Biomedical subjects
Publications and source records attributed to W H Elliott.
Artifacts formed by methylation of chenodeoxycholic acid with diazomethane are identical to the synthetic 3alpha- and 7 alpha-monomethyl ethers of methyl chenodeoxycholate. The extent of formation of these artifacts with time has been studied. No detectable artifacts were found with 2,2-dimethoxypropane as the methylating agent.
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To aid in the identification of trihydroxy acidic metabolite(s) derived from beta-sitosterol, 3alpha,7alpha,12alpha-trihydroxy-24-nor-5beta-cholan-23-oic acid was prepared and its methyl ester was treated with Raney nickel in boiling p-cymene to provide methyl 3-oxo-7alpha,12alpha-dihydroxy-24-nor-5alpha-cholanate, 3-oxo-7alpha,12alpha-dihydroxy-24-nor-5beta-cholante and 3-oxo-7alpha,12alpha-dihydroxy-24-norchol-4-enoate. The latter compound was synthesized from the 3-oxo-5beta-derivative with SeO2 to provide a product with identical properties. Catalytic reduction of either saturated 3-oxo-derivative provided the appropriate 3,7,12-triols isomeric at C-3. Results from gas liquid and partition chromatography, mass spectrometry, and otherr physical properties of the acids, their methyl esters and other derivatives are compatible with the assigned structures.
Tauroallocholate is the major bile salt of the lizard, Uromastix hardwickii. Alkaline hydrolysis of bile from 25 gallbladders provided 1.21 g of acidic material, about 90% of which was allocholic acid. Analyses by gas-liquid chromatography, and mass spectrometry verified the presence of almost 10% of deoxycholic acid and smaller amounts of other 5alpha and 5beta-bile acids.
A comparison of the activity of rabbit liver microsomes fortified with 0.1 mM NADPH to promote 12alpha-hydroxylation over 60 minutes with appropriate sterols provided the following relative order of activities: kalpha-cholestane-3alpha, 7alpha-diol, 100; 7alpha-hydroxy-5alpha-cholestane-3one, 76; 7alpha-hydroxycholest-4-en-3-one, 64; 5alpha-cholestane-3-3beta, 7alpha-diol, 26; allochendoexycholate, 22. If the more polar products formation. Investigation by gas-liquid chromatography-mass spectrometry of the nature of these more polar products derived form 5alpha-cholestane-3alpha, 7alpha-diol showed that a series of tetrols was dormed; i.e., 5alpha-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol as the major product and lesser amounts of 5alpha-cholestane-3alpha, 7alpha, 12alpha, 24- and 3alpha, 7alpha, 12alpha, 23tetrols. No significant amount of 26-hydroxylated product was formed.
Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3beta-3H]- or [24-14C]allochenodeoxycholic acid or [5alpha,6alpha-3H2]5alpha-cholestane-3alpha,7alpha-diol to their respective 12alpha-hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal 12alpha-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the 14C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.
[5alpha,6alpha-3H2]-5alpha-Cholestane-3alpha,7alpha-diol (A), (25 R)-3alpha,7alpha-dihydroxyl-[5alpha,6alpha-3H2]-5alpha--cholestan-26-oic acid (B),(25R)-[5alpha,6alpha-3H2]stane-3alpha,7alpha-26-triol (C), and [3beta-3H]allochenodeoxycholic acid (D) were prepared, characterized, and studied with a rat liver microsomal preparation fortified with 1 mM NADPH. The 12alpha-hydroxylated product formed from each of these substrates was identified by isotopic dilution; the relative reactivity of the four substrates was (A) 100; (B) 87; (C) 135; and (D) 40. The microsomal system showed a requirement for added NADPH and other properties similar to those shown for 12alpha-hydroxylation of 7alpha-hydroxycholest-4-en-3-one, which is ultimately converted to cholic acid. Since the coplanar 5alpha-cholestane-3alpha,7alpha-diol is virtually superimposable upon 7alpha-hydroxycholest-4-en-3-one, and the enzymic requirements are comparable, it is suggested that a single enzyme system may be responsible for 12alpha-hydroxylation of these substrates.
[25R]3beta,7alpha-Dihydroxy-[5alpha,6alpha- -3 H2]-5alpha-cholestanoic acid was prepared, purified and 0.34 mg was administered intraperitoneally as the potassium salt to each of three adult male rats with cannulated bile ducts. Bile collected in the first 24 h, containing 97% of the administered 3-H was hydrolysed and the free bile acids were separated by acetic acid partition chromatography. Of the chromatographed tritium 58% was associated with allochenodeoxycholic acid and 14% with its 3beta-isomer; only 5% of the 3-H was found in allocholic acid and 1% with the substrate or more polar unidentified materials. Thus, this dihydroxy-5alpha-cholestanoic acid is metabolized in the rat primarily to dihydroxy-5alpha-cholanic acids, comparable to the metabolism of 3alpha, 7alpha-dihydroxy-5beta-cholestanoic acid in man.
The role of steroids in regulation of delta-aminolevulinic acid synthetase has been studied in isolated rat liver cell suspensions under conditions previously shown to support inducation of the enzyme by drugs. Addition of a variety of C-19 and C-21 steroids to cell suspensions resulted, after 4 to 6 hours of incubation, in 2- to 5-fold increase in the activity of delta-aminolevulinic acid synthetase as measured in liver cell homogenates. The increase was prevented by cycloheximide. The most active steroid inducers tested were pregnene or pregnane derivatives with keto or hydroxyl groups at C-3 and C-20; in particular a beta-hydroxyl group at C-20 enhanced activity. These C-21 steroids at optimal initial concentrations caused 3- to 5-fold induction over 4 hours. A number of C-19 androstene and androstane compounds caused 2- to 3-fold inducation over the same period. Hydrocortisone had no effect. For a variety of androstane and pregnane derivatives, inducation by 5alphaH steroids was as great as or greater than that by 5betaH compounds, in contrast to previous findings in chick embryo liver. Induction of delta-aminolevulinic acid synthetase by steroids in isolated liver cells was shown to be subject to feedback repression by hemin.
The bile acids derived from [4-14-C]cholesterol administered intracardially to rats with cannulated bile ducts were identified and quantitated. Over a period of 28 days about 90% of the administered 14-C was found in bile of which 73% was retained in the biliary acid fraction. [7beta-3-H]cholic acid, alpha-muri[3beta-3-H]cholic acid, beta-muri[3beta-3-H]cholic acid and litho[3beta-3-H]cholic acid were prepared with specific activities of about 30 muCi/mg by reduction of appropriate ketonic precursors with NaB3H4 and were added to the biliary acid fraction. After separation and purification of the bile acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids accounted for 70, 16, 7.5 and 6.1%, respectively, of the 14-C in the biliary acid fraction. The specific activities of these isolated 14-C-labeled acids were almost identical. Lithocholic acid accounted for a maximum of 0.2% and ursodeoxycholic acid and 7-oxolithocholic acid could account for no more than 2% of the biliary 14-C. Gas-liquid chromatography on 3% OV-17 of the trimethylsilyl ether derivatives of the methyl esters of the common bile acids of rat bile results in their complete separation and provides a convenient means of estimating the relative proportions of these acids in rat bile. By this method, the relative amounts of the four major acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids were 63, 20, 8 and 6%, respectively.
Washed-cell suspensions of Bacillus amyloliquefaciens secrete significant amounts of the extracellular enzymes alpha-amylase and protease for about 15 min in the almost complete absence of protein synthesis. This apparently represents release of preformed enzyme en route to secretion. The release was independent of energy but was affected by temperature. Pulse-labeling experiments showed that newly synthesized enzyme molecules are either immediately released into the external medium or equilibrate with the preformed enzyme prior to eventual secretion. The results are compatible with a model of secretion whereby enzyme molecules emerging from the cell membrane become temporarily restricted by the cell wall so that a small pool of active enzyme accumulates in this region.
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