CLIN.
CHEM.
27/10,
1704-1707
(1981)
Liquid-Chromatographic Determination of Bilirubin and Its Conjugates in Rat
Serum and Human Amniotic Fluid
Philip Rosenthal, Norbert Blanckaert, Pokar M. Kabra, and M. Michael mater1
This newly developed and highlyspecificand sensitive
procedure was applied to the determination of unconjugated billrubin and its ester conjugates in rat serum and
human amniotic fluid. Bilirubin conjugates in biological
samples are converted to methyl esters by alkaline
methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are
fractionated by “high-performance”
liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are
readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2
.tmol/L for unconjugated bilirubin, 0.07 to 34.6 mol/
L
for the C-8 monoconjugate,
0.06 to 69.3 tmol/L for the
C- 12 monoconjugate, and 0.17 to 43.7 jimol/L for the diconjugate fraction. The detection limit was 0.03 tmol/L
for unconjugated bilirubin and for each monoconjugate,
and 0.1 mol/L
for the di-conjugated pigment.
Current methods are insufficiently sensitive for accurate
determination of bilirubin and its conjugates in biological
fluids at pigment concentrations in the physiological range,
and are susceptible to interference from other yellow or
diazo-positive compounds (1,2). These methodological limitations present serious obstacles to studies of bilirubin metabolism in most laboratory animals and in humans. Bilirubin
has been impossible to measure accurately, without the use
of radioactive tracers, in serum from normal rats and in human
amniotic fluid contaminated with hemoproteins, flavins, or
maternally-derived
carotenoids, or made turbid with fatty
components (3). The usefulness of conventional techniques
is further diminished by their nonspecificity toward bilirubin
conjugates, caused by variables such as bile acids, which influence the “direct-reacting”
fraction in assays based on the
diazo reaction (4, 5). The errors in estimation of conjugated
and total bilirubin by current methods are reflected in the
unconjugated (“indirect”) bilirubin fraction, which is calculated by subtracting results for direct-reacting from those for
total biirubin.
A highly specific and sensitive method for measurement of
bilirubin and bilirubin mono- and di-ester conjugates in
human serum has been recently developed in our laboratory
(6, 7). Bilirubin mono- and di-conjugates are converted by
alkaline methanolysis to methyl ester derivatives, which can
be readily separated from each other and from unconjugated
pigment
by
“high-performance”
liquid
chromatography
photometrically
at 430 nm.
Here, we present an adaptation
and verification
of the alkaline methanolysis-HPLC
procedure for measuring
bilirubin
(HPLC), and quantitated
and its conjugates in concentrations as low as 30 nmol/L in rat
serum and in human amniotic fluid. The data presented
Departments of Pediatrics and Laboratory Medicine, University
of California, San Francisco, CA 94143.
‘Address correspondence to thisauthorat:U587, Universityof
California, San Francisco, CA 94143.
Received
1704
April
7, 1981; accepted
CLINICAL CHEMISTRY,
June 19, 1981.
Vol. 27, No. 10,
1981
the accuracy,
of the method.
demonstrate
precision,
sensitivity,
and range
Materials and Methods
instrumentation
A high-performance liquid chromatograph,
Model 3B
(Perkin-Elmer Corp., Norwalk, CT 06856), equipped with a
Model
7105 injector
value (Rheodyne,
Berkeley,
CA 94710)
was used in these investigations.
The silica column (250 mm
X 4.6 mm, LiChrosorb
Si 60, 5-urn particle size;
Merck A.G.,
E.M. Laboratories,
Elmsford,NY 10523)was maintained at
45 #{176}C
inan LC-100 column oven (Perkin-Elmer).
A PerkinElmer LC-55 variablewavelength detectorand a 3380A
electronic
integrator
(Hewlett-Packard,
Palo Alto,CA 94304)
were used toquantitatetheseparatedpigments.Absorption
spectrawere recordedwitha Cary 118C spectrophotometer
(Varian,Palo Alto, CA 94303).Referencecompounds were
weighed withan electrobalance
(Cahn InstrumentCo.,Par-
amount, CA
90723).
Reagents
Bilirubin
(#{128}452
in chloroform: 61.0 X 10 L mol
cm’;
containing1% lIla,93% IXa, and 6% XIIIa isomers)was
obtainedfrom Koch-LightLaboratories,
Colnbrook,Bucks,
U.K. Chloroform(containing
1% ethanol)and methanol were
purchased from Burdick and Jackson Laboratories,Inc.,
Muskegan, MI 49442.
methyl ester of xanthobilirubic
acid (#{128}410
Imax
L mol
cm’), prepared as described
by Grunewald etal.(8) was used as internal standard. (This
Crystalline
in methanol:
34.0 X i0
ester was generously provided by D. Lightner, University
Nevada, Reno.) The internal standard solution consisted
of
of
50 sg of the ester in 100 mL of methanol.
All other reagents were analytical-reagent grade.
Reference compounds.
Crystalline preparations of bilirubin
monomethyl
esters and bilirubin
dimethyl
ester were synthesized
as described
(6, 7).
Procedures
Preparation
of reference
solutions.
We prepared
the solutions used to calibratethe resultsof the alkalinemethanolysis-HPLC assay by dissolvingunconjugated bilirubinin
pooled specimens of rat serum or human amniotic fluidthat
had been cleared of endogenous bilirubin
by exposure to fluorescent lighting for 4 h, resulting in degradation of the photosensitive
pigment to non-detectability
by this assay (9).
Reference solutions were prepared by dissolving various
amounts of the unconjugated pigment (determined by weight)
in cleared serum or amniotic fluid, as described by Billing et
al.(5).
Reference solutions for the conjugated pigments were
prepared from rat-bilerichin conjugated bilirubin,because
mono- and di-conjugatesof bilirubinof adequate purityand
stability
arenot currentlyavailable.
Rat bile was enriched with
conjugated pigments by intravenousinfusion of unconjugated
biirubin
into Sprague-Dawley rats whose common bile ducts
were cannulated for collection of bile, which was used as stock
for subsequent dilutions (7). The concentrations
of mono- and
di-conjugated
bilirubin in this stock bile were determined as
described previously and their identity was confirmed, with
synthetic mono- and dimethyl esters of bilirubin used for
reference (7). Reference solutions were obtained by serial
dilution with cleared (light-treated)
rat serum or amniotic
fluid,
asfortheunconjugatedpigment.
#{149}
Preparation of rat serum. Rats of the Sprague-Dawley and
Gunn strains (350 g mean body weight) were used as sources
of serum. After anesthesia with pentobarbital
administered
intraperitoneally
(0.1 mg/i#{174}
g body weight), the abdominal
aorta was exposed surgically, and blood was aspirated at the
bifurcation of the aorta. The serum was either analyzed immediately or stored in the dark at -12 #{176}C
for processing within
a week.
Collection of human amniotic fluid. Amniotic fluid was
obtained by abdominal amniocentesis for diagnostic evaluation of pregnancies suspected of blood-group incompatibility
or for determination
of the lecithin/sphingomyelln
ratio as an
index of fetal maturity. Specimens were protected from light
in containers wrapped in aluminum foil, and were assayed
either immediately
or after storage in the light-shielded
containers at -12 #{176}C
for less than a week.
Alkaline met hanolysis.
All procedures were performed in
dim lighL About 60mg of the sodium salt ofascorbic acid, 2-3
mg of disodium ethylene diamine tetraacetate,
4 mL of
methanol, and 2 mL of methanol containing the internal
standard were mixed with 0.6 mL of rat serum or amniotic
fluid. The mixture was treated with 6 mL of a 20 g/L solution
of KOH in methanol and immediately vortex-mixed. After
reaction for 60-90 s at 20-25 #{176}C,
6 mL of chloroform and 12
mL of glycine/HC1 buffer (0.4 molfL HC1 brought to pH 2.7
with solid glycine) were added sequentially.
Organic and
aqueous phases were separated by brief centrifugation,
the
organic phase was transferred to a dry tube and the extract
evaporated to dryness under nitrogen at30 #{176}C.
The residue
was stored under argon at -12 #{176}C
and analyzed within a
week.
Liquid chromatography.
The pigment residue obtained
from alkaline methanolysis
was redissolvedin 50 zL of the
solution injected in the liquid chromatograph. The pigments
were separated by using a convex gradient (curvature 0.2Perkin Elmer Manual for Series 3 Liquid Chromatograph) at
a flow rate of 1.5mL/min, starting with chloroform/acetic acid
(199/1 by vol) and ending with chloroform/methanol/acetic
acid (197/2/iby vol) in 6 mm. The lattersolventwas continued for 8 mm, and the column was then re-equilibrated for 10
min with the initial solvent before injection of the next sample.
The absorbance of eluted pigments was recorded at 430 nm,
and the area under each peak integrated electronically.
Calibration
and calculation of results. The amount of each
bilirubin
fraction
in specimens was calculated
with the
equation:
IS(ug)
X (area pigment/area
IS) X RF X (0.2/n) X 5
=
P
where IS = amount of internal standard, RF = area 1 #{128}g
internal standard/area
1 umol pigment, n = volume of sample
(0.6 mL), P = umol of pigment per liter of sample.
RF values for unconjugated
bilirubin
and its conjugates
were determined
by chromatography
of pure reference compounds, as previously described (6). Values obtained were:
bilirubin,
1.541; monoester,
endovinyl
isomer C-8, 1.647;
monoester, exovinyl isomer C-12, 1.683;diester
2.264.
Assignment
of peaks. Assignment of peaks for the bilirubins was determined by comparing retention times to those
for reference unconjugated
bilirubin,
bilirubin
monomethyl
esters, dimethyl
esters, internal standard, and carotenoids
(6).
A.
B. METHANOL
ALKALINE
METHANOL
2
.
E
C
0
.
a)
S
a
#{149}
I
0
0
S
t
OT
C
:1
0
+
a
o
itililLil
O
3
2
#{149}
III
6
9
12
5
0
3
Time (mm)
6
9
12
15
Time (mm)
FIg. 1. Chromatograms of a divided sample of rat serum pre-
pared in alkaline methanol (A) or methanol (B)
Blllrubin-conjugate
methanol
peaks
appear only in the sample treated with alkaline
Tests for interference.
Carotenoids (lycopene and
and
a-carotene), which are often present in biological samples and
may interfere with bilirubin determinations, elute in a single
-
peak that emerges before bilirubin (6) (Figure 1). Heminwhich occurs in hemolytic serum and may potentiallyinterfere-was found to be immobile in the chromatographic system used (6). An unidentifiedcarotenoid in serum has been
found to elute in close proximity
with bilirubin
C-8 monoconjugate, depending on the individual
sample and column
used (6). This compound persists, whereas the bilirubin C-8
monoconjugate is absent from chromatograms of samples
reacted with plain methanol instead of alkaline methanol
(Figure 1). Thus, we could appropriately
assign peaks and
areas for the bilirubin
monoconjugate
and the carotenoid by
comparison of chromatograms
obtained from divided samples
processed with and without alkaline methanol.
Results
Evaluation
of Analytical
Variables
Accuracy. Serial dilutions of known amounts of unconjugatedand mono- and di-conjugated
bilirubin
were analyzed
by alkaline methanolysis-HPLC,
and the results evaluated
by linear regression analysis. For each individual
pigment
fraction, a linear relation was shown between amount of pigment injected and corresponding
peak area in the chromatogram. The method yielded linear results throughout the range
of bilirubin
concentrations
tested (0.07-121.1 umol/L
unconjugated bilirubin,
0.07-34.6 umol/L C-8 monoconjugate,
0.06-69.3 umol/L
C-12 monoconjugate,
0.17-43.7 umol/L
di-conjugate)
(Table 1).
Average (±SD) analytical
recovery of injected material
through the chromatographic
procedures with ‘4C-labeled
reference pigments was 92
4% for unconjugated
bilirubin,
92 ± 6% for a mixture of the monomethyl ester, and 87 ± 5%
for the dimethyl ester (6).
CLINICAL CHEMISTRY,
Vol.
27,
No. 10.
1981
1705
Table 1. Elements of Linearity Equations
(y
b + mx)
=
b
m
r
Unconjugated bilirubin
C-8 monoconjugated
C-12 monoconjugated
0.01
-0.05
0.98
1.0
1
1
-0.04
0.97
1
Diconjugate
-0.13
-0.40
1.0
1.0
1
1
Higher concentration (ref. 6)
A.
B.
C
Precision. The reproducibility of the assay was assessed by
calculating the coefficient of variation (CV) in determination
of each individual bilirubin fraction repeated 10 times on a
single sample (Table 2). The precision was satisfactory at these
low concentrations
(CV <4.5% for within-day,
14% for dayto-day analyses).
Sensitivity.
At 0.07 tmol/L,
the signal-to-noise
ratio was
3, using 0.6 mL of sample. The detection limit was approximately 0.03 zmol/L for unconjugated
bilirubin
and the monoconjugates and 0.1 zmol/L for the diconjugates.
Bilirubins
In
0
4-
4
2
0
in Rat Serum (Figure 2A)
Bilirubins
in Human Amniotic Fluid (Figure 2B)
Amniotic fluid was obtained from women with normal
pregnancies and with pregnancies at risk from maternal-fetal
blood-group incompatibility. A total of 13 “normal” specimens
and 39 “at-risk” specimens were analyzed. Amniocenteses
were performed between the 25th and 40th week of gestation.
Bilirubin was detected in all samples. Mean total biirubin in
amniotic fluid from normal pregnancies was 1.5 (SD 1.5)
smol/L, with a range of 0.03 to 4.8 zmol/L. Mean total bilirubin in amniotic fluid from Rh-sensitized pregnancies was
4.0 (SD 2) tmol/L, with a range of 0.7 to8.6 mol/L.
Bilirubin
concentrations
tended to diminish with increasing gestational
age, and displayed considerable individual
variability
at any
given age. Whereas no conjugated pigment was detected in
specimens from normal pregnancies,
amniotic
fluid from
blood-group-incompatible
pregnancies occasionally contained
Table 2. Reproducibility of the Assay Bilirubin
Concentration, jmol/L (and CV, %)
Unconjugated
bilirubin
C-8 monoconjugated
C- 12 monoconjugated
Diconjugate
CLINICAL CHEMISTRY,
Within-day
Day-to-day
0.6 (10)
0.7 (11.5)
0.7(7)
0.6 (9.7)
0.8(10)
0.7 (13)
0.7 (13.5)
0.6(14)
Vol. 27, No.
10. 1981
a
a
Three strains of rats were investigated:
Sprague-Dawley
(n=24), heterozygous Gunn (9), and homozygous Gunn rats
(4) with unconjugated hyperbilirubinemia due to an inherited
defect in bilirubin
conjugation.
Mean total serum bilirubin
in Sprague-Dawley
rats was 0.77 (SD 0.2) smol/L,
with a
range of 0.3 to 1.3 zmol/L.
Serum bilirubin
concentrations
were comparable in male and female Sprague-Dawley
rats,
and in heterozygous
Gunn rats. Bilirubin
conjugates accounted for approximately
4% of the total serum bilirubin
in
both Sprague-Dawley
and heterozygous Gunn rats.
The mean serum bilirubin in homozygous Gunn rats unable
to conjugate bilirubin,
owing to complete deficiency of bilirubin UDPglucuronyltransferase
(EC 2.4.1.17),
was 99.6(SD
2.0) imol/L.
Biirubin
conjugates were not detectable inserum
from homozygous Gunn rats.
1706
I-
C
0
2
-.
F
Fig.2. (A) Totalbilirubin
concentrations
inserum from three
strains of rats, as determined by alkaline methanolysis (B)
Concentration of total bilirubin In human amniotic fluid from
Rh-sensitized and non-sensitized pregnancies
conjugated bilirubin amounting to 2-3% of total pigment.
However,amnioticfluidfrom one severely
affected pregnancy
contained 15% of the pigment in the form of conjugates (Figure 3). No apparent relation was observed between the total
bilirubin
values and presence or absence of bilirubin
conjugates.
Discussion
Alkaline methanolysis-HPLC
provides a highly sensitive,
specific,
and reproducible
procedure for direct estimation of
bilirubins
inbiological
fluids.
Resultsofanalysesperformed
athigherserum biirubinconcentrations
inhuman serum are
reportedelsewhere(6).The datapresentedhereinextendthe
applicability
ofthis method to measurement of bilirubin and
itsconjugatesat extremelylow concentrationspreviously
undetectableby non-isotopic
methods.The results
indicate
thatbilirubins
inratserum and amnioticfluidcan be measuredatconcentrations
rangingfrom 0.07to 121.2imol/L for
unconjugatedbilirubin,
0.07to 34.6mol/L forthe C-8 monoconjugate,
0.06 to 69.3 mol/L
for the C-12 monoconjugate,
and 0.17 to 43.7 mol/L for diconjugated bilirubin. Conjugated
bilirubins
were shown to be present in the serum of these animals, albeit in relatively slight concentrations,
accounting for
about 4% of total serum bilirubin.
Analysis of human amniotic fluid obtained by amniocentesis in normal pregnancies and in pregnancies at risk from
maternal-fetal
blood-group
incompatibility
demonstrated
that measurable quantities
of unconjugated
bilirubin
were
present in all instances. The occasional detection of conjugated bilirubin
in amniotic fluid from pregnancies with Rh
sensitization
providesfirm evidence of antenatal conjugation
ofbilepigment by human fetusesthatareformingexcessive
bilirubin.
The quantitative
results
obtainedwithalkaline
methano-
w
method will allow investigations
of bilirubin and its conjugates
even at extremely low concentrations, as in experimental
studies of bilirubin metabolism in normal humans and rats,
and in determination of bilirubin conjugates in amniotic fluid.
The usefulness of alkaline methanolysis-HPLC
for detection
and measurement of bilirubins from other biological sources
such as urine, cytosol, and cerebrospinal fluid remains to be
investigated.
0
0
rd)
cj
0
0
C
a
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(I)
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a
This work wassupported by grants HD-03148 and AM-07 179
the NIH, USPHS.
4.-
a
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0
References
0
1. Killenberg, P. G., Stevens,R. D., Wilderman, R. F., and Wilderman, N. M., The laboratory method as a variable in the interpretation
of serum bilirubin fractionation. Gastroenterology
78, 1011-1015
(1980).
2. Doumas, B. T., Perry, B. W., Sasse, E. A., and Straumfjord, J.U.,
Jr., Standardization in bilirubin assays: Evaluation of selected
methods and stability of bilirubin solutions. Clin. Chem. 19,984-993
+
e
0
4-
0
0’
.0
I
z
h
(1973).
3. Liley, A. W.,
0
I-
Liquor amnii analysis in the management
of the
complicated
by rhesus sensitization.
Am. J. Obstet. Gynecol. 82,1359-1370 (1961).
pregnancy
-)
z
4. Heirwegh, K. P. M., Fevery, J., Meuwissen, J. A. T. P., eta!., Recent
advances in the separation and analysis of diazo-positive
bile pigments. Methods Biochem. Anal. 22, 205-220 (1974).
5. Billing, B. H., Haslam, R., and Wald, N., Bilirubin standards and
the determination of bilirubin by manual and Technicon AutoAnalyzer
methods.Ann. CUrt. Biochem. 8, 21-30 (1971).
Time (minutes)
Fig. 3. Chromatogram
from
of human amniotic
fluid containing,
per
lIter, 5.6 mol of unconjugated billrubln, 0.05 smol of C-8 monoconjugated, 0.12 zmol of C-12 monoconjugated, and 0.74
lLmol of diconjugated blllrubln
Numbers in parentheses:retention times ofbillrubin metabolites, mm
6. Blanckaert, N., Kabra, P. M., Farina, F. A., et al., Measurement
ofbilirubin
andits monoconjugates and diconjugates in human serum
by alkaline methanolysis
and high performance
raphy. J. Lab. Clin. Med. 96, 198-212
(1980).
liquid
7. Blanckaert,
N., Analysis of bilirubin and bilirubin
conjugates. Biochem. J. 185, 115-128 (1980).
chromatog-
mono- and
di-
8. Grunewald, J. 0., Cullen, R., Bredfeldt, J., and Strope, E. R., An
efficient route to xanthobilirubic acid, an oxydipyrryl methene. Org.
Prep. Proced. mt. 7, 103-110 (1975).
lysis
and HPLC provide a basis for future applications of this
procedure ina varietyofclinical and experimental situations 9. McDonagh, A. F.,Bile pigments: Bilatrienes and 5,15-biladienes.
in which accurate determination of total, unconjugated,
moIn The Porphyrins 6, D. Dolphin, Ed., Academic Press, New York,
noconjugated, and diconjugated bilirubin is important. The
NY, 1979, p 345.
CLINICAL CHEMISTRY,
Vol. 27, No. 10, 1981
1707