Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats.

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.     

Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.     

Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.     

Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates.

Caged dideoxyribosylthymine triphosphate, dideoxyadenosine triphosphate and arabinosylcytosine triphosphate were prepared in high yield by reaction with 1-(2-nitrophenyl)diazoethane at pH 4 and room temperature for 24 h. Synthesis of caged alpha-32P-labelled dideoxyadenosine triphosphate (approx. 5000 Ci/mmol) in 85% yield was achieved by a modification of the method used for the synthesis of the unlabelled compounds. ATP was shown to be an excellent buffer in the synthesis of alpha-32P-labelled material, and in caged form to be an effective carrier in h.p.l.c. purification. Preparative h.p.l.c. was used to achieve purification of unlabelled caged compounds to greater than 98% purity and 32P-labelled material to 97% purity. Photolysis of unlabelled and 32P-labelled caged compounds by using XeF-excimer laser irradiation at 351 nm was characterized by using difference spectrophotometry and h.p.l.c. analysis. The stability of caged dideoxyadenosine [a-32P]triphosphate in the presence of cultured mammalian cells was evaluated; the adenosine derivative is essentially stable for 1 h.     

Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.     

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Wavelength-dependence of the relative rate constants for the main geometric and structural photoisomerization of bilirubin IX alpha bound to human serum albumin. Demonstration of green light at 510 nm as the most effective wavelength in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha via (EZ)-bilirubin.

The kinetics for the quantitatively important reaction: (Formula: see text) that is, the photochemical interconversion between bilirubin and its geometric and structural photoisomers bound to human serum albumin in aqueous solution when various wavelengths of monochromatic light were used, were assayed by h.p.l.c. In order to clarify the wavelength-dependence of the relative rate constants in the individual steps, a light-source with a half-bandwidth of 10 nm was used at increments of 20 nm, in the range from 410 nm to 550 nm. We describe for the first time studies on the wavelength-dependence of rate constants in geometric and structural photoisomerization reactions in vitro of (ZZ)-bilirubin or (EZ)-bilirubin bound to human serum albumin, especially the relative rate constants of cyclization of (EZ)-bilirubin into (EZ)-cyclobilirubin. Because studies in vitro have demonstrated that the wavelengths from 350 to 450 nm are mutagenic, the results obtained indicated that the safest and ideal light-source for phototherapy is green light of 510 nm, which keeps (ZE)-bilirubin concentrations as low as possible, as shown by a maximal value of k2 at 510 nm and a relatively low value of k1 at 510 nm. This light-source still ensures the substantial absorption of (ZZ)-bilirubin, which is the precursor of (EZ)-bilirubin, the intermediate in (EZ)-cyclobilirubin formation and, furthermore, as shown by the maximal value of k5 and a considerable value of k4 at 510 nm, promotes the cyclization of (EZ)-bilirubin derived from (ZZ)-bilirubin even though k3 at 510 nm also shows a peak value.     

Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.     

The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.     

The Biological Effects of Bilirubin Photoisomers

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.     

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.     

Detection of radical species in haematin-catalysed retinoic acid 5,6-epoxidation by using h.p.l.c.-e.p.r. spectrometry.

E.p.r. signals were detected in an all-trans-retinoic acid/haematin incubation mixture by using an e.p.r. spin-trapping technique. The spin adducts are presumably attributable to some intermediates in haematin-catalysed retinoic acid 5,6-epoxidation, since addition of nitrosobenzene to the reaction mixture dose-dependently inhibited the epoxidation. Analysing the reaction mixture by h.p.l.c.-e.p.r. spectrometry resulted in the detection of three peaks (III-1, III-2, IV) ascribable to the radical species. Two (peaks III-1 and -2) of the three peaks, which appeared 10 min after the reaction had started, seem to be attributable to the radical species directly participating in the epoxidation. The radicals trapped by nitrosobenzene do not appear to be derived from active oxygen, since none of these peaks were detected in a similar h.p.l.c. analysis of O2- and OH.-generating systems. They are presumably derived from retinoic acid. This view is also supported by the following results: none of these peaks were detected in the h.p.l.c. elution profile of the reaction mixture when retinoic acid was absent; peaks III-1 and 2 were detected even under anaerobic conditions, and their peak heights were unchanged under aerobic conditions.     

用组织光学特性鉴别诊断人离体的正常膀胱和膀胱癌组织

研究正常人膀胱和膀胱癌组织在Kube lka-Munk二流模型下对476.5 nm,514.5 nm和808 nm波长的激光的光学特性的差异。采用双积分球系统和Kube lka-Munk二流模型进行测量研究。实验结果表明,正常膀胱和膀胱癌组织在Kube lka-Munk二流模型下对476.5 nm,514.5 nm和808 nm波长的每一个波长的激光的吸收、散射、总衰减、有效衰减系数都有非常显著性的差异(P<0.01)。膀胱癌组织对476.5 nm,514.5 nm和808nm波长的激光的吸收系数明显地较正常膀胱组织对相应波长的激光的吸收系数要大(P<0.01),膀胱癌组织对476.5 nm和514.5 nm波长的激光的散射系数明显地较正常膀胱组织对相应波长的激光的散射系数要小(P<0.01),而膀胱癌组织对808 nm波长的激光的散射系数明显地较正常膀胱组织对同一波长的激光的散射系数要大(P<0.01)。膀胱癌组织对476.5 nm,514.5 nm和808 nm波长的激光的总衰减系数明显地较正常膀胱组织对相应波长的激光的总衰减系数要大(P<0.01),膀胱癌组织对476.5 nm,514.5 nm和808 nm波长的激光的有效衰减系数明显地较正常膀胱组织对相应波长的激光的有效衰减系数要大(P<0.01)。提示使用双积分球系统和Kube lka-Munk二流模型来确定离体的正常膀胱组织和膀胱癌组织对476.5 nm,514.5 nm和808nm波长的激光的光学特性的差异鉴别诊断病变的膀胱组织是一个有效的方法。     

The non-fluorescence of 4-fluorotryptophan.

The derivative 4-fluorotryptophan was confirmed to have negligible fluorescence at 25 degrees C and 285 nm (tryptophan/4-fluorotryptophan quantum-yield ratio greater than 100:1). However, photolysis experiments on tryptophan and 4-fluorotryptophan, in which loss of starting material was measured by reverse-phase h.p.l.c., demonstrated that 4-fluorotryptophan was significantly more photochemically active than the parent tryptophan, with the 4-fluorotryptophan photolysis quantum yield being 7 times larger than that of tryptophan at 25 degrees C and 285 nm. In addition, at 77 K and 275 nm 4-fluorotryptophan displayed strong fluorescence and phosphorescence, with emission quantum yields comparable with those of tryptophan at 77 K and 275 nm.     

Structural comparison of two esterases from Drosophila mojavensis isolated by immunoaffinity chromatography.

Antibodies raised against esterase-4 and esterase-5 from Drosophila mojavensis were coupled to Protein A-Sepharose CL-4B to prepare high-efficiency immunomatrices used for their purification. Final purification was achieved by anion-exchange h.p.l.c., in the case of esterase-5 followed by gel-filtration h.p.l.c. The resultant esterase preparations were homogeneous, as judged by gel-filtration h.p.l.c., SDS/polyacrylamide-gel electrophoresis and non-denaturing gel electrophoresis. Esterase-4 and esterase-5 are the products of a duplicated gene. They are differently localized in the insect's body and expressed in different periods during development. Although both enzymes exhibit little immunological cross-reactivity, their amino acid compositions show few significant differences and their N-terminal sequences are largely identical, which clearly show their common origin.     

Bilirubin glucuronyltransferase. Specific assay and kinetic studies

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.     

Isolation and analysis of human parathyrin in parathyroid tissue and plasma. Use of reversed-phase liquid chromatography.

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1--84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.     

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.