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In this paper the production of the R/APfd-j/j strain which is congenic with the R/APfd strain is reported. The R/APfd-j/j completely lacks hepatic bilirubin UDP-glucuronyltransferase activity, as do our GUNNXR/Pfd-j/j rat strain and various other stocks of GUNN rats (j/j) described in the literature. Our recombinant inbred strain GUNNXR/Pfd-j/j was produced from non-inbred GUNN (j/j) rats. This GUNNXR/Pfd-j/j rat was used as a donor of the jaundice gene j, the R/APfd rat serving as the recipient. After eight backcross-intercross cycles (16 generations) the R/APfd-j/j strain was obtained which is congenic with the R/APfd strain. Congenicity was demonstrated by various techniques including transplantation of skin tissue, strain-specific tumour cells and hepatocytes, the mixed lymphocyte reaction, and comparison of biochemical markers. The potential of the novel inbred strain of jaundiced rat, R/APfd-j/j, and the corresponding control strain R/APfd for biochemical and clinical studies of bilirubin metabolism are briefly discussed.

A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.

Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.

1. In aqueous solution above pH7 bilirubin-IXalpha 1-O-acylglucuronide rapidly isomerizes to the non-C-1 glucuronides by sequential migration of the bilirubin acyl group from position 1 to positions 2, 3 and 4 of the sugar moiety. The transformations are enhanced by increasing the pH. Compared with the rates at 37 degrees C the transformations are rather slow at 0 degrees C. Virtually complete inhibition is observed at values below pH6. The isomerization at 25 degrees C and pH 7.4 is not affected by the presence in the solutions of a molar excess of human serum albumin. 2. Isomerization in bile kept at 37 degrees C at pH7.7-7.8 is probably non-enzymic, as the rates of change are similar to those observed under comparable conditions for aqueous solutions of glucuronides of bilirubin-1Xalpha and of azodipyrrole. 3. Analysis without delay of normal biles of man and rats collected at 0 degrees C over a maximum period of 10 min shows that the bilirubin-IXalpha mono- and di-glucuronides consist exclusively of the 1-O-acyl isomers. 4. The mixtures of the four positional isomers of bilirubin-IXalpha glucuronide found in freshly collected biles of man and rats with cholestasis probably originate from initially synthesized 1-O-acylglucuronide by the same mechanism of sequential migration as has been observed in aqueous solutions of conjugated bilirubin-IXalpha.

Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were critically dependent on the presence of UDP-glucuronic acid. Pretreatment of the animals with phenobarbital stimulated both reactions. When 33 microM bilirubin was incubated with microsomal preparations from phenobarbital-treated rats, 80-90% of the substrate was converted to bilirubin glucuronides; the reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or when the substrate concentration was increased to 164 microM bilirubin, proportionally more BMG and less BDG were formed. Homogenate and microsomes from homozygous Gunn rats neither synthesized BMG nor converted BMG to BDG. These findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically decreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17) activity, proportionally more BMG and less BDG are excreted in bile.

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

The ß, ? and d isomers of bilirubin-IX show a decreased stability as compared with the IXa isomer; characteristic mass spectra are obtained only for the tetrakis-(trimethylsilyl) derivatives of the IXa and IX? isomers. Hydrogenation of the vinyl substituents increases the thermal stability of the bilirubins and gives rise to a characteristic mass spectrum for the tetrakis-(trimethylsilyl) derivative of meso-bilirubin-IXd. The ethyl anthranilate azopigments derived from the four bilirubins yield characteristic mass spectra, except for the two unstable divinyl substituted azodipyrroles (mol.wt. 416), derived from bilirubin-IXß and IXd. The corresponding mol.wt. 420 azopigments derived from the hydrogenated bilirubins are thermally stable. Elucidation of mass spectral fragmentation pathways is facilitated by the varying positions of the substituents and by deuterium labelling, and permits the assignment of structures to the various isomeric azodipyrroles.

A recently developed reverse-phase high-performance liquid chromatography procedure was applied to the analysis of bilirubins present in serum of neonates. In normal newborns, unconjugated (UCB) and conjugated bilirubins (CB) rose in parallel from day 2 to day 4: the average conjugated fraction remained between 1.2 and 1.6% of total pigment, significantly below the 3.6% documented in adults. However, the absolute concentration of conjugates was 2- to 6-fold higher than the serum levels in adults. Bilirubin conjugates were detected in all samples, but only 20% of these were bilirubin diconjugates, in contrast to a mean of 54% in the adult population. The increased absolute concentrations of UCB and CB together with the decreased fractional amount of conjugates and with the decreased diconjugates seem to be the result of an increased bilirubin production combined with a relative deficiency of the conjugation in the newborn. The bile pigment analysis did not point to a defective biliary secretion. Serum bile acids were high but did not correlate with the levels of bilirubin conjugates. The pattern of serum bilirubins was not different in 4 newborns with hemolytic syndromes. Cholestasis occurred in another child with hemolysis; this was evident from the high concentration and the high fractional amount of conjugates and from the high serum bile acids level.

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.

The biliary excretion of the four isomers of bilirubin-IX was studied in Wistar rats (JJ) and homozygous Gunn rats (jj). Synthetic preparations of 14C-labelled pigments were used. 1. After intravenous administration, the alpha-isomer was rapidly excreted in conjugated form in bile of Wistar rats. In Gunn rats excretion was insignificant. In contrast, both rat species promptly excreted the non-alpha-isomers at rates that were comparable with that found for bilirubin-IXalpha in Wistar rats. 2. In normal rats about 16% of the beta- and delta-isomers and at least 50% of the gamma-isomer were excreted as ester conjugates of the injected parent bile pigments. Conjugation of the beta- and delta-isomers had occurred exclusively at the carboxyl groups of pyrrole ring D and C respectively. For bilirubin-IXgamma no preference for any carboxyl group could be established. 3. In homozygous Gunn rats the non-alpha-isomers were apparently excreted chemically unaltered. This suggests that, as for bilirubin-IXalpha, conjugation of the non-alpha-isomers is also deficient in Gunn rats.

Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX α differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXβ allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXα- and non-α isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.