Sulfobromophthalein (BSP) is normally cleared from the blood by the liver and excreted into the bile. Brauer, Krebs and Pessotti (1) originally reported that part of the pigment appearing in dog bile was chromatographically different from the injected dye. Our studies using differenlt chromatographic techniques indicate that in man the bulk of the recovered pigments (65 to 75 per cent, according to estimates based on their absorption at 575 nu) consists of two metabolites. Similar metabolites are normally found in the urine and trace amounts also appear in the serum. The absorption spectra in the visible range and the indicator characteristics of BSP and the metabolic products were indistinguishable (2).The Isolationt of the cruide mtetabolites. In initial experimenits, the metabolites were isolated as follows. The total BSP pigments were extracted from bile with three volumes of acetone. The acetone extract was taken to dryniess ini vacuo, then dissolved in water (approximately 0.2 ml. per ml. of initial bile). The solution was chromatographed on Whatman 3 MM paper in an ascending system employing tert-butanol-water (1.73: 1 v/v) for six to 15 hours. The BSP bands were developed with ammonia fumes (or if permanent records were desired, by dipping the paper in a fresh saturated solution of sodium hydroxide in 90 per cent ethanol). After the position of the metabolites had been determined with ammonia, the bands were cut out and individually eluted with water. The resultant solutions were then taken to dryness.Putrification of the inetabolites. Purified pigments were prepared by chromatography in three systems. Activated alumina (Alcoa, 80 to 200 mesh) was washed 10 times in two volumes of water and once in acetone. During each washing period, the larger particles were allowed to settle for one minute; the supernatant was then decanted and discarded. The dried alumina was suspended in a loading solution consisting of two volumes of acetone to one volume of water. Columns, 3.4 cm. (outer diameter) by 6 cm., were prepared by gravity packing.The dried acetone extract from 60 ml. of bile was sus-1981
Physiologic amounts of bilirubin-H3 were injected into normal rats and the distribution of isotope was determined in serum, subcellular fractions of the liver, and bile at intervals during the phases of uptake and excretion. Recovery in the three spaces after 2, 5, 15, and 30 min was 75–85%, indicating little bilirubin entered the extrahepatic tissues. At 5 min, 50% of the dose (excluding trapped serum bilirubin-H3) was in the liver, a bilirubin concentration greater than in serum; biliary excretion of bilirubin was less than 3%. By 30–60 min, most of the label was excreted in the bile as intact, diazotizable and crystallizable bilirubin. Bilirubin-H3 appeared principally in the hepatic cell sap at a rate and concentration too high for an albumin-linked transfer. No differences in the distribution of isotope in subcellular fractions were detectable during the phases of uptake or excretion or when the quantity injected was increased 20–40 times. Thus, intracellular distribution was primarily a constant function of intracellular bilirubin concentration. When compared to in vitro experiments, significant amounts of bilirubin accumulated within the microsomes and the acid hydrolase-rich fraction (lysosomes) but not in the mitochondrial or nuclear fractions.
The quantum yields for photochemical addition of glycine and the L-amino acids commonly occurring in proteins (excluding proline) to polyuridylic acid have been determined in deoxygenated phosphate buffer at pH 7, using a tluorescamine assay technique. All twenty amino acids were found to be reactive. with cysteine. tryptophan, phenylalanine, tyrosine, arginine, lysine and methionine being the most reactive. The analogous quantum yields for a series of eighteen dipeptides of the form glycyl X (X being one of the commonly occurring amino acids, including proline), of L-alanyl-L-tryptophan, of the tripeptides L-seryl-L-seryl-L-serine and L-threonyl-L-threonyl-L-threonine, of the tetrapeptide 1.-cystine-bis-glycine, and of the lysine derivative N"-acetyllysine were also measured. All were found to be reactive toward photoaddition to poly U.
Sulfobromophthalein (BSP) clearance hasproved a sensitive index of hepatic function. Although dosage, sampling intervals and techniques of measurement have been modified (1)(2)(3)(4), the procedure remains essentially as originally described (5, 6). Basically it conisists of the intravenous injectioni of a stanidard amiiount of BSP and determination of residual BSP in the plasma from blood samples collected at prescriled intervals.Since clearanice of BSP is (lecreased 1)oth in hlepatocellular dysfunction anid biliary obstruction, its determination is of limited usefulness in differentiating these two conditions. Recent studies, however, have shown that BSP is conjugated in the liver and excreted primarily as conjugates into the bile and to a lesser extent into the urine (7-11). Chemical analysis indicates that the dye is conjugated witlh cysteine and possibly with the peptide, glutatlhione (12). These metabolites are colorimetrically indistinguishable from free BSP. The demonstration that sulfobromophthalein is conjugated by the liver suggests the following possibilities: 1) where uptake or hepatic conjugation of BSP is impaired due to parenchymal dysfunction, the circulating dye should remain in the unconjugated form; 2) in biliary obstruction, where conjugation of the dye proceeds normally but secretion from the liver is impaired, increasing amounts of conjugated dye should appear in the blood. If such is true, the determination of the metabolized BSP, in addition to total BSP, in serum miglht serve to distinguish impairment of uptake or conjugation from intrahepatic or extrahepatic biliary obstructioln.In this study the levels of free and conijugated * Supported by a grant (A-2455) from the National Institutes of Health, United States Public Health Service, Bethesda, Md. BSP wvere investigated in sera and urine from nornmal subjects and patients with hepatic disease. SUBJECTS AND METHODSThe 87 subjects selected for study consisted of 17 patients with normal liver functioni, 10 patients in whom liver funictioni tests and clinical work-up (including liver biopsy wheni feasible) indicated viral hepatitis, 33 patients with portal cirrhosis and varying degrees of hepatic decompenisationi, 10 patients with extrahepatic obstruction as indicated by liver function studies and coInfirmed by surgical explorationi, anid 17 patients initially with niormal liver functioni who were given methyltestosterone (67 mg. per day) for 14 days.The following liver function tests were carried out in each group: total serum protein and albumin (13); alkaline phosphatase (14) (upper limit of normal in our laboratory is 8 units) ; one minute and total bilirubin (15) and 45 minute BSP retentioni (16). In addition, serum glutamic oxalacetic transaminase levels (17) were determined in the subjects treated with methyltestosterone.The circulating level of BSP metabolites was determined in fasting subjects as follows. Commercial BSP (5 mg. per Kg. body weight) was injected intravenously. Blood was drawn from the opposite arm at specific int...
The photochemical quantum yields for addition of glycine and the L-amino acids commonly occurring in proteins (excluding proline) to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer at A 254 nm and pH 7, using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid.The quantum yields for photoaddition of eighteen dipeptides of the form glycyl X (X being one of the amino acids commonly occurring in proteins, including proline), and of L-alanyl-L-tryptophan, L-seryl-L-seryl-L-serine, L-threonyl-L-threonyl-L-threonine, L-cystine-bis-glycine. and N"-acetyllysine to polyadenylic acid, polycytidylic acid and polyguanylic acid were measured. All of these were found to add photochemically to each of these polymers. Polyribothymidylic acid, tested with eleven of these peptides and with N"-acetyllysine, was found to be reactive with all.
Sulfobromophthalein (BSP) was injected into rats, and the livers were excised at timed intervals. The ratio of free to mercaptide-conjugated BSP in the liver was determined after extraction and chromatographic separation. After 2 minutes, 12.5% of the total dose was concentrated in the liver; of this, 65% was free BSP. At 5 minutes, 33% of the total dose appeared in the liver, of which approximately 50% was still in the free form. Increasing concentrations of BSP were infused or administered as a single injection, and free and conjugated pigment appearing in the bile was measured. The maximum secretion rate for free BSP was 3–7 µg/ min/gm liver. Maximum capacity for secretion of conjugates was not reached, even when 60 mg BSP/kg body weight was administered. When purified metabolites were injected, they were rapidly secreted into the bile. It was concluded that uptake of BSP from the blood is a dynamic process which precedes and is relatively independent of the conjugation step. In contrast, conjugation may be the rate-limiting step in determining the secretion rate of the pigment from the hepatic cell.
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