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Hemoglobin digestion in the midgut of hematophagous animals results in the release of its prosthetic group, heme, which is a pro-oxidant molecule. Heme enzymatic degradation is a protective mechanism that has been described in several organisms, including plants, bacteria, and mammals. This reaction is catalyzed by heme oxygenase and results in formation of carbon monoxide, ferrous ion, and biliverdin IXalpha. During digestion, a large amount of a green pigment is produced and secreted into the intestinal lumen of Aedes aegypti adult females. In the case of another blood-sucking insect, the kissing-bug Rhodnius prolixus, we have recently shown that heme degradation involves a complex pathway that generates dicysteinyl-biliverdin IX gamma. The light absorption spectrum of the Aedes purified pigment was similar to that of biliverdin, but its mobility on a reverse-phase chromatography column suggested a compound less hydrophobic than biliverdin IXalpha. Structural characterization by ESI-MS revealed that the mosquito pigment is the alpha isomer of biliverdin bound to two glutamine residues by an amide bond. This biglutaminyl-biliverdin is formed by oxidative cleavage of the heme porphyrin ring followed by two subsequent additions of glutamine residues to the biliverdin IXalpha. The role of this pathway in the adaptation of this insect vector to a blood-feeding habit is discussed.

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When the larvae of a saturniid silkmoth, Antheraea yamamai, are maintained under high intensity light (5000 lux), they produce green cocoons whereas the cocoons produced under light of low intensity (e.g., 50 lux) or in darkness are yellow. The green colour of the cocoon is due to the presence of a blue bilin pigment in combination with yellow pigment, and light stimulates the accumulation of blue bilin. In the present study, we show that two blue bilins, with similar characteristics to the sarpedobilin in the green cocoon, can be induced in larval haemolymph both in vivo and in vitro. In both conditions, the amount of these bilins increased with increasing intensity or duration of light exposure. Induction also occurred at 0 degrees C. In contrast, the chromophore of the constitutive biliprotein of the haemolymph did not change depending on light conditions. Size fractionation of the haemolymph indicates that the precursor of the blue bilins induced by light is bound to a protein with a molecular mass of 5000 Da or more. Thus, in these insects, the blue bilin responsible for green colouration is facultative under photochemical stimulation.

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Seven bilirubin metabolites negative to the diazo reaction were identified in the urine of healthy persons by enzyme-linked immunosorbent assay (ELISA) using the anti-bilirubin monoclonal antibody 24G7. Two of the seven metabolites were isolated and their chemical structures were determined using fast-atom bombardment-mass spectroscopy (FAB-MS) and 1H-NMR. The two metabolites are 1,14,15,17-tetrahydro-2,7,13-trimethyl-1,14- dioxo-3-vinyl-16H-tripyrrin-8,12-dipropionic acid and 1,14,15,17-tetrahydro-3,7,13-trimethyl-1,14-dioxo-2-vinyl-16H- tripyrrin-8,12-dipropionic acid. They are regioisomers of each other. The two bilirubin metabolites are novel tripyrrole biocompounds and belong to a third group of bile pigments following biliverdin and bilirubin. We gave these compounds the generic names biotripyrrin-a and biotripyrrin-b, respectively.

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i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)-methanol (25:75). The detection limit was 0.2 microgram/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250-2500 times more sensitive than conventional methods.

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The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.

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A method for improving the assay of beta-glucuronidase in hepatic and gallbladder bile is described. The method uses ion-pair extraction with N,N,N-triheptyl-1-heptanaminium bromide to remove pigments and bile acids. Conjugated bilirubin, unconjugated bilirubin, and taurine and glycine conjugates of deoxycholic and chenodeoxycholic acids are extracted efficiently from bile by the procedure. The sensitivity of the spectrophotometric assay of beta-glucuronidase in bile using phenolphthalein glucuronide is increased significantly.

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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.

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Aldosterone secretion rate (ASR) is utilized as a rapid screening procedure to detect subtle forms of hypertension. A rapid and robust chromatographic method has been developed, based on easily prepared, rigid matrices, which permit the flow of urine by suction through sequential purification columns. Both major metabolites, aldosterone glucuronate and tetrahydroaldosterone glucuronate, are isolated in ca. 75% yield. They are hydrolyzed and quantitated by high-performance liquid chromatography-radioimmunoassay (HPLC-RIA) or, for bulk preparation, further purified by preparative chromatofocusing. These two polar conjugates are isolated in nearly pure form by HPLC on a C2 column with a two-step gradient. An ASR determination can be completed in one to two working days; preparative-scale work takes somewhat longer.

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Mixed lipid micelles were isolated from rat bile on taurocholate-equilibrated Sephadex G100 and G200 columns (5-60 mM) to study relationships between lipids and other constituents of bile. Phospholipid, cholesterol, and a bile salt peak co-eluted as mixed micelles at all taurocholate concentrations. The micellar radius, derived from the elution profile, increased progressively from approximately 1.6 nm to approximately 3.5 nm when the column taurocholate concentration was reduced from 40-60 mM to 5 mM (the physiological range for rat bile). Biliary bile pigment and bromsulphthalein, added in vivo, eluted as self-aggregates that were smaller than the lipid micelles. In contrast, on bromsulphthalein-equilibrated columns, unconjugated bromsulphthalein associated weakly with lipid micelles but this association accounted for less than 10% of the unconjugated dye in bile. No associations were found between lipid and proteins when SDS-polyacrylamide gel electrophoretic polypeptide patterns of column fractions were compared with the lipid elution profiles at different taurocholate concentrations. Two high molecular weight protein aggregates were demonstrated in bile (greater than 222,000 Mr) by Sephadex G200 chromatography. These studies provide a reliable estimate of rat bile lipid micelle size and suggest that bile pigment, bromsulphthalein and proteins do not form strong associations with biliary mixed micelles but exist in bile predominantly as self-aggregates.

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Over a three-year period 10 patients were observed with acute and relapsing pancreatitis for which no conventional cause could be identified. In all 10 patients the gallbladders visualized on oral cholecystography and no stones were identified by cholecystography or ultrasonography but biliary drainage revealed aggregates of bile pigment granules. In eight of the 10 patients, cholecystectomy was eventually performed, recommended on the basis of the abnormal findings on biliary drainage. None of the eight patients who underwent cholecystectomy and followed for up to two years have had further bouts of pancreatitis. It is proposed that: 1. aggregates of bile pigment granules in the passage from gallbladder to duodenum can cause acute and relapsing pancreatitis; 2. preoperative identification of such aggregates can only be made by biliary drainage; 3. aggregates of pigment granules and cholesterol crystals should be looked for in the bile of all patients with pancreatitis without a clearcut etiology and 4. in the absence of other overt causes, the presence of crystals and pigment granules in bile of patients with pancreatitis justifies therapeutic cholecystectomy with a realistic expectation that pancreatitis will not recur after this operation.

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Brief reduction of bilirubin with dilute sodium amalgam was shown to give chromogens containing both vinyl and ethylidene beta-substituents. Acid-catalysed rearrangement of these chromogens with bilirubin or mesobilirubin and subsequent dehydrogenation gave a range of new violins containing unconjugated ethylidene and vinyl substitutents. Rearrangements between mesobilirubinogen, bilirubin and mesobilirubin gave dihydrobiliviolins, mesobiliviolins, biliverdins, dihydrobiliverdins and mesobiliverdins of the IIIalpha, IXalpha and XIIIalpha series. In this way 30 compounds were prepared, purified by t.l.c. as dimethyl esters, and characterized by n.m.r., mass and electronic spectroscopy, and by chemical interconversion and degradation.

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The blue-green bile pigments of Actias selene (Attacidae) have been investigated at different stages of its development. Coproporphyrinogen-14-C, protoporphyrin-IX3-H, and pterobilin-14-C, injected to larvae are metabolised into phorcabilin I, the main neopterobilin in this animal. It is concluded that phorcabilin I is a bile pigment of the IX gamma series and that pterobilin is its direct precursor. A method for the preparation of labelled protoporphyrin from quail egg-shell is reported.

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