Separation of the urinary ester-form bilirubin was attempted, and the results obtained may be summarized as follows: 1. A brown pigment was obtained from jaundiced urine by the following procedures; namely, salting out, methanol extraction, chloroform flocculation, and separation on cellulose column. The pigment has been found to be easily soluble in water, displaying the absorption maximum at 420 - 410 m&#956; at pH 7.0, and it also gave a positive reaction both to GMELIN's and EHRLICH's diazo reagents within a minute without the addition of alcohol. These characteristics agree well with those of the socalled
ester-form bilirubin. 2. On the basis of the results of paper chromatography and paper electrophoresis, the pigment has been determined to contain no amino
acid, steroid, nor reducing substance. Moreover, no glucuronic acid could be detected whether examined in vitro or by paper chromatography together with paper electrophoresis, either.</p

Hirano, Hiroshi

Kaneda, Kohtaro

Komuta, Kiyoatsu

Kondo, Tadasuke

Monobe, Taisei

Sakamoto, Takeshi

Okayama University Digital Information Repository

Studies on Bile Pigments VIII. A Form of Direct Reacting Bilirubins Appearing in Jaundiced Urine

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 11, Issue 2 1957 Article 4JUNE 1957Studies on Bile Pigments VIII. A Form ofDirect Reacting Bilirubins Appearing inJaundiced UrineTakeshi Sakamoto∗ Kiyoatsu Komuta† Tadasuke Kondo‡Hiroshi Hirano∗∗ Taisei Monobe†† Kohtaro Kaneda‡‡∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,‡‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on Bile Pigments VIII. A Form ofDirect Reacting Bilirubins Appearing inJaundiced Urine∗Takeshi Sakamoto, Kiyoatsu Komuta, Tadasuke Kondo, Hiroshi Hirano, TaiseiMonobe, and Kohtaro KanedaAbstractSeparation of the urinary ester-form bilirubin was attempted, and the results obtained may besummarized as follows: 1. A brown pigment was obtained from jaundiced urine by the followingprocedures; namely, salting out, methanol extraction, chloroform flocculation, and separation oncellulose column. The pigment has been found to be easily soluble in water, displaying the absorp-tion maximum at 420 - 410 mµ at pH 7.0, and it also gave a positive reaction both to GMELIN’sand EHRLICH’s diazo reagents within a minute without the addition of alcohol. These charac-teristics agree well with those of the socalled ester-form bilirubin. 2. On the basis of the resultsof paper chromatography and paper electrophoresis, the pigment has been determined to containno amino acid, steroid, nor reducing substance. Moreover, no glucuronic acid could be detectedwhether examined in vitro or by paper chromatography together with paper electrophoresis, either.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa Med. Okayama 11, 81-87 (1957)STUDIES ON BILE PIGMENTSVIII. A FORM OF DIRECT REACTING BILIRUBINSAPPEARING IN JAUNDICED URINETakeshi SAKAMOTO, Kiyoatsu KOMUTA, Tadasuke KONDO,Hiroshi HIRANO, Taisei MONOBE, & Kohtaro KANEDAFrom the First Department of Internal Medicine, Okayama UniversityMedical School, Okayama, JAPAN(Director: Prof. Dr. K. Kosaka)Received for Publication March 29, 1957The following is a preliminary report on an investigation being car-ried out upon the reaction of natural pigments against EHRLICH's diazoreagent, especially, on that of urinary direct reacting pigment appearingin jaundice.Almost all the pigments giving a positive direct diazo reaction couldbe separated from diazo-negative urinary pigments by salting out withammonium sulphate. These pigments were successively extracted withmethanol, and brown crystalloid as shown in Table 1, was obtained byfurther purifying procedures.This crystalloid has been found to be easily soluble in alcohol andmore soluble in water, whereas it is insoluble in chloroform, carbon tetra-chloride, benzene, or ether. Its aqueous solution displayed the absorp-tion maximum at 410-400 mp. at pH 7.2, and showed no fluorescenceunder ultraviolet ray. It was negative to both EHRLICH'S aldehyde andSCHLESINGER'S reactions, but without addition of alcohol acted positivelyto EHRLICH'S diazo reaction within one minute, showing a reddish violetcolor, and likewise positively the Gmelin test. It is well known that di-basic acid indirect bilirubin is soluble in chloroform and insoluble inwater or alcohol, and that the chloroform solution displays the absorptionmaximum at 450 mfl and does not act positively on EHRLICH'S diazo rea-gent without addition of alcohol. In view of these, it may be said thatthe pigment thus obtained, still possessing the chracteristics of bilirubinas it does, is clearly different from th~ dibasic acid bilirubin, judgingfrom their positive reactions to both GMELIN and EHRLICH'S diazo rea-gents.811Sakamoto et al.: Studies on Bile Pigments VIII. A Form of Direct ReactingProduced by The Berkeley Electronic Press, 195782 T. SAKAMOTO, K. KOMUTA, T. KONDO, H. HIRANO, T. MONOBE & K. KANEDATable 1. Operation to separate a form of direct reactingbilirubins from jaundiced urineJaundiced urineISalting out with ammonium sulphate,then filteredI IResidue Filtrate(brown) Direct diazo (+) (yellw) Diazo (-)IGmelin (+) I Gmelin (-)Dried, then extracted with tmethanol Discarded(Methanolic solution JIIFiltered, then dr1ied in vacuo, and Repeated thisredissolved in methanol procedureseveraltimesResidue Methanolic solution(yellow) Direct diazo (+) 'I~ Gmelin (+) Dried in vacuoDiscarded Powder(brown) Direct diazo (+)IGmelin (+)vy-ashed with chloroform severaltimesIChloroform extract(almost colorless)~DiscardedWashed with chloroform well,then crystalled out frommethanolic solutioncontaining chloroformCrystalloid(brown)11Crude Ester-Form BilirubinIResidue(brown)I Dissolved again in methanOlMethanolic solutionIAdd double the quantity of chloroform,and let it stand for 1/2-1 hourIFlocculate(brown) Direct diazo (+)I Gmelin (+)ISupernatant(brown) Direct diazo (+)~ Gmelin (+)DiscardedEven after thoroughly mlxlDg this aqueous solution with an equalvolume of N/IO hydrochloric acid in an attempt to extract pigment withchloroform, it has been found that no pigment is tranferred to chloroform,but that almost all the pigments are transferred to chloroform after2Acta Medica Okayama, Vol. 11 [1957], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss2/4Studies on Bile Pigments 83KOSAKA'S saponification procedure.2 The chloroform solution so obtainedshows no fluorescence under ultraviolet ray and acts negatively toEHRLICH'S aldehyde or SCHLESINGER'S reactions, while presenting a posi-tive GMELIN reaction as well as a positive indirect reaction to diazoreagent. These data seem to suggest that the direct bilirubin possessesall the characteristics of the so-called ester-form bilirubin.1,2On examining this aqueous solution by paper chromatography withthe spray of EHRLICH's diazo reagent, no spot such as o-aminophenol-, na-phthoresorcin-, antimony trichloride-, phosphoric acid-, or phosphomolyb-dic acid-positive spot could be detected on the chromatographic paper otherthan a yellow bilirubin spot and amino acid spot with different Rf value,presenting a reddish violet direct diazo reaction within a minute. At thisphase of the experiment, various developing solvents and aspects of timeelements involved in the preliminary treatment of the paper with thesolvent were studied, and as the result, the supernatant of n-butanol-ethanol-water in the proportion of 4: 1 : 2 has been found to be mostsuitable as the developing solvent for this purpose. However, the questionwhether or not the pure ester-form bilirubin can successfully be separatedat this stage is of interest inasmuch as it depends on whether or notamino acid is eradicatable from the crude ester-form bilirubin. For thepurpose of ascertaining this point in question, the crude ester-form bili-rubin had been developed on a cellulose column by using the aforemen-tioned supernatant of n-butanol-ethanol-water; and succeeded in chroma-tographic separation of a single color, yellow pigment as shown in Fig. 1.This yellow pigment was found to possess all the characteristics ofthe crude ester-form bilirubin, namely, readily soluble in methanol, andstill more soluble in water; but a paper chromatography revealed thatits aqueous solution had the absorption maximum at 420-410 mlfL at pH7.0 and that it was free of amino acid. From these results, it may be saidthat the ester-form bilirubin so separated on the cellulose column is a di-rect bilirubin and that it contams lesser impurity than any bilirubins se-parated from jaundiced urine by other methods.Recently it has been reported that the direct reacting bilirubin is theone consisting of bilirubin glucuronide3•4•5 as its principal component, andC. J. WATSON, agreeing to this opinion, has informed in a personal commu-nication to one of the present authors, SAKAMOTO, that "Thus, the formerconcept that the prompt direct birubin was sodium bilirubinate as advan-ced by HUNTER and others and that the indirect bilirubin was bilirubin-globin as proposed by DUESBERG, also by POLONOVSKI and FIESSINGER,both of these concepts having been favored earlier by me, must now beabandoned."6 Further experiments are now being carried out, but from3Sakamoto et al.: Studies on Bile Pigments VIII. A Form of Direct ReactingProduced by The Berkeley Electronic Press, 195784 T.5AKAMOTO, K. KOMUTA, T. KONDO, H. HIRANO, T. MONOBE & K. KANEDA1.5....cv:g 1.0........v0uc.!a....u.5....I<~ 0.5,.I.\""'I, ,: .,,,,,IColumn : 18 X 200 mm.Eluent (by 543)• • • • • •• Azo-pigment(by 563)5 10 15 20Fraction numbers (2 ml. each)Fig. 1. Elution curve of the crude ester-form bilirubin on cellulose columnthe results obtained so far, no evidence has been found to prove that thedirect reaction of the ester-form bilirubin is dependent upon a combinationof the bilirubin with glucuronic acid. Moreover, since glucuronic aciddoes not always present itself in the direct reaction of other form bilirubin,it requires further studies to solve the question whether or not bilirubingluconide is essential in the direct reaction.? The definitive explanationon the role of glucuronic acid in the direct reaction of bilirubin, however,must await the results of more comprehensible studies on the point that alarge quantity of direct bilirubin, other than the bilirubin separated bythe present method, still remains in the direct bilirubin extracted fromjaundiced urine. On the other hand, when the crude ester·form bilirubinhad been examined by paper electrophoresis (Fig. 2), two bands presentingdirect reaction were obtained. Of the two, the less stable pigment wasreadily dissolved in water and its aqueous solution had been found to havethe absorption maximum at 420-410 m/l at pH 7.0. And reacting withinone minute with diazo reagent without adding alcohol, this solution turnedreddish violet, but this pigment proved to be different from the pigmentpossessing the absorption maximum at 565 m/l, characteristic of naphtho-4Acta Medica Okayama, Vol. 11 [1957], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss2/4Studies on Bile Pigmentse85!! I [ Without colodn.!P(. ~ ..,. ~___....... y....~::;-llr-W----y...:;el~.lz:..O-W-. _[orangei -=- ~ Dicect di.,o ceacdonlight Violet violet. .---...l-------l:-1I~~ [ Ninhyddne ccactionvioletFig. 2. Paper electrophorograms of the crude ester-form bilirubinrescin reaction by the ISHIDATE and NAMBARA method.9EXPERIMENT1. Material. Jaundiced urine from the respective patient describedin Table 2 was used. Administration of glucuronic acid preparate wasdiscontinued at least a week before taking their urine.Table 2. Jaundiced urine employed (in mg.jdl.)Acute hepatitisGallstone0.18(1,200 m!.)2.24(1,700 ml.)9.17 0.07 Cancer of bile(2.21) (800 m!.) duct6.57 !AldehYde ( -) Mechanical(0.24) (600 m!.) jaundice1.14(0.05)2.04(0.28)37.99(20.00)22.75(15.91)20.15(15.15)15.34(11.16)15.10(12.10)4.46(4.08)5.60(5.24)15/Wl(1956)8/X(1956)9/XI(1956)27/XI(1956)25/XlI(1956)8/II(1957)13/II(1957)224841495449531 Y.K.I (;I2 ToNol ~3 J. H.i (;I4 K. Y,[95 IT. S.[9I6 T. V'I(;_~_Ko I.l~~~----'--~-'---------'~-::-:------'-'._---------~-_.-I I II Bilirubin V . I! S - I V . nnary INo. '\Name Sex Years, Date erum_ rmary urobilinogen i Diagnosis_____1 1___ (l5i~~~t) ~~t~\ (volume) I _6.64 Aldehyde (-): Cancer of the(1.15) (750 ml.) I amp. of Vater11.10 0.09 Chronic hepatitisCl.18) (1,100 ml.) I15.74 [Aldehyde (-) Mechanical(3.25) (1,050 ml.) jaundiceAbb.: Direct: Direct reacting bilirubinI. D.: Indirect reacting bilirubin5Sakamoto et al.: Studies on Bile Pigments VIII. A Form of Direct ReactingProduced by The Berkeley Electronic Press, 195786 T. SAKAMOTO, K. KOMUTA, T. KONDO, H. HIRANO, T. MONOBE & K. KANEDA2. EHRLICH'S diazo reaction. The solutions used for EHRLICH'S diazoreaction were divided into two groups, namely, solution I which wasprepared by dissolving 1 g. of sulfanilic acid and 15 ml. of concentratedhydrochloric."acid in 1,000 mI. of distilled water, and the solution 11 pre-pared by dissolving 0.5 g. of sodium nitrite in 100 ml. of distilled water.By mixing 10 mI. of the solution I and 0.3 ml. of the solution 11 beforethe use, the solution so mixed was used within 5 minutes.3. Detection of glucuronic acid. For the determination of glucuronicacid in vitro, barium naphthoresorcin carbonate method8 and naphthore-sorcin picrate method9 were employed and an appearance of the absorp-tion maximum at 565 m/.l was then considered to be specific to the productsof these reactions while on filter paper, o-aminophenol method10 and theordinary naphthoresorcin methodll were used for the detection.4. Paper chromatography. Paper chromatography was carried outby one dimensional ascending method as described in the preceding re-port.125. Column chromatography. Column chromatography has beenconducted by the method, the details of which are described in the pre-ceding report.B Two ml. of each effluent was collected with fraction col-lector, and after evaporating the solvent under a low pressure the residualpigment was dissolved in 2 ml. of methanol and its extinction coefficientwas determined with the filter S-13' Then adding 3 ml. of the diazo mix-ture to this solution the extinction coefficient of the resulting azo pigmentwas determined with the filter S"3 30 minutes afterwards.6. Paper electrophoresis. The sample was manipulated under thefollowing conditions; using a paper electrophoresis apparatus, ModelKOBAYASI (NATSUME Co.), veronal buffer solution, pH 8.5, 180 V/ 20cm., 12 mA/cm. on SCHLEICHER u. SCHtlLL Nr. 2043a paper, for 5 hours.7. Calibration of the absorption. For the qualitative determination,recording spectrophotometer, Model DK (BECKMAN) was used, and forthe quantitative determination, PULFRICH's photometer.SUMMARYSeparation of the urinary ester-form bilirubin was attempted, andthe results obtained may be summarized as follows:1. A brown pigment was obtained from jaundiced urine by the fol-lowing procedures; namely, salting out, methanol extraction, chloroformflocculation, and separation on cellulose column. The pigment has been6Acta Medica Okayama, Vol. 11 [1957], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss2/4Studies on Bile Pigments 87found to be easily soluble in water, displaying the absorption maximumat 420 - 410 mp. at pH 7.0, and it also gave a positive reaction both toGMELIN'S and EHRLICH's diazo reagents within a minute without the addi-tion of alcohol. These characteristics agree well with those of the so-called ester· form bilirubin.2. On the basis of the results of paper chromatography and paperelectrophoresis, the pigment has been determined to contain no aminoacid, steroid, nor reducing substance. Moreover, no glucuronic acidcould be detected whether examined in vitro or by paper chromatographytogether with paper electrophoresis, either.REFERENCES1. YAMAOKA, K.: Jap. J. Gastro·Enterol. 48, No.3 & 4, 1, 1950 (in Japanese); J. Jap.Soc. Int. Med. 42, 531, 1953 (in Japanese)2. KOSAKA, K.: Tokyo-Iji-Shinshi 67, No. 11, 14, 1950 (in Japanese)3. TALAFANT, ED.: Chem. listy 50,817,1956.4. BILLING, B. H. & LATH, G. H.: Biochem. J. 63, 6P, 1956.5. SCHMID, R.: Schweiz. med. Wschr., 86, 775, 1956; Science 124, 76, 1956.6. WATSON. C. J.: Personal Communication, May 1. 1956.7. SAKAMOTO, T.: Igaku Kenkyu 27, 376, 1957.8. OGA TA, A. & YAMANOUCHI, T.: Nihon Yakugaku Shi 49,553,1929; 50, 1063, 1930.(in JapanelO;e)9. ISHIDATE, M. & NAMBARA, T.: Report from the Glucuronic Acid Assembly.The Glucuronic Acid Assembly, Tokyo, 1956.10. HIRASE, 5., ARAKI, C., & NAKANISHI, S.: Bull. Chem. Soc. Japan 26, 183, 1953.11. BRYSON, J. L., & MITCHELL, T. J.: Nature 167, 864, 1951.12. SAKAMOTO, T. : Acta Med. Okayama 10, 227, 1956.13. SAKAMOTO, T. : Acta Med. Okayama 10, 253, 1956.7Sakamoto et al.: Studies on Bile Pigments VIII. A Form of Direct ReactingProduced by The Berkeley Electronic Press, 1957

Studies on Bile Pigments VIII. A Form of Direct Reacting Bilirubins Appearing in Jaundiced Urine

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