CLIN. CHEM.

29/1, 31-36(1983)

Assessmentof the Fundamental ccuracyof the Jendrassik-Gr#{243}fand A Total Direct Bilirubin Assays
Donald H. Lo and Tai-Wing Wu23
Using unconjugated bilirubin (Be) and authentic human diconjugated bilirubin (dB) supplemented in low-bilirubin serum pools, we have directly verified for the first time that the Jendrassik-Gr#{244}f bilirubin assay modified after total Doumas et al. (Gun. Chem. 19: 984-993, 1973) detects B and dB quantitatively, in Ba-equivalent concentrations relative to gravimetric concentration assignment reinforced with quantification by nuclear magnetic resonance (Wu et al., Gun. Chem. 26: 1323-1335, 1980). By contrast, the Jendrassik-Gr#{243}f direct bilirubin assay (also modified after Doumas) quantifies only 70 5% of the gravimetrically determined dB as B equivalents. By using B and dB (instead of B only, as is routinely done) to calibrate the responses of the total and direct bilirubin assays, the analyses of B and dB become much more quantitative. We mathematically explored the meanings of the conventional terms total, direct, and indirect bilirubins. The clinical and diagnostic implications of our observations are
discussed. AddItional Keyphrases: Ehrllch reaction and indirect reacting biirubins
.

not yet recommended a procedure of choice) the serum is incubated, in the absence of promoters, with the diazo reagent at pH 1.7-2.0, ending with ascorbic acid addition and tartrate treatment as in the TBIL assay. The assumptions implicit in these variants are that the TBIL assay is believed to measure all the bilirubin subspecies known in serum, each component equally (i.e., in chemical reactivity and molar absorptivity)
and additively. According (Be), to more recent includes literature the (6, 7) these

subspecies
gated

consist of unconjugated
which

bilirubin

(Be) and conjumonoconjugated

bilirubin

(mBa) and diconjugated (dB) bilirubins; we will discuss the fourth (delta) bilirubin fraction in serum (8) later. The
DBIL assay is presumed, however, to measure only conjugated

bilirubin (Be). Because B is extremely labile and difficult to isolate in pure form, both the total and direct diazo assays are routinely calibrated only with the commercially available B,,.5
This, plus the lack of a definitive reference method for the

measurement of total bilirubin or its subspecies, has raised questions about the fundamental accuracy of all diazo methods, including the Jendrassik-Gr#{243}fprotocols (2, 6, 9, 10).
Against conjugate that background, we recently isolated the chief of bilirubin from human bile (11). Using 270-MHz

total, direct,

#{176}H nuclear Of the different methods for determining bilirubin, those based on the diazo reaction first described by Ehrlich in 1883 (1) are the most widely used (2). In this reaction, the diazo reagent (e.g., diazotized sulfanilic acid) splits bilirubin into two azodipyrrolic fragments, which can then be quantified spectrophotometrically. There are numerous versions of the diazo method, differing in their reaction conditions and reagent compositions. Of these, the method according to Jendrassik and Gr#{243}f 4) has been recommended (3, by the National Committee for Clinical Laboratory Standards (NCCLS) as the procedure of choice for total bilirubin measurement (5). As with many other diazo-type assays, the Jendrassik-Gr#{243}f method has two variants-the total bilirubin (TBIL) and direct bilirubin (DBIL) assays.4 In the TBIL assay, caffeine and sodium benzoate are added to serum as promoters, followed by incubation and subsequent reaction of bilirubins with p-benzenediazonium sulfonate at pH -5.6. Then ascorbic acid is added, followed by alkaline tartrate, which shifts the absorption maximum of the azobilirubin from -500
to 600 nm, where absorbance varies directly with the analyte

magnetic

resonance

(NMR), we directly verified

the molecular structure of this compound to be that of diconjugated bilirubin. We also devised a protocol for quantifying it, based on the fingerprinting NMR signals of functional

groups unique to the tetrapyrrole backbone. Using this authentic diconjugate (dB) and quantitative NMR as a guide, we added to low-bilirubin serum pools known amounts of B,
dB, or both. With these samples, we began an inquiry into the

validity of some of the assumptions underlying the Jendrassik-Gr#{243}f total and direct diazo methods (12). This paper summarizes our experience with B,, and dB. Our conclusions may not apply directly to icteric patients sera, in which the other less-characterized bilirubin subfractions might be increased.

Materials and Methods

B,, and B test samples. These were prepared by dissolving known weights of unconjugated bilirubin (Sigma Chemical

Co., St. Louis, MO 63178) after standard treatment with NaOH, 0.1 mol/L (4), and (or) the bile-isolated dB (11) in
aliquots of the same human serum pool. The dB preparation we used was estimated to be 88-95% pure according to NMR (11), augmented by further analyses with thin-layer chromatography (8) and liquid chromatography (13). Typically, we prepared in serum a concentrated stock (e.g., 100-200 mg/L) of each bilirubin subspecies, then serially diluted the stock with the same basal matrix (containing 3-4 mg of B,,

concentration.

In the DBIL assay (for which the NCCLS has

1 Clinical Chemistry Technical Center, Kodak Park, and 2 Biosciences Division, Research Laboratories, Eastman Kodak Co., Rochester, NY 14650. to whom correspondence should be addressed. Nonstandard abbreviations: B,,, unconjugated bilirubin; B, conjugated bilirubin or bilirubin conjugates; dB, diconjugated bilirubin or bilirubin diconjugate(s); mBa, monoconjugated bilirubin or bilirubin monoconjugate(s); TELL, total bilirubin in the diazo methodology; DBIL, direct bilirubin in the diazo methodology; IBIL,

per liter) to generate

a concentration

series. Mixtures

of B,,

indirect bilirubin in the diazo methodology; S or S bilirubin, delta bilirubin; NMR, nuclear magnetic resonance. Received Apr. 26, 1982; accepted Aug. 27, 1982.

of B per portion of the bilirubin molecule is the tetrapyrrole backbone containing an intact central methylene group, regardless of the presence or absence (or type) of esterifying moieties linked to the propionic side chains.
#{176}

The results are conventionally

reported

in milligrams

liter. The inherent assumption is that the only diazo-positive

CLINICAL CHEMISTRY, Vol. 29, No. 1, 1983 31

I
#{163}

20

#{149} Bu
#{163}

dB

#{149} dB Bu+
00
-

-J

a, E
V +

-J

80-

a
U S

40

U U U

20
U

.
I
I U

[TBIL] mg/L

I_

.1
-

20

40

60

100

120

140

160

[Bu] mg/L

3. Gravimetric total bilirubin concentrations (expressed in vs total bilirubin measured by the diazo method ([B,,]+ [d80])nB,,.equlvalent concentrations were compared with total blikiln i
Fig.

B,, equivalents)

Fig. 1. Gravimetric

concentration

assignment

in B,, and dB test

fluids
Known weiits of B..were ed to a senimbased pool to provide six concentrations of B,,test fluids (#{149}). Similarly, known weights of authentic dB0were added to fow concentrations of dB test fluids (A). In addition, nine concentrations of B,,+ dB0 mixtures were prepared (U)

concentration ETBIL]as determinedby the ,Jendrassik-G#{244}f assay. avlTBIL metric [d80] is multiplied by the ratio of molecular masses for B,,/B0(584/936) to convert it to B,,-equivalent units. Typical linear regressionstatistics are: y = 0.995x - 1.126. Si..,,= 2.342. r = 0.999. Symbols as In Fig. 1

and dB were obtained by mixing different volume proportions of the B,,- and dB-supplemented fluids. B was weighed
either on a microbalance (Model H542; Mettler Corp.,

for total and direct bilirubin were performed in our laboratory by Dr. W. D. Fellows. Table 1 presents the reaction conditions (private communication, Dr. B. T. Doumas, 1978).

AG, Zurich, Switzerland)

26 automatic

or on an electrobalance
Ventron

electrobalance;

Instruments (Cahn Model Cahn Instru-

Results
Manual Diazo Method
In the first study, 19 B,,, dB0, or both in various gravimetrically assigned concentrations (Figure 1) were analyzed by the manual Jendrassik-Gr#{243}f TBIL assay. When the total bilirubin concentrations ([B,,] + [dB0]),6 assigned grayimetrically in mg/L, were plotted in the vertical axis against the total bilirubin concentration [TBIL] measured conventionally (i.e., against Be-only calibration standards and reported in milligrams of B per liter) in the TBIL assay (Figure
Total bilirubin measurement.

ments Div., Cerritos, CA 90701). For best results with the hygroscopic and labile dB, however, we weighed the freshly lyophilized bile-isolate in a strongly desiccated and dim environment, e.g., with an electrobalance placed inside a dry box and in yellow light. All pipetting was done rapidly with Pyrex glass volumetric pipettes. Each concentration of sample to be tested was prepared in several aliquots of identical volumes separately sealed in brown glass vials under nitrogen and stored at -60 #{176}C.vial was reused after thawing. No The gravimetric preparation of dB-containing fluids was based on the weight percent of the bilirubin diglucuronide predetermined in the solid isolate by quantitative NMR (11). The resulting dB concentrations were expressed in milligrams per liter of serum. Manual diazo methods. The Jendrassik-GrSf procedures

serum-based

fluids containing

2), two trends

became

obvious.

For Ba-only

fluids,

the two

concentrations

agreed closely, but for dB fluids or mixtures

concentration of

6The convention of using brackets to symbolize is used here.

-j -J

a,

a
E
0

B
V

[TBIL]

mg/L

00

[TOIL] mg/L

Fig. 2. Gravimetric total bilirubin concentration vs total bilirubin measured by the diazo method
Valuesof ([B,,] + [dB]) derived from Fig. 1 were compared with total bilirubin concentration ITBLI as determined by the Jen&assilr-&#{243}f assay. Symbols TBIL as in Fig. 1

Fig. 4. [DBIL] concentration compared with the [TBIL] concentration as determined by the Jendrassik-G-#{244}f for the assays 19 B and dB test fluids
Symbols as in Fig. 1.B,,in test fluids is largely taidetected in the Jen&assik-Gof D6t assay, as evidenced by the typical regression statistics: [DeL] = 0.005 [TelL] + 2.185, S = 0.293, r = 0.670. The DBIL assay detects 66% (2.5%) of B in test fluids, as compared with the TBIL assay results; typical regression statistics are: [DBIL] = 0.66 1 . ETBILI - 3.005, S,..,,= 1.459, r = 0.996

32 CLINICALCHEMISTRY, Vol. 29, No. 1,

1983

Table 1. Reaction Conditions for Total and Direct Bilirubin Assays

Reagent 1. Caffeine/benzoate reagent Anhydrous sodium acetate, 56 gIL Sodium benzoate, 56 gIL Disodium EDTA, 1 gIL
Total Direct
-

assay. Again, these conclusions

2.0 mL

Caffeine, 37 gIL
2. HCI, 50 mmolIL 3. Sample (calibrator, a unknown, or waterb)
-

control,

0.2 mL 0.5 mL

1.0 mL 0.2 mL 0.5 mL

4. Diazotized sulfanilic acid c Sulfanilic acid, 4.88 g/L Sodium nitrite, 0.12 g/L

Mix and incubate at 25#{176}C 10 mm for

5.

Ascorbic acid, 40 g/L

0.1 mL 1.5 mL

0.1 mL 1.5 mL

were supported by subsequent studies where [DBIL]/[TBIL] was 65-75%. Thus, using authentic B,, and dB0, gravimetrically referenced, we have verified the accuracy of the Jendrassik-Gr#{243}f TBIL method modified after Doumas et al. (12). In contrast, the DBIL method, which detects less than 10% of B,., as B, consistently underestimates the dB. Although the reason for the incomplete recovery is not known, one could speculate that the reaction conditions (e.g., pH, incubation time) might not be optimal for the detection of B in the DBIL assay. Furthermore, one may infer from our findings that the TBIL assay might indeed be standardized with B,, calibrators alone, as is conventionally done, but the DBIL assay, routinely calibrated with B,,, should be more rigorously standardized with authentic B or with both B,, and dB (see below). In the last option, the method should theoretically become more accurate by calibrating out the incomplete response of the DBIL reaction (as compared with the TBIL reaction). In the following section, this inference will be examined empirically.

6. Alkaline tartrate Sodium hydroxide, 75 gIL Sodium tartrate dihydrate, 263 gIL 7. Nd, 50 mmol/L 8. Caffeine/benzoate

Effect of Calibrating the TBIL and DBIL Diazo Tests with Both B and dBc Calibrators
Calibrating the TBIL and DBIL diazo assays with grayimetrically prepared B,,- and dB0-fluids simultaneously should permit an accurate quantification of [B,,], [dB], and hence [B.r] (the sum of [B,,] + [dB]) relative to the gravimetric reference. Principle of the experimental approach. According to Beers law, for a single analyte in solution:
AA intensity of the log(Io/IA)
=

1.0 mL
-

2.0 mL

Zero spectrophotometer with reagent blank,b measure absorbance at 600 nm, ca!ibrate,d and predict concentratlone in unknown sample
a Six B,, calibrators:

5, 10, 20, 50, 100. and 200mg of B. per liter of bovine

serum albumin (Cohn Fraction V). 40 g/L. b For reagent blank. #{176}Prepared freshly by mIxing 20 ml of a stock solution of sulfanilic acid (5.0 g/L) with 0.5 ml of the nitrite stock (5.0 gIL); for sample blank, use 0.5 mL of sulfanilic acid (5 g/L) in place of diazotized sulfanilic acid. d Computecorrected absorbance [A(corr)] by subtracting absorbance in each sample blank tube. Plot A(corr) vs assigned concentrations of B. In mg/I for the six calibrators. Compute slope and intercept of calibration line by least-squares regression analysis. 0 Given an unknown sample with measured A(corr), compute total bilirubin concentration as [TelL] = [A(corr) - b]/a and report as milligrams of B,, per liter.

ai[A] + 71

where AA is absorbance

due to solute or analyte A, jo is the beam, A is the intensity of the transmitted beam, a1 is the molar absorptivity (e) times the pathlength (L), [A] is the molar concentration of analyte A, and y is the background density. For two noninteracting and distinct analytes A and B in the same solution, we can expand Beers law to the relationship described in equation 1
incident (1)

of the two components, there were linear, systematic departures from the ideal slope of 1. Such deviations were abolished, however, if the gravimetric total bilirubin concentration was replotted in Be-equivalent concentrations5 [i.e., using the molecular mass of 584 for B,, and 936 for dB to convert the gravimetric concentration of [dB] to [dB] X 584/936 vs [TBIL] (Figure 3)]. Similar results had been obtained over a three-year period from different batches of serum-based fluids supplemented with B from the National Bureau of Standards (SRM No. 916) or Kodak Laboratory Chemicals, Rochester, NY 14650, and with separate preparations of dB. These results collectively demonstrate that the Jendrassik-Gr#{243}f BIL assay can T and does measure [TBILJ as the sum of B,, and dB when both are expressed in B-equiva1ent concentrations. Direct bilirubin measurement. To assess the performance of the DBIL method relative to its TBIL counterpart, we subjected the set of fluids described above to both assays simultaneously and plotted [DBIL] against [TBIL] (concentrations reported as B,,, mg/L) (Figure 4). Ideally, if the direct assay detects dB0 specifically and quantitatively, there should be a one-to-one correspondence in the [DBIL] and [TBIL] results for dB-fluids and no detection (or low [DBIL] result) of B,-fiuids in the DBIL assay. The actual results strongly indicate that although the DBIL assay indeed detects a small fraction (2-6%) of B,,, as evidenced by the near-zero slope for the B,, series, it measures only about 66% of the dB, as compared with the [TBIL] measured by the TBIL assay. Again,

is the total absorbance given by analytes A and B (or by their respective stoichiometrically converted reaction products) in the same solution mixture, [A] and [B] refer to the concentrations of A and B, respectively, a1 and /3 refer to the molar absorptivities of A and B each multiplied by the pathlength L (ifL = 1cm, then a1 = forAand /3 = s for B), and 7i is the background as defined above. If we substitute [Be] and [dB], respectively, for [A] and [B]

where A1

in equation 1 and denote (by the subscript 1 throughout) that this equation is for the DBIL assay, we obtain equation 2: A1(DBIL)
Similarly,
=

aj[B,,] + /31[dB] + 7i
reaction:

(2)

we can write for the TBIL

A2(TBIL)

a2[B,,] + /32[dB1 + 72

(3)

where the subscript 2 refers all parameters to the TBIL assay and the other symbols bear their usual significance as detailed above. By combining equations 2 and 3, we can solve for [Be] and [dB] as follows. [dB] [B,,] where a
b c
= = =

a[a2
-a[/32

A1(DBIL) Ai(DBIL)

a1 A2(TBIL)
-

b]
-

(4)
(5)

fit . A2(TBIL)

c]

1/(a2/31
(71a2
-

a1$2) 72a1)

= (71/32

72/31)

CLINICALCHEMISTRY, Vol. 29, No. 1, 1983

33

and

[B,,] + [dB]

= [BT]

a[(a2

/32)

(fir

aj)

A1(DBIL) A2(TBIL) + (c
.

b)]

(6)
0

If a set of B,., and dB calibrators is available (each calibrator with its gravimetric concentrations assigned to [B,,] and [dB) in mg/L, respectively) and A1(DBIL) is the measured absorbance for each fluid in the DBIL assay condition, the calibration parameters (aj, fir, and 7) can be determined by a least-squares regression analysis. With the same set of B,, and dB calibrators improvised above and A2(TBIL) relating to the absorbance measured in the TBIL assay, a different set of three calibration parameters (a2, /32, and 72) can be determined, describing the second discrete assay condition for the TBIL method. Experimental results. To assess how well this principle could be a,plied to practice, we subjected the 19 (B,, and dB) calibrators studied earlier (Figure 1) to this treatment. First, the calibration parameters in equations 2 and 3 were established. Second, [dB] and [B,,] can be predicted from the dualassay measured responses of A1(DBIL) and A2(TBIL) in equations 4 and 5, respectively, and [Br] can be calculated as the sum of [B,.,] and [dB] in equation 6. The results of this analysis are shown in Figures 5-7, illustrating the good quantitative agreement between the predicted and the grayimetrically assigned concentrations of these fluids. The chemical principle describing the dual-analyte detection system is expressed mathematically by equation 1. This generic equation had been discussed by Hahn et al. (14). Moreover, the concept of using two discrete reaction kinetic profiles (e.g., reaction rates) to determine unconjugated and conjugated bilirubin simultaneously had been described by Landis and Pardue (15). However, our study presents two unique characteristics: (a) authentic and gravimetrically determined dB ar incorporated, and (b) B,, and dB are determined simultaneously from two independent and discrete assay conditions, corresponding to the TBIL and DBIL assays.

100

-J

[Bu] mg/L expected

Fig. 5. Predicted [B,,] compared with expected (i.e., gravimetrically assigned) [B,,] In the 19 B and dB test fluids described
in Fig. 1
Typical

regression statistics: y

O.999x

0.380, S,.,, =

2.3 16, r

0.999

0 xj

100

-J

E
50

[dB]

mg/L expected

What Do Total

and Direct

Bilirubin

Mean? Fig. 6. Predicted [dBc] compared with expected (i.e., gravimetrically assigned) [dBc]
Representative regression statistics: y
0.996
=

Thus far, we have delineated the quantitative performance of the two diazo variants (total vs direct) when these are calibrated with B,, only (conventional practice) or when they are calibrated with both B,., and authentic dB samples (twocalibrator approach). The obvious question that emerges is:

0.871x + 3.010, Sr,,

2.835, r

How do the assay results of [TBIL] and [DBIL] as conventionally determined relate to [B1] and [dB] as defmed by the
two-calibrator approach? We shall attempt to answer this

question when

mathematically.

approach. In the conventional practice, B,., serves as the only calibrator in the diazo tests, equation 2 is reduced to equation 7: Mathematical

A1(DBIL) and equation 3 is reduced A2(TBIL)

a2[DBIL]

+72

(7)

to equation
=

8:
+
72

a2[TBIL)

(8)

With simple mathematical transforms of these equations, the two concentrations, [TBIL] and [DBIL], can be predicted from the resulting equations 9 and 10, respectively: [TBIL] [DBIL]
=

[A2(TBIL)

y2]/a2

(9)
(10)

([Bj+

[dBJ)

mg/L

expected

[A1(DBIL) bilirubin

Also, the term indirect lated as:

34

[IBIL] might be calcu-

Fig. 7. Total bilirubin concentration prediction compared with the expected (i.e., gravimetrically assigned) concentration
Typical regression statistIcs: y
=

0.979x

0.335, Sr,,

5.002,

0.996

CLINICAL CHEMISTRY, Vol. 29, No. 1, 1983

[IBIL]

[TBIL]

[DBILJ

(11)

Our experience with the TBIL and DBIL assays is summarized as follows: [TBIL] provides a quantitative measure of B,., and dB in B,, equivalents, and [DBILj provides a systematic measure of dB, which is 0.65x.B,,-equivalent, where x <1. To assess the magnitude of B,., in serum, one computes the parameter [IBIL] = [TBIL] [DBIL],which is now equal to (1 X [B,,]) + ([1 x].[dB]). In other words, the intuitive assertion that [IBIL] provides a 100% indicator of the presence and magnitude of [B,,] alone is analytically inaccurate, since this measurement is contaminated by a contribution of (1 x).[dB] with whatever amount of dB is present in the clinical sample. When laboratory results are interpreted, the clinical significance of indirect bilirubin (as currently reported by the laboratory) might have to be modified. Three assay results, [TBIL], [DBIL], and [IBIL], are reported from the dual assays of TBIL and DBIL in equations 9, 10, and 11, respectively, with use of B,, calibrators alone. If B,., and dB calibrators are improvised for calibrating the dual assays, [Br], [dB] and [B,,] can be predicted from equations 6, 4, and 5, respectively. The corresponding relationships between [TBIL] and [Br], [DBIL] and [dB], and [IBIL] and [B,,] can be mathematically derived, and are described in equations 14, 12, and 13, respectively:
,

in question gave a DBIL/TBIL ratio of 0.75-0.85. Until authentic, purified standards of mB and b bilirubin have been tested, we caution against drawing further conclusions about the overall validity of the Jendrassik-Gr#{243}f ssays for quana tifying truly total serum bilirubin (Bq = B,, + mB + dB+ #{244} bilirubin. . .) or its direct-reacting subfractions (B = mB + dB + bilirubin). Partly because of these intrinsic uncertainties, our probing of the meanings of total and direct bilirubins in the last section of this paper must be viewed merely as a lead to further inquiries as more facts unfold. Our preliminary mathematical deductions also suggest that the term indirect bilirubin, often computed as ETBIL] [DBIL], is not a simple function of B,,, and hence has unknown clinical diagnostic significance. Nevertheless, this issue should be reexamined by concrete assays
-

experimental

testing.

[dB] [B,.,] [BT]

=

a[a-

[DBIL]
-

(a1 A2)
.

b + (cx2

72)]
-

(12)
71)]

a[a2 - f31[IBIL] a[a2(13j

[DBIL]a2(/32
+ a2(a2
-

f3)

/32(72

(13)
=
-

ai)[TBIL]
+ 72(a2

/32 +

/3

/32)[DBIL] ai) + c

b]

(14)

Simple inspection of equations 12-14 shows that [DBIL], [TBIL], and hence [IBIL] in the conventional approach may not be linearly related to [dB], [B1], and [B,,], respectively. This mathematical inference might be particularly true for [B,.,] vs [IBIL]: equation 13 shows that [B,,] is not a simple linear function of [IBIL].

Discussion
We have demonstrated that the Jendrassik-Gr#{244}f ethod m for total bilirubin (12) measures B,, and dB quantitatively when these analytes are expressed in B,,-equivalent concentrations. The corresponding method for direct bilirubin, however, is flawed in two respects. First, it underestimates the dB relative to the TBIL assay. Second, it detects a variable fraction of B as dB (2-6%). Thus, from a strictly analytical standpoint, the current TBIL assay might be adequately calibrated with B,., alone, but the DBIL assay should be calibrated with dB or in conjunction with B,,. The benefits of the latter approach are illustrated by the quantitative recoveries from artificial mixtures of these two bilirubin subspecies. However, from the data presented, we cannot infer how quantitative both variants of the diazo method are in measuring two other bilirubin subspecies that are beginning to receive attention, namely, the monoconjugated bilirubin (mBa) (6, 7, 13) and the direct diazo-reacting, strongly protein-linked (delta) bilirubin (8). Indeed, we have recently shown (8) that the 5 biirubin partly purified from adult serum gives, in the diazo variants used in this study, a DBIL result that is typically 80-90% of the TBIL value. Should this relation hold with more highly purified preparations of #{244} bilirubin in the future, one would strongly suspect that the different bilirubin derivatives in serum have different direct diazo reactivities. This may also help explain why for some highly icteric sera shown by liquid chromatography (13) to contain 80% of the total bilirubin as #{246} bilirubin, the Jendrassik-Gr#{243}f

Given the current uncertainties as enunciated above, especially in regard to the DBIL assay, one may legitimately question how valid and useful is the direct bilirubin value in the differential diagnosis of jaundice. Although we cannot definitely answer this, for the reasons stated earlier, we point out that the quantity direct biirubin is still highly regarded, primarily because of the favorable clinical correlations demonstrated between above-normal [DBIL] values in serum and many hepatobiliary conditions (4, 16-18). Also, in some clinical environments, a particular DBIL assay may be used to monitor the jaundiced patients disease state, in which case the direct value is assumed to be a semiquantitative reflection of the total B fraction. As we learn more about the various bilirubin conjugates and how to measure them, we may optimize existing diazo methods or develop more quantitative measurements by other means. These efforts may lead to much more incisive, specific, and sensitive diagnosis of jaundice than has been available so far. Finally, we wish to re-emphasize that the quantitative conclusions in this paper apply to B,, and dB supplemented in low-bilirubin serum pools and only to one variant of the Jendrassik-Gr#{243}f ethods (12). As noted by other informed critics m (2, 6, 9, 16), different direct diazo methods have been noted to give widely disparate [DBIL] predictions, not only from icteric serum, but also from fluids containing only or mostly unconjugated bilirubin (6, 9). Our own experience is in accord with these observations.

We acknowledge the excellent technical assistance of Ms. S. S. Sullivan. We also thank Drs. W. D. Fellows and R. N. Rand for helpful
discussion and diazo bilirubin analyses. Finally, we acknowledge the critical reading of this manuscript by Drs. J. Mauck and J. Novros.

References
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CLINICALCHEMISTRY, Vol. 29, No. 1, 1983 35

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