel electrophoresis buffer,

7

OF PIG BLOOD

stained

of hemolysates with

benzidine). shown

from

maternal

Human

adult

Human

and and

fetal

cord

pig

blood

blood

(pH

hemolysates

8.3, are

for comparison.

The hemoglobin of the miniature pig in late fetal life and in the neonatal period is indistinguishable from adult pig hemoglobin on the basis of starch gel electrophoresis at pHs of 8.6 and 7.0 (fig. 2). The spectral absorption curves of oxyhemoglobin from adult and fetal pig red cells are identical in the visible as well as in the ultraviolet range. In both, the ultraviolet absorption spectra of oxyhemoglobin shows an identical tryptophan band at 291 nanometers. Hemoglobin solutions from adult and fetal pig red cells have the same high resistance to alkali denaturation (fig. 3). The peptide patterns of tryptic hydrolysates of unfractionated hemoglobin solutions from adult and fetal pig red cells are illustrated in fig. 4, and no difference is apparent. Discussion

The importance for the fetus of blood with a high affinity for oxygen needs experimental examination. Recent studies of human fetuses who received intrauterine transfusions of adult blood indicate that a higher fetal than maternal oxygen affinity is not a prerequisite for normal growth and development in man (Novy et al., 1971). Furthermore, the domestic cat (and the elephant in early fetal life) have blood with an oxygen affinity which is not significantly higher than maternal blood (Novy and Parer, 1969; Riegel et al., 1967). The quantitative importance for placental oxygen transfer of fetal blood oxygen affinity apparently differs among species and should be related to the rates of maternal and fetal placental blood flow, oxygen carrying capacities, and placental vascular geometry in evaluating its effect on the placental transfer of oxygen. The chemical mechanisms responsible for the high oxygen affinity of fetal blood have evolved along different lines in different species. In some animals, fetal hemoglobin has an intrinsically higher oxygen aflinity(goat and sheep); in others, fetal hemoglobin responds differently than adult hemoglobin to 2,3-DPG. In man,

8

M. J. NOVY, A. S. HOVERSLAND,

MINIATURE AAdult o

0.5

D. S. DHINDSA

AND J. METCALFE

PIG

Fetus

HbOP,

35°C

I

I

i

0.145

1

Il.0

1

I

I

I

13.0

12.0

14.0

PH

3. Alkali

Fig.

denaturation

rates

of hemoglobin

solutions

half-life is plotted

as a function

MINIATURE

from

adult

and

fetal

pig

blood.

The

of pH.

PIG

ADULT

g

FETUS

11’ L 03 02

I t

L

100

50 ML

Fig. 4. Peptide patterns of tryptic fetal pig blood. The absorbance

150

200

EFFLUENT

hydrolysates of unfractionated hemoglobin solutions from adult and of the reaction products of ninhydrin and peptides was measured continuously

at 570 mp.

hemoglobin A and hemoglobin F exhibit an identical intrinsic oxygen affinity but hemoglobin A is affected by 2,3-DPG to a greater extent than is hemoglobin F (Bauer, Ludwig and Ludwig, 1968), and the same is true of rhesus monkey hemoglobins (Metcalfe et ol., 1973).

HEMOGLOBIN TYPE AND OXYGEN AFFINITY OF PIG BLOOD

9

Our data suggest that in the pig the same hemoglobin is synthesized during late fetal and adult life and that differences in the intraerythrocytic milieu account for the different oxygen affinities of maternal and fetal blood. The observation that hemoglobin solutions of maternal and fetal pig blood do not differ significantly in oxygen affinity after 2,3-DPG is removed by dialysis against phosphate or bicarbonate buffers and the identical response to the addition of exogenous 2,3-DPG to the hemoglobin solutions indicates a functional identity of the hemoglobin molecules. The higher oxygen affinity in the blood of fetal and just-born piglets seems to be largely due to the low concentration of 2,3-DPG in their red cells. Duhm and Kim (1972) have shown that the erythrocyte concentration of 2,3-DPG at birth is low in pigs (approximately 2 pmoles/g of erythrocytes), rises to about 8 pmoles/g by the second week of newborn life, and reaches the adult level of about 10 pmoles/g at 6 weeks. Whether the functional similarity of the hemoglobins from adult and fetal pig red cells is due to the presence of the same hemoglobin or two structurally different hemoglobins with identical functional properties is controversial (Novy et al., 1967; Glauser and Glauser, 1971; Kleihauer and Tautz, 1972). Glauser and Glauser (1971) compared several properties of hemoglobin from adult pig blood with those of blood obtained from fetuses during the second month of gestation. They demonstrated a slower electrophoretic mobility, a greater resistance to alkali denaturation, and a difference in the peptide “fingerprint” of the hemoglobin solution from fetal pig erythrocytes in early gestation. In contrast, Kleihauer and Tautz (1972) applied similar tests and found no difference between adult hemoglobin and that obtained from fetuses with crown-rump lengths ranging between 20 and 230 mm. Our data pertain only to fetuses of greater than 120 mm crown-rump length and delivered in the last weeks of gestation. It is possible that a different hemoglobin is synthesized earlier in the development of the pig and is then completely replaced by adult hemoglobin in the latter part of gestation. Such a transition is known to occur, for instance, in the elephant fetus (Kleihauer et al., 1965). However, a fetal hemoglobin in the pig during the second month of gestation would need to be distinguished from the embryonic hemoglobin Gower, and hemoglobin Gower, which are still detectable in the pig during the second month of intrauterine development (Kleihauer and Stoffler, 1968). Recent work also by Kleihauer (personal communication, 1972) suggests that the amino acid content of alpha and non-alpha chains of hemoglobins from adult and fetal red cells of pigs is identical. If the higher oxygen affinity of fetal pig blood is maintained by a lower 2,3-DPG concentration in their red cells, the next question is, “What factors regulate intraerythrocytic 2,3-DPG levels?” Intracellular pH is known to be a potent regulator of the intracellular 2,3-DPG concentration (Duhm and Gerlach, 1971). The low level of 2,3-DPG in fetal pig erythrocytes may depend upon a continuing mild acidosis of the fetus. Alternatively, the persistence of 2,3-DPG in the blood of newborn pigs during incubation suggests an enzymatic difference between the red cells of the mother and the neonate. Differences in the glycolytic activity of red

10

M. J. NOW,

A. S. HOVERSLAND,

D. S. DHINDSA

AND J. MBTCALFE

cells from adult pigs compared with those from neonatal pigs have been described and traced to different permeability characteristics (Kim and McManus, 1971; McManus, Kim and Valle, 1971). This raises the possibility that differences in the red cell membrane are responsible for the different intraerythrocytic environments of hemoglobin in fetal and adult blood and thereby for the resultant differences in blood oxygen affinity. Acknowledgements

The authors are indebted to Dr. Morris E. Weaver for providing the miniature pigs used in this study and to Dr. Richard T. Jones and Dr. Robert D. Koler for assistance with determinations of peptide patterns and alkaline denaturation of pig hemoglobin. We thank Mrs. Zelma Stocklen and Miss Dietrun Kamp for expert technical help. References Bartels,

H. and Ch. Barter (1969).

Forsvarsmedicin Bauer,

C., I. Ludwig

triphosphate Bauer,

and M. Ludwig

on the oxygen

Ch., M. Ludwig,

human Duhm.

adult

Duhm.

I. Ludwig

H. D. Kim

and

(1972).

in erythrocytes

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on Metabolism

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curve and tissue oxygenation.

effects of 2,3-diphosphoglycerate (1969).

Factors

and adenosine

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governing

the oxygen

affinity

of

of the hypoxia-induced

increase

of 2,3-diphospho-

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of the rapid

postnatal

increase

on the oxygen affinity of pig blood. and

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(1966).

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(1971). On the mechanisms

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(1971).

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adult pigs. Proc. Sot. Exp. Biol. Med. 137: 1449-1451. Grisolia,

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hemoglobins es ein fetales

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McManus,

T.,J.,

H. D. Kim and D. Valle (1971).

development. Metcalfe.

TYPE AND OXYGEN

Abstract,

Meeting

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NOW

In the Proceedings

AFFINITY

changes

of General

(1973).

General

of the Satellite

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type in the adult

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(1969).

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neonatal

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(1967).

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A comparison

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Physiologists.

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11

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blood

oxygen

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M. J., F. D. Frigoletto,

umbilical-cord

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transfusions

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a normal

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in

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W. D. and

C. R. Kemp

(1944).

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solutions

by