Respiration Physiology (1973) 19, 153-161; North-Holland Publishing Company, Amsterdam

CHANGES IN RED CELL 2,3-DIPHOSPHOGLYCERATE

CONCENTRATION AS CAUSE OF THE POSTNATAL DECREASE OF PIG BLOOD OXYGEN AFFINITY’

R. BAUMANN, F. TEISCHEL, R. ZOCH and H. BARTELS

Physiologisches Institut der Medizinischen Hochschule Hannover, Karl- Wiechert-Allee 9, Hannover 3,

Germany

Abstract. Oxygen affinity (expressed as oxygen half saturation pressure P,,) and red cell 2,3-diphos-

phoglycerate (2.3 DPG) concentration of miniature pig blood was examined in fetuses (955110 days

gestational age) and piglets from birth until 50 days post partum. Fetuses had the highest oxygen

affinity (P,, 23 mm Hg) and lowest 2.3 DPG concentration (2.40 mM 2.3 DPG/l, RBC). Both P,,

and 2.3 DPG concentration increased rapidly during the first week after birth towards adult levels.

Haemoglobin electrophoresis did not show different behaviour of haemoglobin obtained from foetal,

postnatal or adult pig blood. The oxygen equilibrium curves of the respective dialysed haemoglobin

solutions were identical and upon addition of 2,3 DPG the same decrease in oxygen affinity was ob-

served. It is concluded that the postnatal decrease in pig blood oxygen affinity is caused by a con-

comitant increase in red cell 2,3 DPG concentration.

Blood

Foetal

Haemoglobin

Miniature pig

Neonatal

Oxygen affinity

2.3 DPG

In most mammalian species foetal blood has a higher oxygen affinity than adult blood. Transition towards adult oxygen affinity usually takes place during the early postnatal period and is accomplished by different mechanisms. Either like in sheep or goats (Blunt et al., 197 1; Baumann, Bauer and Rathschlag-Schaefer, 1972) foetal haemoglobin is replaced by adult haemoglobin of intrinsically lower oxygen affinity or as for example in man (Bauer et al., 1969; Tyuma and Shimizu, 1969) foetal and adult haemoglobin show the same oxygen affinity but react differently with 2,3- diphosphoglycerate (2,3 DPG) with regulates the oxygen affinity of several species (Benesch and Benesch, 1969; Bunn, 1971). In addition variations in red cell pH can also play a role as cause for postnatal changes in oxygen affinity (Hilpert et al.,

1963).

Accepted for publication 23 June 1973.

’ Financial support by Deutsche Forschungsgemeinschaft is gratefully acknowledged.

153

154 R. BAUMANN. F. TEISCHEL. R. ZOCH AND H. BARTELS

If a haemoglobin reacts with 2.3 DPG like human haemoglobin, changes in the

concentration of 2,3 DPG should also lead to affinity changes. The possible role of

2,3 DPG concentration changes as cause for postnatal oxygen affinity changes was

investigated in miniature pigs, since Novy et ~1. (1967) found identical functional

characteristics of dialysed foetal and adult pig haemoglobin, while on the other

hand whole blood oxygen affinity differed largely. Like Tautz and Kleihauer (1972)

he was unable to detect any difference between the haemoglobin type present in

adult or foetal red cells, which would rule out a change in haemoglobin type as

cause for the different whole blood oxygen affinities. This led Novy et al. (1967)

to the conclusion that differences in red cell environment had to be responsible for

this phenomenon. The results of our study confirm his view and show that 2,3 DPG is

the only factor responsible for postnatal changes in pig blood oxygen affinity in

accordance with the study of Duhm and Kim (1972) which came to our attention

while this investigation was in progress.

Material and methods

Three miniature pig sows of mixed breed (Giittingen) and their offspring (21

piglets) were used for the investigation. In addition blood samples from eight

miniature pig fetuses2 of 95-110 days gestational age of the same breed were

examined.

Blood samplirlg

Blood was taken from the piglets by heart puncture in light halothane anesthesia

and by puncture of the superior vena cava from the sows. Sampling started immedia-

tely after birth and was repeated at daily intervals during the first week using 2-6

animals alternately. Sampling was continued at larger intervals until the 50th day

post purtum. Powdered sodium heparinate was used to prevent clotting.

Blood gus anulyses

Whole blood: Samples of blood were equilibrated for 20 min at 37 “C and a Pco2

of 40 mm Hg with water vapor saturated gas mixtures of known composition.

The pH of the equilibrated samples was measured with a Radiometer microglass

electrode Type G 297/G2 and pH meter 4 calibrated with Radiometer precision

buffers. Micromanometric analyses for 0, and CO, were made on 0.05 ml samples

after Van Slyke and Plazin (1961).

Oxygen capacity and oxygen affinity (expressed as oxygen half saturation

pressure P,, at plasma pH 7.4, 37 “C in whole blood) were calculated after Bartels

and Harms (1959). Haemoglobin was measured as cyanmethaemoglobin (Kleihauer

and Betke. 1957) and methaemoglobin with the method of Evelyn and Malloy (1938).

Haematocrit values were determined in duplicate with the Hawskley micro-method.

For assays of 2.3 DPG one volume of freshly drawn blood was added to nine

volumes of icecold 69/i perchloric acid and centrifuged after stirring. Aliquots of the

’ Kindly provided by Dr. R. Ruefer ( Max-Planck-Institut fiir experimentelle Medizin. G6ttingen)

2.3 DPG AND PIG BLOOD OXYGEN AFFINITY 155

supernatant were neutralized with 2 M K&O,. 2,3 DPG was measured using the

enzymatic test of Krimsky (1962). All enzymes and substrates for the test were

purchased from Boehringer Co., Mannheim.

Prepuration of haemoglobin solutions

Packed erythrocytes were washed three times with 0.9% NaCl and haemolyzed by

addition of one volume of distilled water to one volume of packed cells and centri-

fuged thereafter for 1 hr at 100,000 g. The clear supernatant was placed in Visking

bags and dialysed at 4 “C for three days against several changes of 0.05 M Bis-Tris

buffer in 0.1 M NaCl (pH 7.2 at 20 “C).

After dialysis the haemoglobin solution was diluted with the buffer to a final

concentration of 1 x lo-’ mM/Hb, and divided into three parts: A) no 2,3 DPG

added, B) 30 PM 2,3 DPG/g Hb added, C) 45 PM 2,3 DPG/g Hb added. Oxygen

equilibrium curves were then determined with the method of Benesch, MacDuff and

Benesch (1965) using the Hitachi 1247 Spectrophotometer. Haemoglobin electro-

phoresis was carried out on cellulose acetate strips (Tris-EDTA buffer pH 8.2) with

the Boskamp microphor electrophoresis system.

Results

BLOOD 2.3 DPG CONCENTRATION AND P,,(table 1)

Fetuses showed the lowest P,, (23.0 mm Hg) and 2,3 DPG concentration (2.40

mM/L RBC). During the first day after birth both values increased to 27.6 mm Hg

P,, and 5.68 mM 2,3 DPG/L RBC respectively. By the fifth day post partum the

piglets had already established P,, and 2,3 DPG values comparable to adult ones.

Haemoglobin and haematocrit values (table 1) decline during the first five days after

birth and reflect a genuine decrease, since during this period no animal was bled

twice. In the later course however group 2 animals became markedly more anaemic

than group 1 animals since in the former group blood sampling had started at

birth whereas in the latter group it started by the third day posf partum. P,,

and 2,3 DPG values of group 2 animals were markedly elevated at the fortieth

day as compared to group 1 animals. whereas at all other dates no significant

difference was observed.

The P,O and 2,3 DPG of piglets 50 days after birth were slightly elevated in

comparison to the adult values like in all other species so far investigated. No

statement can be made about the significance due to the small sample size. Figure 1

shows the time course of the postnatal change in P,, and 2,3 DPG concentration.

DIALYSED HAEMOGLOBIN SOLUTIONS

Table 2 summarizes P,, values of dialysed haemoglobin solutions prepared from

adult blood and from foetal, neonatal and postnatal piglet blood respectively, with

and without 2,3 DPG added. As can be seen from fig. 2 the oxygen equilibrium

curves of both groups are identical and upon addition of 2,3 DPG the same

TA

BL

E

1 5

Mea

ns

(X)

of

P,,

mm

H

g 0,

. 2.

3 D

PG

mM

/L

RB

C,

haem

oglo

bin

8%

and

haem

atoc

rit

valu

es

of

foet

al

and

neon

atal

pi

glet

s un

til

50

days

af

ter

birt

h.

For

n 2

4 th

e st

anda

rd

devi

atio

ns

(SD

.)

are

give

n,

for

n <

4 th

e si

ngle

va

lues

(x

) ar

e re

cord

ed.

Sinc

e on

e lit

ter

deve

lope

d a

mar

ked

anae

mia

it

was

nece

ssar

y to

sp

lit

the

pigl

ets

into

2

grou

ps

(i.e

. gr

oup

1 an

d gr

oup

2 fr

om

day

20 p

ost

part

um

onw

ards

).

Age

n

P,a

mm

H

g O

2 pH

7.

4,

37

‘C

x +S

.D.

x

2,3

DPG

m

M/L

R

BC

x

Hae

mog

lobi

n 8%

H

aem

atoc

rit

y0

&SD

. x

%

_+S.

D.

x x

&SD

. x

Fetu

ses

95-l

10

day

s

gest

atio

nal

age

8

1 hr

af

ter

birt

h 2

17-2

4 hr

af

ter

birt

h 6

2nd

day

4

3rd

day

3

4th

day

4

5th

day

2

10th

da

y 3

13th

da

y 5

15th

da

y 4

20th

da

y to

tal

5

grou

p 1

3

grou

p 2

2

23.0

23.8

27.6

29.7

32.3

31.7

32.5

33.2

33.1

33.3

32.8

32.2

33.6

1.0

25.8

22.9

3.2

1.6

34.4

34.6

28.4

1.5

33.4

31.5

32.5

33.2

31.8

1.7

0.5

0.9

32.0

32.4

32.3

33.2

34.1

2.40

3.25

5.68

6.9

1

7.66

7.20

8.21

9.24

8.00

8.46

6.82

7.32

6.17

0.92

1.06

0.8

1

2.01

1.07

0.96

0.83

3.59

2.92

9.06

8.24

5.68

7.94

8.48

8.42

10.8

5

8.47

7.86

6.87

7.24

5.61

6.73

11.5

11.9

10.5

9.2

8.5

8.2

7.7

10.0

9.5

9.3

8.8

10.5

6.9

1.0

12.3

11.5

.

1.5

0.5

6.9

8.6

10.0

0.5

8.3

1.2

9.0

10.6

10.3

0.8

0.5

2.1

10.3

9.8

11.3

6.7

7.1

39.6

39.0

32.3

29.6

28.0

26.6

26.3

32.3

32.1

30.6

30.7

35.7

23.2

4.0

40.0

7

38.0

;;

I

3.2

K

2.4

!z

22.0

.p

27.5

7

34.6

2.

6 ! z

29.0

>

23.5

3

32.0

?

31.0

34

.0

$

2.2

E

2.1

2

6.3

36.5

33.0

37.5

23.0

23.5

30th

da

y to

tal

grou

p 1

grou

p 2

40th

da

y to

tal

grou

p 1

grou

p 2

50th

da

y to

tal

grou

p 1

grou

p 2

Adu

lt

3 32

.8

6.86

2 32

.7

33.6

6.

88

31.9

1 32

.9

4 33

.7

3.0

8.60

2 32

.5

6.99

30.0

2 36

.0

10.2

0

36.3

3 34

.4

9.72

1 33

.6

2 34

.8

33.9

10

.22

35.7

2 31

.1

30.6

8.

48

31.7

8.6

7.16

10

.3

6.60

6.81

1.99

10

.7

7.47

11

.4

6.51

10.9

5 8.

8

9.46

9.5

8.70

11.1

0 7.

7

9.35

8.42

13

.1

8.53

__

31.0

8.4

32.5

29

.0

12.2

36

.0

5.2

28.0

1.4

32.9

5.

8

11.2

37

.2

37.0

11.6

36

.5

10.0

29

.0

32.0

7.5

26.0

t3

31.3

13.0

39

.5

;s

7.7

27.2

27

.5

8

7.7

27.0

12.9

38

.7

38.0

39.5

w

‘t

158 R. BAUMANN, F. TEISCHEL, R. ZOCH AND H. BARTELS

PSO I 10 mM2.3-DPGJL RBC

_*.

mmHs 9 ,,T _.-’

35 ,’ ‘\ _.*’

-----F----- ~___,._________________________--__,.-_:~___Adu[~

8 7’ “.: \\

8’ o-a+o 0

‘;_________--4’ .’

Adult .7

30 .6

-5

-4 25

.3

. DPG

0 650

d 0.

t 2 ietol

20’ 0 10 20 30

, 40 50

days past partum

Fig. 1. Postnatal changes in oxygen affinity (P,, at pH 7.4. 37 “C) and red cell 2,3 DPG concen-

tration of miniature piglets.

TABLE 2

Ps, mm Hg 0, at pH 7.2, 20 “C of dialysed haemoglobin solutions prepared from either maternal or

piglet blood during different stages of development (i.e. foetal, neonatal, postnatal) referred to as “foetal”

blood during different stages of development (i.e. foetal, neonatal, postnatal) referred to as “foetal”

in the graph with and without 2,3 DPG added.

Piglets sow

P,, mm Hg O,, pH 7.2 P,, mm Hg O,, pH 7.2

x +S.D. x x &SD. x

dialysed Hb solution 2.99 + 0.05 3.02 0.03

no 2.3 DPG added n=5 n=4

+30 PM 2.3 DPG/g Hb 5.77 0.09 5.79 0.08

n=5 n=4

+45 PM 2,3 DPG/g Hb 6.52 6.55 6.53 6.53

n=3 6.45 n=2 6.53

6.55

decrease in oxygen affinities is observed. The methaemoglobin concentration in these

experiments varied between 224%.

HAEMOGLOBIN ELECTROPHORESIS

Samples of foetal, postnatal and adult haemoglobin were repeatedly analysed for

electrophoretic mobility, no difference between adult and foetal or postnatal

haemoglobin samples could however be detected.

Discussion

The results clearly demonstrate that the postnatal increase in red ceil 2,3 DPG

2,3 DPG AND PIG BLOOD OXYGEN AFFINITY 159

SOz% h 100

90.

80.

70,

60.

50.

I dialysed Hb solution no 2.3-DPG added . 20. II

30. ,y

with 2.3- DPG/gHb added III with

30pM 15pM 2%DPG/gHb added

10.

,

1 2 3 I 5 6 7 8 9 10 11 12

mmHg& Fig. 2. Effect of 2.3 DPG on oxygen equilibrium curves of haemoglobin solutions prepared from

maternal and piglet blood during different stages of development (i.e. foetal. neonatal: postnatal).

PSO mmHg

35.

y=209844083x

r--O.8481

20 > 1 2 3 4 5 6 7 8 9 10 11 12

mM2.3-DPGJL RBC Fig. 3. Correlation between whole blood oxygen half saturation pressure (P5,,) and red cell 2.3 DPG

concentration.

160 R. BAUMANN, F. TEISCHEL, R. ZOCH AND H. BARTELS

concentration is responsible for the decrease of pig blood oxygen affinity during this period. as shown by fig. 3 which demonstrates the close correlation between P,, of whole blood and red cell 2,3 DPG concentration, The oxygen equilibrium curves of dialysed foetal, postnatal and maternal blood were identical and upon addition of 2,3 DPG the same shift in oxygen affinity is observed in agreement with Duhm and Kim (1972). Although some authors (Glauser and Glauser, 1971) hold the view that there is a foetal haemoglobin in pigs, this does not affect our conclusion, since as far as oxygen affinity and response to 2,3 DPG are concerned haemoglobin solutions derived from fetuses and adult pigs show the same characteristics.

The reason for the sudden increase in red cell 2,3 DPG concentration during the first few days after birth is unknown. While for human red cells it is established that an increase in red cell pH leads to increased synthesis of 2,3 DPG (Asakura et al., 1966), it is not clear if the large increase in blood pH of piglets during the first 48 hr after birth (,Randall, 1972) might act as a stimulus towards higher 2,3 DPG synthesis since no detailed data are available about glycolysis of neonatal pig red cells. Moreover, the fact that the glycolytic activity of adult pig red cells is by far the lowest of all animals so far investigated (MacManus and Kim, 1968) makes it difficult to apply the same principles that have been established for the regulation of 2,3 DPG metabolism in the human red cell.

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