Reduced albumin binding of MADDS — a measure of bilirubin binding — during pregnancy and delivery

E~rrotpcur~Jourrrd of Oh.stc~tr~~ & ~;yn~olo~~ and Rqm~ductir~~ Bido~y?, I6 (1 YY2) OS- IO0 ,I’# lYY3 lllscvicr Science Publishers B.V. All rights reserved 002X-2243/Y2/$OS.O0

ElJROBS 01301

Reduced albumin binding of MADDS - a measure of bilirubin binding - during pregnancy and delivery

Summary It was the purpose of the present study to investigate whether the decrease in the ability of plasma albumin to bind bilirubin, noted in pregnancy and during delivery, takes place in one step, during pregnancy alone, or in two steps, during both pregnancy and delivery. Furthermore, it was investigated whether a possible decrease during labor was related to the method of delivery itself. The material comprised (a) 17 pregnant women who delivered vaginally; (b) 25 women who were delivered by Cesarian Section; and (c) a group of 25 non-pregnant women. The reserve albumin concentration for binding of MADDS (a measure of the binding of unconjugated bilirubin), the total albumin concentration and the ratio between them were constant in the h-week period up to birth and were significantly lower than the corresponding values in the non-pregnant group. During labor a significant decrease in reserve albumin and ratio of reserve albumin to total albumin was observed, while no change in the total albumin concentration was noted. No significant difference in reserve albumin concentration, total albumin concentration and ratio between them was found when the group of mothers who delivered vaginally was compared to the Cesarian Section group. It is concluded that the decrease in the ability of plasma albumin to bind unconjugated bilirubin takes place stepwise. The first step is a significant reduction during pregnancy followed by a further decrease during labor. The method of delivery is of no significance. Plasma albumin: Biliruhin; Pregnancy; Delivery: Monoacetyldiaminodiphenyl sulphone

Introduction Unconjugated bilirubin is toxic to nerve cells. In plasma it is tightly but reversibly bound to albumin. The toxicity probably depends on the deposition of bilirubin acid in phospholipid struc-

(‘orrespc’rltlcnc~10; Finn Ebbesen, Department of Pediatrics, University Ilospital of Aalborg. DK-Y000. Aalborg, Denmark.

tures [I]. In humans, it was found that besides being positively correlated to the unconjugated bilirubin concentration, the existence of bilirubin encephalopathy was inversely correlated to the binding properties of plasma albumin [2,3]. In healthy neonates, the binding properties of plasma albumin are reduced as compared to adults [4-61. In pregnant women the binding of bilirubin to albumin is also reduced [4,7-g], although to a lesser extent [4]. Thus, a relation may

96

exist between the reduction in the ability of albumin to bind bilirubin observed among pregnant women and that observed among newborn infants. Therefore, the bilirubin-albumin binding during pregnancy and delivery has been the source of much investigation [4,7-91. Using the MADDS (monoacetyldiaminodiphenyl :ulphone) method [lo] Esbjiirner et al. [7] found that the bilirubin binding ability of plasma albumin was decreased during pregnancy from the 29th to the 31st gestational week. Using the peroxidase method, Karp et al. [S] found that the binding ability at term, prior to delivery, probably was reduced compared to non-pregnant women. Ritter et al. [9] found that it was significantly reduced during the second stage of labor. Using the MADDS method, we [4] have previously found that the binding ability was significantly reduced immediately after delivery but normalized during the first 72 hours after delivery. However, it is uncertain whether the decrease in the ability of albumin to bind bilirubin takes place in one step, during pregnancy, or in two steps, a reduction during pregnancy with a further reduction during delivery. The cause of this uncertainty was that for practical reasons in the individual study, it was chosen to focus on a limited period of time during pregnancy or delivery. TABLE Data (group

It was the purpose of the present study to investigate whether the reduction in binding ability takes place in two steps as mentioned above and to investigate whether a possible reduction was related to the method of delivery, either vaginally or by Cesarian Section. The reserve albumin concentration for binding of MADDS was used as a measure of the reserve albumin concentration for binding of unconjugated bilirubin [lo]. MADDS is used as a deputy ligand for bilirubin, based upon the findings that MADDS and bilirubin competitively bind to human serum albumin, and that many drugs have similar effect on MADDS and bilirubin binding [lo]. However, there is evidence indicating that the MADDS and bilirubin binding functions of albumin are not quite identical [11,12]. Materials

and Methods

The material comprised three groups of women. Group one consisted of 19 healthy pregnant women with uncomplicated pregnancy. Blood samples were drawn weekly from 35 completed weeks to delivery and also just after delivery ( < 1 h after delivery). Seventeen of the women delivered vaginally without complications. Two delivered by Cesarian Section, because of compli-

I

and plasma

parameters

for women,

who delivered

vaginally

(group

0, and for women

who delivered

by Cesarian

2) Median

Maternal age (years) Gestational age (days) Birth weight (kg) Apgar score 1 min 5 min Plasma concentrations (~mol/l) total bilirubin reserve albumin for binding of MADDS total albumin Ratio of reserve albumin to total albumin NS, not significant.

P values

(range)

Vaginal delivery (n = 17)

Cesarian Section (n = 2.5)

28 (21-41) 288 (267-298) 3.90 (3.40-4.62) 9.7 (7- 10) 10 (9-10)

30 (19-37) 276 (259-288) 3.45 (2.36-4.49) 9.8 (7-10) 10 (10-10)

NS < 0.01 < 0.01 NS NS

9 (4-15)

9 (5-13)

NS

206 (140-320) 397 (324-500)

217 (96-303) 368 (309-456)

NS NS

0.52 (0.34-0.87)

0.60 (0.28-0.73)

NS

Section

97

cations during labor. These two were excluded. In the remaining group, none of the newborns were asphyctic at birth (1 min Apgar score 2 7 and 5 min Apgar score > 9). Data are given in Table I. Group 2 consisted of 25 healthy pregnant women who were delivered by elective Cesarian Section at term (gestational length > 259 days). A blood sample was obtained just after delivery. No operative complications were observed. None of the newborns were asphyctic. Data are given in Table I. The indications for the elective Cesarian Sections were (number of pregnancies in brackets): fetopelvic disproportion (121, malpresentation (51, repeat Cesarian section (2), placenta previa (l), soft tissue dystocia (fibroma uteri) cl), condyloma acuminata (1) and fetal hydrocephalus (1). In 15 of the pregnant women, general anesthesia was used for the Cesarian Section. The following medicaments were used: dinitrogenoxide, succinylcholine, thiopental and ketobemidone. In the remaining IO women, epidural anesthesia (bupivacaine) was used. Of the medicaments mentioned, ketobemidone does not affect the binding

TABLE

II

The plasma concentration

of bilirubin,

reserve

between the reserve albumin concentration

I)

of bilirubin to albumin [13]. Thiopental has in vitro been found to influence the binding of bilirubin to albumin. However, at therapeutic in vivo concentrations this influence is insignificant [13]. The plasma concentrations of succinylcholine and bupivacaine are so small that any influence on the binding of bilirubin to albumin seems unlikely. Group 3, the control group, consisted of 25 healthy non-pregnant women of the same age as the women in groups 1 and 2. The median age was 29 (range: 21-39) years. From this group, one blood sample was obtained. All blood samples were drawn by venepuncture. The following concentrations were determined: the total bilirubin concentration, the reserve albumin concentration for binding of MADDS and the total albumin concentration. The total bilirubin concentration was considered identical with the concentration of unconjugated bilirubin, since the concentration of conjugated bilirubin in plasma must be considered to be negligible in this group of healthy women [14]. As the plasma concentration of bilirubin is

and in non-pregnant

albumin

for binding

of MADDS,

and the total albumin concentration

the total albumin

concentration

in pregnant women who delivered

and the ratio vaginally (group

women (group 3)

Non-pregnant

Vaginal

(group 3)

(group

birth II

(weeks before delivery) -6

-5

(S-12)

NS-T----NS~PNS--T-7.5 (h-17) (3-11)

B (~mol/l) 5.4 -NS:.O (l--IS) p f~mol/l)

a (~mol/ll

p/a

(ri

1

-3

7.‘) (5~16)

NS-%(247-312)

NS;X? 7234-328)

f--P < 0.001 s44 --K “+z (456618) (324-412)

NS-&(324-471)

NS:‘)? (338-485)

“+G(324-471)

““32 (62-87)

NS--K (55-82)

““--G(46-X4)

r

P < 0.001

Values are median,

7

range in brackets.

NS is not significant.

-I

7.1 (h-16)

(215-322)

0 < O.O4l X.6 (4~15)

(5-16) NS-T-$

(197-321)

< 0.001 -, (159%345)

206 (140-320)

(353-456)

397 (324-456)

< 0.031 6X (42-90)

52 (34-87)

NSTNSI

NS 69 (57-85)

7 NS
yP < 0.001 --l---NS 480 253 --K (226-339) (38X-536)

F:Y- 102)

concentration.

““7

-4

(324-485) NS-P

:4

B is the bilirubin

concentration.

(54-83)

p is the reserve albumin concentration.

a is the total albumin

98

negligible compared to the reserve albumin and the total albumin concentrations, the ratio of reserve albumin to total albumin can be regarded as an expression of the binding ability of albumin for MADDS and thus for unconjugated bilirubin. The plasma unconjugated bilirubin concentration was determined by the diazo-method 1151. Bovine bilirubin (Seronorm@) was used for the standardization. The coefficient of variation of the analysis was 9% by duplicate determinations. The plasma reserve albumin concentration was determined by the [14C]MADDS method [lo]. Human serum albumin (KABI) was used for the standardization. By triplicate determinations, the coefficient of variation of the analysis was 5%. The total plasma albumin concentration was determined by the bromcresol purple method [161. Human serum albumin (KABI) was used for the standardization. The coefficient of variation of the analysis was 2% by duplicate determinations. The pH was determined on an Acid-Base Laboratory 2 (Radiometer). Informed consent was obtained from the parents. Statistical analysis was performed using Wilcoxon-Pratt’s test for paired observations and Mann-Whitney’s test for unpaired observations. Friedmann’s test was used to test whether the concentrations of reserve albumin for binding of MADDS and total albumin, and the ratio between them, showed any tendency to increase or decrease during the 6-week period prior to delivery. The level of significance chosen was 5%. Results

The results of the measurements of the plasma concentrations of total bilirubin, reserve albumin for binding of MADDS and total albumin and the ratio of reserve albumin to total albumin in group 1 and group 3 are shown in Table II and Fig. 1. The pregnant and non-pregnant women had similar bilirubin concentrations. However, a small increase was observed during labor. In the last 6 weeks prior to delivery, the concentrations of reserve albumin, total albumin and the ratio between them remained unchanged and were significantly lower compared to the group of non-

Ratio reserve albumin / total albumin tM*dlan. 96%conll**no~ Ilmltd

t

I

I

-6

-5

-4

-3

-2

-1

0

Weeks before delivery

Fig. 1. The ratio between the plasma concentration of reserve albumin for binding of MADDS and total albumin during the 6 weeks period prior to delivery and immediately after delivery in group 1.

pregnant women. By Friedmann’s test, neither the concentrations of reserve albumin and total albumin nor the ratio between them showed any tendency to increase or decrease during the 6week period prior to delivery ((Chi(R’)) 0.32 and 0.41, respectively). Similarly, the total albumin concentration showed no tendency to increase or decrease during the last weeks of pregnancy ((Chi(R2)) 0.21). A further decrease in the reserve albumin and ratio of reserve albumin to total albumin was observed during delivery, while the total albumin concentration was unchanged. A comparison of the mentioned plasma parameters obtained in the group of women who delivered vaginally (group 1) and the group of women who delivered by Cesarian Section (group 2) is presented in Table I. No significant difference was found in the plasma concentrations of total bilirubin, reserve albumin for binding of MADDS, total albumin and the ratio between the two last mentioned. The gestational length was slightly shorter in the group of women who delivered by Cesarian Section than in the women who delivered vaginally. However, this difference was not considered to influence the results, as the plasma parameters measured were unchanged in the 6-weeks period immediately prior to delivery.

YY

Discussion We found in the present study that the ratio between the reserve albumin concentration for binding of MADDS and the total albumin concentration - a measure of the ability of plasma albumin to bind MADDS - was (1) constant during the last 6 weeks of pregnancy and significantly lower than among non-pregnant women, and (2) further reduced during delivery. Presuming that the ability of albumin to bind MADDS is identical with the ability of albumin to bind unconjugated bilirubin, the reduction in binding ability during pregnancy and delivery takes place stepwise. First, a considerable reduction occurs during pregnancy followed by a further reduction during delivery. When these results are added to the information previously available [4,7-91, it is concluded that the ability of plasma albumin to bind bilirubin is reduced during pregnancy and is further reduced during delivery and that the binding ability is normalised to non-pregnant level 72 h after delivery. The binding ability immediately after delivery was not influenced by the method of delivery, vaginally or by Cesarian Section. Thus, the reduction in binding ability seen during delivery was independent of the method of delivery itself. There is no proven explanation for the reduced binding ability for MADDS of albumin in pregnant women and newborn infants. Possibly, a common cause may exist. For example, the fetus may produce an unknown ligand interfering competitively or allosterically in the binding of MADDS to albumin, and this ligand can cross the placenta membrane. However, it has not been possible to demonstrate the presence of a competing ligand in newborn infants [17]. Another possibility is the presence of acetaldehyde adducted to albumin [17]. During the pregnancy the plasma concentrations of the steroid hormones increase strongly, but as the amounts of the hormones are very small and only a minor fraction is bound to albumin an influence on the binding of bilirubin to albumin seems impossible. Furthermore, the changes in the hormone concentrations do not follow the changes in the bilirubin-albumin binding. However, the reason(s) for the

reduction in the binding ability of albumin do not have to be the same in pregnant women and neonates. Other possible causes of the reduced binding ability might be conformational or covalent changes in the albumin molecule. Possibly, the reduced ability of albumin to bind bilirubin during pregnancy and delivery is part of a greater connection. Also, the ability of plasma albumin to bind many medicaments is changed during pregnancy. The binding conditions of the antiepileptic drug valproate during pregnancy and delivery seem to be comparable to the binding conditions of bilirubin. The free fraction of valproate in plasma increases during pregnancy and increases further during delivery [lg]. Presumably, this is, at least partly, explained by an increase in the plasma concentration of free fatty acids [ 191. Acknowledgments The study was supported by Aalborg Stiftstidendens research foundation, the Ville Heise foundation and Nordjylland Amtskommunes research foundation. Signe Andersen. Institute of Medical Biochemistry, University of Aarhus, Denmark, has kindly determined the reserve albumin concentrations. References Eriksen

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