CLIN. CHEM.

26/6, 738-740 (1980)

Bilirubin Binding Capacity of Albumin Isolated from Cord-Blood Serum Is

Less Than That from Serum of Adults
Aviva Alayoff, Jaime Kapitulnik, Avraham Konljn, N. A. Kaufmann, and S. H. Blondheim

As estimated by Sephadex gel filtration, the bilirubin Albumin was isolated from serum by a modification of the
binding capacity of albumin isolated from cord-blood serum method of Taylor and Schimke (JO), as follows. During 30 mm
by ion-exchange chromatography is less than that of al- add 100 mL of a saturated solution of ammonium sulfate (pH
bumin isolated from serum of adults. Albumin isolated from 7), with gentle stirring, to an equal volume of serum. Centri-
cord-blood serum showed increased bilirubin binding as fuge (11 000 X g, 4 #{176}C, 15 mm), adjust the pH of the supernate
compared with the albumin in the native serum. These to 4.8 (the isoelectric point of human serum albumin) by
findings suggest that the lower bilirubin binding capacity adding acetic acid (100 g/L solution), and again centrifuge.
of serum from newborns as compared with serum from Dissolve the precipitate in 100 mL of phosphate buffer (10
adults is a result of both an intrinsic deficiency in binding mmol/L, plus NaC1, 150 mmol/L; pH 7.2), and apply the so-
capacity of neonatal albumin and the presence of sub- lution to an upward-flow column (50 X 1000 mm) of Sephadex
G-100 (Pharmacia, Uppsala) by use of a penistaltic pump.
stances in neonatal serum that interfere with bilirubin
Elute the protein with the same buffer and collect 5.5-mL
binding.
fractions of effluent. Pool the central fractions of the highest
protein peak and dilute the pool to give a concentration of 1.8
AdditIonal Keyphrases: newborns neonatal jaundice
g of protein per liter, as determined with a Gilford M 240
spectrophotometer (Gilford Instrument Labs., Inc., Oberlin,
The availability and affinity of plasma albumin binding
OH 44074) set at 280 nm; an absorbance of 1.0 is equivalent
sites for bilirubin are critical factors in the pathogenesis of
to 1.8 gIL. Apply this solution to a 25 X 600 mm column of
brain damage resulting from neonatal jaundice. There are
diethylaminoethyl-cellulose 52 (Whatman, Inc., Clifton,
substantial differences in bilirubin binding between serum
NJ 07014) previously equilibrated with 30 mmol/L tnis(hy-
from neonates and that from adults (1-3).’ the high-affinity
droxysnethyl)aminomethane . HC1 buffer (pH 7.4). Elute the
bilirubin binding site (4) of albumin in cord-blood serum re-
protein with a concentration gradient (30 to 300 mmol/L) of
tains less bilirubin that that of albumin from the serum of
the buffer. Pool the central fractions of the albumin peak,
adults. In addition, once this high-affinity binding site is
concentrate by perevaporation, and dialyze the concentrate
saturated, more bilirubin dissociates from the low-affinity
vs phosphate buffer (67 mmol/L, pH 7.45). Again perevapo-
binding site(s) of cord-blood albumin than from albumin
rate the dialysate to reach a protein concentration of 30 to 40
derived from serum of adults (2). The gradual transition from
gIL. Divide this solution into 10-mL samples and store them
the infant to the adult pattern of bilirubin binding is complete
at 18 #{176}C.
by about the fifth postnatal month (5). It has been suggested This protein fraction was electrophoresed on polyacryl-
(6-8) that endogenous compounds that compete with bilirubin
amide gel, 75 g/L (pH 8.9). We stained the gels with Coomassie
for binding to serum albumin are present in higher concen- Brilliant Blue, scanned at 570 nm with a linear transport unit
trations in the serum of infants and that this impairs bilirubin
attached to the Gilford spectrophotometer, and measured the
binding in newborns. On the other hand, albumin from the
areas under the resulting peaks.
serum of infants and of adults, isolated by affinity chroma-
We used a Sephadex gel-filtration method (2) to study the
tography, differs in amino acid composition and in isoelectric
binding of bilirubin by serum and by the isolated albumin
focusing pattern (9).
preparations. Crystalline bilirubin (ICN Pharmaceuticals,
It was therefore of interest to examine the possibility that
Inc., Cleveland, OH 44128) was added in increasing amounts
such qualitative differences between albumin from infants and
to serum or isolated albumin to give solutions with final bili-
adults might by themselves account for, or at least affect, their
rubin/albumin molar ratios of up to 2 (2). These molar ratios
bilirubin binding characteristics. In this study, we isolated
were calculated by assuming that 8.5 mg of bilirubin per gram
albumin by ion-exchange chromatography (10) under gentle
of albumin approximates the 1/1 ratio. Bilirubin was deter-
conditions and studied its bilirubin binding by a Sephadex
mined by a diazo method (11), albumin by the method of
get-filtration method (2).
Fernandez et al. (12). We diluted the above solutions with
Materials and Methods equal volumes of the phosphate buffer and applied 1.5 mL of
the final solutions to columns of Sephadex G-25 (fine)
Blood from fasting normal adult volunteers and from the
(Pharmacia, Uppsala), equilibrated with the phosphate buffer.
umbilical cord of full-term normal newborns was allowed to
Bilirubin that remained adsorbed on the gel column (after
clot at room temperature and the serum was separated by
complete elution of serum proteins) was eluted with 0.1 mol/L
centrifugation (4000 X g, 10 mm). Pooled specimens of serum
NaOH and then finally extracted into 1.5 mL of CHC13 (2).
from adults (each derived from three to five individuals) and
Bilirubin in the CHCI3 was quantitated by its absorbance at
of cord-blood serum (from 10 to 20 deliveries) were kept at
450 nm (1.0 A = 10mg of bilirubin per liter of CHC13).
-18 #{176}C for two to five days until studied.
A commercial albumin preparation derived from serum
from human adults (Immuno AG, Vienna; 200 g/L, and con-
taining 20 mmol of sodium caprylate and 20 mmol of sodium
Metabolic Laboratory and Department of Medicine B, Hadassah
University Hospital, and the Departments of Pharmacology and
acetyltryptophan per liter) was subjected to the albumin
Nutrition, Hebrew University-Hadassah Medical School, Jerusalem, isolation procedure described above. The commercial prep-
Israel. aration and the albumins we isolated from both types of serum
Received Nov. 14, 1979; accepted Feb. 12, 1980. were treated with neutralized activated charcoal (C5385;

738 CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980

 9.0 7.5 B

V
0
-c
x
a,

0.
a,
U.)
>
75

6.0
/ /
V
x
a)
0
.c
0.
a) 3.0

>..

V
a)
6.0

4.5

1.5

0
I.-
0
4.5 0
4,5 A
.0 V 0”
0 3.0 -

0
U)
V 3.0 A
1.5
0
I ci,__._r#{149}#{176}I0

Oh 0.8 12 1.6 2
1.5 BR/Mb motor ratio
Fig. 2. Relation between amount of bilirubin adsorbed onto a
column of Sephadex and the bilirubin/albumin molar ratio of:
#{176}‘---‘r A -A, cord-blood serum (final concn of albumin, 33.5 gIL);
0 04 OS 1.2 1.6 20 - , albumin isolatedfrom cord-blood serum (final corscn
BR/AIb molar ratio 28.8 gIL); #{149}
#{149},
- adult serum (final concn of albumin, 39.5
gIL); and 0-0, albumin isolated from adult serum (final concn
Fig. 1. Relation between the amount of bilirubin adsorbed onto 37.2 g/L)
a column of Sephadex and the bilirubin/albumin molar ratio of:
albumin isolated from pool of cord-blood serum (final
concn 28.8 gIL); and 0-0, albumin isolated from pool of adult
serum (final concn 34.1 g/L)
Bilirubin was eluted from the column with 0.1 mol/L NaOH and extracted into
1.5 mLof cHcl3
24
a)
Sigma Chemical Co., St. Louis, MO 63118) (13). Free fatty
acid content was determined by the method of Ho, with 63Ni 0
-c
(14). 0.
a) 18
Results 11’l
The bilirubin binding capacity of albumin isolated from >
0
cord-blood serum was less than that of albumin isolated from
serum of adults, as shown by the greater amounts of bilirubin 12
a)
adsorbed by Sephadex from the solutions of isolated cord- .0
5-
blood albumin. This difference in binding capacity (Figure 0
1) was found at all bilirubin/albumin molar ratios we studied. U’)
Both of the isolated albumins were essentially homogeneous
0
on polyacrylamide gel electrophoresis, the main densitometric
peaks constituting 97.2 and 98.5% of the total areas for cord- cx
blood serum and adult blood serum, respectively.
The isolation procedure improved the bilirubin binding
capacity of the albumin of neonatal origin, but not that of
albumin of adult origin (Figure 2). Subsequent treatment with 0.8
activated charcoal of isolated albumin of either neonatal or BR/Mb molar ratio
adult origin did not further improve bilirubin binding ca- Fig. 3. Relation as in Fig. 1 for a commercial preparation of adult
pacity. The bilirubin binding capacity of the commercial albumin #{149}
-#{149},before
- charcoal treatment (final concn of al-
preparation of serum albumin also improved when the isola- bumin, 51.0 g/L; free fatty acids: 3742 tmoI/L); 0- -0, after
tion procedure used in this study was applied to it. Treatment charcoal treatment (final concn of albumin, 50.0 gIL; free fatty
of the original commercial albumin preparation with charcoal acids: 178 tmol/L); C) - - C), after re-isolation by ion-exchange
chromatography (final concn of albumin, 35.3 gIL; free fatty
removed 92% of its free fatty acids, with a concomitant im-
acids: 201 jzmol/L)
provement in bilirubin binding, an improvement identical in
degree with that achieved as a result of’ the isolation procedure
(Figure 3). stances such as free fatty acids and heme pigments (6), which
interfere with bilirubin binding by albumin.
#{149}
Discussion In the present study we used a technique for isolating al-
The lower bilirubin binding capacity of native neonatal bumin that minimizes damage to the protein. This procedure
serum as compared to adult serum (1-3) has been attributed probably removed substances that interfere with the binding
to the presence in neonatal serum of larger amounts of sub- of bilirubin by albumin in native cord-blood serum, thus im-

CLINICAL CHEMISTRY, Vol. 26, No. 6, 1980 739

proving its bilirubin binding capacity. However, bilirubin adsorption of bilirubin from neonatal and adult serum. Clin. Chem.
binding capacity of the albumin from adults was not improved 18,43-47 (1972).
when it was isolated from serum. 3. Gartner, L. M., and Lee, K. S., Bilirubip binding, free fatty acids
The improvement in bilirubin binding capacity of com- and a new concept for the pathogenesis of kernicterus. Birth Defects
mercial albumin derived from adult serum, after either re- 12, 264-271 (1976).
isolation or treatment with activated charcoal, can be attrib- 4. Jacobsen, C., Chemical modifications of the high-affinity biliru-
bin-binding site of human-serum albumin. Eur. J. Biothem. 27,
uted to the extensive removal of free fatty acids from the al-
513-519 (1972).
bumin. Such removal of free fatty acids and possibly of other
5. Kapitulnik, J., Horner-Mibashan, R., Blondheim, S. H., et al.,
interfering substances was probablythe reason for the increase Increase in bilirubin-binding affinity of serum with age of infant. J.
in bilirubin binding capacity that resulted from jsolation of Pediatr. 86, 442-445 (1975).
the neonatal albumin. 6. Odell, G. B., The dissociation of bilirubin from albumin and its
The fact that the bilirubin binding capacity of the isolated clinical implications. J. Pediatr. 55, 268-279 (1959).
neonatal albumin was less than that of the isolated adult al- 7. Melichar, V., Polalek, K., and Novak,M., The relationship between
bumin may be ascribed to intrinsic differences in the isolated bilirubin concentration and the level of non-esterified fatty acids in
albumins themselves. These are evidenced by marked diver- the blood of newborp infants. Biol. Neonat. 4,94-101 (1962).
gences in amino acid composition and in isoelectric focusing S. Starinsky, R., and Shafrir, E., Displacement of albumin-bound
patterns of albumin isolated by affinity chromatography from bilirubin by free fatty acids. Implications for neonatal hyperbiliru-
binemia, Clin. Chim. Acta 29,311-318 (1970).
cord-blood as compared with serum from adults (9). It would
be of great interest to determine whether the transition from 9. Wallace, S., Altered plasma albumin in the newborn infant. Br. J.
Clin. Pharmacol. 4, 82-85 (1977).
the fetal to the adult pattern of bilirubin binding (5) involves
10. Taylor, J. M., and Schimke, R. T., Synthesis of rat liver albumin
a gradual change in the proportion of an adult to a fetal type in a rabbit reticulocyte cell-free protein-synthesizing system. J. Biol.
of albumin, as is true of hemoglobin (15). Chem. 248, 7661-7668 (1973).
11. Malloy, H. T., and Evelyn, K. A., The determination of bilirubin
with the photoelectric colorimeter. J. Biol. Chem. 119, 481-490
Supported by grants from the Miles Laboratories, Elkhart, IN, and (1937).
Ames-Yissum, Ltd., Jerusalem. The skillful technical assistance of 12. Fernandez, A., Sobel, C., and Goldenberg, H., An improved’
Ruth Shimron and Ruth Levy is much appreciated. method for determination of serum albumin and globulin. Clin. Chem.
12, 194-205 (1966).
13. Chen, R. F., Removal of fatty acids from serum albumin by
References charcoal treatment. J. Biol. Chem. 242, 173-181 (1967).
1. Jacobsen, J., and Fedders, 0., Determination of non-albumin- 14. Ho, R. J., Radiochemical assay of long-chain fatty acids using ssNi
hound bilirubin in human serum. Scand. J. Clin. Lab. Invest. 26, as tracer. Anal. Biochem. 36, 105-113 (1970).
237-241 (1970). 15. Trought, H., The specificity of haemoglobins including embryonic
2. Kapitulnik, J., Blondheim, S. H., and Kaufmann, N. A., Sephadex haemoglobin. Arch. Dis. Child. 7,259-262 (1932).

740 CLINICALCHEMISTRY,Vol. 26, No. 6, 1980