Bile Pigment Excretion: a Comparison of the Biliary Excretion of Bilirubin and Bilirubin Derivatives

Home , Bilin (biochemistry), Bilirubin glucuronide, Urobilin

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 obtained 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 employed, 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 mesobilirubinogen-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 crystallization. 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 diazo 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-glucuronidase 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 under 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. 2. RADIOACTIVITY IN BILE AFTER INTRAVENOUS lation spectrometer, and the results were analyzed sta- ADMINISTRATION OF BILIRUBIN-5H. tistically by methods described previously (20). were administered to two Gunn Results of bilirubin-3H rats, less than 10% of the radioactive dose was Rate of tetrapyrrole excretion in normal and excreted (Figure 2, Table I). Similar results Gunn rats. After the intravenous administration were obtained with mesobilirubin-3H (Figure 3, of 23 or 238 pg of bilirubin-gH to two normal Table I). From 71 to 80% of administered meso- (Sprague-Dawley, C.D.) rats, excretion of the bilirubin-3H was excreted by four normal rats label was evident during the first 15 minutes, and within 90 minutes, whereas 10 and 2% was ex- by 90 minutes 79 to 86% of the radioactive dose creted by two Gunn rats during an identical time had appeared in bile. When comparable amounts period.

TABLE I Biliary excretion of labeled tetrapyrroles

Characteristics of dose Radioactivity Type of excreted in Experiment rat Wt Tetrapyrrole administered Vol Radioactivity Amount bile in 90 min 5 ma dpm %g% dose 1 S.D.* 430 Bilirubin-3H 1.1 458,000 238 79 2 S.D. 400 Bilirubin-'H 2.0 759,000 23 86 3 Gunn 350 Bilirubin-5H 1.2 196,0Q0 25 8 4 Gunn 400 Bilirubin-tH 1.0 179,000 22 8 5 S.D. 460 Mesobilirubin-3H 1.9 137,000 369 76 6 S.D. 430 Mesobilirubin-'H 2.8 245,000 660 74 7 S.D. 400 Mesobilirubin-3H 4.4 336,000 906 70 8 S.D. 460 Mesobilirubin-8H 1.5 46,000 124 71 9 Gunn 400 Mesobilirubin-'H 1.5 101,000 272 2 10 Gunn 400 Mesobilirubin-'H 1.1 811,000 29 10 11 S.D. 340 Mesobilirubinogen-'H 0.7 513,000 3 74 12 S.D. 330 Mesobilirubinogen-'H 2.6 497,000 3 75 13 S.D. 330 Mesobilirubinogen-5H 0.3 11,654,000 70 85 14 S.D. 360 Mesobilirubinogen-'H 0.4 313,000 2 79 15 S.D. 460 Mesobilirubinogen-H 3.4 746,000 498 83 16 S.D. 450 Mesobilirubinogen-'H 3.4 4,753,000 3,146 95 17 Gunn 410 Mesobilirubinogen-H 0.9 780,000 10 92 18 Gunn 400 Mesobilirubinogen-'4C 1.4 197,000 1,900 88 19 S.D. 430 i-Urobilin-3H 1.2 1,020,000 310 69 20 S.D. 430 i-Urobilin-3H 1.7 272,000 333 72 21 Gunn 350 i-Urobilin- H 1.2 218,000 93 82 22 Gunn 400 i-Urobilin-3H 1.3 461,000 197 85 23 Gunn 420 i-Urobilin-3H 1.1 495,000 606 83 * Sprague-Dawley. 1842 ROGER LESTER AND PETER D. KLEIN normal rats was more rapid for mesobilirubino- CUMULATIVE % gen-3H and i-urobilin-3H than for bilirubin-8H ADM. RADIOACTIVITY EXCRETED IN BILE and mesobilirubin-3H. This observation is in ac- IOC ~Normal Rats cord with previous findings obtained during double label studies of the rates of of 80- relative excretion bilirubin-3H and mesobilirubinogen-14C (11). 60- Identification of excreted tetrapyrroles. After 40- administration of bilirubin-3H, 85 to 100% of the radioactive label appearing in bile could be hy- 20 Gunn RAots drolyzed and then crystallized as bilirubin-8H. Chromatography of the diazo pigment derivative 15 30 45 60 75 90 of this material established that 90 to 92%o was MINUTES AFTER IN. INFUSION OF MESOIBLIRUSIN-SiH present in bile in the form of conjugated bilirubin. FIG. 3. RADIOACTIVITY IN BILE AFTER INTRAVENOUS Comparable results were obtained after injection ADMINISTRATION OF MESOBILIRUBIN-8H. of mesobilirubin-3H. Seventy to 80% of the ex- In contrast to the findings with bilirubin and mesobilirubin, mesobilirubinogen-8H and i-uro- CUMULATIVE % ADM. RADIOACTIVITY bilin-3H were excreted readily by both normal EXCRETED IN BILE 4 and The and Gunn rats (Figures 5, Table I). lOC) r.1nA low cost and the high specific activity of mesobilirubinogen-3H made it possible to examine the 80 rate of hepatic excretion for an extremely wide 60 :: Normiun Rats range of doses. Within 90 minutes of intravenous injection, the normal rats excreted 74 to 95%, and 40 two Gunn rats 88 to 92% of microgram to milli- 20 gram amounts of mesobilirubinogen-3H.2 After / IIa administration of 310 to 333 ,ug of i-urobilin-3H 15 30 45 60 75 90 to two normal rats, 69 to 72% of the radioactive MINUTES AFTER W.V. INFUSION OF i-UROBILIN-SH dose appeared in bile, and three Gunn rats ex- FIG. 5. RADIOACTIVITY IN BILE AFTER INTRAVENOUS creted 82 to 85 % of a comparable dose. As shown ADMINISTRATION OF I-UROBILIN-3H. in Figures 2 to 5, the early phase of excretion in creted material was diazo reactive and could be a CUMULATIVE % crystallized from bile in mixture with endoge- ADM. RADIOACTIVITY nous bilirubin. When this mixture was chro- EXCRETED IN BILE matographed in system I, and compared with look crystalline samples of bilirubin-3H and mesobili- O.-- -- 80 le .- rubin-3H, more than 85% of the material applied of led lb 60 to the paper was identified as mesobilirubin-8H. 0 The diazo pigment derivative of excreted mesobili- 40 0-----o Normol Rots rubin was chromatographed in the system devised 0- - - 0 Gunn Rots 20 by Schmid for the chromatography of unconjugated and conjugated bilirubin (4). Eighty-eight I I I I a to of 15 30 45 60 75 90 92% the identifiable pigment migrated with MINUTES AFTER IV. INFUSION OF MESOBILIRUBINOGEN-H an Rf identical to that of bilirubin glucuronide. FIG. 4. RADIOACTIVITY IN BILE AFTER INTRAVENOUS Control markers of the diazo derivative of uncon- ADMINISTRATION OF MESOBILIRUBINOGEN-8H. jugated mesobilirubin migrated at the same rate as the diazo derivative of unconjugated 2 Data from one Gunn rat study performed with mesobilirubin. bilirubinogen-14C and described previously (10) are After incubation of the material presumed to be included. the diazo derivative of conjugated mesobilirubin BILE PIGMENT EXCRETION 11843 with f8-glucuronidase, all of the identifiable pig- The techniques for the isolation and identifica- ment migrated with an Rf identical to that of un- tion of mesobilirubinogen and i-urobilin are by no conjugated bilirubin and unconjugated mesobili- means as satisfactory as those employed for bilirubin. rubin. Whereas the availability of labeled com- In bile samples taken from rats 15, 16, and 18 pounds permitted accurate assessment of rates of given intravenous mesobilirubinogen-3H, 95 to excretion, identification of excreted metabolites 103% of the administered radioactive dose was was limited to those experiments in which more present in the form of Ehrlich-reactive chromogen. than 150, Lg of mesobilirubinogen-3H or i-urobilin- Seventy to 84% of the Ehrlich-reactive chromogen 3H was administered. It should be emphasized could be extracted from acidified bile into pe- that, because of the limitations of methodology, troleum ether: ethyl ether (9: 1). This recovery we made no attempt to identify minor fractions equaled that obtained in three control experiments, of labeled compounds excreted in the bile. Our in which crystalline mesobilirubinogen-3H was dis- studies do not exclude the possibility that some solved directly in rat bile and extracted into pe- mesobilirubinogen or i-urobilin is excreted conju- troleum ether-ethyl ether. Thirty to 45%o of the gated with glucuronide or other sugar moieties as radioactivity in bile samples treated with ferric has been suggested previously (21). chloride and hydrochloric acid could be crystal- Within these limitations, we established that the lized as urobilin. Chloroform extracts of acidi- major proportion of mesobilirubinogen was ex- fied bile obtained from experiments 15, 16, and 18 creted in bile intact and unaltered. The excreted (Table I) were chromatographed in systems II material was Ehrlich reactive and could be oxi- to IV, and, because of oxidation of mesobilirubino- dized to urobilin. That most of this material was gen to urobilin on exposure to air and the solvent not mesobilirubinogen glucuronide was indicated system, the point of migration could be identified by the fact that two-thirds to three-quarters could by spraying the chromatogram with methanolic be extracted into the nonpolar solvent petroleum zinc acetate. The Rf obtained on chromatography ether-ethyl ether (9: 1). Mesobilirubinogen ex- of bile extracts equaled the Rf of control samples tracted from the bile of rats receiving the tritiated of crystalline (unconjugated) mesobilirubinogen chromogen intravenously migrated chromatograph- and were comparable to values previously re- ically at the same rate as crystalline (unconju- ported for i-urobilin (18). gated) mesobilirubinogen. Finally, when meso- Similar results were obtained with i-urobilin-3H. bilirubinogen-3H was administered to Gunn rats, Fifty'to 60% of the radioactivity appearing in bile 88 and 92% of the dose was excreted in bile within could be extracted into chloroform and identified 90 minutes of injection. Although it is conceiv- spectrophotometrically and by crystallization as able that Gunn rats have a normal complement of urobilin. When the chloroform extracts of bile "mesobilirubinogen glucuronyl transferase," the obtained in experiments 19, 20, 22, and 23 (Table possibility appears remote. I) were developed chromatographically in solvent The relative stability and ease of crystallization systems II to IV, the Rf obtained matched the of i-urobilin permitted a more direct examination Rf of control i-urobilin markers. of the mechanism of its excretion. After intravenous administration of the tritiated pigment, ap- Discussion proximately one-half of the material appearing in the bile could be identified spectrophotometrically On the basis of earlier reports and the results and by crystallization as intact i-urobilin. Pig- described above, it can be concluded that signifi- ment extracted from bile migrated chromato- cant differences exist in the mechanisms by which graphically in three solvent systems with Rf iden- bile pigments and their derivatives are excreted. tical to those of crystalline i-urobilin. As with On the one hand, bilirubin is excreted by the liver mesobilirubinogen, Gunn rats excreted three-quar- as the diglucuronide (3-5); on the other, the ex- ters of the administered i-urobilin-8H over a dose periments described above suggest that both meso- range of 93 to 606 /Lg. In fact, during the period bilirubinogen and i-urobilin are excreted, at least from 30 to 90 minutes after injection, Gunn rats in part, as the intact unconjugated tetrapyrrole. appeared to excrete i-urobilin-3H more effectively 1844 ROGER LESTER AND PETER D. KLEIN than normal rats, and a similar but less pronounced bilirubin the second and eighth side chain groups trend was observed in studies of mesobilirubino- are unsaturated (vinyl), whereas in mesobiliru- gen-3H excretion (Figures 4 and 5). The ap- binogen and i-urobilin these side chains are satu- parent lower rate of excretion among normal rats rated (ethyl). The intermediate compound meso- might be explained as the result of competitive in- bilirubin appeared to provide a test of whether the hibition of mesobilirubinogen and i-urobilin ex- presence of unsaturated outer bridges determines cretion by endogenous bilirubin (22). Com- which tetrapyrroles form glucuronides, or whether petitive inhibition would be of a much lower order unsaturation of the side chains is the determining of magnitude in Gunn rats, which are incapable of factor. Mesobilirubin cannot be isolated from bio- excreting appreciable quantities of bilirubin (8). logic materials (17) but can be synthesized by the The limited availability of Gunn rats prevented the catalytic reduction of bilirubin in vzitro (12a). The performance of an adequate number of experi- vinyl side chains of bilirubin are saturated by the ments for valid statistical confirmation of this reduction, but the outer carbon bridges remain un- trend, but it is clear that Gunn rats excrete meso- saturated (Figure 1). bilirubinogen and i-urobilin at least as effectively The pattern of excretion of mesobilirubin-3H as normal rats. was strikingly similar to that of bilirubin-8H. In view of their apparent similarity (Figure 1), Excretion by Gunn rats was defective, and nor- it is somewhat surprising that bilirubin is excreted mal rats excreted mesobilirubin-3H as the glu- almost exclusively as the glucuronide, whereas the curonide conjugate. This result would be pre- major fraction of mesobilirubinogen and i-urobilin dicted if it were assumed that the conjugation of is excreted without conjugation. The side chains tetrapyrroles depends upon the presence or ab- of each of the tetrapyrroles are in so-called IXa sence of unsaturation in bridges a and c. It sequence (14d), and a carbonyl group is attached should be noted that the results do not exclude to the free a position of each of the outer pyrrole the possibility that, rather than unsaturation at rings. The formula of each tetrapyrrole differs positions a and c being significant, the point of from the others only in the degree of saturation of differentiation between the two groups of com- its double bonds. Each is weakly acidic at physio- pounds is the presence of two unsaturated bridges logic pH; with the exception of i-urobilin, each is per se. Thus, it would be necessary to administer maximally soluble in chloroform and minimally labeled tetrapyrroles with a,b or b,c unsaturation soluble in water. Bilirubin and i-urobilin have (e.g., mesobiliviolin, mesobilierythrin) to prove chromophoric centers consisting of systems of that the a,c configuration is specific. Unfortu- resonating conjugated double bonds. Bilirubin nately, the structure of these compounds and their and mesobilirubinogen have four protonated pyr- preparative chemistry have not, at present, been role nitrogens, and i-urobilin has three. The developed sufficiently to permit purification and physiologic behavior of the members of the series physiologic investigation. has several features in common. After intravenous Any explanation of the relationship between the injection, each is cleared rapidly from the plasma, conjugating mechanism and the presence of taken up by the hepatic parenchyma, and secreted methyne a,c bridges must remain speculative. at high concentrations in bile. Nevertheless, one can distinguish several major Despite the apparent similarities, however, an effects of unsaturation at these positions on the re- examination of the molecular formulas shown in sultant pigment and pigment derivatives. By Figure 1 reveals that essentially two differences constructing Dreiding molecular models of the exist between the depicted structures of bilirubin, tetrapyrrole series one can readily demonstrate on the one hand, and mesobilirubinogen and i-uro- that the limitation of rotation introduced by un- bilin on the other. In the former, the inner and saturation of these bridges (as in bilirubin and outer pyrrole rings are joined by unsaturated mesobilirubin) results in a marked degree of (methyne) carbon-carbon linkages [bridges a and molecular "rigidity." Since free rotation can c according to accepted nomenclature (14e, 23)], occur around saturated carbon-carbon bonds, how- whereas in mesobilirubinogen and i-urobilin these ever, molecular models of mesobilirubinogen and positions are saturated (methene). Secondly, in i-urobilin exhibit a much higher degree of flexi- BILE PIGMENT EXCRETION 1845 bility. Several lines of evidence suggest that in- accepted that bilirubin is conjugated by esterifica- ternal hydrogen bonding occurs in some or all of tion of its propionic acid carboxyls (28). On the molecules of the series. The rigidity intro- the other hand, final proof of this assumption by duced by methyne a,c bridges would be expected the isolation of bilirubin glucuronide and direct to influence the ease with which hydrogen bonds analysis of its structure has not yet been advanced. form between the side chain carboxyls and the hy- When a comparison is made to steroid conjugates, drogen atoms attached to ring nitrogens (24), or the extreme instability of conjugated bilirubin between the carbonyl oxygens of the outer rings more closely resembles an enol- than an ester- and the hydrogen atoms of the ring nitrogens glucuronide (29). Unsaturation of the a,c bridges (25). Moreover, the presence of unsaturation at extends the conjugation of the double bond system positions a,c should further influence hydrogen to include both the outer and inner pyrrole rings. binding at the carbonyl oxygen atoms by extend- Because of this and because of their closer prox- ing the system of resonating conjugated double imity to the carbonyl (keto-enol) group than to bonds to include both the inner and the outer pyr- the propionic acid side chains, the effect of un- role rings. By effecting changes in internal (or saturation of bridges a and c is more likely to be external) hydrogen bonding, the presence of on the former. Our evidence that alterations in methyne or methene bridges might further modify these bridges influence the mechanism of conjuga- molecular configuration and thereby influence the tion suggests that the question of the site of bili- biologic behavior of individual tetrapyrroles. rubin conjugation should be re-examined. It is tempting to speculate that the configura- tional differences conferred by saturation or un- Summary saturation of the a,c bridges influence the "fit" of The mechanism of excretion of four tetrapyr- the tetrapyrrole to the secretory carrier. The roles-bilirubin, mesobilirubin, mesobilirubinogen, more flexible molecules (mesobilirubinogen and and optically inactive (i)-urobilin-was studied in i-urobilin) might fit the secretory carrier directly, normal and Gunn rats. For this purpose 3H-la- whereas the more rigid members of the series beled tetrapyrroles were prepared, and their radio- (bilirubin and mesobilirubin) might have to be chemical purity was established. On intravenous modified by conjugation in order to permit a fit. administration, each was rapidly excreted in bile On the other hand, the physical-chemical charac- by normal rats, but only mesobilirubinogen and teristics of all four tetrapyrroles are altered by the i-urobilin were excreted in substantial quantities by degree of saturation of the outer bridges. Those Gunn rats. In the normal species studied, 90% of tetrapyrroles that are saturated at positions a,c the bilirubin and mesobilirubin excreted was in have lower melting points and are more water the form of the glucuronide conjugate. In con- soluble than the other members of the series. trast, a major fraction of excreted mesobilirubino- Rather than specifically modifying secretory car- gen and i-urobilin appeared in the bile intact rier fit, the physical-chemical changes introduced and unaltered. by alteration of the outer bridges may influence the These findings suggest that glucuronide conju- mechanism of excretion by limiting penetration of gation of bile pigments may be governed by a the molecules to the site of the carrier or by in- specific molecular configuration: unsaturation of fluencing other stages in the complex excretory the outer (a,c) tetrapyrrole bridges. process. To determine the relative contribution of each of these factors, we are now studying the Acknowledgments excretion of other polypyrrolic compounds. The authors wish to thank Dr. Rudi Schmid and Dr. One additional observation should be made on Sidney R. 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