Measurement of bile acid synthesis in man by release of14CO2 from [26-14C]cholesterol: Comparison to isotope dilution and assessment of optimum reference cholesterol specific activity

Bile acid synthesis can be measured as release of¹⁴CO₂ from [26-¹⁴C]cholesterol divided by cholesterol specific activity, but this method has not been validated in human subjects. We made twelve comparisons of this CO₂ method to standard isotope dilution in six normal subjects and found a mean discrepancy of 6%. Linear regression analysis of one value with respect to the other revealed a correlation coefficient of 0.83 (P<0.001), a Y-intercept close to zero (−4.98) and a slope close to 1 (1.06), suggesting good correspondence between the two methods. To assess the potential for error arising from use of serum cholesterol to estimate specific activity of cholesterol used for bile acid synthesis, we compared synthesis measured using serum free cholesterol specific activity to that measured using bile cholesterol specific activity, which is known to be near isotopic equilibrium with the precursor pool used for bile acid synthesis. Synthesis calculated in these two ways differed by less than 10%. The data indicate that the CO₂ method using either serum or bile cholesterol specific activity provides a valid estimate of bile acid synthesis in man.     

Fatty acid and sterol synthesis by rat small intestine in vitro

Slices of rat jejunum were incubated with [2-¹⁴C]pyruvate, [1-¹⁴C]acetate, or [³H]H₂O to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P≤0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p≤0.05) in lipogenic activity. The highest levels of lipogenesis were observed when [³H]H₂O + glucose at a concentration of 20 mM was used. From such experiments, the absolute rate of fatty acid synthesis in the tissue preparation was calculated: 734±54 nmoles acetyl units incorporated into fatty acids/g tissue/hr.     

Psyllium, not pectin or guar gum, alters lipoprotein and biliary bile acid composition and fecal sterol excretion in the hamster

Different soluble dietary fibers known to alter cholesterol metabolism were fed to golden Syrian hamsters, and their specific impact on lipoproteins, biliary bile acid profile, and fecal sterol excretion was evaluated. Semipurified diets containing 20% fat; 0.12% cholesterol; and 8% of psyllium (PSY); high (hePE) and low (lePE) esterified pectin; or high (hvGG) and low (lvGG) viscous guar gum were fed for 5 wk. Compared to control, PSY caused a significant reduction in plasma cholesterol (2.9±0.5 vs. 5.5±0.5 mmol/L), whereas hePE, lePE, hvGG, or lvGG had no apparent effect on plasma lipids. Hepatic total and esterified cholesterol were substantially decreased with PSY, pectin and guar gum, whereby PSY produced the most pronounced effect. Distinctive changes existed in the bile acid profile related to the different fibers. In contrast to pectin and guar gum, PSY caused a significant increase in the cholate:chenodeoxycholate and the glycine:taurine conjugation ratio. Pectin and guar gum did not alter daily fecal neutral sterol excretion while PSY caused a 90% increase due to a higher fecal output. Daily fecal bile acid excretion and total fecal bile acid concentration were significantly increased by PSY, whereas hePE, lePE, hvGG, and lvGG revealed no or only minor effects. Taken together, the disparate hypocholesterolemic effects of PSY, pectin, and guar gum on cholesterol and bile acid metabolism in the hamster are possibly related to different physicochemical properties, e.g., viscosity and susceptibility to fermentation, affecting the fiber-mediated action in the intestine.     

Quantification of key odorants formed by autoxidation of arachidonic acid using isotope dilution assay

Six odor-active compounds generated by autoxidation of arachidonic acid (AA) were quantified by isotope dilution assay (IDA), i.e., hexanal (1), 1-octen-3-one (2), (E,Z)-2,4-decadienal (3), (E,E)-2,4-decadienal (4), trans-4,5-epoxy-(E)-2-decenal (5), and (E,Z,Z)-2,4,7-tridecatrienal (6). Compound 1 was the most abundant odorant with about 700 mg/100 g autoxidized AA, which corresponds to 2.2 mol% yield. Based on the odor activity values (ratio of concentration to odor threshold), odorants 3 (fatty) and 5 (metallic) showed the highest sensory contribution followed by 1 (green), 2 (mushroom-like), 6 (egg white-like), and 4 (fatty). For the first time, reliable quantitative results are reported for odorants 1–6 in autoxidized AA, in particular odorant 6, which is a characteristic compound found in autoxidized AA. Synthesis of deuterated 6, required for IDA, is described in detail. The formation of odorants 1–6 by autoxidation of AA is discussed with respect to the quantitative data.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

Stimulation of bile acid synthesis by dibutyryl cyclic AMP in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to examine the effects of dibutyryl cyclic AMP on the incorporation of¹⁴C-acetate and¹⁴C-cholesterol into bile acids. After an initial lag period, both precursors were incorporated into cholic and chenodeoxycholic acids at a linear rate for the subsequent 60 min. An apparent stimulation of bile acid formation from¹⁴C-acetate by dibutyryl cyclic AMP was complicated by the concomitant inhibition of cholesterol synthesis. In experiments with¹⁴C-cholesterol, dibutyryl cyclic AMP (1 mM) increased the labeled cholic and chenodeoxycholic acids in the medium by 83 and 224%, respectively, but cellular levels of labeled bile acids were unchanged. As a result, the nucleotide stimulated the overall incorporation of¹⁴C-cholesterol into cholic acid by 39% and into chenodeoxycholic acid by 123%. The mean ratio of labeled cholic to chenodeoxycholic acid declined from 55∶45 in control cells to 41∶59 in cells incubated with dibutyryl cyclic AMP. The results demonstrate that label incorporation can be used to study the regulation of bile acid synthesis in isolated hepatocytes. We propose that dibutyryl cyclic AMP enhances bile acid production by phosphorylating, and thus stimulating the activity of, cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis.     

Comparison of UF synthesis by alkaline‐acid and strongly acid processes

This work discusses two processes for producing urea‐formaldehyde (UF) resins. One is the alkaline‐acid process, which has three steps: usually an alkaline methylolation followed by an acid condensation and finally the addition of a final amount of urea. The other process, the strongly acid process, consists of four steps, in which the first step involves a strongly acid condensation followed by an alkaline methylolation, a second condensation under a moderately acid pH and finally, methylolation and neutralization under a slight alkaline pH. Two resins were produced using the two above described processes. The molecular weight distribution (MWD) of the resins was monitored off‐line by GPC/SEC and the final resins were characterized by GPC/SEC and HPLC. These studies showed that the two resins differ greatly in chemical structure, composition, viscosity, and reactivity. The monitoring of MWD indicated that the first condensation under a strongly acid environment leads to the production of a polymer with a distinctly different chemical structure, therefore increasing the flexibility of polymer synthesis and opening the way to the improvement of end‐use properties. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Programming of initial steps in bile acid synthesis by breat-feeding vs. Formula-feeding in the baboon

We tested the hypothesis that breast-vs. formula-feeding differentially affects the enzymatic activity of three sterol hydroxylases critical in the initial steps of bile acid formation. Thirty baboons were either breast-fed or formula-fed for the first 14 wk of life before weaning to baboon chow. At 14 and 34 wk of age, liver biopsies were assayed for cholesterol 7α-hydroxylase (CYP7A1), 27-hydroxycholesterol-7α-hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27A1). We also determined the kinetics of ³H-27-hydroxycholesterol (27-OHC) turnover in vivo at both ages. At 14 wk of age, hepatic CYP7A1 activity was low but sevenfold higher among formula-fed vs. breast-fed baboons. By 34 wk, CYP7A1 activity had increased nearly 10-fold in both infant diet groups, and the sevenfold difference in CYP7A1 between previously breast-and formula-fed animals persisted. There were no differences in CYP7B1 activities between infant diet groups at either 14 or 34 wk of age although the activity increased in both groups by about 50% from 14 to 34 wk. CYP27A1 activity also increased between 14 and 34 wk of age, and, compared with CYP7A1, relatively small differences in CYP27A1 activity due to infant diet were observed at each age. Plasma 27-OHC turnover had a half-time of 2–4 min. We had previously reported that after weaning, the total bile acid synthesis rate was higher among baboons that were formula-fed than among breast-fed animals. The present results suggest that this difference is most likely due to significantly higher CYP7A1 activity among formula-fed vs. breast-fed animals.     

Effects of phenobarbital upon bile acid synthesis in two strains of rats

Rats of the Wistar and Sprague-Dawley strains were injected with sodium phenobarbital (100 mg/kg body wt/day) for 8 days. Fecal bile acid excretion was measured on days 6 and 8 of the experiment, and biliary bile acid composition, hepatic microsomal cholesterol, 7α-hydroxylase, and 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase were determined at the end of the study. In the Wistar rat, injection of phenobarbital produced a doubling of fecal bile acid output (controls, 5.3 mg/rat/day; treated rats, 10.6 mg/rat/day) and a two-three fold increase in cholesterol 7α-hydroxylase. The fecal bile acid output of Sprague-Dawley rats increased 20% in response to phenobarbital (controls, 9.5 mg/rat/day; treated rats, 11.6 mg/rat/day). The activity of cholesterol 7α-hydroxylase remained unchanged. In both strains, phenobarbital treatment produced a decrease in the proportion of cholic acid in total biliary bile acids (controls, 85%; treated groups, 65%). This was associated with a decrease of 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase activity by ca. 50%. Biliary cholesterol concentrations were reduced in phenobarbital treated rats of both strains, but liver cholesterol concentrations remained unchanged. The drug produced a 25% increase in liver wt, on the average.     

The metabolism of lithocholic acid and lithocholic acid-3-α-sulfate by human fecal bacteria

Both lithocholic acid and lithocholic acid-3α-sulfate are metabolized by mixed fecal bacteria and by pure strains of the genusClostridium. Mixed fecal bacteria metabolized lithocholic acid to 3-ketolithocholic acid; lithocholic acid-3α-sulfate was metabolized to isolithocholic acid, 5β-cholanic acid and Δ³-cholenic acid under both aerobic and anaerobic conditions. The results indicate that a specific genus, theClostridium, has a primary role in the metabolism of lithocholic acid-3α-sulfate to Δ³-cholenic acid     

Effect of chronic ethanol administration on cholesterol and bile acid synthesis in vivo

Chronic administration of ethanol failed to stimulate the hepatic rate of cholesterol synthesis in meal-fed rats. In contrast, chronic ethanol feeding caused a 50% inhibition in the rate of incorporation of [4-¹⁴C] cholesterol to bile acids in the bile-duct cannulated rats. It is, therefore, suggested that the decreased rate of cholesterol degradation to bile acids may play an important role in ethanol-induced accumulation of cholesterol in liver.     

Tissue-selective inhibition of sterol synthesis in mice by pravastatin sodium after a single or repeated oral administrations

Pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exhibits liver-selectivity in inhibiting sterol synthesis, when administered as a single oral dose to mice or rats, whereas lovastatin and simvastatin do not. This may be due to the fact that pravastatin is distributed intracellularly, to a large extent, in the liver and extracellularly in nonhepatic tissues. In the present study, we examined whether the difference in liver-selectivity among these three HMG-CoA reductase inhibitors observed in single-dose studies was preserved after repeated oral administrations of drugs to mice.De novo sterol synthesis in different tissues of mice was examinedin vivo three hours after the last dose of drug by measuring incorporation of intraperitoneally injected [¹⁴C]acetate into total sterols. Pravastatin administered orally for 11 consecutive days at 5 and 10 mg/kg exhibited a greater liver-selectivity than lovastatin and simvastatin: sterol synthesis was inhibited more than 60% in the liver by all three drugs, whereas that in nonhepatic tissues was inhibited less than 10% by pravastatin and more than 30% by lovastatin and simvastatinin in most of the nonhepatic tissues examined. Pravastatin administered orallyfor 11 consecutive days at 10 mg/kg caused more selective inhibition of sterol synthesis in liverex vivo than two other inhibitors at the same dose. Pravastatin inhibitedde novo sterol synthesis from [¹⁴C]acetate into sterol fraction in the liver slicesin vitro, but minimally in those of the spleen and testis, whereas lovastatin and simvastatin inhibited in those of all three tissues. Since the drug concentrations determined in the same tissue samples of the liver, spleen, and testis were almost comparable among the three drugs, it was suggested that the cellular distribution profiles of pravastatin observed in a single-dose study were preserved in the multiple-dose study. We conclude that the difference in tissue-selectivity between pravastatin and the other two inhibitors to inhibit sterol synthesis in mice is maintained, regardless of the duration of administration.     

Short-term effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor on cholesterol and bile acid synthesis in humans

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short-term effect of one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7α-hydroxy-4-cholesten-3-one level 9which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 ± 1.1 ng/ml vs. 6.7 ± 2.5, mean ±SD; P<0.05). This decrease continued for 8 h (2.5 ± 0.8 vs. 5.2 ± 1.5; P<0.05). On the other hand, plasma 7α-hydroxy-4-cholesten-3-one level did not change until after 6 h; then at 8 h it was lower than control (15.7 ± 11.8 ng/mL vs. 24.7 ± 16.9; P <0.05). According to three-way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P<0.01) and time-course (P<0.01). On the other hand, the 7α-hydroxy-4-cholesten-3-one level was affected by both individual difference (P<0.01) and time course (P<0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was not influenced by pravastatin, although cholesterol synthesis was inhibited. The shortterm inhibition of cholesterol synthesis did not affect bile acid synthesis.     

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that bile adsorbing resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps? HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps? HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus? HPMC capsules and Coni Snap? gelatin capsules gave less protection.     

Fatty acid composition of the sterol ester fraction of human adrenal cortex in Cushing's syndrome and after treatment with aminoglutethimide

The lipid that accumulated in the adrenal cortex of a patient who had been treated with aminoglutethimide has been compared with the lipid in normal human adrenal cortex and identified as esters of cholesterol with fatty acids. While the concentration of free cholesterol was normal, that of esterified cholesterol was three times greater than that in normal controls. Sterols were analyzed by gas chromatography and found to consist almost wholly of cholesterol. The fatty acid composition of the cholesterol esters in adrenal cortex from patients with abnormal steroid secretion rates was determined. An increased proportion of cholesteryl arachidonate (cholesteryl-3β-[allcis]-eicosa-5,8,11,14-tetraenoate) was found in adrenal cortex from patients with decreased steroid secretion rates and a decreased proportion in adrenal cortex from patients with steroid secretion rates raised sufficiently to cause Cushing's Syndrome. Publication no. 1429 of the Cancer Commission of Harvard University.     

Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats

Fecal bile acids in germ-free rats were analyzed after inoculation withBacteroides vulgatus, Bifidobacterium longum, Escherichia coli orClostridium ramosum. B. vulgatus preferentially deconjugated tauro-β-muricholic acid andB. longum taurocholic acid.C. ramosum deconjugated both bile acids, butE. coli deconjugated neither. 7α-Dehydroxylation of bile acids was negligible even after 18 days of inoculation, but a small amount of 7-oxo-bile acid, less than 5%, was formed. Fecal excretion of bile acids increased after inoculation withB. vulgatus, B. longum andC. ramosum, but not withE. coli.     

三氟乙酸及甲酸体系下重组人促红素液质肽图的比较

目的探讨流动相中添加三氟乙酸(trifluoroacetic acid,TFA)或甲酸(formic acid,FA)时,重组人促红素(erythropoietin,EPO)液质肽图中各肽段的保留时间、峰面积及离子化效率的差异。方法将重组人EPO样品进行酶切,分别在含0.1%TFA及0.1%FA的流动相体系下测定液质肽图。通过分析质谱数据对各色谱峰进行定性,记录并计算各肽段的保留时间、峰面积及质谱信号强度。结果两种添加剂体系下,各肽段的保留时间存在差异,大部分肽段的保留时间变化在1 min左右,个别肽段超过2 min;TFA体系下各肽段的峰面积相对较小,约为FA体系下的84%;FA体系下各肽段离子化效率较高,信号强度一般为TFA体系的6倍,个别肽段高达10倍以上。结论两种体系下重组人EPO的液质肽图存在较大差异,应根据实验目的选择合适的流动相添加物。     

On the role of fatty acid in adsorption and corrosion inhibition of iron by amine—fatty acid salts in acidic solution

A study has been made of the adsorption and corrosion inhibition of iron by triethanolamine salts of 18C unsaturated fatty acids in 0.5 M deaerated H₂SO₄ solution. The adsorption was considered in terms of the competitive coadsorption of acid molecules and amine cations, being the main species in this solution. From measurements of differential double layer capacitance and dissolution kinetic of iron, the preferential adsorption of fatty acids over amine has been found. The orientation of acids appeared decisive for the formation of inhibitory effective adsorbates. Such adsorbates were originated by vertically oriented oleic acid molecules. Less effective were the layers based on salts of acids possessing a greater number of Π bonds. The double, horizontal and vertical orientation was found for these acids. The main role of amine was the cross-linkage of acid chains adsorbed on the iron surface.     

The behavior of glyceride-fatty acid mixtures in bile salt solution: Studies by gel filtration

Bile salt lipid emulsions were prepared which simulated the emulsified oil-micellar phase system of the small intestinal content during fat digestion. Application of such emulsions to gel columns prepared and eluted with 6 mM sodium taurodeoxycholate separated an emulsion phase and a micellar phase. The distribution of lipid solutes into the two phases under these conditions was measured. Micellar dimensions were larger as lipid concentrations were increased. Inclusion of multiple lipid classes resulted in larger micellar particles. Monoglyceride and fatty acids were eluted completely in the micellar phase under these conditions. Minimal measurable amounts of triolein were recovered in micellar solution. This was confirmed by extraction, chromatographic separation and quantitative analysis. As diolein concentration was increased, less was recovered in the micellar phase. When monoglyceride was added, more diolein entered the micellar phase. Addition of triglyceride enhanced the distribution of diolein into the emulsion phase.