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Biomedical subjects

C N Williams

Publications and source records attributed to C N Williams.

At least 73 records · Page 4Linked to original sources
PubMed1980

3 alpha-, 7 alpha-, and 12 alpha-OH group specific enzymic analysis of biliary bile acids: comparison with gas-liquid chromatograpy.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSDH) from P. testosteroni, 7 alpha-HSDH (Escherichia coli ATCC No. 29532) and 12 alpha-HSDH (Clostridium group P, strain C48-50, ATCC No. 29733) were used to directly measure 3 alpha-, 7 alpha-, and 12 alpha-OH groups in extracted human bile-rich duodenal aspirates. Twelve samples chosen from widely differing ratios of cholic/chemodeoxycholic/deoxycholic were computed by solving three simultaneous equations. Comparison of these ratios with those obtained by a) thin-layer chromatography and 3 alpha-, 7 alpha-HSDH assays and b) gas-liquid chromatographic analysis showed no significant difference. Addition of known amounts of pure cholic, chenodeoxycholic, deoxycholic, or lithocholic acid to individual bile extracts gave an appropriate yield of 3 alpha-, 7 alpha-, and 12 alpha-OH groups. The direct (non-chromatographic) enzymic method has the advantages of being rapid, convenient, and inexpensive, and thus suitable for clinical use.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivatives by gas-liquid chromatography.

Quantitative analyses of individual bile acids in biological samples are limited by the lengthy multistep preparations necessary. Using heptafluorobutyric acid anhydride in pyridine as derivatizing agent, we reduced several steps to one. Bile acids and their glycine and taurine conjugates form stable heptafluorobutyrate derivatives, climinating the need for deconjugation and preparation of methyl esters. The derivatives have been characterized by mass spectrometry, and optimum reaction yields have been determined. Operating conditions for analyzing the bile acid heptafluorobutyrates by gas-liquid chromatography on various column packings were investigated, and 0.5% QF-1 or 3% OV-255 was found suitable. The bile acid derivatives were identical whether starting with the bile acid or the glycine or taurine conjugates. The procedure was applied to a quantitative analysis of artificial mixtures of bile acids and bile conjugates, and also of human bile. The results compared favorably to those obtained with a 3 alpha- and 7 alpha-hydroxysteroid dehydrogenase fluorimetric method.

Bile↗
PubMed1977

Prevalence of gallstones and gallbladder disease in Canadian Micmac Indian women.

The prevalence of gallstones and gallbladder disease was studied between October 1973 and June 1976 in Canadian Micmac Indian women aged 15 to 50 years in an inland rural community near Shubenacadie, NS. Of 132 women at risk 98 underwent cholecystography, 6 had a history of cholecystectomy (verified from hospital records) and 3 had cholecystectomy because of cholecystitis during the 3 years of the study. Of the 17 abnormal cholecystograms 10 showed radiolucent gallstones, and repeated studies documented gallstones in 6 of the 7 radiographs on which the gallbladder was not visualized. The prevalence of gallstones was found to be 211/1000, and that of gallbladder disease, 240/1000. The peak prevalence was at 30 to 39 years of age. The women with gallbladder disease were significantly more obese and of greater parity than those without gallbladder disease even when age was controlled. The Micmac Indian women of Nova Scotia appear to be at a much higher risk for the development of cholesterol gallstones and gallbladder disease than Caucasian women in Framingham, Massachusetts.

Adolescent↗
PubMed1977

Nutrient intake and meal patterns of Micmac indian and Caucasian women in Shubenacadie, NS.

North American Indians have a higher morbidity from gallbladder disease, diabetes mellitus and obesity than other North Americans; this may result from their food intake. Nutrient intake and meal patterns were compared in 120 Micmac Indian and 115 Caucasian women in Shubenacadie, NS. Findings were compared with the Canadian Dietary Standard (CDS) and the Nutrition Canada national and Indian survey reports. The diet of Indian women had higher carbohydrate, lower protein and lower fibre content than that of Caucasian women, who derived a higher percentage of energy from protein and had a higher intake of vitamin A, niacin and ascorbic acid. Overnight fast was longer among Indian women. A high percentage of all women studied reported diets that did not reach the CDS for total energy intake in kilocalories or for calcium, iron, vitamin A, thiamin or riboflavin.

Adolescent↗
PubMed1977

Increased lithogenicity of bile on fasting in normal subjects.

The effect of fasting on bile lithogenicity was studied in 19 normal men and 22 normal women. The molar percentages of bile acid, phospholipid, and cholesterol, determined after random overnight fasts of 9, 12, and 16 hr, were plotted on triangular coordinates. The molar percentage of cholesterol increased in both men and women. Analysis using Admirand and Small's criteria for cholesterol saturation revealed that 4.50% of normal women were lithogenic at 9.1 hr and 54.5% at 16.5 hr (P less than 0.005). A similar trend in men was not significant. The mean values for both sexes were lithogenic at 16 hr only. Lithogenic bile was present in 4 men and 1 woman at 9 hr fasting and became more lighogenic with longer fasting. Analysis using the criteria of Hegardt and Dam revealed an increased proportion of both sexes moving into the metastable-labile and supersaturated zones on fasting, again with significant changes for women (P less than 0.01). The duration of fasting is important in interpreting the presence of lithogenic bile; although more pronounced in women, both sexes showed increased cholesterol saturation in bile with fasting.

Adult↗
PubMed1977

Ion pair extraction technique with azure A, for differentiating biles of normal subjects and patients with liver disease from patients with Crohn's disease with small bowel involvement.

The ion pair extraction technique with Azure A solution, chloroform/methanol (equal volumes), and dilute sulfuric acid, was used to study bile-rich duodenal aspirates of (a) healthy volunteers and patients with (b) cholestatic liver disease, (c) Crohn's disease with small bowel involvement, and (d) those with primarily colon involvement. Duplicate aliquots of extracted duodenal aspirates from these four groups were subjected to ion pair extraction, with and without prior acetylation, and the absorbance ratios at 645 nm without acetylation/with acetylation ("acetylation index") compared; values (+/- SD) obtained were 0.052 +/- 0.0098, 0.038 +/- 0.014, 0.136 +/- 0.048, and 0.077 +/- 0.056, respectively, for the four groups. The acetylation index for group c was significantly (P less than 0.001) different from that of groups a and b. All other intergroup comparisons were not significant. The acetylation index correlated positively (r = 0.922) with the ratio of glycine- to taurine-conjugated bile acids, measured independently, implying that it reflects this ratio.

Acetylation↗
PubMed1976

Behavior of 3alpha- and 7alpha-hydroxysteroid dehydrogenases on chenodeoxycholate substituted Sepharose.

Chenodeoxycholate (3alpha-, 7alpha-dihydroxy-5beta-cholanoate) was linked to Sepharose 4B by an ethylenediamine bridge. When 3alpha-hydroxysteroid dehydrogenase and 7alpha-hydroxysteroid dehydrogenase preparations were applied to a column of covalently linked chenodeoxycholate, both enzymes were retarded at pH 6.7; the 7alpha-OH oriented enzyme more than the 3alpha-OH enzyme. Approximately forty-fold purification of 7alpha-hydroxysteroid dehydrogenase was achieved in one step. Although no significant purification of 3alpha-hydroxysteroid dehydrogenase occurred, the background value in the fluorometric enzymatic estimation of bile acids by eluted 3alpha-hydroxysteroid dehydrogenase was markedly reduced. Molecular weight estimation by Sephadex G-200 gave the values of 47,000 for 3alpha-hydroxysteroid dehydrogenase and 105,000 for 7alpha-hydroxysteroid dehydrogenase.

Chenodeoxycholic Acid↗
PubMed1976

Bile-acid metabolism and the liver.

Primary bile acids are exclusively synthesize, and all bile acids are conjugated, in the liver. When hepatic function is altered by disease, not surprisingly the bile-acid metabolism reflects this change. 1. In chronic liver disorders the glycine: taurine ratio of serum and bile is reduced. 2. In cases of relapsing acute hepatitis and relapsing chronic active hepatitis the serum bile acids are increased, providing an aid to early diagnosis. Their pattern often distinguishes chronic cholestatic conditions from Laennec's cirrhosis. 3. In chronic liver disease, the concentration of intraduodenal bile acids is reduced; when the reduction is severe, this probably accounts for co-existent steatorrhoea. 4. In Laennec's cirrhosis, both the synthesis and pool size of cholic acid are markedly depressed; in primary biliary cirrhosis, however, preliminary data indicate a decrease in the chenodeoxycholic acid component.

Barbiturates↗
PubMed1975

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.

Bacteroides↗
PubMed1975

Convenient non-chromatographic assays for the microbial deconjugation and 7alpha-OH bioconversion of taurocholate.

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.

Bacteroides fragilis↗
PubMed1975

Lyophilized 7 alpha-hydroxysteroid dehydrogenase: a stable enzyme preparation for routine bile acid analysis.

Preparations of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Escherichia coli strain 23 can be frozen and thawed without significant loss of activity. 7 alpha-HSDH may then be lyophilized into powder form, which is stable for more than 6 months (3% loss of activity). The lyophilized 7 alpha-HSDH preparation has the additional advantage over previously described preparations of a low and stable fluorescence background when applied to the fluorometric estimation of bile acids, especially in combination with thin-layer chromatography. Analysis of duodenal aspirates from 18 normal subjects gave bile acid ratios identical with those reported earlier and obtained by using gas-liquid chromatography. A significant difference in the glycine: taurine ratio between males and females was observed.

Bile Acids and Salts↗
PubMed1975

Non chromatographic colorimetric assay for total taurine-conjugated bile acids: application of measurements of glycine: taurine ratio in bile.

A direct colorimetric assay for total conjugated taurine from bile-rich duodenal aspirates is described. The method is based on complete acetylation of the free hydroxyl groups by acetic anhydride at 100 degrees C. of both the tri- and di-hydroxy bile acids in Folch extracted bile samples. Taurine-conjugated bile acids are measured by ion pair formation with Azure A and subsequent extraction of the complex into the organic phase of a biphasic system. Absorption at 645 nm. of this complex directly quantifies total taurine-conjugated bile acid. Total bile acids are then estimated by the 3alpha-hydroxysteroid dehydrogenase assay. The difference between the concentration of the total conjugated bile acid and of the total conjugated taurine determines the concentration of glycine conjugates and the glycine:taurine ratio. Potentially interfering materials such as sulphalipids, certain phospholipids, and unconjugated bile acids are removed by Folch extraction. The 3-hydroxysteroid sulfates (cholesteryl sulfate, lithocholate sulfate, and glycocholate sulfate) are not measurable by heating in acetic anhydride and do not interfere. Taurolithocholate-3-sulfate, under identical conditions, gives a measurable but very low color yield and in normal physiologic concentrations would contribute negligible color. As previously reported, this assay under prescribed conditions is selective for long-chain amphipathic sulfates or sulfonates with no measurable color yield with glycine conjugates, unconjugated bile acids, free fatty acids, or lecithin. Values for glycine:taurine ratios by the above-described method in both normal bile extracts and extracts from patients with either elevated or depressed ratios relate closely to those determined by thin-layer chromatography.

Acetates↗