Bile pigment excretion: a comparison of the biliary excretion of bilirubin and bilirubin derivatives. R Lester, P D Klein J Clin Invest. 1966;45(12):1839-1846. https://doi.org/10.1172/JCI105487. Research Article Find the latest version: https://jci.me/105487/pdf Journal of Clinical Investigation Vol. 45, No. 12, 1966 Bile Pigment Excretion: A Comparison of the Biliary Excretion of Bilirubin and Bilirubin Derivatives * ROGER LESTER t AND PETER D. KLEIN WITH THE TECHNICAL ASSISTANCE OF ARMAND M. MATUSEN (From the Fifth and Sixth [Boston University] Medical Services, Boston City Hospital, and the Department of Medicine, Boston University School of Medicine, Boston Uni- versity Medical Center, Boston, Mass., and from the Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Ill.) During the process of hepatic excretion, a di- tween mesobilirubinogen and bilirubin (Figure 1). verse group of exogenous organic compounds and To determine whether a relationship exists among endogenous metabolites is conjugated with polar molecular structure, conjugation, and excretion, anionic molecules (1). Although it is clear that we prepared a series of tritiated bile pigments and the physical and chemical characteristics of these administered it intravenously to rats with ex- compounds can be drastically altered by conju- ternal biliary drainage. We determined the rate of gation, the relationship between conjugation and biliary excretion of these compounds and estab- excretion has not been fully elucidated (2). For- lished whether they are excreted in bile as the glu- mation of the glucuronide conjugate appears to be curonide conjugate. an essential step in the excretion of bilirubin (3-5). Gunn rats, a congenitally jaundiced mu- Methods tant species of Wistar rats, have a partial deficiency Preparation of radioactive compounds. Radiochem- of glucuronyl transferase activity for several sub- ically pure, specifically labeled bilirubin-'H was prepared strates and an essentially complete inability to biosynthetically by techniques described previously (11) form bilirubin glucuronide (6, 7). As a result Mesobilirubin was labeled with tritium by catalytic they are incapable of excreting significant quan- reduction of unlabeled bilirubin (12a) in the presence tities of bilirubin in the bile (8), although their of tritium gas. For this purpose 50 mg of crystalline bilirubin was mixed with 10 mg of palladium black capacity to excrete exogenously administered bili- (99%) in a small hydrogenation vessel equipped with a rubin glucuronide is undisturbed (9), and in other magnetic stirrer and dropping funnel. Into the funnel respects hepatic function is normal. was placed several milliliters of 0.1 N NaOH, and the In the course of studying the enterohepatic cir- apparatus was evacuated and filled with tritium gas ob- tained by the electrolysis of 15% KOH in tritiated water, culation of mesobilirubinogen-14C we observed SA 36 mc per mmole. The catalyst was equilibrated for that a Gunn rat excreted three-quarters of a 1.9- 30 minutes, and 3 ml of the NaOH solution was added mg dose of this bilirubin derivative within 90 min- dropwise with stirring. The deep red mixture was utes of intravenous administration (10). This stirred for 3 hours, during which 40 ml of tritium gas finding, which suggested that mesobilirubinogen (theory 37.9 ml) was taken up, and the solution changed to a light yellow color. The solution was acidified with was not excreted as the glucuronide conjugate, was glacial acetic acid, and the yellow pigment was extracted unexpected in view of the apparent similarity be- into chloroform. The chloroform extracts were washed with water, dried over sodium sulfate, and evaporated to * Submitted for publication May 26, 1966; accepted dryness. The yellow pigment was crystallized and re- August 12, 1966. crystallized by displacement of chloroform with boiling Supported in part by the U. S. Atomic Energy Com- methanol. The yield of crystalline material equaled 34% mission and the U. S. Public Health Service (grants of the starting material. On spectrophotometric examina- AM 07091 and AM 09881). tion, the end product showed maximal absorption at 433 t Recipient of U. S. Public Health Service Develop- nmy [absorption maximum of mesobilirubin in chloroform ment Award 12,127. = 434 mAe (13) ] ; a diazo derivative was prepared (14a) Address requests for reprints to Dr. Roger Lester, with maximal absorption at 540 mnA. The specific ac- Boston University School of Medicine, 80 East Concord tivity of mesobilirubin-'H equaled 7.7 mc per mmole, and St., Boston, Mass. 02118. it remained constant on repeated recrystallization. 1839 1840 ROGER LESTER AND PETER D. KLEIN BILIRUBIN mmole, and within the limits of the methodology em- ployed, the specific activities of amorphous, crystalline, Me V e P P Me me V and recrystallized mesobilirubinogen were equal (10). Mesobilirubinogen-3H dissolved in petroleum ether was oxidized to optically inactive (i)-urobilin-3H hydrochlo- o,=CH CHCH2=Gp ride according to the method of Watson (16). This red- H orange pigment was crystallized and recrystallized by H H H displacement of methanol with boiling ethyl acetate. The I* 4H yield of crystalline end product equaled 10%o of the meso- bilirubinogen-3H. Because of the low yield, it was con- venient to add unlabeled mesobilirubinogen (prepared by techniques similar to those described above) to the MESOBILI RUBIN starting material to obtain enough pigment for crystal- Me Et P Et lization. When i-urobilin-0H hydrochloride was dissolved in 3%o HCl in methanol, maximal absorption occurred at 493 mu [absorption maximum of i-urobilin hydrochlo- 0J77CH H2 CH ride in 3% hydrochloric acid in methanol = 494 m/A (14b)], and characteristic green fluorescence was obtained H H H H on addition of zinc acetate to methanolic solutions (14c). I +4H The maximal SA obtained was 1 mc per mmole, and the specific activity remained constant on repeated crystal- lization. Administration of tritiated compounds. Male Sprague- MESOBILI RUBINOGEN Dawley C.D. rats and congenitally jaundiced Gunn rats Me Et me P P Me Me Et were prepared with an external biliary fistula. Weighed samples of tritiated pigments and pigment derivatives were dissolved in 0.1 N NaOH, and the basic solution ClfllCH2 YJCH2 was added gradually and with stirring to rat serum. The serum was neutralized and administered to the rats H H H H by rapid intravenous injection. With the rats under con- tinued ether anesthesia, the bile was collected during 15- minute periods for a total of 90 minutes with collection tubes changed at 15-minute intervals. All samples were i- collected in subdued light at 40 C, and when possible, UROBILIN analysis of the specimens was performed immediately Me Et Me P P Me Me Et upon completion of the experiment. Analytical methodology. Chromatography of the di- azo pigments of bilirubin and mesobilirubin was per- J7LCH CHI LCH i70 formed according to the method of Schmid (4). After development of the chromatograms, conjugated diazo H H H pigments were eluted from the paper in 1 N acetic acid, FIG. 1. SCHEMATIC REPRESENTATION OF THE CONVER- and the eluates were taken to dryness in vacuo. The SION OF BILIRUBIN SUCCESSIVELY TO MESOBILIRUBIN, MESO- dried pigment was dissolved in a small volume of 1 N BILIRUBINOGEN, AND OPTICALLY INACTIVE (i)-UROBILIN. acetic acid, and 0.1 ml was incubated at 370 C for 6 to 12 Me = methyl; Et = ethyl; V = vinyl; P = propionic acid. hours under nitrogen with 30,000 U of bacterial p-glu- curonidase 1 and 2.5 ml of 0.2 N acetate buffer pH 5.0 Mesobilirubinogen-3H was prepared by sodium amal- (total volume of incubation mixture, 3.0 ml). On com- gam reduction according to the method of Fischer and pletion of incubation, the solution was acidified and ex- Orth (12b) by substituting 2 ml of tritiated water, SA tracted with butanol; the extracted pigment was then 54 mc per mmole, in the reaction mixture. The colorless rechromatographed. Mixtures of crystalline (unconju- amorphous mesobilirubinogen-'H was crystallized from gated) bilirubin and mesobilirubin were applied to What- hot ethyl acetate and stored in evacuated tubes, in the man 3 mm paper in 5- to 10-jg quantities and separated dark, at - 20° C. The yield of amorphous powder by chromatography (17) in system I [ascending, chloro- equaled 60%, but only 10% of the end product was ob- form: petroleum ether (1: 9)]. The chromatogram was tained in crystalline form. On addition of Ehrlich rea- divided into strips and counted in a liquid scintillation gent and saturated sodium acetate, the tritiated chromo- counter. Mesobilirubinogen and i-urobilin were extracted gen formed a characteristic urobilinogen-aldehyde com- from acidified bile into chloroform, and 1- to 4-,ug quan- plex with maximal absorption at 557 mju [absorption tities were chromatographed on Whatman 3 mm paper maximum of urobilinogen-aldehyde = 556 mAu (15) ]. The SA of mesobilirubinogen-8H equaled 42 mc per 1 Sigma Scientific Co., St. Louis, Mo. BILE PIGMENT EXCRETION 1841 in the following solvent systems: system II, ascending, methanol: butanol: ammonia (1: 3: 2) (17); system III, ascending, ethyl acetate: octanol (1:1, saturated with ammonia) (18); system IV, descending, butanol: pyri- dine: water (1: 1: 1). The chromatograms were dried, sprayed with zinc acetate in methanol, and examined un- der fluorescent light to identify the compounds and to calculate their Ra. Bilirubin and mesobilirubin were crystallized from bile according to the method of Ostrow, Hammaker, and Schmid (19). i-Urobilin was extracted into chloroform from acidified bile and crystallized from methanol-ethyl acetate as described above. Mesobilirubinogen in bile specimens was oxidized with ferric chloride and hydro- 15 30 45 60 i5 90 chloric acid and then crystallized as i-urobilin. Bile MINUTES AFTER l.V. INFUSION OF BILIRURIN-3H were counted in a specimens and crystals liquid scintil- FIG.