The measurement of bilirubin fractions in serum.

Volume 28, Issue 5,6 (1991)

415

The Measurement of Bilirubin Fractions in Serum Basil T. Dournas and Tai-Wing Wu

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ABSTRACT Bilirubin fractions are measured by (1) the direct diazo reaction, (2) high-performance liquid chromatography (HPLC), (3) direct spectrophotometry, and (4) enzymatic methods. HPLC, which effects separation and quantitation of the four bilirubin fractions, is the method of choice, but impractical for routine use. A special application of direct spectrophotometry allows the measurement of unconjugated bilirubin and the sum of bilirubin conjugates. This approach, which provides essentially the same information as HPLC, unfortunately is available only in one clinical analyzer. The direct diazo reaction measures bilirubin conjugates plus &bilirubin, albeit not very accurately. Direct diazo methods that measure unconjugated bilirubin as direct could obscure the clinical diagnosis. At acid pH, enzymatic methods measure all direct reacting bilirubins, while at pH 10 only conjugated bilirubins are measured. Because the measurement of conjugated bilirubins is clearly more helpful than that of direct bilirubin in the differential diagnosis of jaundice, direct diazo methods should be replaced by methods specific for bilirubin conjugates. Key Words: bilirubin fractions, direct diazo reaction, high-performance liquid chromatography, direct spectrophotometry, enzymatic methods, conjugated bilirubins, differential diagnosis of jaundice.

I. INTRODUCTION* Until the mid-l950s, bilirubin in serum was described as direct and indirect and the sum of the two as total. At that time, a number of inve~tigatorsl-~ by use of open-column chromatography separated two bilirubin fractions from jaundiced sera and demonstrated that indirect bilirubin was unconjugated bilirubin, while the “direct” fraction was thought to be either bilirubin monoglucuronide, possibly formed from the reaction of unconjugated bilirubin and bilirubin diglucuronide, or a mixture of mono- and diglucuronide. In 1966, Kuenzle and his c o - ~ o r k e r sreported ~.~ that jaundiced sera from adults with liver disease contained a fourth bilirubin fraction. This fraction, f d y bound to protein, was isolated by column chromatography and named 6-bilirubin. Despite four publications in German journals and two in American journals, this discovery received practically no attention for 15 years until it was rediscovered in 1981.6*7Most clinical laboratories still

*

Nonstandard abbreviations: B,, conjugated bilirubin; B,, bilirubin covalently linked to albumin (&-bilirubin); BOX, bilirubin oxidase; BSA, bovine serum albumin, C o b Fraction V; B,, unconjugated bilirubin; CAP, College of American Pathologists; dB,, bilirubin diglucuronide; DBIL, direct reacting bilirubin (the sum of bilirubin monoglucuronide, diglucuronide and &-bilirubin); E, molar absorptivity; HPLC, high-performance liquid chromatography; HSA, human serum albumin, Cohn Fraction V; mB,, bilirubin monoglucuronide; SDS, sodium dodecylsulfate; SRM, Standard Reference Material (from the National Institute of Standards and Technology); t.l.c., thin layer chromatography.

B. T. Dournas, B.S., M.S.,Ph.D., Dept. of Pathology, Medical Collegeof Wisconsin, 8700 W. Wisconsin Avenue, Milwaukee, WI 53226. T.-W. Wu, B.Se., M.Sc., Ph.D., Dept. of Clinical Biochemistry, The Toronto Hospital, 200 Elizabeth Street, Toronto M5G 2C4 Canada.

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Critical Reviews in Clinical Laboratory Sciences


measure total and direct bilirubin in serum or plasma, and until the early 1980s, these were the only bilirubin measurements available. At the beginning of the last decade, by use of high-pressure liquid chromatography (HPLC),serum bilirubin was separated into four fractions.8 At the same time the use of the thin film technology and simultaneous spectrophotometric analysis of a two-component system permitted the measurement of the sum of the two bilirubin conjugates in serum.9

II. CHEMISTRY AND STRUCTURE A. Chemistry and Structure of Bilirubins as a Class of Bile Pigments Bilirubin is a family of bile pigments all containing a common asymmetric tetrapyrrole structure. There are three major classes of bilirubin in blood: unconjugated bilirubin (B,), sugar-conjugated bilirubin (BJ, and bilirubin covalently linked to albumin also called 6bilirubin (B,) or biliprotein. Although the metabolism of bilirubin is not our primary concern here, we should note that B, is a catabolite of heme that is converted to B, mainly in the liver by the enzyme-catalyzed addition of one or two sugar molecules (principally glucuronic acid) to either one or both of its propionate side chains, and that B, is concentrated in the bile before being excreted in the feces (after further metabolism in the intestine). According to current thought, &bilirubin is formed largely from an acyl transfer of the bilirubin moiety from B, (or an acyl-migrated isomer thereof) onto the epsilon-aminogroup of a lysine residue on the albumin molecule.10-12The possibility of an enzymatic route for synthesis of B, in vivo has yet to be ruled out.

B. Chemistry and Structure of Specific Bilirubin Fractions* 1. Unconjugated Bilirubin Unconjugated bilirubin is extremely apolar and practically insoluble in water at physiologic pH and temperature. The apolar nature of B, had puzzled chemists for a long time because the presence of the two propionic acid chains would have indicated otherwise. Fog and Jellum f i t suggested that the main form of B, (the IX-a isomer) is not a linear tetrapyrrole, but a tightly internally folded structure in which the propionate groups are linked to the pyrrole nitrogen^.'^ This kind of structure, which accounts for the poor solubility of B,, was confirmed by X-ray c~ystallography.'~~'~ B, assumes the Z,Z-configuration, the folded conformation being stabilized by six intramolecularH-bonds (Figure l), and from this model it can be deduced that partial or complete rupture of the H-bonds, as in irradiation of B, with blue light, could lead to partially open (Z,E and E,Z) or completely unfolded (E,E) geometric isomers (Figure 2). As expected, these isomers are more polar and more watersoluble than native B,. Thus, geometric isomerization provides a plausible mechanism for the excretion of B, in bile as a soluble pigment upon phototherapy.16It should be noted that, apart from X-ray data, there has been no direct proof that B, actually occurs as a Z,Zisomer in vivo. Also, the photon-induced geometric isomers of B, appear less diazo-reactive and bind to albumin less avidly than native Bu.17,18 On the basis of these findings, one may infer that the overall configuration and conformation of B, (or of its isomers) plays an important role in determining its solubility, binding affinity for albumin and diazo-reactivity. 2. Sugar-Conjugated Bilirubins B, is formed by the enzymatic addition of one to two molecules of a sugar (principally glucuronic acid) onto either one or both of the propionic acid side chains of B,. The resulting mono- (mB,) and di- (dB,) sugar conjugates (Figure 3) are more polar than B,, nontoxic to

*

Concentration of bilirubin fractions are given in mass units and are expressed in mg/dl of unconjugated bilirubin. To convert mg/dl to Fmolll (molar concentraiton units) multiply times 17.1.



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FIGURE 1. Structure of unconjugated bilirubin. (Top) The Z,Z configuration with hydrogen bonds represented by dotted lines; (bottom)structure in simplified form. (From Bonnett, R., Davies, 1. E., and Hursthouse, M. B . , Nature, 262,326, 1976. With permission.)

cells, and are excreted against a concentration gradient across the canalicular membrane of the hepatocyte in the bile. The glucuronide nature of B, was proposed independently by Talafant,’ S ~ h m i d and , ~ Billing’s group’ in 1956 and 1957, and subsequently verified by others.’9-23It appears that while dB, is the predominant bilirubin in human bile, mB, is often the chief bilirubin conjugate in jaundiced sera in which the direct fraction is greater than 50% of total bilirubin.24 The fact that mB, seems to be much less stable than dB, in vitro may be among the reasons why mB, has been particularly difficult to isolate and purify. One should also note that bilirubin can migrate from C-1 to C-2, C-3, and C-4 hydroxyls of glucuronic acid to form acyl-migrated isomers of both mB, and dB,,23 with potentially profound consequences for the formation of delta bilirubin. lo 3. Bilirubin Covalently Linked to Albumin (&Bilirubin) The possible presence of a strongly protein-bonded bilirubin was first proposed by Kuenzle et al.,4.’ but it was not until 1981 that 8-bilirubin was identified6 to be an entity distinct from B, and B,. On the basis of current knowledge, B, is a bilirubin covalently linked to albumin through an arnide bond between one of its two propionic acid side chains and an €-amino group of a lysine residue on albumin.I2B, has been synthesized in vitro by incubating (at 37°C) rat serum with dB, or a mixture of mB, and dB,, or human serum albumin with a mixture of mB, and dB,.I0 Formation of B, in vivo appears to be largely nonenzymatic, and it involves acyl migration of bilirubin from a bilirubin-glucuronic ester to a nucleophilic


418


ANTI-QE, SYN-.152

\\“. H

I

FIGURE 2. Photochemical reaction cycle for bilirubin IX-cr showing Z $ E isomerization and concomitant disruption (or formation) of intramolecular hydrogen bonds. (From Lightner, D. A., Bilirubin, Vol. 1, Chemistry, Heirwegh, K. P. M. and Brown, S. B., Eds., CRC Press, Boca Raton, FL, 1982, chap. 1. With permission.)

site on albumin. Unconjugated bilirubin does not react with albumin to form &bilirubin. Further evidence for the formation and structure of B, is as follows: (1) in unconjugated hyperbilirubinemia (hemolytic jaundice) there is no more than a trace of B, in serum,25and analbuminemic rats are unable to synthesize B,;26 (2) in obstructive hepatobiliary disease the increase in the concentration of B, in serum parallels the increase in B,;27(3) &bilirubin is the slowest fraction to clear from serum following resolution of severe hepatobiliary disease, presumably owing to its albumin moiety, which has a half-life of approximately 19 d; (4) the half-life of bilirubin, covalently bound to rat albumin, injected into the rat was 2 d, identical with that of rat albumin, while the half-life of unconjugated bilirubin linked to rat albumin was 6 min;28(5) when B, is coupled with the diazo reagent, one half of the azopigment is not ultrafilterable presumably because it is still bound to a l b ~ m i n . ~ An interesting attribute of B, is that this irreversible bilirubin-albumin complex reacts readily, as does B,, in the direct diazo rea~tion.’”~That this is so would suggest that the strength of binding to albumin is not a basis for distinguishing between “direct” and “indirect” bilirubins. Rather, such terms may refer to bilirubins of different solubilities in water. From an examination of the azopigments of authentic human B,, Lauff et al.’ deduced that about 65% of the B, molecules are nonesterified (not conjugated to sugar), while 30 to 35% could be sugar conjugated. This point was confirmed recently by Yoshida et al. ,29 who also suggested that B, arising from dB, will tend to be sugar conjugated on the carboxyl group not linked to protein, whereas B, arising from mB, will tend to be unconjugated. Eventually, elucidating the exact binding site(s) of bilirubin on the albumin will be important



419

BILIRUBIN DIGLUCURONIDE

BILIRUBIN MONOGLUCURONIDE (C-12)

BIL IRUB1N MONOGLUCURONIDE (C-8)

FIGURE3. Structures of bilirubin glucuronides. Monoglucuronides can exist as two molecularspecies, depending on whether the (2-12 or C-S propionic side acid chain is esterified. (From Chowdhury, J. R. and Chowdhury, N. R . , Seminurs in Liver Disease, Vol. 3, Phieme Medical Publishers, New York, 1983, 1 1 . With permission.)

to our understanding of its chemistry and structure. Results from chemical and enzymatic cleavage of B, indicate the presence of one or two bonding regions with very similar amino acid sequence on albumin for the covalent linkage.30Because the two regions are located in different sites within the protein, one of the sites might be preferentially involved in the linkage with bilirubin. This remains to be verified.

111.

THE MEASUREMENT OF BILIRUBIN FRACTIONS IN BLOOD

Although the purpose of this article is to review methods for measuring the bilirubin fractions in serum, we feel it is necessary to present the measurement of total bilirubin (the sum of the four fractions) because its value is used quite often for calculating the concentration of bilirubin fractions separated by chromatographic techniques in the same manner the value of serum total protein is used to calculate concentrationsof protein fractions in electrophoresis.

A. Total Bilirubin 1. Reference Method For the sake of brevity, we present the most important aspects of only one method, that is the reference m e t h ~ d ~ developed ”~~ by the Committee on Standards of the American Association for Clinical Chemistry and ‘‘credentialed” by the National Reference System for the Clinical L a b ~ r a t o r y . ~ ~ The method, which is based on the Jendrassik-Gr6f principle,34is as follows: to 0.5 ml

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of sample add 4 ml of caffeine reagent and after 10 min, 1 ml of diazotized sulfanilic acid. After 10 min, add 3 ml of alkaline tartrate reagent and measure the absorbance at 598 nm. Correct the absorbance of the test solution by subtracting that of the sample blank, prepared by substituting sulfanilic acid for diazo reagent. The substitution of dyphylline for caffeine in the Jendrassik-Gr6f method is a matter of choice; reagents containing dyphylline-acetate or caffeine-acetate-benzoate yield identical total bilirubin values in human sera.35 a. CALIBRATION

Bilirubin from the National Institute for Standards and Technology (NET), formerly the National Bureau of Standards, SRM 916a is the recommended material for preparing standard solutions for calibration of bilirubin assays. Bilirubin from other sources may be used provided that it is assayed against the SRM 916a. Aqueous solutions of bilirubin are stabilized by addition of protein, whole serum or 4 g/ dl human serum albumin (HSA) or bovine serum albumin (BSA). For the reference method either human or bovine albumin or human serum may be used for preparing bilirubin standard solutions, because the molar absorptivity of the azopigment at 598 nm is the same in these 3 mat rice^.^' Methods using methanol as promoter of the reaction must be calibrated with bilirubin standards in human serum, because the molar absorptivity of the azopigment in either human or bovine albumin is considerably lower than that in human serum.36 To ascertain that bilirubin standard solutions have been prepared properly, they should be analyzed by the reference method and the molar absorptivity be calculated. For the SRM 916a, the standard solution is considered acceptable when the absorptivity of the azopigment (corrected for the non-bilirubin impurities of the SRM) at 598 nm is between 75,100 and 77,900 1 mol-' * cm-1.37Additional criteria for the acceptability of the standard solution are the molar absorptivity of the azopigment at 530 nm (measured without adding alkaline tartrate), and those of bilirubin in caffeine reagent at 432 nm and 457 nm38.39(Table 1).

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b. LINEARITY, PRECISION, ACCURACY

The absorbance of the azopigment at 598 nm adheres to Beer's law to at least 2.07 A, which under the conditions of the assay corresponds to a serum bilirubin concentration of 27 mg/dl. This linear relationship between absorbance and concentration holds true for solutions of unconjugated bilirubin in human serum, HSA or BSA as well as for sera from patients with hepatic jaundice, which contain all of the bilirubin fractions. Between-run coefficients of variation are less than 4% for bilirubin concentrations within physiologic limits and near 1% for concentrations above 5 mg/d1.32The color yield (absorbance) is so reproducible that the standard deviation of the molar absorptivity of the azopigment at 598 nm, determined in 5 laboratories, was only 610 1 mol-I cm-' (Table 1). In the absence of a definitive method for total bilirubin, it is impossible to assess the accuracy of the reference method. The following evidence, however, indicates that the method is accurate: (1) B, and dB, react quantitatively during the 10-min reaction time,40 and there is no reason why mB, should not also react completely; (2) B,, mB, and dB, yield the same azopigments in the reaction mixture, those of B,, because the conjugated azopigments are rapidly hydrolyzed upon addition of alkaline tartrate.31On the basis of this evidence it is reasonable to conclude that three of the four bilirubin fractions are measured accurately by this method. We do not know, however, if B, is measured accurately; the one half of the azopigment attached to protein could exhibit an absorptivity different from the azopigment not bound to protein. Although the data of Lauff et aL2' suggest that B, is measured accurately, other authors reported a 3% underestimation of total bilirubin when the B, concentration in serum is high,3' an inaccuracy quite small and medically irrelevant.

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Table 1 Comparison of Molar Absorptivities" of SRM 916a with Those of SRM 916 o,

L * mol-' . cm-'

Caffeine reagent


432 nmb SRM 916a Averaged SD of single measurement S R M 916 Average'

'

457 n

Reference method m b

530 nmc

598 nmc

580

48,980 570

56,660 1,030

76,490 610

49,170

48,540

56,790

76,290 (76,260)'

50,060

Corrected for the non-bilirubin impurities of the SRMs, Le., SRM 916, 1.0% (purity, 99%); SRh4 916a, 1.7% (purity, 98.3%). Bilirubin. Azopigments. Mean values from five laboratories. Mean values from one laboratory. Result reported in Reference 3 1.

From Doumas, B. T., Perry, B. W., McComb, R. B., Kessner, A,, Vader, H. L., Vink, K. L. J., Koedam, J. C., and Paule, R. C., Statistical analysis, Clin. Chem., 36, 1698, 1990. With permission.

c. THE lllga AND XIII-cw ISOMERS OF BILIRUBIN

Commercial bilirubins, including SRM 916a, contain variable amounts of the 111-a and XIII-a isomers formed during the preparation of bilirubin from bile and gallstones. In contrast, bilirubin formed in vivo consists almost exclusively of the IX-a isomer;41isomerization is prevented by the presence of albumin.42In the diazo reaction, bilirubin IX-a yields two isomeric azopigments (Figure 4, azopigments A and B), while the symmetrical 111-a and XIII-a isomers yield azopigments B and A, respectively. If isomers 111-a and XIII-a are not present in equimolar quantities, and the two azopigments do not exhibit identical molar absorptivities, calibration with such a bilirubin preparation could lead to inaccuracies in the measurement of total bilirubin in serum. The isomer content of SRM 916 (established by HPLC) is 12.2% III-a, 75.7% IX-a,and 12.1% XIII-IX.~~ Since the IIIa and XIII-(11isomers are present in equimolar quantities, the concentration of azopigment A will be equal to that of B, that is, in diazo methods SRM 916 behaves as if it consisted entirely of bilirubin I X - a . In SRM 916a (released recently by NIST) values by HPLC for isomers 111-a and XIII-a are 7.5 and 9.3%, respectively, and by thin layer chromatography (t.1.c.) 5.3 and 11.5%, re~pectively.~'Although calibration of diazo methods with SRM 916a could introduce an inaccuracy in the measurement of TBIL in serum, this inaccuracy should, as illustrated in the following example, be rather negligible. Assuming that the E values at 598 nm for azopigments A and B are 70,000 and 80,000 1 mol-' cm-I, respectively, then the E value for the azopigments from the IX-a isomer will be 75,000. If the t.1.c. values for isomers 111-and XIII-a are correct, then the E value for the azopigments of 916a would be 74,690; if the HPLC values are correct, the E value would be 74,910 1 mol-I cm-I. Both of these values are within one SD (SD for a single measurement) from the value (75,000) of the IX-a isomer and, therefore, lack statistical significance. The close agreement between the E values at 598 nm of the azopigments from SRM 916 and 916a (Table 1) does not support the likelihood of a very large difference in the E values of azopigments A and B .

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M

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a3 I Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

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FIGURE 4. The mechanism of the diazo reaction. Bilirubin reacts with diazo compound (diazotized sulfanilic acid) forming azopigment and hydroxypymmethene carbinol. A second molecule of azopigment and a molecule of formaldehyde are formed from the reaction of carbinol with a second molecule of diazo compound. Bilirubin M-a yields azopigments A and B; bilibins III-a and XIIIa yield exclusively azopigments B and A, respectively. R = H or sugar moiety.

The three bilirubin isomers exhibit different absorption maxima and E values in CHC1,: 111-a,65,200 1 mol-' cm-' (455 to 458 nm); IX-a, 62,600 1 mol-I cm-' (453 to 455 nm); XIII-a, 52,500 1 * mol-I * cm-' (449 to 453 n~n).~~ Molar absorptivity values for isomers III-a and XIII-a in human serum have not been established. Until this is done, the error, if any, introduced in the measurement of bilirubin by methods based on direct spectrophotometry and calibrated with SRh4 916a, will remain unknown.

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d. INTERFERENCES

Interference from hemoglobin is negligible if its concentration does not exceed 200 mg/ dl,31*"a value occurring rarely even in the serum of neonate^.^' For grossly hemolyzed sera, addition of ascorbic acid (0.1 ml of a 4 g/dl solution) before the alkaline tartrate eliminates or reduces the suppression of the bilirubin values by h e m ~ g l o b i n . ~Data ' . ~ on the interference of hemoglobin at 598 nm and at 530 nm (when addition of alkaline tartrate is omitted) are shown in Table 2. Few exogenous compounds have been reported to interfere in the measurement of bilirubin by this rneth~d;~' significant positive interference is caused by L-dopa and a-methyldopa.48 Changes in both the absorption spectrum and the absorptivity of the alkaline azopigment are induced by a variety of metal ions.49For most of the ions, such changes require relatively high concentrations that are unlikely to be found in reagents used in this bilirubin assay. Zinc, however, even at low concentrations causes a measurable increase in the absorptivity of the azopigment at 598 nm, but at physiologic concentrations its effect is negligible.31

B. Determination of Direct Reacting Bilirubin by the Diazo Reaction Although the diazo reaction described by van den Bergh in 191650cannot be considered a recent development, it is still the most widely used principle for the measurement of direct bilirubin in serum. Depending on the conditions of the assay, the diazo reagent reacts in the absence of a promoter with 8-bilirubin, conjugated bilirubin and some of the unconjugated


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Table 2 Interference by Hemoglobin (Hb) in the Reference Method for Serum Total Bilirubin Bilirubin, mg/dl At 530 nm'


At 598 nm

Hb, mg/dl 0 50 100

200 300 1000

Note:

With ascorbic acid

Without ascorbic acid

10.0 10.0 10.0

10.0

Hb, mg/dl

10.0 10.0 9.5

10.0 10.0 9.9 9.7 8.1

0 50 150 300 700

10.0

10.0 9.9 9.7 9.5

Hemoglobin was added to B. solutions in human serum.

' Without addition of ascorbic acid and alkaline tartrate.

bilirubin. In the typical assay, serum is diluted with either water or 50 mmoVl HCI before addition of diazo reagent. After a variable coupling time the absorbance of the azopigment is measured near 540 nm or, after adding alkaline tartrate, at 598 nm.3s.s1.52 The extent to which unconjugated bilirubin reacts in the direct assay is determined by the pH of the reaction mixture; the lower the pH the lesser the reactivity of the unconjugated bilirubin.s3 For many years, the main reason for measuring direct bilirubin in blood was to distinguish between hemolytic and hepatic jaundice. However, the presence of direct bilirubin in blood does not allow us to differentiate between persisting obstruction or relief in cholestatic jaundice. This is because B,, which persists in blood for many weeks after the obstruction (intrahepatic or extrahepatic) has been relieved, is a direct-reacting bilirubin. The differentiation between persisting obstruction or relief is possible by measuring the sugar-conjugated bilirubins instead of DBIL.54At present, this is possible only through the use of HPLC or the Kodak Ektachem Analyzer. Diazo methods adapted to most, if not all, of the other clinical chemistry analyzers measure variable amounts of B, and, therefore, obscure the clinical condition of the patient. Clearly, diazo methods for DBIL must be substituted by methods that measure only the conjugated bilirubins. However, since the diazo method is still used by a large number of laboratories and because some versions of this method measure substantial amounts of unconjugated bilirubin as DBIL, providing most misleading results to the physician, we present a procedure that allows differentiatingbetween hemolytic and hepatic jaundice by keeping the reaction of B, at a minimum.

1. Preferred Method Keeping the unconjugated bilirubin from reacting requires diluting the serum with HC1 and incubating for at least 5 min before adding diazo reagent.55This preincubation somewhat decreases the reactivity of the direct-reacting fractions, which is a far less serious drawback than measuring unconjugated bilirubin as direct. The 1-min reaction underestimates the direct bilirubin and even after a 10-min coupling time the reaction is not complete. A procedure that combines the most desirable features of several methods is as follows:s6 dilute 0.25 ml of sample with 1.0 ml of 50 mmoVl HCI and let stand for 5 min. Add 0.5 ml of diazo reagent and after a 10-min incubation (room temperature), add 0.1 ml of a 2 g/dl solution of ascorbic acid, followed by 1.5 ml of alkaline tartrate and 2.0 ml of caffeine

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Critical Reviews in Clinical Laboratory Sciences SO3Na

SO, Na

CHz

CHZ

CHZ

CHZ

NH

NH

L=o

c=o

kHz

CH2

I

I

I

I


I

I

0

T

k

H

C

J

H

T

L

C

I

I

I

M = Methyl Group V = Vinyl Group

I

H

2

h

HC

QH o

H

H

FIGURE 5. Structure of ditaurobilirubin disodium salt.

reagent. Prepare a sample blank by substituting sulfanilic acid for diazo reagent. Measure the absorbance of test and blank against water at 598 nm and subtract the absorbance of the blank from that of the test. Calculate the concentration of direct bilirubin in the sample against a standard (unconjugated bilirubin) analyzed by the procedure for total bilirubin, i.e., dilute the sample with caffeine reagent and add HC1 at the end. The caffeine reagent suppresses the ab~orptivity~’*~’ of the azopigment at 598 nm by about 12%. Omitting the caffeine reagent in this assay for direct bilirubin has been responsible for the paradox of direct bilirubin values being sometimes higher than the total; this situation is expected when the percent of B, in the specimen is quite small and serum is diluted with water instead of HCl. Diluting serum with HCl and adding the caffeine reagent (necessary for the reaction of B, in the calibrator) after alkaline tartrate in the direct assay eliminates direct bilirubin values that are higher than the total. Ascorbic acid is added to the reaction mixture to destroy excess diazo reagent, which would react with B, upon addition of alkaline tartrate. Ascorbic acid destroys the diazo reagent at the acid pH of the reaction mixture. Hydroxylamine, used instead of ascorbic acid in some methods, does not destroy the diazo reagent at the acid pH and, therefore, does not stop the coupling reaction until alkaline tartrate is added.56 Before ditaurobilirubin (DTB) became available commercially, B, was used exclusively for calibrating methods for direct bilirubin. However, the use of B, as calibrator in direct bilirubin assays would have been impractical, if at all feasible, in most of today’s automatic clinical analyzers. DTB (Figure 5), a diconjugate of bilirubin with taurine, first synthesized by Jirsa et al.,” and subsequently found in the bile of the chick embryo and young chick by T e n h ~ n e n , ~is*now used almost exclusively for calibrating direct bilirubin assays. Its disodium salt is water-soluble, resistant to acid and alkaline hydrolysis, and, if kept dry, remarkably resistant to oxidation by air. We have kept DTB for more than 5 years at 4°C without any change in the bilirubin content (when analyzed for total bilirubin). Most commercial preparations contain variable amounts of the monoconjugate (10 to 15%).59In the reference method for total bilirubin described earlier, the absorption spectrum and molar absorptivity of the azopigment (at 598 nrn) derived from DTB are identical to those of the B, a~opigment.’~ Because commercial preparations of DTB are not 100% pure, their bilirubin

Volume 28, Issue 5,6 (1 99 1)

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Table 3 Reactivity of Ditaurobilirubin, &Bilirubin and Bilirubin Conjugates in the Direct Diazo Reaction


Bilirubin, mg/dla

Total

Ditaurobilirubind &Bilirubin Bilirubin conjugatesc

a

5.6 16.8 2.3 9.0 5.7 17.0

Direct 4.8 13.9 1.8 8.4 4.4 12.8

Direct (pH 4.75p 5.4 16.3 1.2 5.8 5.1 15.0

Expressed as unconjugated bilirubin. Preferred method. In 0.4 rnoVl acetate buffer, pH 4.75. In human serum. Bile isolate (consisting of 42% mB,, 44% dB,, 9% B,, and 5% B,) used to enrich human serum.

Adapted from Dournas, B. T., Wu, T.-W., Poon, K.-C. P., and Jendrzejczak, B., Clin. Chem., 31, 1677, 1985; Dournas, B. T., Wu, T.-W., and Jendrzejczak, B., Clin. Chem., 33, 769, 1987.

content should be established by comparison with a primary standard (SRM 916a). Solutions of DTB in human serum are stable for at least 6 months at - 70°C; freeze-dried preparations are stable for many years at 4°C. a. REACTIVITIES OF THE BILIRUBIN FRACTIONS IN THE DIRECT DIAZO REACTION

Under the conditions of the preferred assay, the color yield from “bilirubin conjugates”, B, and DTB is less than the theoretical, i.e., values for direct bilirubin are lower than those for total bilirubin (Table 3). The reactivity increases at higher concentrations of the diazo reagent, but the improvement in accuracy is negated by an increase in the reactivity of B,. The “bilirubin conjugates’’ used to obtain these data were a mixture of mB,, and dB, isolated from human bile.@’ This mixture was dissolved in human serum and lyophilized. The percentage composition of the bilirubin fractions in the enriched serum pool (reconstituted material) was mB,, 42%; dB,, 44%;B,, 9%; B,, 5%.61 At pH 4.75 the reaction of DTB is almost quantitative, i.e., direct bilirubin is equal to total. B, reacts more quantitatively, but B, reacts much less at this pH than at acid pH (- 1.6) (Table 3). Perhaps, because albumin being least soluble in water at pH 4.75 (isoelectric for albumin), it may present less of its bilirubin for the coupling reaction. Unconjugated bilirubin reacts very little in this procedure (Table 11); the reactivity of B, is considerably higher when serum is diluted with water instead of 50 mmoYl HCl.56Addition of surface active agents to the reagents should be avoided; they dissociate B, from albumin, keep it in solution inside the surfactant micelles, and hence promote the coupling The purpose for adding ascorbic acid before alkaline tartrate is not for reducing the interference of hemoglobin, but for destroying excess diazo reagent, which would react with unconjugated bilirubin solubilized at alkaline pH. Variation in room temperature, from 20 to 30”C, has no effect on the direct bilirubin values obtained by this procedure.56

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Table 4 Mean Direct Bilirubin Values for CAP" Specimens C-94 and C-95b Obtained by the Most Frequently Used Clinical Analyzers in CAP Surveys Bilirubin, mg/dl


c-94

c-95

~-

Andyzer

1989

1990

1989

1990

DuPont Dimension Beckman Astra Kodak Ektachem Hitachi 700 Series Technicon RA

0.15 0.22 0.26 0.43 0.60

1.79 2.72 2.74 2.69 3.16

0.63 1.16 1.13 1.67 1.63

0.28 0.59 0.51 0.63 0.72

Olympus Demand Coulter Dams Abbott Spectrum' DuPont ACA' Technicon RA 1 m

0.61 0.53 1.04 1.08 1.13

3.46 4.04 4.12 4.51 4.08

2.26 2.34 2.88 2.62 2.15

0.85 0.91 1.40 1.45 1.51

Preferred method

0.28

1.98

0.94

0.36

loo0

a

College of American Pathologists. Specimens C-94 and C-95 were semm pools enriched with ditaurobilirubin and unconjugated bilirubin. Bichromatic mode.

Methods that measure a substantial percentage of B, as direct bilirubin could in certain clinical conditions lead to erroneous diagnoses. Gilbert's syndrome is distinguished from Dubin-Johnson or Rotor's syndrome by the absence of direct bilirubin in Gilbert's syndrome. Direct bjlirubin concentrations of 1.5 mg/dl or more in neonates warrant a diagnostic evaluation, for they are usually associated with serious clinical situations, including biliary atresia, sepsis, severe infection, or hepatitis.6sThus, falsely elevated direct bilirubin values resulting from poorly designed methods could mislead the physician and prolong hospitalization and therapy. b. LINEARITY, ACCURACY, PRECISION

The absorbance-concentrationrelationship does not adhere strictly to Beer's law, but the deviation is small. The deviation is negative for DTB and bilirubin c o n j ~ g a t e sand , ~ ~positive for The measurement of DBIL is inherently inaccurate because the reaction of the three directreacting bilirubins does not go to completion. Small inaccuracies, however, are tolerable because once the diagnosis of conjugated hyperbilirubinemia is made, knowledge of the exact concentration of DBIL does not provide the physician with useful information. The intralaboratory precison of this method is good; between-run coefficients of variation for two control sera with direct bilirubin concentrations of 2.5 and 5 . 1 mg/dl were 1.6 and 2.1%, re~pectively.~~ Different methods, however, yield widely variable results. Listed in Table 4 are mean values for direct bilirubin reported by nine clinical instruments in the Chemistry Survey of the College of American Pathologists, and also results obtained by the preferred method. Values for total bilirubin on the same specimens (Table 5 ) are shown in order to demonstrate the extent to which unconjugated bilirubin reacts as direct in various methods. It is clear that B, appears to react to a large extent as DBIL in some of the methods adapted to clinical instruments. This reactivity of B, could be due to a high pH of the


427

Table 5 Mean Total Bilirubin Values for CAP" Specimens (2-94 and C-9!jb Obtained by the Most Frequently Used Clinical Analyzers in CAP Surveys Bilirubin, mg/dl c-94


Analyzer

c-95

1989

1990

1989

1990

DuPont Dimension Beckman Astra Kodak Ektachem Hitachi 700 Series Coulter Dacos Technicon RA loo0 Olympus Demand Abbott Spectrum' DuPont ACA' Technicon RA 1OOCF

1.08 1.04 1.05 1.13 1.16 1.06 1.17 1.59 1.29 1.j61

5.86 5.86 5.33 5.72 5.48 5.57 5.97 6.28 5.60 6.56

5.09 5.05 5.05 4.99 5.11 4.93 5.51 5.74 5.17 5.35

1.64 1.66 1.31 1.78 1.52 1.53 1.68 I .97 1.67 2.24

Reference method3'

1 .OO

5.22

4.85

1.42

' College of American Pathologists. Specimens C-94 and C-95 were serum pools enriched with ditaurobilirubin and unconjugated bilirubin. Bichromatic mode.

reaction mixture, the presence of surfactants (which promote the reaction of B,) in the reagents, inaccurate calibrators, or all of the above. In at least one instrument, which is known to use the bichromatic mode without a sample blank (Technicon RA lOOO), the excessive turbidity of these specimens was partly responsible for the higher DBIL and TBIL values. The differences in the results (Tables 4 and 5) obtained with the RA 1OOO used at a single wavelength (with sample blanking) and the bichromatic mode (without sample blanking) are undoubtedly due to turbidity. Turbidity introduces an error in bichromatic analysis without sample blanking because light scattering is inversely proportional to the wavelength. The error is positive when the primary wavelength (the one at which the chromogen is measured) is shorter than the secondary, and negative when it is longer; in the case of the RA 1000 the primary wavelength is shorter than the secondary.

C. Mechanism of Interference by Hemoglobin 1. Chemical Interference a. REFERENCE METHOD FOR TOTAL BILIRUBIN

In the Jendrassik-Gr6f method for total bilirubin, hemoglobin causes a suppression of bilirubin values owing to destruction of azopigment during the coupling reaction (addition of diazo reagent) and upon addition of alkaline tartrate.= Prevention of the destruction of azopigment by reducing agents (i.e., potassium iodide, ascorbic acid) suggests an oxidative process. In both steps the oxidizing species is most likely hydrogen peroxide formed when heme is oxidized to femheme6' by the diazo reagent or alkaline tartrate. With femheme acting as a pseudoperoxidase, hydrogen peroxide could oxidize the azopigment to colorless product(s). Ferriheme or hydrogen peroxide alone does not oxidize the azopigment, but H,O, in the presence of hemoglobin oxidizes both the azopigment6' and b i l i r ~ b i n . ~ ~ . ~ ~ b. PREFERRED METHOD FOR DIRECT BILIRUBIN

The mechanism of interference by hemoglobin in the direct diazo reaction appears to be the same as described earlier.71Destruction of the azopigment, however, is more pronounced


428


at acid than at neutral pH, because at low pH conversion of hemoglobin to ferriheme is rapid and quantitative, resulting in a high yield of H,O,, while at neutral pH only a small portion of hemoglobin is oxidized to methemoglobin Interference by hemoglobin also occurs at the point where the sample is added to HCl; sugar-conjugated bilirubins (also DTB are oxidized to the corresponding biliverdins,* which are diazo-negative, and the extent of B, oxidation is proportional to the concentration of hemoglobin. The oxidation of B, is negligible. Interference by hemoglobin in the direct diazo reaction can be substantially reduced by addition of KI to the serum diluent (HCl).71KI reduces hydrogen peroxide but not the diazo reagent. However, because KI in HCl is oxidized by air to iodine, it must be added just before the sample.

2. Spectral Interference In addition to chemical interference, spectral interference from hemoglobin may cause an increase or decrease in the actual bilirubin values. Spectral interference should be absent when the diazo reaction takes place at acid pH and absorbance is measured at a single wavelength. This is because hemoglobin in both the test and sample blank solutions is converted completely to acid femheme and, therefore, exhibits the same absorbance. For the same reason, there is no spectral interference in the Jendrassik-Gr6f method because at the alkaline pH of the final solution (pH - 13) hemoglobin is converted to alkaline femheme in both test and blank. The situation becomes complicated in the so-called “bichromatic” measurements, a misnomer for the approach used in certain clinical analyzers for correcting the absorbance of the test for the background absorbance contributed by hemoglobin, bilirubin, or turbidity. This amounts to measuring the absorbance at two wavelengths (selected so that the measured chromogen exhibits maximum absorptivity at the fxst and negligible at the second) and subtracting the absorbance at one of the wavelengths from the other. The correction is based on the assumption that the absorbance of the interferant is identical at both wavelengths, something that is quite uncommon. Whether the interference by hemoglobin on the measurement of bilirubin would be positive, negative, or no interference is determined by the selection of the wavelengths, and the concentrations of bilirubin and hemoglobin. An example of spectral and chemical interference combined is shown in Table 6. The data were obtained by supplementing human serum pools with known amounts of bilirubin conjugates and hemoglobin, and then analyzing for direct bilirubin by the preferred method and by the bichromatic approach. In the single-wavelength method hemoglobin causes a negative bias in all specimens because the interference is strictly chemical. In the bichromatic method (corrected absorbance = A,, M1 - &,,,), the bias is the net difference between the spectral and the chemical interferences. The bias is positive at low bilirubin concentrations because methemoglobin absorbs more strongly at 540 nm than at 600 nm and, therefore, spectral interference predominates. At a high bilirubin concentration (11 mg/ dl) the bias is negative because the predominant interferenceis chemical, i.e., there is enough bilirubin to be destroyed. The bias is close to zero at 6 mg/dl because the two interferences, acting in opposite directions, cancel each other. D. Normal Reference Values Most reports agree that the upper normal limit for total bilirubin in healthy human subjects is from 1.0 to 1.2 mg/dl (Table 7). Total bilirubin values in serum depend on age and sex,72-74but the upper limits of reported ranges are so close that, in view of the large intraindividual variation, which is as high as the interindivid~al,~’ and the analytical error of routine methods, it would be difficult to make a clear distinction between males and females or among age groups. Reference values for serum total bilirubin in neonates are shown in Table 8, and additional information may be found in a recent p ~ b l i c a t i o n It .~~


429

Table 6 Hemoglobin Interference in Direct Bilirubin Methods Preferred Method (Single Wavelength) Bilirubin, mg/dl


(Hb,m d d ) Pool Pool Pool Pool Pool

1

2 3 4 5

(0)

(50)

(100)

(250)

0.5 4.1 6.2 8.0 11.9

0.3 3.4 5.8 7.0

0.2 2.9 5.4 6.3 9.5

0.1 2.2 4.6 5.2 7.9

0.1

1.1 4.0 5.6 1.0 10.1

1.8 4.3 6.0 7.1 9.6

10.6

(500)

1.9 3.9 4.4 6.9

Bichromatic Method Pool Pool Pool Pool

0.4 3.8 5.7 7.6 11.2

1

2 3 4 Pool 5

Note:

0.5 3.9 5.7 7.6 11.2

0.7 4.0 5.7 1.7 11.2

Aliquots of human serum were supplemented with various amounts of bilirubin glucuronides and hemoglobin. They were analyzed for direct bilirubin by the preferred method to demonstrate chemical interference by hemoglobin, and by the Du Pont ACA to present an example of combined chemical and spectral interferences.

Table 7 Normal Reference Values for Serum Total Bilirubin in Adults Bilirubin, mg/dl Subjects 139 6 99 P 603 d 816 P 6740 8 11215 P Note:

a

x

2.5 percentile

97.5 percentile

Ref.

0.7 0.5 0.4' 0.3 0.5

0.2 0.2 0.1 0.1 0. I 0. I

1.2

31

0.4

1.1

1.1 0.9 1.2 0.9

72 73

Reference 31 = reference method; References 71 and 72 = Jendrassik-Cr6f method adapted to the Sh4A 12/60 and SMAC systems (Technicon Instruments, Inc.), respectively.

Median.

should be pointed out that some of the upper limits of bilirubin concentrations shown in Table 8, although usually harmless to term and healthy babies, may be dangerous (risk of kernicterus) to premature or sick infants, especially when such high concentrations are associated with the presence of drugs that displace bilirubin from its albumin binding sites. The widely accepted upper normal limit for direct bilirubin is 0.2 mg/dl,77-79 most of it being unconjugated bilirubin reacting as direct.80Analysis of 109 serum samples from healthy adults by the preferred method gave the following results: TE, 0.12 mg/dl; SD, 0.04 mg/dl; observed range, 0.02 to 0.28 mg/dl; only two samples had direct bilirubin concentrations greater than 0.2 mg/d1.77 Normal values obtained with methods that measure substantial amounts of B, as DBIL are expected to be higher than those obtained by the preferred method.

430


Table 8 Reference Values for Serum Total Bilirubin in Neonates


Bilirubin, mg/dl

Cord &I d 1-2 d 3-5 d Thereafter

Premature

Full-term

Methods for the measurement of serum delta-bilirubin.

Azopigments from serum biliprotein and accuracy of total bilirubin measurement.

The measurement of bilirubin in liver.

Serum bilirubin fractions in healthy subjects and patients with unconjugated hyperbilirubinemia.

The analysis of bilirubin in serum.

Alteration of serum bilirubin level in schizophrenia.

albumin ratio as predictors of bilirubin encephalopathy.

Flow cytometric measurement of intracellular bilirubin in human peripheral blood mononuclear cells exposed to unconjugated bilirubin.

Estimation of unbound serum bilirubin by the peroxidase assay method: effect of exchange transfusion on unbound bilirubin and serum bindings.

The albumin binding of unconjugated bilirubin in serum.

A rapid and simple method for simultaneous measurement of serum free thyroxine and triiodothyronine fractions.

Effects of serum bilirubin on atherosclerotic processes.

Does serum bilirubin predict incident metabolic syndrome?

Transcutaneous bilirubin measurement in healthy Saudi term newborns.

Transcutaneous bilirubin measurement in healthy Saudi term newborns.

Serum bilirubin and hepatic enzyme induction.

Serum bilirubin and hepatic enzyme induction.

Serum bilirubin and hepatic enzyme induction.

Fluorescent protein-based detection of unconjugated bilirubin in newborn serum.

Amylase, hepatic enzymes and bilirubin in serum of chronic alcoholics.

The relationship between serum bilirubin and Crohn's disease.

The relationship between serum bilirubin concentration and coronary artery ectasia.

THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS.

The relationship between serum bilirubin concentration and coronary slow flow.