Effects of cholestyramine and squalene feeding on hepatic and serum plant sterols in the rat

Hepatic and serum phytosterol concentrations were compared in the rat under basal conditions and during activated cholesterol and bile acid production due to squalene and cholestyramine feeding. Both treatments consistently decreased hepatic and serum levels of sitosterol and campesterol and, unlike esterified cholesterol, esterified plant sterols were not increased in liver during squalene feeding. Serum levels of phytosterols were decreased quite proportionately to those in the liver. The hepatic levels of sitosterol and campesterol closely correlated with each other, but not with cholesterol levels. The percentage esterification of both phytosterols was lower than that of cholesterol. The results indicate that activation of hepatic sterol production leads to depletion of hepatic plant sterols. It is suggested that poor esterification of plant sterols may contribute to this decrease.     

Identification of sterols and bile acids by computerized gas chromatography-mass spectrometry

In mass spectrometry, sterols and bile acids form fragment ions characteristic of certain steroid structures. After separation of derivatized sterols or bile acids by the gas chromatograph and fragmentation in the mass spectrometer, data collected by the computer can be collated to provide a reconstructed gas chromatogram and a series of fragment ion current chromatograms in which the relative abundances of characteristic fragment ions are plotted vs time or scan number. Intensities of these fragment ions will be greatest and hence coincide with peak elution of unidentified sterols or bile acids. The use of known amounts of labeled appropriate sterols or bile acids permits quantitation of the identified sterol or bile acid.     

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 10¹⁰ counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7α-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-β-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an imporant role in the formation of secondary bile acids and coprostanol in rats.     

Dietary protein effects on cholelithiasis in hamsters: Interaction with amino acids and bile acids

The objective of this experiment was to study the effects of dietary cottonseed protein and casein on plasma and biliary lipids, plasma amino acids and gallstones in hamsters. Thirty-four male hamsters (60 ± 5 g) were fed either the lithogenic “Dam Diet” (containing 20% casein, 74.3% sucrose and 5.7% vitamin-mineral mix) or a similar diet that contained 20% cottonseed protein for 30 days. Both diets contained protein as a protein isolate. The concentration of alpha-aminobutyric acid was significantly elevated in the casein-fed group. Significant differences in the total plasma cholesterol or lipoprotein cholesterol concentrations were not observed between the two dietary groups. A significant elevation in the absolute concentration of biliary cholesterol was observed in the casein-fed hamsters. Cottonseed protein-fed animals exhibited a significantly elevated concentration of bile acids. The ratio of glycochenodeoxycholic:glycocholic acid was significantly higher in the cotton-seed protein-fed group. This study reports that an elevated concentration of biliary cholesterol with a concomitant decrease in bile acid concentration yields a condition favorable to gallstone formation. It is proposed that cottonseed protein may have a specific effect on the bile acid pool by increasing the ratio of glycochenodeoxycholic acid:glycocholic acid which, in turn, prevents formation of cholesterol gallstones.     

Analysis of fecal neutral steroids and bile acids in humans on constant fat diet

A method is described for the separation and quantification of fecal neutral steroids and fecal bile acids. The fecal extract is separated into the neutral steroid fraction and bile acid fraction with ionexchange resin columns. The principal neutral steroids and bile acids are then separated and quantitated by thin-layer chromatography. Values for the fecal neutral steroids, cholesterol, coprostanol and coprostanone and fecal bile acids, deoxycholic acid and lithocholic acid of 5 subjects on a constant fat diet for a 3-week period are presented.     

Nature of fecal sterols and intestinal bacterial flora

Sterol excretion in the spontaneously atherosclerosis-susceptible White Carneau (WC) pigeon, the Silver King (SK) pigeon and the Show Racer (SR) pigeon was studied by thin layer chromatography (TLC), argentation TLC and gas liquid chromatography. Unlike man and the chicken, these pigeons excreted no coprostanol or coprostanone derivatives of sterols. Moreover incubation of¹⁴C-labeled cholesterol with pigeon feces indicated that, also unlike man and the chicken, these pigeons are unable to convert it to coprostanol. Bacteriologic examination revealed the absence of gram-negative anaerobic flora and of members of the genusBifidobacterium in both the WC and SR pigeons. On the other hand, one of the two SK pigeons examined showed evidence of the presence of bothBacteroids fragilis andB. bifidum in the upper intestinal tract. Although no qualitative experiments were performed, no unusual characteristics of the aerobic flora were noted in these pigeons. In addition, analysis of human stool specimens indicated a “normal” bowel flora. The flora of the intestinal tract of the chicken is similar to that of the human. Because of this similarity, it appears that differences in environment (living conditions, diets) between the human and the chicken are of little consequence. The results obtained in this study suggest the possibility that the anaerobic gram-negative flora and sponsible, at least in part, for the chemical conversion of cholesterol to coprostanol. Presented in part at the Fourth International Symposium on Drugs Affecting Lipid Metabolism, Philadelphia, September 1971, and at the AOCS Meeting, Los Angeles, April 1972. The following nomenclature has been used for the steroids referred to in this paper: cholesterol, cholest-Δ⁵-en-3β-ol; coprostanol, 5β-cholestan-3β-ol; campesterol, 24-methylcholest-Δ⁵-en-3βol; stigmasterol, 24-ethylcholest-Δ^5,22-dien-3β-ol; β-sitosterol, 24-ethylcholest-Δ⁵-en-3β-ol; coprocampestanol, 24-methyl-5β-cholestan-3β-ol; coprostigmastenol, 24-ethyl-5β-cholest-Δ²²-en-3β-ol; coprostigmastanol, 24-ethyl-5β-cholestan-3β-ol; coprostanone, 5β-cholestan-3-one; campestanone, 24-methyl-5β-cholestan-3-one; stigmastenone, 24-ethyl-5β-cholest-Δ²²-en-3-one; and β-sitostanone, 24-ethyl-5β-cholestan-3-one.     

Effects of clofibrate and tiadenol on the elimination of lipids and bile acids in rat bile

The aim of the work presented here was to compare the biliary elimination of cholesterol and the different bile acids of rats that had been made hypolipidemic by short-term treatments with clofibrate or tiadenol. Both treatments induced a significant decrease in cholesterol output in the bile. The analysis of the different bile acids showed a decrease in dihydroxylated acids elimination (especially CDC acid) without any difference between the 2 sexes. This decrease was associated with an increase in cholic acid excretion. These results are directly correlated with the dose of the administered hypolipidemic drug. The drugs caused a significant increase in the ratio of trihydroxylated acids to dihydroxylated acids. The maximal effect on the concentration of the biliary acids of the bile and on the output was obtained, for both drugs, with a treatment of 200 mg/kg/day. Clofibrate had a greater effect than tiadenol at this dose. Both drugs show a greater effect on lowering serum lipid levels in female animals when compared to males, whereas elimination of bile cholesterol and modifications of bile acids were greater in male animals than female animals.     

Dietary induced alterations in the fatty acids of rat bone marrow

Weanling rats were fed fat free diets supplemented with 10% added fatty acids so that dietary effects on bone marrow fatty acids could be determined. The addition or deletion of linoleic acid from the fatty acid supplement resulted in alterations of the fatty acid patterns of bone marrow lipids but to a lesser degree than in erythrocyte lipids. With myristic acid supplementation, increased amounts of stearic acid were found in the lipid fractions, the difference between the bone marrow and erythrocyte lipids being less marked than when linoleic acid was fed. The activities of the bone marrow lipases varied with the dietary treatment. When linoleic acid was fed, higher rates of hydrolysis were observed with saturated fatty acid substrates. The reverse occurred when saturated fatty acids were fed.     

The effects of polyoxyethylated cholesterol on fecal bile acids and nitrogen and on cholesterol balance in rats

Polyoxyethalated cholesterol (POEC) is a water soluble derivative of cholesterol which decreases cholesterol absorption in rats without affecting body weight, fatty acid excretion, or intestinal histology. In the present study rat feces were analyzed for cholic, deoxycholic, chenodeoxycholic, muricholic and lithocholic acid following 3 months of feeding a standard or a 2% enriched cholesterol diet with or without 1.5% POEC. In rats maintained on the cholesterol free diet, POEC increased total bile acids (mg/day) by 50% from 14±3 to 21±3 (mean ±SEM) but only the increase in chenodeoxycholic acid was significant (P<0.05). The corresponding POEC effect in the 2% cholesterol diet was 31% (70±8 to 93±3, P<0.01). Fecal nitrogen and serum cholesterol did not vary among groups. Comparing these data with neutral steroid excretion previously determined showed that POEC in the cholesterolfree diet increased the negative cholesterol balance more than three-fold (34±7vs 118±13 P<0.01). In rats fed 2% cholesterol, POEC caused a negative cholesterol balance of 222±8 compared to the control of 27±52 (P<0.01). The data indicate that POEC exerts complex effects in the intestinal tract which increase both bile acid and cholesterol excretion.     

Effect of plant sterols,fatty acids and lecithin on cholesterol absorption in vivo in the rat

The inhibitory effect of plant sterols, fatty acids and lecithin on cholesterol intestnal absorption was studied in the unanesthetized rat using a single pass perfusion technique. Bile was excluded from the perfused intestine. Cholesterol absorption did not change following the additions of cholestanol, cholestanone, lanosterol, stigmasterol and β-sitosterol. A 3-fold increase in the molarity of cholestanol and β-sitosterol or the separate additions of the saturated short and medium chain fatty acids, butyric and octanoic, also did not change cholesterol absorption. The unsaturated long chain fatty acids, oleic, linoleic, linolenic and arachidonic, inhibited cholesterol absorption. Lecithin additions at concentrations of 0.1–1.5 mM caused a progressive, dose-related inhibition of cholesterol absorption. The inhibitory effect of these agents on cholesterol absorption is likely to have been caused by changes in cholesterol solubility in the micelle and shifts in the partition coefficient of cholesterol away from the cell membrane to the micelle.     

Action of three bile acids on hepatic and intestinal cholesterogenesis in the rat

Incorporation of [1¹⁴−C] acetate into cholesterol by subcellular particles from the liver and the small intestine of rats with a biliary diversion and a duodenal perfusion of sodium taurocholate, taurochenodeoxycholate or taurodehydrocholate, was studied in vitro. In the liver, taurochenodeoxycholate prevented the increase of cholesterol synthesis induced by biliary drainage. Taurocholate had no action on cholesterol synthesis at any time, day or night. Intestinal synthesis of cholesterol was reduced by taurocholate and taurochenodeoxycholate but was not modified by taurodehydrocholate infusion.     

Effect of age on plasma bile acids and lipid components in the rat

With increasing age, total plasma bile acid contents increased in rats over a period of 11 months, and also total plasma cholesterol and carcass fat contents increased in the same manner. Plasma showing high bile acid levels at 11 months was found by means of high performance liquid chromatography to contain cholic acid as one of the major components, chenodeoxycholic acid and trace deoxycholic acid. These results suggest that there are close relationships between the plasma bile acids and age-dependent changes of lipid components in the rat.     

Dietary arachidonic and linoleic acids: Comparative effects on tissue lipids

The effects of preformed dietary arachidonic acid (AA, 20∶4n−6) on murine phospholipid fatty acid composition in tissues capable (liver) and incapable (peritoneal exudate cells, PEC) of desaturating and elongating linoleic acid (LA, 18∶2n−6) to AA were investigated. The results were compared with those obtained on matched animals on LA diets by either substituting or supplementing dietary LA with AA. Modest amounts of AA ethyl ester (0.5 wt%) included in the diet significantly increased tissue phospholipid AA levels by 39% and 57% in the liver and in PEC, respectively. The changes were further enhanced when dietary LA and AA intakes were equivalent,i.e., 57% and 68% in liver and PEC, respectively. This enrichment was observed in all phospholipid classes analyzed, with the greatest impact on phosphatidylcholine. In addition, the doubling of dietary LA had little effect on tissue phospholipid AA levels. The data suggest that while the level of n−6 PUFA may have an important effect on tissue fatty acid composition, the type of n−6 PUFA in the diet could be of greater significance.     

Fatty acids and sterols ofCronartium fusiforme basidiospores

The hydrocarbons, fatty acids, and sterols ofCronartium fusiforme basidiospores were examined by gas chromatography and mass spectrometry. Trace quantities of aliphatic hydrocarbons were detected, but these were probably not fungal products. Principal nonsubstituted fatty acids were palmitic (11.7%), linoleic (17.2%), and linolenic (16.1%) acids; the predominant acid was 9,10-cis-epoxyoctadecanoic acid (40.7%). Four sterols were detected, three of which were identified as stigmast-7-enol (57.6%), stigmasta-5,7-dienol (22.7%), and ergost-7-enol (16.0%). The fourth sterol (4%) is a C₂₈ diene. Lipids of the aeciospores and basidiospores ofC. fusiforme are compared.     

Fatty acids and sterols in oils from canola screenings

One sample of canola seed (variety Tower) and five samples of screenings were commercially processed to yield first an “expeller oil” and subsequently an “extractor oil” by the hexane extraction of the residue. The screening samples contained 25–50% intact or broken canola seed. The balance included 21–31% weed seeds (especially lambsquarter and stinkweed), hulls, fragments of the embryo, and chaff. All the oil samples were analyzed for sterol and fatty acid composition. The extractor screening samples had slightly higher sterol contents than the corresponding expeller samples, while the Tower samples gave the lowest values. The averages (in mg/g oil or extract) for the extractor screening samples were: brassicasterol, 1.0; campesterol, 4.1; and β-sitosterol, 7.3. For expeller screening samples the average were: 0.9, 3.6 and 6.2 and for the Tower oils they were, respectively, 0.9, 3.8, 5.3 and 0.9, 3.5, 4.7. The fatty acid compositions of the screening samples for both extractor and expeller oils were similar to that of the Tower oil except for the higher proportions of docosenoic acid (22:1) and eicosenoic acid (20:1) and the more obvious presence of three C₁₈ conjugated dienes totalling up to 0.6% of one screening oil sample. The docosenoic acid level (mainly erucic acid) ranged from 3.0 to 7.0% for the extractor oils and from 2.5 to 8.0% for the expeller samples, compared to 0.1% for the two Tower oils. The oil contents of the screenings ranged from 20 to 30%, and the fatty acids and sterols appear to be nutritionally useful and innocuous in all respects. Presented in part at the ISF/AOCS World Congress, New York, April–May 1980.     

Dietary triacylglycerol structure and saturated fat alter plasma and tissue fatty acids in piglets

Human and pig milk triacylglycerols contain a large proportion of palmitic acid (16:0) which is predominately esterified in the 2-position. Other dietary fats contain variable amounts of 16:0, with unsaturated fatty acids predominantly esterified in the 2-position. These studies determined if the amount or position of 16:0 in dietary fat influences the composition or distribution of liver, adipose tissue, lung, or plasma fatty acids in developing piglets. Piglets were fed to 18 d with sow milk or formula with saturated fat from medium-chain triglyceride (MCT), coconut or palm oil, or synthesized triacylglycerols (synthesized to specifically direct 16:0 to the 2-position) with, in total fatty acids, 30.7, 4.3, 6.5, 27.0, and 29.6% 16:0, and in 2-position fatty acids, 55.3, 0.4, 1.3, 4.4, and 69.9% 16:0, respectively. The percentage of 16:0 in the 2-position of adipose fat from piglets fed sow milk, palm oil, and synthesized triacylglycerols were similar and higher than in piglets fed MCT or coconut oil. Thus, the amount, not the position, of dietary 16:0 determines piglet adipose tissue 16:0 content. The effects of the diets on the plasma and liver triacylglycerols were similar, with significantly lower 16:0 in total and 2-position fatty acids of the MCT and coconut oil groups, and significantly higher 16:0 in the plasma and liver triacylglycerol 2-position of piglets fed the synthesized triacylglycerols rather than sow milk or palm oil. The lung phospholipid total and 2-position 16:0 was significantly lower in the MCT, coconut, and palm oil groups, but similar in the synthesized triacylglycerol group and sow milk group. The lung phospholipid total and 2-position percentage of arachidonic acid (20:4n-6) was significantly lower in all of the formula-fed piglets than in milk-fed piglets. The physiological significance of this is not known.     

Simultaneous quantification of free fatty acids,free sterols,squalene, and acylglycerol molecular species in palm oil by high-temperature gas chromatography-flame ionization detection

This paper discusses a rapid GC-FID technique for the simultaneous quantitative analysis of FFA, MAG, DAG, TAG, sterols, and squalene in vegetable oils, with special reference to palm oil. The FFA content determined had a lower SE compared with a conventional titrimetric method. Squalene and individual sterols, consisting of β-sitosterol, stigmasterol, campesterol, and cholesterol, were accurately quantified without any losses. This was achieved through elimination of tedious conventional sample pretreatments, such as saponification and preparative TLC. With this technique, the separation of individual MAG, consisting of 16∶0, 18∶0, and 18∶1 FA, and the DAG species, consisting of the 1,2(2,3)- and 1,3-positions, was sufficient to enable their quantification. This technique enabled the TAG to be determined according to their carbon numbers in the range of C₄₄ to C₅₆. Comparisons were made with conventional methods, and the results were in good agreement with those reported in the literature.     

Bile acid metabolism in mammals: VI. Effect of ethionine upon bile acids of rat bile

Sex differences in the effect of ethionine upon rat liver metabolism prompted our investigation into possible sex differences in the effect of ethionine upon bile acid metabolism. The bile ducts of 24 rats, 12 male and 12 female, were cannulated. After 1 hr of bile collection, 6 rats of each sex were given ethionine, 1 mg/g body wt, by feeding tube. The bile acid composition of the bile collected during the subsequent 4 hr was analyzed by combined thin layer and gas chromatography. Ethionine induced a reduction in bile flow (3rd and 4th hr) and in bile acid concentration (4th hr) in female rats. The amino acid had no effect upon bile flow but did increase biliary secretion of bile acids (1st and 2nd hr) in male rats. Cholic acid accounted for the bulk of the reduction in total bile acid secretion in the female studies. The increase in total bile acid secretion in the male studies involved all bile acids. The effects of ethionine upon bile acid secretion were delayed in the female studies, immediate in the male. The changes in bile acid secretion involved only the taurine conjugates in the female studies, both taurine and glycine conjugates in the male. There are substantial sex differences in the effect of ethionine upon bile acid metabolism in the rat.     

An improved procedure for bile acid extraction and purification and tissue distribution in the rat

Two bile acid extraction procedures were compared using endogenously radiolabeled tissues and feces. The method of Setchell et al. (J. Lipid Res. 24, 1085–1100, 1983) resulted in essential complete extraction, whereas that of Manes and Schneider (J. Lipid Res. 6, 376–377, 1971) gave recoveries between 56–82%. The time requirement for the method of Setchell et al. could be drastically reduced with no loss in extraction efficiency. Using extracts from endogenously labeled material, a purification procedure using C18 solid-phase extraction cartridges was developed that recovers >90% of bile acids. The distribution of bile acids within the intestinal tract and liver of the rat was determined.