Sterol synthesis from biliary squalene in the jejunal mucosa of the rat in vivo

Because bile contains substantial amounts of cholesterol precursors, e.g., squalene and differnet methyl sterols, the fate of biliary squalene was studied by incubating isolated jejunal loops of the rat in vivo with bile containing³H-squalene and¹⁴C-cholesterol. After 90 min, no radioactivity was found in plasma lipids. Closer analysis of gut epithelium revealed that both labeled compounds were preferentially taken up by the villous cells. Biliary³H-squalene was absorbed almost completely and was further cyclized to free and esterified methyl sterols and cholesterol. Whereas squalene not cyclized to sterols stayed in the mucosa, the newly synthesized sterols were transferred to lumen. The lipid patterns of gut lumen and mucosal cells were quite different, suggesting that the transfer of the newly synthesized lipid to intestinal lumen was not due to the desquamation of epithelial cells alone. The results suggest that biliary cholesterol precursors can contribute to the cholesterol production of the jejunal villous cells bypassing the rate-limiting step of the cholesterol synthesis pathway, and to the “nonexchanging” fecal neutral sterols of the rat.     

Dietary squalene increases tissue sterols and fecal bile acids in the rat

Feeding 1% squalene increased markedly the concentrations of squalene and methyl sterols in each serum lipoprotein class, intestinal mucosa, liver and also in adipose tissue. It also increased cholesterol concentration of the liver and serum VLDL, and esterified cholesterol in serum LDL as well as fecal bile acids. The results suggest that absorbed dietary squalene contributes to some extent to the squalene content of adipose tissue, effectively increases the overall cholesterol synthesis and enhances cholesterol elimination preferentially as fecal bile acids.     

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.     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.     

Inhibition of rat hepatic sterol formation from squalene by plasma lipoproteins

The conversion of³H-squalene to sterols by rat liver microsomes and cytosol was inhibited by individual rat and human plasma lipoproteins at various concentrations. This inhibition was also observed with added human high density apolipoprotein, but triglycerides, cholesterol or cholesteryl esters had no inhibitory effects. Lipoproteins and apo high density lipoprotein (HDL) were demonstrated to bind³H-squalene in vitro. The binding of³H-squalene by apo HDL could be reversed by increasing concentration of liver cytosol containing sterol carrier protein.     

Influence of dietary fat on bile acid secretion of rats after portal injection of3H-cholesterol and [4-14C] cholesteryl esters

Labeled cholesterol and its esters were injected via the portal vein into bile duct-cannulated rats, subsequent to a 7 week regimen of either 5% safflower oil or 5% beef tallow in a hypercholesterolemic diet. Analysis of bile collected over a 6 hr period from the safflower group showed 8.6% higher output of bile acids, 13.6% higher conversion of³H-cholesterol to bile acids and 40% higher conversion of [4-¹⁴C]cholesteryl oleate to bile acids than bile collected from the tallow group. During the 1st hr conversion of both oleyl and linoleyl esters of¹⁴C-cholesterol to bile acids was much slower than conversion of the free³H-cholesterol to bile acids, thus eliminating these esters as preferred substrate for bile acid formation. However at 6 hr two-thirds of the injected¹⁴C of oleyl ester was recovered in the liver, and about half of this was in the form of free cholesterol. Thus cholesterol ester hydrolase, though inhibited by dietary cholesterol, evidently did not impose limitations on formation of free cholesterol for subsequent oxidation reactions. Specific radioactivities were of doubtful significance because of uncertainities as to “active” pool size. The data suggest that dietary linoleate exerts its hypocholesterolemic effect in some manner other than ester formation and that its point of action involves stimulation of cholesterol oxidation to bile acids. Journal Paper No. 4938 EAS, Purdue University.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Inhibition of sterologenesis in brain and liver of fetal and suckling rats from dams fed di-2-ethylhexyl phthalate plasticizer

The effect of di-2-ethylhexyl phthalate (DEHP) on lipid metabolism was studied in liver and brain from fetal rats taken 3 days before parturition from dams receiving dietary DEHP during gestation. In fetuses from rats receiving 0.5% or 1.0% DEHP in a stock diet, the incorporation of¹⁴C-acetate and labeled mevalonate (³H or¹⁴C) into the C₂₇ sterols, C₃₀ sterols, and squalene fractions of brain tissue incubated in vitro was significantly reduced between the confidence limits P<0.05 to P<0.001. When liver from fetuses was incubated with labeled mevalonate, incorporation of label into the C₂₇ sterol and C₃₀ sterol fractions was significantly reduced as well (P<0.02 to P<0.001), whereas incorporation of labeled mevalonate into the squalene fraction was not significantly altered. The incorporation of¹⁴C-acetate into total hepatic lipids of the fetal rats was also studied, and statistically significant reductions in incorporation were observed in the lanosterol fraction (P<0.001), the combined fraction of sterol esters + squalene (P<0.02), and the combined fraction of cholesterol + diglycerides (P<0.01). No significant changes were observed in the incorporation of¹⁴C-acetate into phospholipids, free fatty acids, or triglycerides. In 8-day old suckling rats delivered from dams fed 0.5% DEHP for the last 16 days of gestation and maintained on the same diet during the nursing period, the incorporation of¹⁴C-mevalonate into hepatic C₂₇ sterols, in vitro, was significantly depressed (P<0.05) whereas in corporation into C₃₀ sterols and squalene was similar to control values. In these same suckling rats, body weights were significantly lower in the control group (21.7 vs. 18.8 g, P<0.01), whereas liver weight as a % of body weight was significantly higher (P<0.01) in rats nursing from the DEHP-fed dams. The results indicate that the inhibitory effect of dietary DEHP on lipid metabolism in the mature rat is transmitted across the placental barrier to the developing fetus and that the abnormal pattern of lipid metabolism in rats delivered from DEHP-fed females is only partially restored to normal during the suckling periods.     

Arylsulfonate esters as hypocholesteremic agents: III. Mechanism of action studies

The mechanism responsible for the hypocholestermic action of arylsulfonate esters of long chain fatty alcohols has been studied with rats fed either normocholesteremic or hypercholesteremic (1% cholesterol plus 0.5% glycocholate) diets. Linoleyl tosylate is more effective in lowering plasma and liver cholesterol levels of rats on the hypercholesteremic diet than several other hypocholesteremic agents tested. Linoleyl tosylate does not redistribute cholesterol to extrahepatic tissues nor inhibit hepatic cholesterol biosynthesis. Linoleyl tosylate is not effective in counteracting Tritoninduced hypercholesteremia nor in lowering plasma cholesterol levels of the suckling rat. Linoleyl tosylate increases the fecal elimination of dietary [4-¹⁴C] cholesterol and prevents its accumulation in blood and liver. Oleyl p-(n-decyl) benzene sulfonate also prevents the apparent absorption of [26-¹⁴C] cholesterol from the gastrointestinal tract. Linoleyl tosylate increases the fecal excretion of neurtal sterols but not of bile acids. The results indicate that the arylsulfonate esters of long chain fatty alcohols lower body cholesterol levels by inhibiting cholesterol absorption from the gastrointestinal tract. Exactly how absorption is inhibited is not clear, but linoleyl tosylate was found to stimulate the activity of cholesteryl esterase prepared from the intestinal mucosa. Published as Journal Paper No. 6698 Agricultural Experiment Station, Purdue University, Lafayette, IN.     

Effect of phthalate esters on serum cholesterol and lipid biosynthesis in liver,testes, and epididymal fat in the rat and rabbit

Lipid biosynthesis was studied in vitro in liver, testes, and epididymal fat obtained from rats and rabbits fed di-(2-ethylhexyl)phthalate for 4 weeks at levels of 0.5% and 1.0%, respectively. Several differences in response of the two species to DEHP feeding were observed. In rats, but not in rabbits, DEHP feeding significantly reduced the incorporation of labeled mevalonic acid into total sterols (p <0.02), digitonin-precipitable sterols (p<0.01), and squalene (p<0.05). Inhibition of hepatic sterologenesis previously observed with DEHP feeding in the rat was also observed in the rabbit. In liver minces from the DEHP-fed rabbits, incorporation of³H-mevalonic acid into C₂₇ sterols (cholesterol) and C₃₀ sterols (lanosterol) was significantly reduced by about 40% (p<0.05 and p<0.01, respectively), whereas the incorporation of¹⁴C-glycerol 3-phosphate into phospholipids, and the combined fraction of monoglyceride + diglyceride, was significantly increased (p<0.001 and p<0.01, respectively). In studies with epididymal fat, DEHP feeding did not affect the total incorporation of¹⁴C-acetate or³H-mevalonate into total saponifiable and nonsaponifiable lipids of either the rat or rabbit. However, in the rat, significantly less of the¹⁴C-acetate (p<0.02) and³H-mevalonate (p<0.01) that was incorporated appeared in the combined fraction of cholesteryl ester + squalene. In addition, DEHP feeding significantly reduced serum cholesterol (p<0.01) in the rat but not in the rabbit. The results of this study indicate that DEHP feeding is associated with alterations in tissue lipid metabolism and that there are species differences in the response of tissues to DEHP.     

Site of bile acid absorption in the rat

Bile acid absorption was measured in the small intestine of the rat using⁹¹Y as a nonabsorbed reference substance. Some 50% of the secreted bile acids were absorbed in the proximal half of the small intestine. In situ incubations of ligated intestinal segments into which tauro(¹⁴C-carbonyl)cholic acid was introduced confirmed the considerable uptake of bile acids in the jejunum. The in situ experiments indicated that serosal transport is the limiting stage of bile acid absorption in the jejunum but not in the ileum. Increasing bile acid concentrations in the in situ experiments did not affect the percentage disappearance of dose from the jejunum but reduced the percentage mucosal uptake in the ileum. It is concluded that, in the rat, the proximal small intestine is as important in the absorption of bile acids as the distal small intestine.     

The excretion and characterization of intravenously administered olestra

Although sucrose octaoleate that is consumed is neither digested or absorbed, following intravenous injection it is found mainly in the liver. Olestra is a mixture of the hexa-, hepta-, and octaesters of sucrose. To follow the metabolic fate of intravenously administered [¹⁴C]sucrose-labeled olestra, we measured its urinary elimination, and the rate of excretion of¹⁴C in the feces, and characterized the¹⁴C-labeled material that appeared in bile. The fecal excretion for days 4–14 after dosing was found to be first order with the half-life of the injected olestra being 5.0±0.5 days. The¹⁴C recovered in the bile was soluble in chloroform. Two-dimensional thin-layer chromatography autoradiograms of the biliary lipid showed the pattern of the biliary¹⁴C to be essentially the same as that of the dosed olestra. Biliary excretion and subsequent fecal egestion of essentially unhydrolyzed sucrose esters is the principal route for the removal of intravenously administered olestra. Only traces of¹⁴C were found in the urine.     

Incubation of human fecal homogenates with 4-14C-cholesterol

Fresh fecal homogenates from nine subjects consuming solid diets have been incubated for seven days at 37 C with 4-¹⁴C-cholesterol. One series of incubations was conducted under nitrogen, another under air. The extent of bacterial modification of cholesterol to known fecal metabolites varied considerably among the subjects, as expected, but when present such bacteria were shown to be highly active in the conditions used. Production of¹⁴CO₂ was essentially zero in all incubations. Recovery of added¹⁴C from the incubated homogenates following extraction with chloroformmethanol (2∶1 v/v) and evaporation to dryness was quantitative in all cases. About 4% of the labeled cholesterol added appeared to be present in acidic components following incubation. It is concluded that in the incubation system used vigorous bacterial conversion of 4-¹⁴C-cholesterol to metabolites known to be produced in the human intestine could occur readily, in either aerobic or anaerobic conditions. However, CO₂ or other small, relatively volatile fragments labeled with¹⁴C could not be detected. Part of this work was carried out at the Institute for Metabolic Research, Oakland, Calif.     

The distribution of14C-labeled cholesterol in the dog: Effect of long-term epinephrine administration

A time course study of¹⁴C-cholesterol distribution in dogs was performed after the intravenous administration of 4-¹⁴C-cholesterol. Liver cholesterol attained isotopic equilibrium with plasma cholesterol within 24 hr after the administration of the labeled cholesterol. The tissue cholesterol pools of most organs attained isotopic equilibrium with plasma cholesterol between the second and ninth day after 4-¹⁴C-cholesterol had been given. Thoracic aorta cholesterol equilibrated most slowly with plasma cholesterol. Thirty-seven to 75 days after the¹⁴C-cholesterol had been given, the specific radioactivity of thoracic aorta cholesterol was 2 to 5 times greater than that of plasma cholesterol and 1.73 and 1.65 times greater than that of the abdominal and terminal aortic segments respectively. Adrenal gland cholesterol attained specific radioactivities greater than that of plasma cholesterol between the fourth and ninth day after 4-¹⁴C-cholesterol had been given, and this relationship was maintained over the 75-day period of observation. The specific radioactivity of bile cholesterol was less than that of plasma cholesterol at all time periods. The daily administration of epinephrine in oil over a period of four to seven weeks was accompanied by a more equal distribution of¹⁴C-cholesterol throughout the length of the aorta. An increase in the specific radioactivity of kidney cortex cholesterol, relative to that of plasma and kidney medullary cholesterol, also was observed in epinephrine-treated dogs.     

The biosynthesis of cholesterol and other sterols by brain tissue: I. Subcellular biosynthesis in vitro

The biosynthesis of cholesterol by subcellular particles from rat brain was studied with several labeled cholesterol precursors as substrates. Rats from two age groups were used for preparation of the subcellular fractions: 15-day-old and adult. Microsomes and a soluble fraction were required for maximum biosynthesis of¹⁴C-nonsaponifiable material. The latter was synthesized in good yield by subcellular fractions from both age groups, but 90% or more was present as¹⁴C-squalene, when either U-¹⁴C-glucose, 2-¹⁴C-sodium acetate or 2-¹⁴C-mevalonic acid was the radioactive substrate. Neither³H-squalene oxide nor¹⁴C-lanosterol was converted to sterol when incubated with microsomal+soluble preparations, but some 4% of¹⁴C-desmosterol was converted to cholesterol by adult preparations. Thus a metabolic block, largely between squalene to desmosterol, exists in isolated microsomal+soluble preparations from both 15-day-old and adult rat brain. One of 12 papers to be published from the Sterol Symposium presented at the AOCS Meeting, New Orleans, April 1970.     

Effect of chlorpromazine on rat arterial lipid synthesis,in vitro

The effect of chlorpromazine, a major tranquilizer, on arterial lipid metabolism was studied in vitro in rat aortas incubated with [¹⁴C] acetate and [¹⁴C] mevalonate as lipid precursors. Chlorpromazine at a level of 0.25 mM in the incubation medium significantly reduced the incorporation of [¹⁴C] acetate into free fatty acids (p<0.01) and total phospholipids (p<0.001) but not triglycerides. Chlorpromazine also altered the pattern of arterial phospholipids synthesized from [¹⁴C] acetate by significantly increasing the relative proportion of phosphatidylinositol plus phosphatidylserine (p<0.02) and reducing the relative proportion of sphingomyelin (p<0.001). [¹⁴C] Acetate incorporation into the combined fractions of steryl esters plus hydrocarbons and sterols plus diglycerides was also significantly reduced (p<0.001) by 0.25 mM chlorpromazine. Studies with [¹⁴C] mevalonate showed that chlorpromazine is also an inhibitor of sterol biosynthesis in arterial tissues as evidenced by 35–40% reductions (p<0.05) in the formation of¹⁴C-labeled squalene and C₂₇ sterols.     

Metabolism of an n-paraffin,heptadecane, in rats

¹⁴C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed extensive¹⁴CO₂ excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was ω-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7–10%). Heptadecanoic acid was desaturated to heptadecenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these functional lipids.     

Probable sources of plasma cholesterol during phosphatide induced hypercholesterolemia

Rats in isotopic steady state with respect to 4-¹⁴C-cholesterol were infused intravenously with massive amounts of lecithin and also injected once with Na acetate³H. During the following 8 hr their plasma gained an average of 11.3 mg of cholesterol; the specific activity of¹⁴C-cholesterol fell in plasma while total¹⁴C-cholesterol and³H activity doubled as compared to controls. The specific activity of¹⁴C-cholesterol diminished in livers of rats receiving lecithin but not in controls. Specific activity of either isotope in cholesterol of intestine, lungs, muscle, skin and brains was the same in control and experimental groups. Total activity of¹⁴C and³H fell in cholesterol of liver. The results show that plasma accumulation of cholesterol during lecithin infusion derives from both cholesterol pre-existing prior to infusion and from that newly synthesized after the start of infusion and that about one third of this cholesterol of mixed origin is supplied from the liver. The authors speculatively suggest skin as a likely source for most of the remainder, with a small additional contribution from brain.     

The absorption and transport of dietary cholesterol in the presence of peanut oil or randomized peanut oil

Peanut oil has been shown to be unexpectedly atherogenic for cholesterol-fed rats, rabbits and rhesus monkeys. However, randomization (rearrangement of fatty acids to random distribution) of peanut oil significantly reduced its atherogenicity for rabbits and monkeys. This study was conducted to investigate whether the absorption and transport of dietary cholesterol was altered in the presence of peanut oil or randomized peanut oil, thereby accounting for the difference in the atherogenicity of the two diets. Intestinal lymph fistula rats were infused intraduodenally with a lipid emulsion at a rate of 3 ml/hr. The lipid emulsion contained either peanut oil (control) or randomized peanut oil (experimental) (10 mM),¹⁴C-cholesterol (1.3 mM) and sodium taurocholate (19 mM) in phosphate-buffered saline, pH 6.4. Lymph triglyceride, cholesterol and phospholipid outputs were similar in both groups of rats during fasting and subsequently during lipid infusion. Comparable recovery of¹⁴C-cholesterol from the intestinal lumen and the intestinal mucosa of the control and the experimental rats showed that the absorotion and transport of dietary cholesterol were similar in both groups of rats. Analyses of thefatty acid of both lymph and intestinal mucosal lipid again failed to reveal a difference between the 2 groups of rats. It is concluded that the difference in the atherogenicity between the peanut oil and the randomized peanut oil is probably caused by events subsequent to the release of cholesterol containing chylomicrons and very low density lipoproteins by the small intestinal epithelial cells.     

Origin of the high saturated fatty acid content of rat fecal lipids

The biohydrogenation of unsaturated fatty acids and the preferential absorption of unsaturated fatty acids over long chain saturated fatty acids from the gut have been investigated to find the origin of the high saturated fatty acid content of the facal lipids of rats fed soybean oil. Label from dietary (1-¹⁴C)-linoleic acid was recovered in the saturated and monounsaturated fatty acids of the fecal lipid. However, when (9,10-³H)-stearic acid and (1-¹⁴C)-linoleic acid were fed together, the isotope ratio (³H/¹⁴C) of the fecal lipid was 1.9 times that of the diet. It is concluded that both processes occur.