High-performance liquid chromatography of bile acids.
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Animals↗
Biomedical subjects
W H Elliott
Publications and source records attributed to W H Elliott.
Bile acids. LXXVII. Large-scale preparation of 5 alpha-anhydrocyprinol from carp bile.
An improved method of preparation of 5 alpha-anhydrocyprinol from large quantities of carp bile is reported. The following materials were obtained by this method: 5 alpha-anhydrocyprinol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 alpha-cholestane 3 alpha,7 alpha,12 alpha,26-tetrol, an unidentified sterol from the neutral lipid fraction, allocholic acid and allochenodeoxycholic acid from the bile acid fraction. Yields of 5 alpha-anhydrocyprinol and bile acids vary in different batches. The identity and purity of these materials were verified by thin-layer chromatography, gas liquid chromatography and gas chromatography-mass spectrometry.
Animals↗
Electron microscopic studies on liver 5-aminolaevulinate synthase.
The structure of chick embryo liver 5-aminolaevulinate synthase has been examined by electron microscopic studies using negative staining. From the different projections of the enzyme particles observed in electron micrographs, a model for the enzyme molecule has been proposed. In this model, an enzyme molecule consists of two curved and identical subunits associated in opposite polarities. From the dimensions of an enzyme molecule subunit measured from electron micrographs, the relative molecular mass of each subunit is estimated to be 70 000.
5-Aminolevulinate Synthetase↗
Molecular cloning of hepatic 5-aminolevulinate synthase.
Hepatic 5-aminolevulinate synthase was induced in chick embryos by administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. A cDNA library was constructed from drug-induced liver mRNA and clones containing sequences coding for 5-aminolevulinate synthase were identified by hybrid-selected translation. The identity of these clones was confirmed by comparing DNA sequence data with the amino acid sequence of peptides from purified 5-aminolevulinate synthase. From Northern blot analysis the size of the mRNA for 5-aminolevulinate synthase was estimated to be 2800 base pairs, approximately 600 base pairs more than that required to code for the primary translation product of relative molecular mass 74000.
5-Aminolevulinate Synthetase↗
Effect of lead ions on chick-embryo liver mitochondrial delta-aminolaevulinate synthase.
Pb2+ activated native chick-embryo liver mitochondrial delta-aminoaevulinate synthase (EC 2.3.1.37). This result contradicted with the inhibitory effect observed by earlier workers who used degraded enzyme preparations. Enzyme activation was biphasic. An initial activation phase was observed with Pb2+ concentrations up to 200 microM, and a secondary phase with concentrations from 200 microM to at least 2mM. Maximum primary activation was 2.5-fold at 200 microM-Pb2+, with a further 2-fold activation observed at 2mM-Pb2+. Primary activation was not affected by a 10-fold molar excess of dithioerythritol, but the secondary activation was abolished by dithioerythritol. Secondary-phase activation was lost upon increasing time of incubation of the enzyme with Pb2+. The implications of these findings are discussed with reference to lead poisoning and the mechanism of delta-aminolaevulinate synthase.
5-Aminolevulinate Synthetase↗
Effect of heme on the activity of chick embryo liver mitochondrial delta-aminolevulinate synthase.
We have examined the effect of heme on the activity of native delta-aminolevulinate synthase isolated from drug-induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1 mM and this finding makes it improbable that heme acts physiologically to control mitochondrial delta-aminolevulinate synthase activity.
5-Aminolevulinate Synthetase↗
Bile acids. LXXIII. Synthesis of analogs of 7 alpha-hydroxy-4-cholesten-3-one as substrates for hepatic steroid 12 alpha-hydroxylase.
Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.
Animals↗
Serum concentrations of alpha-tocopherol after ingestion of various vitamin E preparations.
The rat-fetal-resorption test currently is used to assess the biological activity of vitamin E compounds. Previous studies in humans, however, suggest that rat assays underestimate the potency of free tocopherol relative to the acetate ester form and of RRR-alpha-tocopheryl acetate relative to all-rac-alpha-tocopheryl acetate. Therefore, we studied the serum concentrations of alpha-tocopherol after ingestion of RRR-alpha-tocopheryl acetate, all-rac-alpha-tocopheryl acetate, RRR-alpha-tocopherol, RRR-alpha-tocopheryl succinate, and RRR-alpha-tocopheryl acetate plus apple pectin by each of 20 adult human subjects. Measurements were made at 0, 8, 24, and 48 h after ingestion of 800 IU of the various preparations. The results at 24 h were representative of the differences observed. The mean increase in concentration of alpha-tocopherols (mg/g lipid) in 24 h was 71.2% after RRR-alpha-tocopherol, 63.3% after RRR-alpha-tocopherol acetate, and 41.2% after RRR-alpha-tocopherol succinate. Animal assay data do not correlate with data from studies of absorption and retention in serum of alpha-tocopherols ingested by humans.
Adult↗
Hemin inhibits transfer of pre-delta-aminolevulinate synthase into chick embryo liver mitochondria.
Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-delta-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.
5-Aminolevulinate Synthetase↗
Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver.
The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.
Acetamides↗
Evidence for a cytosolic precursor of chick embryo liver mitochondrial delta-aminolevulinate synthase.
Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.
5-Aminolevulinate Synthetase↗
Purification of 5-aminolaevulinate synthase from liver mitochondria of chick embryo.
5-Aminolaevulinate synthase from chick-embryo liver mitochondria has, for the first time, been purified to homogeneity in its native non-degraded form by molecular sieve chromatography, chromatofocusing and affinity chromatography. The enzyme has a minimum molecular weight of 68000 as determined by sodium dodecylsulphate/polyacrylamide gel electrophoresis and a specific activity of 35000 units/mg of protein. This result conflicts with the previous report of Whiting, M.J. and Granick, G. [(1976) J. Biol. Chem. 251, 1340-1346] that the chick embryo enzyme has a molecular weight of 49000. We show here that the purified form can be degraded proteolytically to a smaller form of molecular weight around 50000 while retaining full enzymatic activity. It seem evident, therefore, that the enzyme isolated by Whiting & Granick (1976) was degraded. We have further established by pulse-labelling studies and immunoprecipitation that the enzyme isolated by our new and rapid procedure has the same minimum molecular weight as that which exists in vivo.
5-Aminolevulinate Synthetase↗
Bile acids. LXVIII. Allylic and benzylic photo-chemical oxidation of steroids.
To provide 7-oxocholesterol derivatives in yields superior to those obtained by chemical oxidation, the preparation of steroidal allylic or benzylic ketones was studied. Air-induced oxidation was investigated with a highly selective low energy UV lamp in the presence of mercuric bromide. With this procedure cholesterol acetate, 5-cholestene-3 beta, 27-diol diacetate, 24(R)-24-methyl-5-cholesten-2 beta-yl acetate, 24(R)-24-ethyl-5-cholesten-28-yl acetate and 24(R)-24-ethyl-(22E)-cholesta-5,22-dien-3 beta-yl acetate were oxidized to the allylic keto-derivative in good yields; estradiol-17 beta diacetate was similarly converted to the 6-oxo-product in improved yield. This method can be very useful in the synthesis of 7-oxocholesteryl acetate and its analogs and 6-oxo-estratrienes.
Allyl Compounds↗
Purification of rat liver mitochondrial delta-aminolaevulinate synthase.
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5-Aminolevulinate Synthetase↗
Radiochemical assay for gamma-aminolevulinate synthase.
We describe here a standard protocol for the radiochemical assay of gamma-aminolevulinate synthase from 17-day chick embryo liver mitochondria and starved male rat liver mitochondria. The assay is sensitive to at least 15 pmol of the gamma-aminolevulinate produced and can be adapted to suit most tissue types. A number of possible modifications to the procedure are discussed.
5-Aminolevulinate Synthetase↗
Bile acids. LXVII. The major bile acids of Varanus monitor.
The major bile acids of gall bladder bile of Varanus monitor have been separated by thin-layer chromatography and shown to be derivatives of taurine. After alkaline hydrolysis, the free acids were separated by thin-layer and partition chromatography. Identification or characterization of the free acids was facilitated by gas-liquid chromatography and gas-liquid chromatography-mass spectometry of the methyl esters or methyl ester-trimethylsilyl ethers. About 13% of the total bile acids was represented by the C24 acids cholic, deoxycholic, allocholic, chenodeoxycholic, and 12-oxo-3 alpha-hydroxy-5 beta-cholanic acids, of which cholic acid constituted about 50%. The remainder of the bile acids consisted of eight C27 acids of which varanic acid was the major constituent; an isomer of varanic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid were also identified. By chromatographic behavior and mass spectral fragmentation, the structures of four C27 acids with unsaturated side chains were elucidated as follows: 3 alpha, 7 alpha-dihydroxy-5 beta-cholest-23-enoic, 3 alpha, 7 alpha-dihydroxy-5 beta-cholest-24-enoic, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-23-enoic, and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoic acids. Similarly, the structure of the 12-deoxy analog of varanic acid, 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid, was suggested for the component that constituted 7% of the total.
Animals↗
Abnormal lipid composition of fat tissue in human mesenteric panniculitis.
Mesenteric fat tissue obtained at autopsy from 6 patients with mesenteric panniculitis (MP) were found to contain significant amounts of cholesteryl esters (CE). In addition, samples from 3 of these cases were found to contain 0.5-1.3% free cholesterol, 0.9-1.9% free fatty acids (FFA), 0.6-2.5% l-alkyl glyceryl ether diesters and small amounts of squalene. Two of these tissues also contained alk-l-enyl glyceryl ether diesters. The fatty acid compositions of the CE, FFA, triacylglycerides and glyceryl ether diesters (GEDE) were determined and oleic acid (18:1) was found to be the major fatty acid. The alkyl group composition of the GEDE consisted essentially of 16:0 and 18:0 and 18:1 carbon atoms in both types of ethers.
Adipose Tissue↗
Bile acids. LXIV. Synthesis of 5 alpha-cholestane-3 alpha, 7 alpha, 25-triol and esters of new 5 alpha-bile acids.
Interest in the structural requirements of a sterol or bile acid for maximal activity by an hepatic microsomal steroid 12 alpha-hydroxylase prompted the preparation of 5 alpha-cholestane-3 alpha, 7 alpha, 25-triol and 5 alpha-analogs of 3 alpha, 7 alpha-dihydroxy-5 beta-cholane-24-carboxylic acid. Methyl 3 alpha, 7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from methyl chenodeoxycholate via the Arndt-Eistert reaction was allomerized with Raney nickel in boiling p-cymene to provide a number of product of which methyl 3, 7-dioxo-5 beta- and 5 alpha-cholane-24-carboxylates, methyl 3-oxo-7 alpha-hydroxy-5 beta-and 5 alpha-cholane-24-carboxylates, were identified. Reduction with K-Selectride of methyl 3-oxo-7 alpha-hydroxy-5 beta-cholane-24-carboxylate, provided a high yield of methyl 3 alpha, 7 alpha-dihydroxy-5 alpha-cholane-24-carboxylate. Treatment of this ester with an excess of methyl magnesium iodide afforded 5 alpha-cholestane-3 alpha, 7 alpha, 25-triol. The products were characterized by thin-layer and gas liquid chromatography, proton resonance, infrared and mass spectrometry.
Bile Acids and Salts↗