CLIN. CHEM.
33/10, 1822-1825 (1987)
Determination of Conjugated and Total Bilirubin in Serum of Neonates, with Use of Bilirubin
Oxidase
ClaudyJ.-P.MuIIon1and Robert LangerlZ3
A new, simple, and rapid assay for conjugated bilirubin that
does not require serum-matrix standards was developed by
using the enzyme bilirubin oxidase (EC 1.3.3.5). This proce-
dure can also be modified to measure total bilirubin. Mea-
surements from these assays were compared with results
obtained with the Sigma 605 (Jendrassik-GrOf method),
Sigma 550/551 (Walters-Gerarde method), and Kodak Ekta-
chem (BuBc) bilirubin assays. The one-year study involved
serum specimens from 283 infants younger than 30 days.
Linear-regression analysis of data for conjugated bilirubin
collected by this assay and by the Kodak Ekiachem assay
yielded a slope of 0.975, an intercept of 0.088, an S of 1.47
mg/L, and a correlation coefficient of 0.94 for a total of 49
specimens. Correlation was also good (r = 0.95) between
results for total bilirubin by this assay and both the Sigma 605
and the Kodak Ektachem methods.
Additional Keyphrases: enzyme methods multilayer film anal-
ysis compared
Many common in vitro assay methods for bilirubin in-
volve use of Ehrlich’s diazotized sulfanilic acid (1). The
Jendrassik-Gr#{243}fprocedure, in which the diazo reagent is
used, is recommended for total bilirubin determination (1,
2). In contrast to total bilirubin measurement, no recom-
mended method exists for conjugated bilirubin determina-
tion. Recently, an Ektachem neonatal bilirubin slide and a
“Dri-stat”enzymatic bilirubinassay have been developed by
Kodak and Beckman Instruments, respectively. The Kodak
Ektachem (BuBc) method is based on the binding of biliru-
bin species to a cationic polymeric mordant, which allows
measurement of both unconjugated and corjugated biliru-
bin (3, 4). The “Dri-stat” bilirubin reagent uses the enzyme
bilirubin oxidase (EC 1.3.3.5) for determination of total
bilirubin (5). Currently the only approach suggested for
determining conjugated bilirubin by using bilirubin oxidase
is in a patent (6).
We propose here a novel assay for conjugated bilirubin by
using bilirubin oxidase; in contrast to other procedures, this
assay requires no serum-based standards and also permits
measurement of total bilirubin. Bilirubin oxidase, derived
from the fungus Myrothecium verrucaria, catalyzes the
oxidation of bilirubin to biliverdin and the oxidation of
biliverdin to a pale purple pigment without catalyzing the
oxidation of hemoglobin (7, 8). This degradation causes the
Department of Applied Biological Sciences, MIT, Cambridge,
MA.
2 Harvard-MIT Divisionof Health Sciencesand Technology,
Cambridge, MA; and Depart-
Whitaker CollegeofHealth Sciences,
ment of Surgery, Boston Children’s Hospital, Boston, MA.
3To whom correspondenceshould be addressed (atMIT).
ReceivedApril20,1987;acceptedJune 15,1987.
1822 CLINICALCHEMISTRY, Vol. 33,No. 10,1987
characteristic yellow absorption band for bilirubin (400-470
nm) to disappear. Because the products of the enzymatic
reaction do not absorb in the yellow band (7, 8), we have
exploited this loss in absorbance to estimate the concentra-
tions of total and direct bilirubin. In the former assay, we
use sodium cholateto dissociate unconjugated bilirubin
from serum albumin. In the latter assay, we use acidic
conditions, under which bilirubin oxidase catalyzes only
conjugated bilirubin oxidation in serum (6). We also com-
pared results by three commercially available methods for
total and conjugated bilirubin: diagnostic kits and reagents
550/551 and 605 from Sigma Co., St. Louis, MO, and the
Ektachem slide assay from Eastman Kodak, Rochester, NY.
In the Sigma methods, diazotized sulfanilic acid is used.
Sigma diagnostic kit 605 is based on the Jendrassik-Gr#{243}f
procedure (9), the recommended procedure for estimation of
total bilirubin (1, 2).
MaterIals and Methods
Chemicals, Standards, Preparation Procedures
All reagentswere analytical grade.Phosphate buffer(50
mmol/L, pH 7.45) was prepared from mono- and di-basic
sodium phosphate salts. Sodium cholate solution (-40
mmolJL) was prepared by dissolving 1.72 g of cholic acid
sodium salt (Sigma) in 100 mL of phosphate buffer. Citric
acid buffer (0.1 mollL, pH 4.5) was prepared by dissolving
citric acid monohydrate and adjusting the pH with a 1.0
mollL NaOH solution.
Bilirubin ditaurine conjugate, disodium salt (Porphyrin
Products, Logan, UT), was used without further purifica-
tion. Its absorptivity (a) in citric acid buffer, calculated
according to Caraway (10), was 54 L g”1 cm’ at 460 nm.
Lyophilized bilirubi#{241}
oxidase was from Amano Interna-
tional Enzyme, Troy, VA; its vendor-specified activity was
2.27 kU/g. We first dissolved the enzyme in NaHCO3 (20
mmolfL, pH 8.3) buffer, then desalted it by passing the
enzyme solution over a 25-mL G-25 Sephadex column pre-
equilibrated with the buffer. Desalted enzyme, examined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
as described by Yang et al. (11), showed a single protein
band indicating high enzyme purity. The enzyme protein
concentration was estimated by the assay of Lowry et al.
(12). Protein concentrations of stock bilirubin oxidase solu-
tions were 13 g/L and 70 mgfL.
Bilirubin diagnostic kits and reagents, no. 550/551, based
on the method of Walters and Gerarde (13, and no. 605,
based on the method of Jendrassik and Gr#{244}f (9), were from
Sigma.
Lyophilized bilirubin standards-total bilirubin concen-
trations of 20, 50, 100, and 150 mg/L in a buffered human
serum albumin solution-were from Sigma. To reconstitute
the standards, we dissolved the contents of each vial in 3.0
mL of distilled water. After reconstitution, we prepared 75
and 125 mgIL bilirubin solutions by mixing equal volumes
of the 50 and 100 mg/L bilirubin standards and of the 100
and 150 mgIL bilirubin standards, respectively. All biliru-
bin standard solutions were kept in the dark at 4#{176}C for a
week or frozen at -20 #{176}Cif stored for longer than that.
All specimens were blood-serum samples from newborn
infants, as provided by the Chemistry Laboratory of the
Brigham and Women’s Hospital, Boston, MA. Because of
the lability of bilirubin to light, the specimens were kept in
the dark until analysis. If not analyzed within 2 h after
specimen collection, the serum samples were stored at
-20#{176}C.
Apparatus
Bilirubin absorbance and initial enzymatic rates of biliru-
bin oxidation were measured spectrophotometrically at 460
nm with a Perkin-Elmer 553 Fast Scan UV/Vis spectropho-
tometer and R 100 recorder (Perkin-Elmer Corp., Analytical
Instruments, Norwalk, CT). The measurement cuvette was
a 1.4-mL, 1-cm-lightpath, quartz, standard ultraviolet-
transmitting cell with a fitted Teflon stopper (Spectrocell
Inc., Orlando, PA). We also used a Kodak Ektachem analyz-
er equipped with a data logger and both a 400- to 420-nm
and a 460-nm wavelength filter.
Conjugated Bilirubin Assay Procedure
Add 10 JLL of bilirubin oxidase solution (13 g/L) to a
cuvette containing 0.94 mL of citric acid buffer; use this to
adjust the spectrophotometer to zero. Then add 50 L of
serum specimen, mixing quickly by inverting the cuvette,
and record the absorbance for 3 mm (the absorbance usually
plateaus within 1 to 2 mm). Estimate the conjugated
bilirubin concentration from the decrease in absorbance
(10), using the absorptivity of ditaurate bilirubin conjugate,
a surrogate for conjugated bilirubin [the chromatographic
and chemical properties of commercially available ditaur-
ate-conjugated bilirubin are similar to those of human-
conjugated bilirubin (14), and its absorptivity is also used in
the conjugated Kodak Ektachem assay]:
concn, gIL = AA/(a x 1) x d
where A = absorbance change
#{128}
= absorptivity
(LOg’ cm’)
I = cuvette light path
(cm)
d = specimen dilution
(= 20)
Total Bitirubin Oxidase Assay Procedure
After adjusting the spectrophotometer tozeroabsorbance
with 0.95 mL of sodium cholate phosphate buffer, add 50 j.tL
of serum or bilirubin standard (in serum albumin) to the
cuvette, quickly invert to mix, and record the absorbance at
460 nm. Then add 10 ILL of bilirubin oxidase (original
concentration, before addition, 70 mg/L), quickly mix, and
record the absorbance change for 3 mm. Construct a stan-
dard curve by plotting the absorbance of the total bilirubin
standards after 3 mm vs their initial concentrations. Esti-
mate the total bilirubin concentration of the test specimens
by comparing their absorbance after 3 mm with the refer-
ence curve.
Data Analysis
All the serum specimens were from infants younger than
30 days. Because of the small amount of serum collected
from each blood sampling, not all bilirubin assays were
carried out for each serum specimen. All data were analyzed
by least-squareslinear regression analysis (15, 16).
Results
Conjugated Bilirubin Oxidase Assay
Figure 1 shows a typical example of the decrease in
absorbance and the absorbance plateau after mixing 50 ILL
of serum (with 63 mg/L total bilirubin and about 18 mg/L
conjugated bilirubin) with 0.95 mL of citric acid buffer
containing bilirubin oxidase. Table 1 summarizes the com-
parison studies and shows good correlation between the
conjugatedenzymaticassay and the Kodak Ektachem assay
for 49 specimens. The conjugated bilirubin concentrations of
the specimens were 2 to 18 mgfL, a typical range during the
neonatal period, with concentrations exceeding 14 mg/L
considered indicative of liver disease (17). The Sigma 605
and 551 assays, which are based on the reaction of diazo-
tized sulfanilic acid reagent and conjugated bilirubin to
form azobilirubin in the absence of unconjugated bilirubin
displacers, were unreliable in our hands. The absorbance for
azobilirubin kept increasing; the failure to reach an eventu-
al plateau in absorbance precluded attempts to estimate the
conjugated bilirubin concentration. After about 10 mm,
depending on the sample, the specimen’s azobilirubin absor-
bance eventually reached a value reflecting the total biliru-
bin concentration of the specimen, which suggested that
unconjugated bilirubin was slowly dissociated from serum
albumin and interfering with the conjugated bilirubin con-
centration measurement.
Total Bilirubin Oxidase Assay
Table 1 shows good agreement between the bilirubin
oxidase, the Sigma 605, and the Kodak Ektachem assays of
total bilirubin. The range of the totalbilirubinconcentra-
tions in the specimens was about 30 to 200 mg/L, which is
typical of total bilirubin concentrations in neonates under-
0.27
0.25
A
0.23
0.21
0.
2 3 4 5 6
time (mm)
Fig. 1.Absorbancechange with time for 10 ILLofbilirubin oxidaseatan
oflginal concentration of 13 gIL added to 0.94 mL ofcitric acid buffer
(0.1moIIL,pH 4.5)and 50 LL ofserum (63mg oftotal bilirubinand 18
per liter)
mg ofconjugatedbilirubin
A = absorbance at 460 nm
CLINICAL CHEMISTRY, Vol.33,No. 10,1987 1823
 Table 1. Linear Regression Equations and Parameters of Conjugated and Total Bilirubin Assays
S, Range, SI,
y x Regressionequation mgIL r n mg/L S., mg/L
Box.Bc Kod.Bc y =0.088+ 0.975 x 1.47 0.94 49 2-18 0.057 0.34
Box.Bt 605.Bt y =2.57 + 0.934 x 12.3 0.95 50 40-200 0.045 5.6
Box.Bt Kod.Bt y= -6.56 + 1.024x 12.4 0.95 75 30-200 0.040 4.8
Kod.Bt 605.Bt y = 13.60 + 0.827x 9.8 0.96 66 30-200 0.028 3.2
550.Bt Kod.Bt y =-23.93 + 1.229x 31.0 0.84 43 40-210 0.126 15.4
bilirubin; Bt
Bc = conjugated = total bilirubin; Box = bilirubin oxidase assay; Kod = Kodak Ektachem assay; 605 = Sigma 605 assay; 550 = Sigma 550 assay.
8,,, = standard error estimate; S, = standarddeviation of slope; S = standarddeviation of intercept. n = no. of serum specimens.

going phototherapy (17). In contrast, there was poor agree-
ment between the Sigma 550 and the Kodak Ektachem
assays.
Discussion
Conjugated Bilirubin Assays
The goal behind the development of a conjugated bilirubin
assay is to separate conjugated bilirubin and unconjugated
bilirubin. The immediate problem in dealing with bilirubin
oxidaseas a means of determining the amount of conjugated
bilirubin in any given blood plasma sample is that the
enzyme reacts favorably with both the conjugated and the
unconjugated species.Because of this, the focus of develop-
ment of the conjugated assay must lie with the determina-
tion and the creation of a suitable environment in which the
conjugated species of bilirubin reacts with the enzyme but
the unconjugated species does not. Takayama et al. (6)
observed that, in the pH range 3.5-4.5, bilirubin oxidase
catalyzes only the oxidation of conjugated bilirubin in icteric
plasma. In the present assay we used a buffer of pH 4.5. As
Table 1 shows, there was very good agreement between the
conjugated enzymatic assay and the conjugated Kodak
Ektachem assay, the latter having been judged (4, 18) the
best of several commercially available methods for estimat-
ing the conjugated bilirubin species-as compared with
liquid chromatography, a very specific method.
Total Bilirubin Assay
Unconjugated bilirubinis poorly soluble;it circulates
the blood bound to serum albumin (19). This binding to
serum albumin protects it from oxidation by bilirubin
oxidase (20). To speed the assay, sodium cholate is used to
disrupt this protective binding and therefore increase the
rate of reaction.
Results by the bilirubin oxidase, the Sigma 605, and the
Kodak Ektachem total bilirubin assays correlated well
(Table 1). The major difference between these assays may be
their sensitivity to &.bilirubin. This form of bilirubin, which
reacts with diazotized sulfanilic acid reagent, is included in
the total bilirubin concentration value measured by the
Jendrassik-Gr#{243}fmethod (Sigma kit 605) but is not mea-
sured by the Kodak Ektachem assay (3). Bilirubin oxidase
probably does not catalyze the degradation of the protein-
bound fraction, #{244}-bilirubin,
because uncortjugated bilirubin
bound to serum albumin is protected from degradation (20).
In our study, however, the effect of the difference in sensitiv-
ity for &bilirubin between the Jendrassik-GrOf, Kodak
Ektachem, and enzymatic methods should be minimal be-
cause in the plasma of infants younger than 28 days, &
bilirubin is <2% of total bilirubin (21).
In conclusion, the use of bilirubin oxidase for measure-
ment of total and conjugated bilirubin concentrations pro-
vides a simple and rapid micromethod. In considering a
procedure for the measurement of conjugated bilirubin, the
bilirubin oxidase and Kodak Ektachem methods should be
further investigated, being potentially free of interference
from &bilirubin and hemoglobin as compared with the
diazotized sulfanilic acid reagent.
We thank W. R. Grace & Company for financial support. We also
thank Drs. M. Epstein and R Lopez from the Brigham and
Women’s Hospital of Boston, MA, for providing us with newborn
serum samples, and J. Kadam, E. Rizzotto, G. Baier, B. O’Neil, and
C. Pelley for technical assistance.
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