J. clin. Path. (1961), 14, 271.

A study of six representative methods ofplasma bilirubin analysis

DEREK WATSON AND JANICE A. ROGERS

From the Biochemistry Department, The Royal Women's Hospital, Melbourne, Australia

SYNOPSIS The large number of modern methods which have been published for the determination,of total bile pigments in the blood plasma indicates the difficulties of the estimation and suggeststhe need for a reliable standard method. In view of the present importance of plasma bilirubinlevels in the diagnosis and treatment of jaundiced subjects, six analytical procedures have beenstudied, each chosen as far as possible, as a representative of a group of methods each embodyingdifferent principles.A spectrophotometric method (White, Haidar, and Reinhold, 1958) and five diazo coupling

procedures (Jendrassik and Grof, 1938; Powell, 1944; Perryman, Richards, and Holbrook, 1957;Stoner and Weisberg, 1957; Lathe and Ruthven, 1958) were investigated, and the results obtainedby carrying out duplicate analyses by different methods on 54 stored and 64 fresh plasmas or seraare presented and discussed in this paper.An amount of haem pigment which can be detected visually in plasma is sufficient to cause a

considerable underestimation of bilirubin by the method of Powell (1944) and overestimation bythe method of Stoner and Weisberg (1957). Inferences drawn from serial estimations by thesemethods can therefore be misleading, and this is especially pertinent when results obtained frominfant blood containing variable amounts of haem pigments are being assessed. Methods basedon the original van den Bergh procedure with various preliminary steps of protein precipitation inalcoholic solutions at about pH4 give low and poorly reproducible results; losses due to the co-precipitation of bilirubin esters with protein have been confirmed. In our opinion such methodsshould be abandoned.

Determination of total bilirubin by colour (White et al., 1958) is reliable only for plasma knownto contain no lipochrome or 'directly-reacting' pigment; total bile pigment may be overestimatedby this technique in plasma containing conjugated bilirubin.We consider that the method of Lathe and Ruthven (1958) is to be preferred for use on a routine

basis. It is conveniently simple, and under all conditions normally encountered gives results suf-ficiently accurate and reproducible for clinical use. When this or any other procedure is used, aprimary standardization between different laboratories with sera containing known amounts ofadded bilirubin is essential if comparable results are to be obtained.

There is no reliable correlation between the con- disease, the cellular diffusion of the protein-boundcentration of bile pigments in the plasma and the pigment appears to be hindered, and a plasma levelintensity of clinical jaundice (With, 1954; Culley, of 5 to 9 mg. bilirubin per 100 ml. is often requiredWaterhouse, and Wood, 1960). Both the free and to produce jaundice.the conjugated forms of bilirubin are bound to Serum or plasma bilirubin assays are thereforeplasma albumin which, because it pervades cutaneous used to assess the progress of jaundiced subjects,and other tissues, can cause clinical jaundice. This and also to detect small increases of pigment suchoccurs in adults (with obstructive jaundice) at levels as may occur in the plasma of cases of early hepatitis,of between 2 and 4 mg. bile pigment per 100 ml. in carriers of homologous serum hepatitis, in someplasma. In infants with or without haemolytic macrocytic anaemias, and in patients receiving treat-Received for publication 12 October 1960 ment with various drugs. A knowledge of bilirubin

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levels is important at the present time for directingthe treatment of jaundiced newborn babies, and,partly for this reason, many new micro methods fordetermining bilirubin levels have recently beendevised.With (1954) reviewed the methods available up

to the end of 1951, but since that time no less than23 new or adaptive procedures have been reported.Although a few of these publications include amention of a small number of analytical resultsobtained by the proposed and the parent method,usually that of Malloy and Evelyn (1937) or Kingand Coxon (1950), no substantial comparativestudies appear to have been made.The work now being reported was undertaken to

find out whether appreciable or significant differ-ences in the results of bilirubin analyses existbetween different methods, and also to assess theextent and to discover the cause of such variationswhen present.

THE METHODS

The methods employed were as follows.I The method of Powell (1944) for plasma containing

up to about 5 mg. bilirubin % was used as published. Inthis method, the conventional diazo reagent is added tothe plasma, followed by an excess of sodium benzoate-urea solution. A plasma blank test is carried out in whichdilute HCI replaces the diazo reagent. Colour densitieswere read in an EEL colorimeter with Ilford filter 625.Analyses on the more deeply pigmented plasma weremade with 0-5 ml. of 1 in 5 saline dilutions of plasma.This method was chosen as a representative of the classemploying a non-alcoholic coupling promoter. It was themethod routinely used in this hospital at the beginningof our study.

2 The method of Jendrassik and Gr6f (1938) wasused as published; this is also a non-alcoholic couplingprocedure. In it, a solution containing sodium acetate,caffeine, and sodium benzoate is added to plasma fol-lowed by a diazo reagent prepared by mixing 0-25 ml.nitrite solution with 10 ml. of 0 5% sulphanilic acid in015 N-HCI. Upon the addition of excess alkali, thepink azo pigments are converted to blue salts. A plasmablank was carried out. The reagent blank was zero. Theextinction of the final green solution was read in an EELcolorimeter using an Ilford 607 filter. This was the onlydeparture made from the original method of Jendrassikand Gr6f who employed a Pulfrich photometer withfilter S61. The method has been re-published in Englishby Fog (1958a).

3 The method of King and Coxon (1950) modifiedin a manner suggested by Perryman et al. (1957) wasused. Ammonium sulphamate (0-15%) is incorporated inthe diazo reagent and a few crystals of sodium azide areadded after the addition of ethanol (85% v/v). Readingsare made at 530 m,u and 425 m,u, and the azo pigmentextinction is then corrected for interference by haempigments by means of a simple formula.

As no photomultiplier was available, we used 0-2 ml.plasma for every test, giving a final plasma dilution ofI in 50 compared with 1 in 100 stipulated by Perrymanet al., and 1 in 10 as used in the original procedure byKing and Coxon. The initial plasma dilution was madewith water. Optical densities were read on a UnicamSP 600 photoelectric spectrophotometer. This methodwas chosen as a representative of the class of methodsinvolving deproteinization before diazo coupling.4 Rice (1957) has suggested that the method of Stoner

and Weisberg (1957) for plasma bilirubin estimation may'fulfil reasonably well the criteria of an ideal analyticalprocedure'. This technique also involves deproteinization,but uses the measurement of acid blue azo pigments as anend-point device. It was on this latter account chosen forstudy. Regrettably, it was not found possible to employthe technique as published, because we were unable toobtain a cuvette of 1 cm. stratum length suitable formeasuring optical densities with only 17 ml. solution.The method had therefore to be scaled up. Care wastaken to keep the concentration of the reagents as nearlyas possible identical to those of the original procedure.We used 0-25 ml. diazo reagent and 0-1 ml. plasma for alltests. Coupling was allowed to proceed in a constrictedcalibrated centrifuge tube for 10 min., after which 0 3 ml.10 N-HCI and 0 5 ml. aqueous ammonium sulphate(saturated at the time of use) were added. The volume ofsolution was made up to 5-0 ml. with ethyl alcohol(absolute), the mixture shaken vigorously for 1 min.,and then allowed to lie almost horizontally for 15 min.After filtration through Whatman No. 40 paper theoptical density of the supernatant fluid was measuredat 580 myA against a reagent blank. No serum or plasmablank is used with this test.

5 The method of Lathe and Ruthven (1958) wasused as published. The diazo reagent consists of a mixtureof 0 3 ml. of sodium nitrite (0-5 %) and 10 ml. of I %sulphanilic acid in 0-2 N-HCI. Coupling takes place in50% methanolic solution. A plasma blank is performedin which 1% sulphanilic acid in 0-2 N-HCI replaces thediazo reagent. The azo pigment colour usually reaches itsmaximum intensity about 5 min. after the alcohol hasbeen added, but with some grossly pigmented specimensup to 9 min. are required. Tests were routinely read at10 min. in an EEL colorimeter with an Ilford 624 filter(525 m,u). The method was chosen as a representativeof the class of non-precipitation methods employing analcohol as the coupling promoter.

6 Direct spectrophotometric assays were made induplicate on fresh samples of heparinized plasma by themethod of White et al. (1958). We used, for all tests, I in50 dilutions of plasma in M/15 phosphate buffer (174 g.KH2 PO4 and 14-4 g. Na2 HPO4 7H.0 per litre), pH 7-4.Extinctions were read at 455 and 575 mju in a UnicamSP 600 with 1 cm. matched cells.

STANDARDIZATION OF METHODS The original method ofPowell is calibrated from one bilirubin solution (4 mg. %),but, as usually employed, it is standardized from adensity/concentration curve obtained by testing serialdilutions of a chloroform-alcohol solution of bilirubin inplace of serum (Varley, 1958). We adopted the former

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procedure for specimens containing less than about4 mg. % bilirubin, and the latter for all other tests. Ineither case, alcohol replaces benzoate-urea as a couplingpromoter. The method of Lathe and Ruthven is alsostandardized from one solution prepared by dissolvinga known amount of bilirubin in chloroform. This is thendiluted with alcohol and treated in a similar manner tothat of the test sample. The method of Perryman et al.employs a buffered methyl red solution as a standard foreveryday use. The method of Lathe and Ruthven in-cludes a check on colorimeter responsiveness with theaid of a solution having a constant optical density, i.e.,Thompson's neutral grey solution. With the exceptionof the Stoner and Weisberg and the Jendrassik andGr6f results, our plasma bilirubin results were calcu-lated by reference to the standards advised by the authorsof the respective methods. All results were also calculatedby reference to standard graphs obtained by the pro-cedures now to be described.One of us donated blood from which 200 ml. of sterile

clear yellow serum was obtained. This was used as amedium for stabilizing bilirubin in solution. Our earlierattempts to use discarded transfusion plasma for thispurpose were unsuccessful because sodium citrateinterfered with some methods of analysis, and a loss ofbilirubin was experienced following its addition toglucose-citrated plasma which had been stored for threeweeks at 4°C.The fresh serum was exposed to ultra-violet light for

three periods of two hours on each of three successivedays, at the end of which the bilirubin concentration hadfallen from its original level of 0-7 mg. to one of less than0-2 mg. per 100 ml. Forty milligrams bilirubin wererapidly dissolved by stirring in 2-0 ml. 0-100 N-NaOH,immediately diluted with about 80 ml. serum, mixed with2-0 ml. 0-100 N-HCI, and finally more serum added tomake a volume of 100 ml. Serial dilutions from this stocksolution were made with the original serum diluted with4 ml. 0-1 M-NaCl per 100 ml. The standard sera thusprepared contained 2, 5, 10, 15, 20, 25, 30, and 40 mg.bilirubin per 100 ml. Using 0-1 ml. of these standards,the methods of Powell and Jendrassik and of Gr6f wereapplied. The range between 6 and 35 mg./100 ml. waslater used for the calculation of the patient's plasmabilirubin. With the method of Lathe and Ruthven, 0-1 ml.was used for the range 4 to 16 mg./100 ml., and 0-05 ml.for levels of 15 to 35 mg./100 ml. Of the same standardsera 1 in 50 dilutions were employed to standardize themethods of Stoner and Weisberg, Perryman et al., andWhite et al. for the complete range of bilirubin concen-trations encountered. Before use, the wavelength scale ofthe Unicam SP 600 was calibrated against a didymiumfilter according to the makers' instructions. The wave-length scale was considered to be true to ±0 5 m/L overthe range 420 to 620 mp.A further series of standard sera containing 0-5, 1-0,

1-5, 2-0, 3-0, 5 0, 6-0, and 8-0 mg. bilirubin/100 ml. weresimilarly prepared from a stock serum containing 10 mg.bilirubin/100 ml., and the analyses carried out on thesespecimens using 0-4 ml. (by the methods of Jendrassikand Gr6f and Lathe and Ruthven) and 0-5 ml. (by themethod of Powell). Blank tests were made on the same

volume of the corresponding standard bilirubin serum.All the standardization procedures were completed

within a period of three days. The standard solutions(and subsequently the test plasmas) were analysed atvarious laboratory temperatures between 17 5 and19 5°C. Eastman-Kodak bilirubin (batch No. 23A) witha millimolar extinction coefficient of 59-3 in aldehyde-and phosgene-free chloroform was used throughout.

THE SPECIMENS FOR ANALYSIS Heparinized blood plasma(10 to 20 i.u. of dry lithium heparin per ml.) was obtainedfrom each of two normal healthy women, nine adults withvarious liver diseases, and 30 jaundiced infants aged 1 to7 days. Serum was obtained from the cord blood of eachof 13 infants. The specimens were stored at -10°C. inthe dark until the day before the start of the analyses,when they were transferred to a refrigerator (5°C.).Twenty-four specimens containing 0-2 to 4 mg. % bili-rubin were allowed to warm to room temperature, andthen analysed by each of the diazo methods within aperiod of four days. When not in use, the specimens werestored at 5°C. in the dark. On the last of the four days,some analyses were repeated to check any loss of bilirubinwhich might have occurred. Sixteen specimens containingfrom 4 to 15 mg. % and 15 specimens containing from15 to 35 mg. % bilirubin were then analysed undersimilar programmes.A further series of analyses for pigments was carried

out on 64 fresh specimens of heparinized plasma. Testswere made by a chosen diazo-coupling technique and bythe spectrophotometric method of White et al. (1958);'directly-reacting' bilirubin was also estimated by themethod of Lathe and Ruthven (1958). Forty-four speci-mens were obtained from 15 premature babies, eight ofwhom were jaundiced, and from four infants witherythroblastosis neonatorum, one of whom developedthe inspissated bile syndrome. In addition there weresix specimens derived from the umbilical cord vein atparturition, and 14 specimens from adult female patients,including cases of toxic hepatocellular disease or obstruc-tion of the common bile duct.

RESULTS

We were unable to obtain reproducible results bythe method of Perryman et al. Varying amounts ofunconjugated and conjugated bilirubin were demon-strated by extraction and subsequent diazo couplingto be retained on the precipitate, and the extent ofretention was influenced by the amount of shakinggiven to the mixtures. This variable loss of pigmentwas also observed when the analyses were carriedout as soon as possible after the collection of bloodsamples from infants with haemolytic jaundice. Anexamination of the individual results obtained inthe duplicate analyses by the other four methodsrevealed no obvious difference in the reproducibilityof the methods. With two exceptions these individualfigures were within 3 % of the mean value quoted.The final coloured solutions from the following

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specimens were opalescent:-Nos. 1, 41, 54 (method The mean results by the methods of Perryman etof Lathe and Ruthven); Nos. 1,2,41, 53, 54 (method al., Powell, and Lathe and Ruthven when calculatedof Powell); Nos. 1, 2, 41, 54 (method of Jendrassik from standardizations against bilirubin-containingand Grof). sera are at variance with those calculated as recom-

TABLE IPLASMA BILIRUBIN LEVELS (MG./100 ML.) OBTAINED BY DIFFERENT METHODS

Perrvman et al. (1957) Stoner and Powell (1944)Weisberg (1957)

(a)l (b)2 (b) (a) (b)

Jendrassik and Lathe and Ruthven (1958)Grof (1938)(b) (a) (b)

13 0-123 0033D 3-44D 1.9SD 1.96 137D 2-98 099D 1 6IODH 1 3IIH 1.3123H 1-41 3 1-214D 2 81 5 1.716H 0 817DH 2-318H 0-819 1-320D 3 121 2222D 2 5233 0 4Mean of values 1 824D 3 525DH 8 126 6327 8928 6929DG 2-230H 8-631H 10-832H 10233H 12 834H 11 635H 12-436 10537 11-638D 13-339 14-740H 14 84IDL 17-242H 14-643 14044 15-945 14-546 18747H 15748D 14049 21-950 29051 27-252 29 153DL 13 054DHL 12-2Mean of values 13-7Mean of all values 9-2

0 10-23 015151.02-40-81 2I101 01i21.02-31*307191 21.02 7182-104153 16-35.38 26-1207-810-09.412-010-711-19 610812 614014-116-613-813 215 013-918-014-913 321-329-427-229-712-311-413 08-6

040 54 12-64-21-63.92-22-0242-4

1.52-22-6153 62-12-04-42-54-2122-74 81057-61048 25-311-713-012-413 913 615-112-112-717 613-416-724-118015-518 517-223-017-615-624-532-332-637016615 216 311 2

0-4 0-30-4 034.4 4.43-1 2-94-0 3-919 183-8 3.71-8 1-625 2320 1 823 2-122 201-7 1-522 2021 191-4 1-23-2 3012 1.12-1 1-936 3-525 2-436 3-503 032-6 244-4 4.57-9 857-0 7-49-2 9.97-1 763-3 3.39-6 10-310-2 10-910-9 11-812-1 13-212-0 13-110-8 11-610-9 11-812-4 13-412-9 14-2126 138142 15-716-6 18-814-3 16-314-8 16-415-6 17-415-5 17-320-4 24-013-7 15 013-9 15-321-2 25-2264 32725-8 31*728-0 35-412-8 14-111-6 11-413-5 15-29-4 104

0-20 33.92 73.71*93-61 52 12-02-01 91.51.91 91-43-1131.93-42-43.5

244 28-76-09-27.33-39-710-911 513512413-711-512-915-113-616819-216-315-216-816-823-516 215-224-031*030-835-014-212-515-110-3

'(a) Standardized as recommended by author(s) of method, and 2(b) standardized as described in text.

3Not included in the calculation of mean values.D = containing visually detectable amount of 'direct' pigment.H = containing > 0-2% haemoglobin.L = grossly lipaemic specimen.G = green specimen.

Sample

0-3034-73-04-1213-91-724

- 212-3

- 221-927

- 2-3_ 1-7

3-51-4233-9

- 2-74-1

284.4 4.985 9.563 7-188 9-968 763.5 4-09-1 1028-8 9-910-2 11-511-6 13-012-8 14-212-3 13-710-3 11-511 9 13-212-6 14-313-7 15-315-9 17-818-2 20-214-4 16-114-2 15 915-7 17-615.1 16-921*0 22-714-7 15-813-6 14-821-4 22-628-6 30-8274 29-731*9 34-2133 14-49.7 12-6138 153- 10 5

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TABLE IIEFFECT OF HAEM PIGMENTS AND BILIRUBIN ESTERS IN PLASMA ON SOME TOTAL BILIRUBIN ESTIMATIONS

No ofSpecimens

Mean Y. Deviation from Approximate True Values'

Perryman et al. Stoner and(1957) Weisberg (1957)

Powell (1944) Jendrassik and Grof(1938)

Unhaemolysed plasma'Direct' pigment 14 -34No 'direct' pigment 20 -25Total 34 -28Haemolysed plasma'Direct' pigment 4 -38No 'direct' pigment 12 -22Total 16 -26

'Values obtained by the method of Lathe and Ruthven (1958).

mended by the respective authors. Considering theresults based on our mode of standardization (TableI), it is apparent that those obtained by the methodof Perryman et al. are appreciably lower (O to 60 %)than the results found by the other diazo methods.When subjected to a statistical analysis, the resultsobtained by the remaining methods could be dividedinto two similar groups: those of Stoner and Weis-berg and of Lathe and Ruthven, and those of Powelland of Jendrassik and Grof. The latter group ofvalues were the lower, and for reasons which will bediscussed, it seemed highly probable that the Stonerand Weisberg and the Lathe and Ruthven valueswere close to the correct results.Using the values obtained by Lathe and Ruthven's

method as a basis, a comparison of all results isshown in Table II, from which it will be seen thatbilirubin in haemolysed specimens appears to beoverestimated by the method of Stoner and Weisberg,

and underestimated by that of Powell. With themethod of Perryman et al., total bilirubin is moreincompletely estimated in plasma containing'directly-reacting' pigment than in specimens notcontaining bilirubin in this form.During the period of storage, some of the haemo-

globin originally present in the haemolysed speci-mens was converted to other haem pigments.Because this and possibly other protein changesmight have affected the analyses, three fresh speci-mens of heparinized bile-pigmented plasma, eachslightly haemolysed, were analysed by the five diazomethods. Each gave results which varied in a similarmanner to those obtained on the stored plasma.

Table III summarizes the results obtained for bilepigments on 64 fresh samples of plasma. Thespecimens from the jaundiced adults and one infantcontained large amounts of 'directly-reacting'bilirubin. In these instances the mean pigment

TABLE IIIPLASMA BILE PIGMENT LEVELS DETERMINED SPECTROPHOTOMETRICALLY AND BY DIAZO COUPLING

Lathe and Ruthven (1958)

%Bilirubin Reacting 'Directly'

Lathe and Ruthven (1958)

Adult plasma (obstructive jaundice)

Adult plasma (hepatocellular jaundice)

Infant plasma (inspissated bile syndrome)

Mean of above specimensMean 40 infant plasmasMean 6 cord plasmas

275

+3-I+1

+12+10+10

-4-_-2

-13-7-9

-8-6-7

-6-4-4

Total Bilirubin (mg./100 ml.)

White et al. (1958)

16-51 3811-82-919-18-68-27-65.54.44-12-72-418

19-910-45.95 68-4

12-82-2

15-112-19-21-5

18-07-87-77-35.73-33-32-41-11-7

16-18-35*04-37-2

13-22-3

727259476572756663

>20>20

38>20>20

71526061

>50<10<10

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concentration found by spectrophotometricanalysis (method of White et al.) exceeded thatfound by diazo coupling (method of Lathe andRuthven) by from 0 to 93 % (mean, 16 %).With the exception of the case of inspissated bile

syndrome, all the infant plasma (from capillary andcord blood) contained less than 10% of pigmentwhich reacted 'directly', Total bile pigment rangedfrom 1 8 to 29 1 mg./100 ml. The mean pigment levelfound spectrophotometrically in these specimens was96 5% (S.E. of mean 0-8 %) of that obtained by thediazo method.

DISCUSSION

DIAZO METHODS WITH PROTEIN PRECIPITATION Theclaim of Perryman et al. (1957) that absorption ofbilirubin esters on the precipitated protein can beprevented by decreasing the serum concentration inthe reaction mixture to 1 % has not been confirmedby the present work. Several of our stored plasmaswere tested in parallel on dilutions of 1 in 20 and1 in 100, and were found to give almost identicalresults. Tests on plasma from freshly drawn bloodalso gave low results, so that it is unlikely thatdenaturation of protein during the storage periodwas responsible for the poor results.The time allotted for the development of the azo-

pigment colour in the method of Perryman et al. is5 min. We allowed 10 min. since this was moreconvenient for batch analyses. An extension of thisperiod to 30 min. (the time employed by King andCoxon) produced no appreciable increase in azo-pigment density, and the omission of the 'few crystalsof sodium azide' to destroy excess diazonium com-pound also had no effect on the final pigmentdensity. In the procedure of Perryman et al. theother deviation from that of King and Coxon'smethod is the addition of 1 ml. of 1.5 % ammoniumsulphamate to 10-3 ml. of diazo reagent before use.This modification was first suggested by Patterson,Swale, and Maggs (1952) as a means of destroyingexcess nitrous acid. We were unable to detect excessnitrite in the diazo reagent of King and Coxon,which is not surprising in view of the fact that thereagent contains only 38% of the theoretical nitriterequirement of the sulphanilic acid. The omission ofthe ammonium sulphamate had no effect on theanalysis.Two comparative studies (Patterson et al., 1952;

Lathe and Ruthven, 1958) have previously indicatedthat the bilirubin values obtained by the method ofKing and Coxon (1950) are lower than those foundby some non-precipitation methods. From thepresent work it appears that this type of precipitationmethod is incapable of accurately determining

serum or plasma bilirubin. That this has not beengenerally recognized is partly due to the fact thatwith King and Coxon's method, non-specificchromogens in the aqueous alcohol extract oftencontribute to the final colour, thereby improving theapparent bilirubin result.The methyl red solution of Haslewood and King

(1937) employed as a standard by Perryman et al.(1957) did not in our hands match the shade ofcolour or the optical density of the azo pigmentsderived from the quoted concentration of our bili-rubin preparation when the latter was prepared as astandard bilirubin serum. We found (with the methodof Perryman et al.) very close agreement betweenthe corrected and the uncorrected E530 m,u valueson each of the standard bilirubin sera, and thebilirubin results obtained for haemolysed and non-haemolysed plasma seem to indicate that the haem-pigment correction formula is adequate (see TableII).

In the case of Stoner and Weisberg's method, acidhaematin when present in the final solution absorbsappreciably at 580 m,u, the wavelength at which theblue pigments are measured. This would account forthe higher values obtained by this rather than byLathe and Ruthven's method, since the latter isrelatively unaffected by haem pigments (Watson,1960). We noted that small but variable opticalreadings could be obtained with blank tests ondifferent plasma containing approximately the sameamount of bilirubin, but the matter was not furtherinvestigated.

DIAZO METHODS WITHOUT PROTEIN PRECIPITATIONThe methods of Jendrassik and Grof (1938) and ofPowell (1944) are modifications of a method devisedby Enriques and Sivo (1926) in which the couplingpromoter is sodium benzoate. Such methods havebeen criticized on the grounds that the final coloursare sometimes 'impure' (King and Coxon, 1950),and that they require several hours for the completecoupling of serum bilirubin in cases of haemolyticjaundice (Gray, 1953; Stoner and Weisberg, 1957).In our experience these criticisms apply to thosemodifications of Enriques and Sivo's method inwhich sodium benzoate has been omitted in favourof a less efficient coupling promoter, e.g., caffeineor citrate (Rappaport and Eichhorn, 1943; Grayand Whidborne, 1946; Chabrol, B6sz6rmenyi,and Fallot, 1949). During the course of severalthousand bilirubin analyses at this hospital byPowell's method, we have never observed 'impure'colours, nor have we been able to detect any furtherincrease in azo pigment density with time. Neverthe-less, false low bilirubin values are obtained byPowell's method if the plasma contains traces of

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haemoglobin or haem pigments. This effect iscaused by interference with coupling, rather thanas was suggested by Sims and Horn (1958)-excessive'blank' correction. Since with fresh haemolysedplasma (naturally or artificially bile pigmented) we

have observed larger underestimates of bilirubin byPowell's method (Watson, 1960) than were actuallyfound with the stored specimens, it is possible thatsome conversion products occurring in our specimensas a result of storage in deep-freeze interfere to a

lesser extent with bilirubin coupling than haemo-globin does.The final solutions of the Malloy and Evelyn (1937)

procedure derived from lipaemic plasma or speci-mens containing normal concentrations of bilirubinare occasionally opalescent, and it is generally agreedthat the intensity of this turbidity may vary betweenthe 'blank' and the 'test' determinations. As pre-

viously stated, we observed opalescence with Latheand Ruthven's method, but the benzoate methods ofPowell, and Jendrassik and Grof were also equallysubject to this source of error.

Several methods of bilirubin analyses, includingthose of Powell (1944), Dangerfield and Finlayson(1953), Sims and Horn (1958), and Bruckner (1959),employ insufficient diazo reagent to hydrolyse andcouple all the bilirubin in deeply bile-pigmentedplasma, unless a smaller volume of plasma than thatstipulated is taken for analysis (Watson, 1961).Lathe and Ruthven in their method suggest usinghalf the quantity of plasma for specimens containingmore than 15 mg. total pigment per 100 ml. However,there is nothing obligatory or distinctive in thechange at 15 mg., since the method employs permillilitre of plasma much higher final concentrationsof diazotised and undiazotised sulphanilic acid thanis used in the parent method of Malloy and Evelyn(1937). On the other hand, when inadvertently using0-1 ml. plasma containing more than 20 mg. bilirubinper 100 ml., we have encountered some plasmaspecimens from which the appearance of maximumazo pigment colour was delayed for 15 to 20 min.Contrary to the contention of Meites and Hogg(1959), we found it an advantage to use differentplasma concentrations because it enabled us to readthe final optical densities more accurately. For thisreason also, we used a volume of 0 4 ml. for plasmawhich appeared to contain normal or only slightlyabove normal concentrations of bile pigment.

Meites and Hogg (1959), using the method ofMalloy and Evelyn (1937), have shown that when thetemperature at which the analysis is performed isdropped from 220C. to 10°C., the production of azo

pigments from serum bilirubin is decreased by about50 %. The density of the azo pigments produced fromchloroform solutions of bilirubin under similar

conditions, however, does not vary by more than 5 %.Since in our laboratory, room temperatures fluctuatefrom 13°C. to 26°C. during the year, Malloy andEvelyn's method would require careful temperaturecontrol, or standardization at different temperatureswith bilirubin sera, procedures inconvenient forroutine use. A modified reagent which reacts maxi-mally in this temperature range has been proposed,but unfortunately the results obtained with it are veryconsiderably affected by subvisible amounts ofhaemoglobin in serum (Meites and Hogg, 1959).Using the method of Lathe and Ruthven we haveobserved that no decrease occurs in the production ofazo pigment from plasma containing bilirubin as aresult of a fall in the laboratory temperature.Moreover, this method has been shown not to besignificantly affected by haemolysis (Watson, 1960).The method of Jendrassik and Gr6f has been

stated to have a high degree of specificity and repro-ducibility (With, 1954; Fog, 1958a). If we assumethat the bilirubin levels found by this method arecorrect, then the results obtained by the method ofStoner and Weisberg, Lathe and Ruthven, andPowell on unhaemolysed specimens will be 5 to 10%too high. The occurrence of side reactions producingyellow and brown diazo products with urobilinogen(Lopez Garcia, 1941) and with some non-proteinnitrogen compounds (Harrison and Bromfield, 1928)have been reported. But these reactions cannot hereaccount for an overestimation of pigment by thethree methods, since, with the exception of plasmaNo. 9, none of our specimens was azotaemic, and it ismore likely that the infant plasma tested containedlower rather than higher than average concentrationsof urobilinogen. Caffeine-benzoate coupling methodshave previously been reported to give lower valuesthan those found by some alcohol coupling methods(Malloy and Evelyn, 1937; Ducci and Watson, 1945).It is therefore possible that some substance presentin the plasma ofjaundiced patients (and absent fromnormal serum fortified with bilirubin) may inhibitthe coupling of bilirubin under the conditions ofJendrassik and Gr6f.

Results based on standardizations from chloro-form solutions and serum solutions of bilirubindiffered by approximately 9% (methods of Powelland of Lathe and Ruthven). In chloroform solutions,the relative extinction of 'azobilirubin' was higherthan in serum solutions; the coupling promoterused in each case was 50% methanol. Using 80%ethanol, O'Hagan, Hamilton, Le Breton, and Shaw(1957) observed 2% lower azo pigment densitieswith choroform bilirubin solutions than with thesame quantities of bilirubin in serum (method ofPowell). Although there is no doubt that changes inthe spectral curves produced by variations in solvent

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and pH are the cause of part of the discrepancies, weare unable to account satisfactorily for the largedifferences. Experiments in which bilirubin wasadded to bile-pigmented plasma have always yielded100 -1- 2%a recovery.

SPECTROPHOTOMETRIC METHOD Higher total bilepigment levels for adult plasma were obtained byspectrophotometry by the method of White et al.than by diazo coupling by the method of Lathe andRuthven. This is contrary to the opinion of White etal. (1958) but in agreement with the findings ofFog (1958b) who measured pigment colour by amore refined procedure (Fog, 1949). On the otherhand, the bile pigment levels obtained by the diazoand spectrophotometric methods showed goodagreement in plasma obtained from infants (bothicteric and non-icteric) during the first week of life.An exception was the infant with the inspissated bilesyndrome (whose plasma bile pigment was mainlyin the conjugated form). Since infant plasma isalmost devoid of lipochromes (Mertz and West,1956), it would appear that estimation by the methodof White et al. (1958) includes in addition to lipo-chromes when present, a measure of non-bilirubinpigments which are exclusively found in plasmacontaining an appreciable percentage of bilirubinreacting 'directly' by the method of Lathe andRuthven. These pigments may be dipyrroles, e.g.,bilifuscin or allied pigments as suggested by With(1954), or possibly bilirubin esters or non-diazocoupling tetrapyrroles with a lhigher specific ex-tinction coefficient than unconjugated bilirubin.

CONCLUSIONS

Haem pigment known to be present in many speci-mens of infant plasma causes the method of Powell(1944) to underestimate and that of Stoner andWeisberg (1957) to overestimate the plasma bilirubinconcentration.

Bilirubin methods incorporating protein precipi-tation, e.g., that of King and Coxon (1950), andmodifications, give low and variable results.

Results obtained by spectrophotometry (White etal., 1958) are satisfactory for infant plasma, providedno appreciable amounts of 'directly-reacting' bili-rubin are present.

Experience with several techniques has led us tochoose that of Lathe and Ruthven (1958) as themost suitable and valid general-purpose method atpresent available for the determination of plasmabilirubin.

We thank Dr. G. Gregory of the University of Melbournefor assistance with the statistical analysis of the results.

REFERENCES

Bruckner. J. (1959). Amer. J. clin. Path., 32, 513.Chabrol, E., Boszormenyi, M., and Fallot, P. (1949). Sen. Hop. Paris,

25, 3437.Culley, P. E., Waterhouse, J. A. H., and Wood, B. S. B. (1960).

Lancet, 1, 88.Dangerfield, W. G., and Finlayson, R. (1953). J. clin. Path., 6, 173.Ducci, H., and Watson, C. J. (1945). J. Lab. clin. Med., 30, 293.Enriques, E., and Siv6, R. (1926). Biochem. Z., 169, 152.Fog, J. (1949). Scand. J. clin. Lab. Invest., 1, 255.-(1958a). Ibid, 10, 241.

(1958b). Ibid, 10, 246.Gray, C. H. (1953). The Bile Pigments. Methuen, London.

, and Whidborne, J. (1946). Biochem J., 40, 81.Harrison, G. A., and Bromfield, R. J. (1928). Ibid, 22, 43.Haslewood, G. A. D., and King E. J. (1937). Ibid. 31, 920.Jendrassik, L., and Gr6f, P. (1938). Biochem. Z., 297, 81.King, E. J., and Coxon, R. V. (1950). J. clin. Path., 3, 248.Lathe, G. H., and Ruthven, C. R. J. (1958). Ibid., 11, 155.Lopez Garcia, A. (1941). Medicina (B. Aires), 1, 219. Quoted by

With (1954) vide infra.Malloy, H. T., and Evelyn, K. A. (1937). J. biol. Chem., 119, 481.Meites, S., and Hogg, C. K. (1959). Clin. Chem., 5, 470.Mertz, J. E., and West, C. D. (1956). A.M.A. Amer. J. Dis. Child., 91,

19.O'Hagan, J. E., Hamilton, T., Le Breton, E. G., and Shaw, A. E.

(1957). Clin. Chem., 3, 609.Patterson, J., Swale, J., and Maggs, C. (1952). Biochem. J., 52, 100.Perryman, P. W., Richards, D. H., and Holbrook, B. (1957). Ibid.

66, 61 P.Powell, W. N. (1944). Amer. J. clin. Path., 14, Tech. Sect., 8, 55.Rappaport, F., and Eichhorn, F. (1943). Lancet, 1, 62.Rice, E. W. (1957). Clin. Chim. Acta, 3, 121.Sims, F. H., and Horn, C. (1958). Amer. J. clin. Path., 29, 412.Stoner, R. E., and Weisberg, H. F. (1957). Clin. Chenm., 3, 23.Varley, H. (1958). Practical Clinical Biochemistry, 2nd ed. Heinemann,

London.Watson, D. (1960). Clin. Chim. Acta, 5, 613.- (1961). Clin Chem., 7, 234.White, D., Haidar, G. A., and Reinhold, J. G. (1958). Ibid, 4, 211.With, T. K. (1954). Biology of Bile Pigments. Frost-Hansen, Copen-

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