Carbonic anhydrase isoenzymes in the erythrocytes of new-born premature and full-term infants

127

Clinica Chimica Acta, 61 (1975) 127-133 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 6929

CARBONIC ANHYDRASE ISOENZYMES IN THE ERYTHROCYTES OF NEW-BORN PREMATURE AND FULL-TERM INFANTS

MOGENS

MONDRUP*

and NIELS ANKER

Department of Clinical Roskilde (Denmark)

(Received

October

Chemistry

and Medicine,

Roskilde

County

Hospital,

31, 1974)

Summary

A specific and quantitative immunological method for the determination of human erythrocyte carbonic anhydrase isoenzymes B and C has been used to determine these enzymes in erythrocytes of umbilical cord blood. The investigations have shown a content of both isoenzymes less than 1 : 10 of that in adults. Premature infants have significantly lower values than full-term infants. The influence of carbonic anhydrase in the respiratory distress syndrome is discussed. No conclusions are drawn. The content of carbonic anhydrase in erythrocytes has been suggested as a laboratory parameter of maturity. In our investigations it does not seem to be useful for the evaluation of maturity.

Introduction

In recent years erythrocyte carbonic anhydrase (HCA) (carbonate hydrolyase, EC 4.2.1.1.) and its various isoenzymes have been extensively studied [l---5] . The enzyme carbonic anhydrase affects the human organism decisively in two areas: (1) In erythrocytes, where HCA catalyses the reversible hydration of CO2 . (2) In secretory organs where the enzyme has an effect on the transfer and accumulation of H’ and HCO;. Two main types of HCA have been isolated from human erythrocytes: Isoenzyme B and C, together with a minor component named A [6--91. Other isoenzymes have also been described [7]. Immunologically, however, they belong either to type B or C. No cross reaction has been observed between type B and C [lo]. In human erythrocytes isoenzyme B is responsible for about 89% of the total HCA activity [ll].In this respect there is no significant difference between males and females [lo] . * To

whom

reprint

requests

should

be addressed.

128

The relation between the determination of HCA isoenzyme activity and the quantitative determination by immunoelectrophoresis has shown a linear dose-response curve for both HCA type B and HCA type C [lo] . Variations in the HCA isoenzyme content in both health and disease have been the subject of increasing interest. However, previous methods of determination, based on measurements of activity are too time consuming for routine investigations. A specific and quantitative immunological method for the determination of HCA type B as well as HCA type C has been described [lo] . Earlier investigators [ 122181 have shown that the HCA enzyme concentration is lower in newborn infants than in adults. In premature infants even lower enzyme concentrations are found. Some authors find the ratio between the enzyme concentration of full-term infants and adults to be about 1 : 4 [ 12,13, 15,161, while others find it to be 1 : 10 [19]. All these investigations are based on measurements of activity. Starting from the earlier established fact that carbonic anhydrase activity in the blood of premature infants is low, it is the purpose of the present investigation to study a large number of infants, using a new immunological method, in order to look for a closer relationship between the concentration of HCA isoenzymes and the degree of prematurity. Materials and Methods Umbilical cord blood was used for the determination of both HCA type B and C in 293 children. In 3 children only HCA type B was determined and in 19 children only HCA type C. The children were grouped according to birthweight in 500-g groups from 1000 g to more than 4000 g. HCA isoenzymes were determined by the method of Ndrgaard-Pedersen and Mondrup [lo]. Antisera against HCA isoenzymes are made by subcutaneous injection of the protein in rabbits [lo] . Pure antigen which is used for immunisation and as standard is made as described by Armstrong et al. [6]. The antigen concentration is determined spectrophotometrically at 280 nm: E Qrn, 1% 280 nm

B = 16.3

c = 18.7

t201

Samples of 5 ml of heparinized blood were centrifuged as soon as possible after withdrawal and the plasma and buffy coat removed. The erythrocytes were washed 3 times with a double volume of cold isotonic NaCl solution. By repeated freezing and thawing (3 cycles) the erythrocytes were lysed. If the determination of HCA type B and C was not made on the same day, the hemolysate was stored at -21°C; HCA isoenzymes were determined in unpurified hemolysate by electrophoresis in antibody-containing agarose gel as described by Laurel1 [21]. Since the charge of HCA isoenzymes is almost identical with that of rabbit antibody, it was necessary to carbamylate both the standard and the hemolysate with 2 M KCNO [22]. In carbamylation, free (Yand e-amino and sulphydryl groups react with KCNO, thus lowering the isoelectric point of the proteins and increasing their anodic migration. As shown in Fig. 1, electrophoresis in 1% agarose was run with 3 V/cm for 16 h. The precipitate was stained with Coomassie Brilliant Blue. The results are calculated as mg enzyme per liter erythrocytes.

129

~~,“,,14,r,l~~llrl,lli ~~~~~~~~~ GA

.*

l *..

*.

Fig. 1. Quantitation of HCA in lysates. l pure standard dilutions 1 : 10.1 : 20 1 : 40.1 solution containing 1 g HCA type B or C per liter. The dilution of the hemolysates is 1 : B and 1 : 4 for HCA type C.

: 80 of a stock 6 for HCA type

Results The concentration of HCA types B and C in erythrocytes of umbilical cord blood has been determined. In our earlier investigations Cl01 we found a mean value in adults of HCA type B of 4.89 g/l erythrocytes. The mean value in full-term infants is less than l/10 of that in adults for both isoenzymes. The enzyme concentrations found in the different weight groups are shown in Tables I and II, Figs 2 and 3. The enzyme concentrations in the premature infants are significantly lower than in the full-term infants. Tables III and IV show the p-values obtained for the differences between the weight groups. In five infants neither HCA type B nor type C could be demonstrated. Three of these infants were born at term, had normal weights and no respiratory problems. One died soon after birth of cerebral lesions and one, weighing 2000 g, developed a respiratory distress syndrome (RDS), but survived.

HCA TYPE B

T

MG/

L ER7

ROCYTES

..

.. *: :.

*. .. .

... ... . .. .

300

:‘..” .*..

T

.* :. .. **

t

:...; *- .: . . . . . . :.... . . . . . . . . . . . . . .

..,. :. . ..

t

:. . . .

. . . . . *. . . . . . . . . . . . . :

.

:.

100

., ..

:.

.* ::... i. ...... .. :.. . .... . ;” .. ..: .. . . ... ..* .*

.. ..

-. .

t

.*.

1000-1500

*..* 1500 -2000

-

2000-2500

:. . ..* 2500-3ooo

‘. :. . :.... . . . . ..

3ooo-3500

i 35004000

;

‘4000

BIRTHWEIGHT

Fig. 2. Concentration of HCA type B in the erythrocytes of newborns with different weights.

:G

130

CONCENTRATION

OF

HCA

TYPE

B

IN

THE

ERYTHROCYTES

IN

THE

DIFFERENT

WEIGH’I

GROUPS Birthweight

NO.

Mtaan (mg/l

(g)

Range erythrocytes) __~~

S.D.

(me/l

erythrocytes)

1000-1500

3

0

1501-2000

I

79

2001-2500

16

175

o-555

i 157

2501-3000

36

209

O-564

k 156

3001-3500

127

274

0-

3501-4000

80

309

24-810

27

250

>4000

TABLE

0

f

O-330

+ 126

654

0

? 167 t 159

o--549

i

156

II

CONCENTRATION

OF

HCA

TYPE

C

IN

‘THE

ERYTHROCYTES

IN

THE

DIFFERENT

WEIGHT

GROUPS ~__ Birthweight

Range

(9)

(mg/l

erythrocytes)

1 ooo-

1500

1501-2000

S.D.

2

14

12-m

17

?

3.6

5

21

ll-

34

i

9.6

2001-2500

17

30

0-m

75

5 22.4

2501-3000

39

40

0-

85

i 16.7

92

+ 20.1

3001-3500

128

47

o-

3501-4000

94

50

O-134

27

48

>4000

I21.5

IS-112

k 20.6

ERVTHRO

t 100 i

i +

1

50 f

t

:.

f 1000-1500

Fig.

3. Concentration

1500-2000

of

HCA

type

2000-

C in the

2500

... : .

. :_. :. .

:: . ... . . . ... :: ... . . ....... . . .. . . ... ... . ::: . .. :... . . ..... . .. .. . 1..

.

.

.

.

.

.

... .. ::.... ... .. ... :: ...... ::..

.. .. . ::

:““’ :::” . . . . . . :: . . . :: :. . .

. . . . . .

:. . .

.

:.

.

.

I 2500-3000

erythrocytes

3000-3500

3500-4000

z= 4000

BIRTHWEIGHT of

newborns

with

different

weights

:G

131

TABLE THE

III

p-VALUES

FOUND

Weight

(HCA

A

tYPe

B)

B

C

D

E

F

G

p < 0.01 p < 0.005 p < 0.05 p < 0.05

p < 0.005 p < 0.001

p< p<

p < 0.005

N.S.

tg1 1000-1500

(A)

1501-2000

(B)

2001-2500

(C)

2501-3000

(D)

3001-3500

(E)

3501-4000

(F)

>4000

N.S..

N.S.

N.S.

P <

0.05

N.S.

P <

0.05

N.S.

p<

0.005

N.S.

0.02 0.05

N.S. N.S. N.S.

(G)

no significance.

Table V shows the values for HCA type B and C in five infants with RDS. The diagnosis was made independently of the enzyme measurements. We shall not attempt any conclusion from these few observations. Discussion The very low HCA activity in the blood of premature infants has been discussed earlier [23] . Some authors believe that the low HCA value results in CO, retention and acidosis and thus contributes to the development of the respiratory distress syndrome (RDS) [ 151, Others have not been able to demonstrate a difference in HCA activity between infants with RDS and other prematures [16,24]. It is obvious that the aetiology of RDS is a complex one, and several hypotheses have been advanced. While the role of HCA deficiency in the causation of the syndrome remains uncertain, it is reasonable to assume that in infants with RDS and consequent retention of CO, , deficient carbonic anhydrase activity may worsen an already precarious situation by inhibiting the conversion of bicarbonate to CO* , leading to inadequate elimination of CO 2 during the rapid passage of blood through the lungs. Absence of carbonic anhydrase is compatible with life and does not necessarily entail respiratory problems, e.g. our 3 cases mentioned above. There are alternative mechanisms for the transport of CO,. It is possible that, in the absence of carbonic anhydrase, increased amounts of CO2 are transported as TABLE THE

IV

p-VALUES

Weight

FOUND

A

(HCA

tYPe

C)

B

C

D

N.S.

N.S.

P < 0.05

N.S.

p<

F

G

0.02

p < 0.025

0.005

p < 0.005

p < 0.05 p < 0.01

0.005

p< p<

E

(9) 1000-l

500

(A)

1501-2000

(B)

2001.-2500

(C)

2501-3000

(D)

3001-3500

(E)

3501-4000

(F)

>4000 N.S..

(G) no significance.

N.S.

0.02

p< p< p<

p < 0.05

N.S.

0.001

p < 0.01

0.02

N.S. N.S. N.S.

7 :32

HCA

TYPE

B

AND

C

CONCENTRATIONS

IN

5

CIIILDREN

WITH

RESPIRATORY

DISTRESS

SYNDROME ~~~~ __...._ Child

Wright

HCA

Type

(9)

(mg/l

ervthrocytes)

B

HCA

Type

(mg/l

erythrocytes)

1

1920

0

2

2000

0

3

2050

105

4

2250

30

17.2

5

2350

0

20.4

C

56.4 0 24.8

______.-

___________~_

dissolved CO2 and carbamino CO,, but this remains to be shown [25]. In anaesthetised dogs, inhibition of HCA only initially decreased the CO, output [26], and acetazolamide-treated glaucoma patients with an almost total inhibition of HCA feel well [27] . These observations support the theory about alternative mechanisms of CO2 transport. As the red cells of adults seem better equipped than those of premature infants to meet the requirements of extrauterine life exchange transfusions have been tried in order to treat RDS. The information is limited and it remains uncertain whether exchange transfusions are of therapeutic value in infants with RDS. It has also been discussed whether the HCA concentration can be used as one of the possible parameters of maturity. The demand for new maturity parameters is increasing because variation in the time incidence of ovulation has been increased by the use of oral contraceptives, which, after discontinuation can cause temporary amenorrhoea. Some investigators [28,29] have found that HCA activity as a maturity parameter is no better than the determination of the hemoglobin F concentration, while others [30] have found the HCA determination better than hemoglobin F. Neither of these methods is demonstrably better than birthweight and length in estimating the maturity of an infant. The magnitude of spread in HCA values in our material, even among full-term infants, makes it most unlikely that the determination of HCA isoenzymes will be of practical value for the evaluation of the degree of maturity. Acknowledgements The skilful technical assistance of Mrs Birte Gylling Pedersen is greatfully acknowledged. This investigation was supported by grants from Fonden til laegevidenskabelig forskning ved sygehusene i region 3 (l-9). References 1

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