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Articles |
1
Medical College of Wisconsin, Department of Pathology, P.O. Box 26509, Milwaukee, WI 53226-0509.
2
Beckman Instruments Inc., 200 South Kraemer Blvd., Brea,
CA 92822-8000.
a Author for correspondence. Fax 414-456-6305; e-mail bdoumas{at}mcw.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum.
Results: Under the conditions of the assay (0.1 mol/L glycine
buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated
bilirubin is oxidized and 
-bilirubin is not oxidized at all. Results
obtained with the bilirubin oxidase method agreed well with those
obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4
mg/L were 20% and 2.6%, respectively. The reference values,
established by analyzing 51 plasma specimens from healthy adults, were
0.0–1.2 mg/L, with a mean value of 0.2 mg/L.
Conclusions: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-bilirubin, and depending on the specificity of
the method, variable amounts of unconjugated bilirubin (UB)
(1). However, high concentrations of DB in plasma do not
necessarily indicate cholestasis because -bilirubin persists in
plasma long after cholestasis is relieved. A better indicator of
hepatobiliary excretion is the presence of abnormal concentrations of
CBs in blood (2).
In 1987, Kosaka et al. (3) published a method for measuring
total serum bilirubin and its fractions by use of bilirubin oxidase
(BO) and the diazo reaction. Among those fractions was the sum of mono-
and diglucuronide (CB). To demonstrate the specificity of the assay for
CB, Kosaka et al. compared the CB values in sera obtained with HPLC
with those of the enzymatic method, using correlation/regression
statistics. The slope of the regression equation indicated that the CB
values obtained with the enzymatic method were 13.3% higher than those
obtained with HPLC, but it is impossible to tell whether this
difference is attributable to the oxidation of UB or -bilirubin or
to different methodological principles.
Our study complements the work of Kosaka et al. (3).
Specifically, we provide quantitative data regarding the extent of
oxidation of UB. Using pure -bilirubin (isolated from human serum),
we demonstrated that under the assay conditions this fraction was not
oxidized at all. We evaluated the rates of oxidation of
ditaurobilirubin (DTB) in human serum, human serum albumin (HSA), and
bovine serum albumin (BSA), and demonstrated that DTB in BSA is not
suitable as a calibrator for this assay. We determined the optimum
activity of BO in the reaction mixture so that the oxidation of UB is
kept at no more than 5%, and by adding mannitol (a free-radical
scavenger) to the reaction mixture, we reduced the spontaneous
oxidation of UB or DTB (a blank reaction) ostensibly by free radicals.
A routine assay for CB is available only to those who have certain types of clinical analyzers. The more generally available measurement is that of DB. The availability of a practical CB method adaptable to a variety of clinical analyzers might increase recognition of the clinical value of routine CB testing. We describe here a two-point manual procedure for CB that could be easily adapted as a rate procedure to automated clinical analyzers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DTB calibrator solutions.
DTB calibrator solutions were
prepared by dissolving DTB (Porphyrin Products) in pooled human
sera. The concentration of bilirubin, expressed as the UB equivalent,
was determined by the reference method for total bilirubin
(4). The DTB calibrator solutions were stored at -70 °C
until used.
Bile isolate.
We prepared solutions of bile isolate, a mixture
of bilirubin mono- and diglucuronide prepared according to the method
of Lucassen (5), in pooled human sera. The percentage of
composition of the bilirubin fractions in the enriched pool,
established by HPLC was as follows: -bilirubin, 6%; bilirubin
monoglucuronide (CBm), 34%; bilirubin diglucuronide (CBd), 57%; UB,
3%.
-Bilirubin.
-Bilirubin was isolated from human serum, as
described previously (6).
BO.
The lyophilized enzyme (Genencor) was supplied in vials
containing (according to the supplier) 200 U of BO. We dissolved the
contents of the vial in 5 mL of distilled water, measured the BO
activity (see "Measurement of BO activity"), dispensed 1-mL
aliquots of the solution into small plastic tubes, and stored the tubes
at -70 °C until used.
Tris buffer.
The composition of the Tris buffer was as
follows: 0.1 mol/L Tris, pH 8.5, containing 40 mmol/L sodium cholate,
15 mmol/L sodium dodecyl sulfate, and 50 mmol/L mannitol.
Glycine buffer.
The composition of the glycine buffer was as
follows: 0.1 mol/L glycine, pH 10.0, containing 50 mmol/L mannitol.
hplc
We used a Hewlett-Packard Model 1090 chromatograph equipped with a
Micronex RP-30 column (Sekisui Chemical Co.). The preparation of the
specimen and the procedure for separation of the bilirubin fractions
was performed as described by Adachi et al. (7) except that
plasma was diluted twofold with saline instead of 0.1 mol/L acetic
acid. The bilirubin concentrations in the fractions were calculated by
multiplying the peak area of each fraction (as ratio of the total area)
by the total bilirubin concentration in the specimen.
enzymatic procedure for bilirubin conjugates
Principle.
At pH 10.0, BO selectively oxidizes bilirubin
conjugates to their corresponding biliverdins. The decrease in
absorbance at 460 nm is proportional to the concentration of bilirubin
conjugates.
We used a Gilford Stasar III spectrophotometer for absorbance measurements.
Test.
Under subdued light, we added to a test tube 1.0 mL of
glycine buffer, pH 10.0, and 50 µL of either heparin-treated plasma
or serum. After a 5-min incubation at 37 °C, we added 10 µL of BO
solution containing 0.6 U (measured with UB as the substrate in Tris
buffer, pH 8.5; see below). We mixed the solution, started a stopwatch,
and aspirated the mixture into the spectrophotometer cuvette, which was
thermostated at 37 °C. The absorbance was recorded automatically by
the instrument every 10 s for 2 min. We analyzed controls and
calibrators (DTB) in the same way. (Note: in this procedure, the volume
of the BO solution may vary, e.g., from 10–50 µL, which is
acceptable as long as the activity of the enzyme per test remains
between 0.5 and 0.8 U.)
Blank.
To measure the blank, we used the same procedure as for
the test but substituted glycine buffer for BO.
Calculations.
The concentration of CB in the specimens was
calculated by comparing the difference in absorbance
(A) between the blank and the test at 2 min
(
A = Ablank -
Atest) to that of the calibrator, or
it was obtained from a calibration curve constructed by analyzing
multiple calibrators; for this study we included four calibrators (25,
50, 100, and 200 mg/L DTB) in every analytical run.
measurement of bo activity
There was a wide variation in the activity of BO (units in vial)
purchased from various sources and between BO lots from the same
source. For example, we assayed vials containing 200 U of BO according
to the manufacturer and found activities from 80 to 650 U. This
may be related to the lack of a uniform procedure for establishing BO
activity.
Because UB is oxidized to some extent in this CB method and because the
extent of oxidation depends on the activity of BO in the reaction
mixture, it was necessary to assay BO preparations so that the activity
of enzyme used in the CB method was between 0.5 and 0.8 U per test. We
used the following procedure for measuring the activity of BO: We
dissolved the contents of the vial in 5 mL of distilled water and used
a small aliquot to make a series of dilutions in water. Each dilution
was assayed for BO activity by use of a 200 mg/L UB solution in
pooled human sera as described under "Enzymatic procedure for
bilirubin conjugates", that is, using 1.0 mL of buffer, 50 µL of UB
solution, and 10 µL of BO. The enzyme dilution that provided a linear
decrease in absorbance between 10 s and 1 min was used to
calculate the activity of BO in U/L from the A/min and
the molar absorptivity of UB in Tris buffer, which is 54 700
L · mol-1 · cm-1.
The BO activity in the vial was calculated by multiplying the measured
activity by the dilution factor and by the volume (in liters) used to
reconstitute the BO in the vial.
We want to point out that the activity of the BO in the oxidation of CB (DTB as the substrate in glycine buffer, pH 10.0) is 34-fold lower than the activity in the oxidation of UB (UB as the substrate in Tris buffer, pH 8.5).
kinetics of bo
The kinetics of BO were determined with DTB as substrate by
varying the DTB concentration while keeping the BO activity and the
initial oxygen concentration constant. All reagents were equilibrated
with air. The assay solution consisted of 1.0 mL of glycine buffer, 50
µL of DTB in pooled human sera, and 10 µL of BO solution (0.6 U).
The reaction was initiated by adding BO and followed by monitoring the
decrease in absorbance at 460 nm. The maximum reaction rates
(A/min) were calculated from the initial linear region of
the reaction curves (<60 s) and corrected for the spontaneous
oxidation of DTB, if there was any. The
Km and
Vmax were estimated from the double
reciprocal (Lineweaver-Burk) plot.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The CBd was almost completely oxidized, and the CBm was
oxidized to a great extent. -Bilirubin was slightly oxidized,
whereas the increase in the peak height of UB could be attributed to
partial hydrolysis of CBm or CBd. When UB solutions in serum were
analyzed by the proposed method for CB, only 5% of UB was oxidized
under the conditions of the assay (Fig. 2
). The absorbance of pure -bilirubin, isolated from human
serum (6), remained unchanged even when the reaction time
was extended to 10 min.
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reaction rates
Shown in Fig. 3
are the reaction rates for DTB in human serum, bile isolate,
-bilirubin, and a typical patient's plasma with a high
concentration of CB. The reaction rates for DTB in three protein
matrices are depicted in Fig. 4
. DTB in BSA was oxidized much slower than DTB in human serum or
HSA.
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dependency of cb values on the oxidation (reaction) time
Patient specimens with high CB concentrations and bile isolate in
human serum were analyzed by the proposed method except that the
absorbance of the reaction mixtures was followed for up to 10 min. CB
concentrations were calculated for various reaction times by use of the
corresponding A values of DTB calibrator solutions. The
results in Table 1
show a dependency of CB values on the reaction time. The 10-min
CB value of each specimen was assumed to represent complete oxidation
of CB and was used to calculate the percentage of CB oxidized at other
times.
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linearity, precision, interference by hemoglobin
The linearity of the proposed method was evaluated by analyzing
dilutions of a patient's plasma specimen with high CB concentration
(232 mg/L) and dilutions of a DTB solution (196 mg/L) in human serum.
Under the conditions of the assay linearity (milligrams of CB per liter
of reaction mixture) extended to at least 9.4 mg/L, corresponding to
200 mg/L CB in plasma or DTB in human serum (Fig. 5
).
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The long-term precision of the assay was established by analyzing over
a period of 6 months two controls prepared from sera with high
concentrations of CB; aliquots of the controls were kept at -70 °C
until used. Shown in Table 2
are precision data for the controls and the reproducibility of
the A460 nm of a DTB solution in
human serum.
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The interference of hemoglobin was evaluated by adding various amounts of a hemolysate to aliquots of a jaundiced plasma specimen. At a hemoglobin concentration of 3 g/L (the highest concentrations tested), the CB concentration of the specimen increased from 73.0 to 75.2 mg/L, a negligible change.
reference values
Fifty-one plasma samples from apparently healthy individuals were
analyzed by the enzymatic method. The mean CB value was 0.2 mg/L, with
a range of 0.0–1.2 mg/L. The total bilirubin values of the 51
specimens were 3–11 mg/L (range), with a mean of 6 mg/L.
comparison of cb values by various methods
Plasma specimens with increased CB values were analyzed by the
HPLC and BO methods. Scatter diagrams of the CB values are shown in
Fig. 6
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formation of biliprotein
Solutions of DTB (220 mg/L) in HSA (40 g/L) and BSA (40 g/L) were
allowed to stand at room temperature (in the dark) for up to 60 h.
The DTB solution in HSA was analyzed by HPLC 1 and 24 h after it
was prepared, and the DTB solution in BSA was analyzed 1 and 60 h
after preparation. The chromatograms showed a bilirubin fraction that
eluted at the same time (14.6 min) as the naturally occurring
biliprotein in human serum. Furthermore, the absorption peak at 450 nm
was superimposed on the peak at 280 nm. The amounts of biliprotein
formed for DTB in HSA were 3.4 and 10 mg/L at 1 and 24 h,
respectively; for DTB in BSA, the amounts of biliprotein formed were
4.8 and 13.8 mg/L at 1 and 60 h, respectively. Because the
absorptivity at 450 nm of the biliprotein formed in human blood from CB
and serum albumin is much higher than that of UB (6), the
amounts of the DTB-biliprotein calculated from the HPLC tracings are
most likely overestimated.
bo kinetics
The BO kinetics experiment was conducted with three DTB solutions
having concentrations from 49.2 to 147.6 mg/L. A double reciprocal
(Lineweaver-Burk) plot provided a straight line (Fig. 7
) described by the equation: y =
1.79x + 0.0105; r2 = 0.999.
The Km and
Vmax values calculated from this
equation were 172 µmol/L and 95 µmol/min, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The procedure may be calibrated with DTB calibrators in human serum or
in HSA; DTB in BSA is oxidized much slower than in human serum or HSA
(Fig. 4
) and, therefore, is not a suitable calibrator. HPLC analysis of
DTB solutions in human serum, HSA, or BSA revealed that DTB reacts
spontaneously with albumin to yield small amounts of a biliprotein
analogous to that formed in the reaction between albumin and bilirubin
glucuronides. Presumably, DTB is linked to albumin through the sulfonic
acid group and a free amino group on the albumin molecule. The
DTB-albumin complex starts forming upon the addition of DTB to the
protein solution and increases with time when the solution is allowed
to stand at room temperature. The reaction stops upon freezing. This
biliprotein is likely present in calibrators and controls that contain
DTB. At this time, we do not know whether this biliprotein in oxidized
by BO in the proposed assay.
Our findings do not support the conclusion of Franzini and Cattozo
(8) that "After prolonged incubation at 37 °C (up to
24 h) of DTB solutions in different protein matrices (HSA, HSR and
BSA) no evidence for biliprotein formation has been obtained"
(8). It appears that these authors considered the small
amounts of biliprotein (~5 mg/L) shown in Table 2
of Ref.
(8) to be artifacts. It should be pointed out that Franzini
and Cattozo used gel filtration to isolate the biliprotein, a technique
that may be less sensitive and specific than HPLC.
The specificity of the CB measurement is very good because
-bilirubin is not oxidized and only ~5% of the UB is oxidized
regardless of its concentration; this small interference for UB is
considered tolerable because it will not lead to erroneous diagnoses.
The interference by hemoglobin is negligible.
The 2-min reaction time was chosen to keep the oxidation of UB at a
minimum. We believe that an essential requirement for a reliable
procedure for measuring CB is specificity, that is, measuring as little
UB and -bilirubin as possible. Because we made these measurements
with a manual spectrophotometer, it would not have been practical to
use a shorter reaction time. If this method were to be adapted to
automated clinical analyzers, the reaction time would most likely be
reduced to 30 s or less.
The precision of the method is comparable to that obtained by the diazo
DB methods and adequate for clinical purposes. Claims about the
accuracy of the method must be qualified. Because of its specificity,
the method can be considered as being accurate, although it is not
certain that it measures the exact concentration of the two bilirubin
glucuronides because there are many potential sources of error. The
molar absorptivities of DTB, CBm, and CBd may not be identical, and the
reaction rate of CB in serum appears to be slower than that of the DTB
calibrator (Table 1
). In addition, the oxidation rate of CBm may not be
the same as that for CBd. Despite these uncertainties the measurement
of CB is still valid because absolute accuracy is not that relevant
because once the diagnosis of hepatobiliary dysfunction is established,
the exact concentration of CB in serum is of little help to the
clinician. It is the disappearance of CB from blood that signals that
the hepatobiliary obstruction has been relieved.
Because the concentration of CB in the serum of healthy adults is too low to be detected by routine laboratory methods (9), the near zero CB values obtained by this method in healthy subjects are not unexpected.
Comparison of values obtained with the BO method and those obtained with HPLC is somewhat puzzling. It appears that samples with a CB concentration <100 mg/L have a slope different from those of samples with a CB concentration >100 mg/L. We have no explanation for this inconsistency.
Under the conditions of the assay, bilirubin is oxidized to biliverdin even in the absence of BO. This spontaneous oxidation has been observed previously (3) and is enhanced by the presence of transition metal ions, which are ubiquitous in plasma. By adding 50 mmol/L mannitol to the glycine buffer (10), we were able to prevent the spontaneous oxidation, which could introduce an inaccuracy in the CB measurement. In the absence of mannitol, we have observed a mean decrease of 1.4 mA/min, which was reduced to 0.15 mA/min when mannitol was added to the buffer.
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Footnotes |
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1 Nonstandard abbreviations: CB, sugar-conjugated bilirubin; DB, direct-reacting bilirubin; UB, unconjugated bilirubin; BO, bilirubin oxidase; DTB, ditaurobilirubin; HSA, human serum albumin; BSA, bovine serum albumin; CBm, bilirubin monoglucuronide; and CBd, bilirubin diglucuronide. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  S.-i. Sakasegawa, H. Ishikawa, S. Imamura, H. Sakuraba, S. Goda, and T. Ohshima Bilirubin Oxidase Activity of Bacillus subtilis CotA Appl. Envir. Microbiol., January 1, 2006; 72(1): 972 - 975. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
= 26.6 mg/L; 
= 20.1 mg/L; n = 34.
Line B, CB >100 mg/L: y = 1.21 (±
0.13)x - 32.5 (± 20.1);
r2 = 0.91; 