Dual-isotope Plasma Ratio Method Research Articles

Multiple mechanisms of hypocholesterolemic action of pactimibe, a novel acyl-coenzyme A:cholesterol acyltransferase inhibitor

Novel acyl-coenzyme A:cholesterol acyltransferase inhibitor pactimibe has been evaluated in vivo; it exhibited significant serum cholesterol lowering activities in hamsters and monkeys without affecting non-high density lipoprotein cholesterol levels. The mechanism of the hypocholesterolemic action of pactimibe was examined in normocholesterolemic hamsters in this study. Results with the dual-isotope plasma ratio method indicated that pactimibe inhibits cholesterol absorption from the intestine, reduces cholesteryl ester formation in the liver, and enhances its elimination from the body. The Triton WR-1339 experiment showed that pactimibe inhibited secretion of very low density lipoprotein cholesterol from the liver. These results suggest that pactimibe is likely to have multiple mechanisms of action responsible for its effectiveness in reducing serum cholesterol.

Effect of silymarin and its polyphenolic fraction on cholesterol absorption in rats

This study evaluated the influence of silymarin (SM) and polyphenolic fraction (PF) of silymarin on cholesterol absorption in rats fed on high cholesterol diet (HCD). HCD induced a remarkable increase in hepatic, plasma, VLDL and LDL cholesterol, a decrease in HDL cholesterol and an elevation in triacylglycerol (TAG) levels in plasma, VLDL and in the liver. SM and PF were administered as dietary supplements (1.0%) in HCD for 18 days. Intestinal cholesterol absorption was measured by dual-isotope plasma ratio method, which calculates percent of cholesterol absorption from the ratio of two labelled cholesterol doses, one given intragastrically ( 14C) and one intravenously ( 3H). Silymarin and PF significantly reduced cholesterol absorption in rats fed on HCD and caused significant decreases in plasma and VLDL cholesterol and content of cholesterol and TAG in the liver. The level of HDL cholesterol was significantly increased after silymarin, but not after administration of PF. The levels of TAG in plasma and VLDL were not affected by either silymarin or PF. These results suggest that the inhibition of cholesterol absorption caused by silymarin and its polyphenolic fraction could be a mechanism contributing to the positive changes in plasma cholesterol lipoprotein profile and in lipid content in liver.

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of 125I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (−85% and −63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum. —Nguyen, L. B., S. Shefer, G. Salen, G. S. Tint, F. Ruiz, and J. Bullock. Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin.

A mutual inhibitory effect on absorption of sphingomyelin and cholesterol

Several studies have shown that there is a strong physical interaction between cholesterol and sphingomyelin (SM). The critical factor is thought to be the high degree of saturation in the very long acyl chains of SM. In this study we examined the effects of SM on cholesterol absorption in the rat and compared them with those of phosphatidylcholine (PC). Cholesterol absorption was studied by use of the dual-isotope plasma ratio method. We also studied the effect of sterols on the fecal excretion of undigested SM and its metabolites after a single oral meal of 3H-dihydrosphingosine-labeled SM. When cholesterol was given dissolved in soybean oil, without addition of SM or other phospholipids, absorption was 68 ± 12% in the rat intestine. As a general feature the absorption was less efficient from the cholesterol/phospholipid dispersions. In dispersions with cholesterol and SM, the lowest cholesterol absorption (9 ± 2%) was seen with a cholesterol:SM molar ratio of 1:1. With dispersions of cholesterol and different PC substrates the absorption of cholesterol was lower with saturated PC (16 ± 8%) than with soybean-PC (22 ± 4%) or dioleoyl PC (23 ± 8%). Uptake of SM in the rat intestine was reduced by sterols. For example, percentage recovery of 3H radioactivity in fecal lipids was 38 ± 8% when SM was given with cholesterol and 16 ± 3% without any sterol. One third of the radioactivity in feces was present as ceramide. Sitostanol had the same effect on uptake of SM as cholesterol. This study shows that when rats are fed mixtures of SM and cholesterol the intestinal uptake of both lipids is decreased. By feeding mixtures of SM and sterols the exposure of the colon to ceramide can be increased.

Cholesterol lowering action of HOE 402 in the normolipidemic and hypercholesterolemic Golden Syrian hamster

The potent hypolipidemic activity of HOE 402 (4-amino-2-(4,4-dimethyl-2-oxo- l-imidazolidinyl)pyrimidine-5- N-(trifluoromethylphenyl)carboxamide monohydrochloride), which was previously demonstrated in rat and rabbit, was investigated in noncholesterol and cholesterol fed male hamsters. In normolipidemic hamsters fed a low cholesterol chow diet containing 0.10% or 0.15% HOE 402 for 3 weeks, the plasma total cholesterol level fell by 13% and 20% respectively, but no effect on hepatic total cholesterol content was detected. Hepatic sterol synthesis was increased 3-fold in hamsters fed 0.15% HOE 402. In hamsters fed a chow diet containing 0.25% cholesterol for 3 weeks, the plasma cholesterol level increased to 226 mg/dl (compared to 123 mg/dl in their chow fed controls) and the liver cholesterol content was 26.2 mg/g compared to 2.3 mg/g in the control group. However, 0.15% HOE 402 led to a 48% reduction and 0.20% HOE 402 to a 80% reduction, in total hepatic cholesterol concentration. There was a 43% fall in plasma cholesterol level being observed with the higher HOE 402 dose. Using the dual isotope plasma ratio method, no inhibition of intestinal cholesterol absorption by HOE 402 was found, either in the noncholesterol fed or in the cholesterol fed hamsters. Cholesterol feeding diminished the whole LDL animal clearance to 393 ± 17 μl/h per 100 g animal (control 666 ± 81 μl/h per 100 g). When treated with 0.20% HOE 402, the whole animal LDL clearance rate was enhanced 2.3-fold to 824 ± 66 μl/h per 100 g. In the hamsters fed 0.25% cholesterol alone whole liver LDL receptor activity was suppressed to 63 ± 5%, compared to that in the untreated controls (100%). The addition of 0.20% HOE 402 to the cholesterol enriched diet not only reversed this suppression, but resulted in a marked stimulation of liver receptor activity to 165 ± 15% (whole body LDL receptor activity 141 ± 10%). These results indicate that HOE 402 exerts its lipid lowering effect by a more direct activation on hepatic LDL receptor activity rather than by an indirect intestinal effect on cholesterol absorption.

Effect of oyster mushroom (Pleurotus Ostreatus) and its ethanolic extract in diet on absorption and turnover of cholesterol in hypercholesterolemic rat.

The effect of the diet containing 5% of powdered oyster mushroom (Pleurotus ostreatus) or an equivalent amount of mushroom ethanolic extract on cholesterol content in serum and liver, on its distribution in lipoproteins, absorption and turnover was studied in male Wistar rats (initial body weight about 70 g) fed a diet with 0.3% cholesterol. 12 weeks of feeding with whole oyster mushroom or mushroom extract reduced cholesterol level in serum by 52 and 33%, respectively. However, cholesterol content in liver was reduced only by whole oyster mushroom (by 20%). Diminished serum cholesterol level was mediated in 60% by reduction of cholesterol in very-low-density lipoproteins. Both whole oyster mushroom and mushroom extract increased the concentration of cholesterol in high-density lipoproteins. Consuming whole oyster mushroom decreased cholesterol absorption (estimated by dual-isotope plasma ratio method) by nearly 16% while no significant effect of mushroom extract could be demonstrated. Feeding the diet containing whole oyster mushroom or its extract reduced the half-times of decay curve of cholesterol-4-14C by 29 and 35%, respectively and reciprocally increased the fractional catabolic rate of plasma cholesterol.

Mechanism of hypocholesterolemic effect of oyster mushroom (Pleurotus ostreatus) in rats: reduction of cholesterol absorption and increase of plasma cholesterol removal.

The content of cholesterol in the serum and liver of male Wistar rats fed, for the period of 8 weeks shortly after weaning, a diet containing 0.3% of cholesterol was reduced by 33 and 27% by the addition of 5% of dried oyster mushroom powder. Although the level of serum triacylglycerols was not affected by oyster mushroom, their content in liver of rats on mushroom diet was reduced by 41%. Very-low-density lipoproteins and low-density lipoproteins participated by 55 and 38%, respectively, in the total reduction of serum cholesterol. Cholesterol content in high-density lipoproteins was not significantly affected by oyster mushroom. Cholesterol absorption as determined by dual-isotope plasma ratio method was significantly reduced by 14% with oyster mushroom diet. Similarly, this diet increased by 42% the fractional catabolic rate of cholesterol determined by the analysis of decay curve of [4-14C]cholesterol.

Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster.

These experiments systematically evaluated the dual-isotope plasma ratio method for measuring intestinal cholesterol absorption in the hamster. It was found that while the ratio of the 3H- and 14C-labeled cholesterol in the plasma, relative to the respective dose of each that was given, became constant by 72 h after their administration, the percent cholesterol absorption was lower in animals that were fasted before dosing (35.7 +/- 5.5%) than in their fed controls (47.5 +/- 3.7%). Furthermore, the percent absorption found 72 h after dosing varied greatly, depending on whether the intragastric dose of labeled cholesterol was administered in medium chain triglyceride (MCT) oil (46.2 +/- 2.3%), olive oil (63.9 +/- 11.2%), or safflower oil (74.6 +/- 4.5%). The level of absorption was not different between hamsters that had unrestricted (46.3 +/- 1.6%) and restricted (43.8 +/- 2.2%) access to their stools during the 72 h after dosing. Other experiments, using only hamsters in the fed state and MCT oil as the intragastric dosing medium, showed that the percent cholesterol absorption could be made to vary over a wide range using treatments known to produce such effects in humans. Thus, feeding either surfomer, cholestyramine, ursodeoxycholic acid, or CI-976, a new inhibitor of acyl-CoA:cholesterol acyltransferase, significantly blocked cholesterol absorption, whereas the addition of either cholic acid or increasing amounts of oil to the diet had the opposite effect. The modified dual-isotope plasma ratio method described here provides a simpler and more physiologic approach to the routine measurement of cholesterol absorption in the hamster and similar small animal models.

Influence of lipid-rich diets on intestinal cholesterol uptake.

The purpose of this work was to determine whether or not cholesterol transport by intestinal brush border is influenced by fat-enriched diets and therefore plays a role in the regulation of cholesterol absorption. This study was carried out on rats divided into three groups according to diet: (1) control with a diet containing 1.2% cholesterol (diet T), (2) diet T plus 28% saturated fat (lard) and (3) diet T plus 28% polyunsaturated fat (corn oil). Uptake of cholesterol and oleic acid from defined mixed micellar solutions was studied on two experimental models: everted sacs and brush border vesicles of the intestinal membrane. In vivo cholesterol absorption was measured by the dual isotope plasma ratio method of Zilversmit. Fat-enriched diets decreased both in vivo cholesterol absorption and in vitro cholesterol uptake without any specific effect of unsaturated fats. This suggests that the mechanisms involved in the transport of cholesterol across the brush border membrane may be rate-limiting for cholesterol absorption. Oleate and butyrate uptakes, in contrast, were unaffected by the fat content of the diet.

A new aspect on the mechanism of intestinal cholesterol absorption in rat.

In order to study the mechanism of intestinal cholesterol absorption, the relationship between the amount of cholesterol administered and the rate of absorption was investigated by the dual isotope plasma ratio method in vivo and the ligated-loop method in situ. The energy requirement of cholesterol absorption was also observed by means of the ligated-loop method. The results obtained are summarized below. 1) Tri-phase absorption was observed by the dual isotope plasma ratio method. When less than 300 microgram of cholesterol was administered, absorption increased linearly, with the coefficient of absorption being more than 80%. When the amount administered was between 300 and 500 microgram, the absorption was constant. With the administration of more than 500 microgram, absorption increased linearly, but the coefficient of absorption decreased to approximately 55%. 2) With the ligated-loop method, a second saturation profile was obtained when between 250 and 400 microgram of cholesterol was administered to a segment. When 50 to 250 microgram of cholesterol were administered, absorption increased in proportion to the increase in cholesterol dosage. 3) The mucosal uptake of cholesterol decreased to 40-60% of the control with the addition of metabolic inhibitors such as NaN3, KCN, 2,4-DNP and ouabain, whereas the uptake of palmitate showed no significant decrease. In addition, the uptake of cholesterol decreased remarkably to 25% of the control with the lowering of body temperature from 37 degrees C to 27 C. These results suggest the existence of an active transport system which has a limited capacity for cholesterol absorption and which requires energy for its operation in the physiological state.

Cholesterol absorption and turnover in rhesus monkeys as measured by two methods.

We compared the absorption of cholesterol in seven rhesus monkeys (four high-responders and three low-responders) as measured by two methods: 1) the dual isotope plasma ratio method of Zilversmit (1972. Proc. Soc. Exp. Biol. Med. 140: 862) and 2) the single isotopic meal feeding technique of Borgström (1969. J. Lipid Res. 10: 331). We also compared the cholesterol pool sizes calculated by kinetic analysis of the plasma cholesterol specific activity decay curves obtained after simultaneous administration of [(3)H]- and [(14)C]cholesterol, one given intravenously and the other orally. The ratio of orally to intravenously administered cholesterol radioactivity in plasma did not attain constancy until 6 weeks after isotope administration. Therefore, the percent absorption of cholesterol was calculated by the Zilversmit method 8 weeks after the administration of isotopes. The mean percent absorption of cholesterol by the Borgström method was 66.3 +/- 5.1 (S.E.) and by the Zilversmit method was 70.3 +/- 7.4. The differences were not statistically significant. However, in two of seven monkeys the percent absorption of cholesterol calculated by the Zilversmit method was higher by 10.4 and 22.6 percentage points than the values obtained by the Borgström method. Cholesterol absorption by either method was higher in the high-responding monkeys than in the low-responding group. The sizes of the rapidly exchangeable pool or the minimum estimate of the total body pool of cholesterol were similar for all monkeys or for either the low-responding or the high-responding animals and were also similar when calculated using the data from either the orally or the intravenously administered radioactive cholesterol. Cholesterol synthesis was significantly higher in the low-responding monkeys (115 mg/day) than in the high-responding (64 mg/day). The present study and our previous studies support the hypothesis that a major factor causing the difference in response of plasma cholesterol to dietary cholesterol between the high- and low-responding rhesus monkeys is a difference in the intestinal absorption of cholesterol.-Bhattacharyya, A. K., and D. A. Eggen. Cholesterol absorption and turnover in rhesus monkeys as measured by two methods.

Inhibition of .BETA.-sitosterol on intestinal cholesterol absorption in rat using in vivo dual isotope ratio method.

The inhibitory effects of beta-sitosterol on intestinal cholesterol absorption were studied by means of a dual isotope plasma ratio method (in vivo), which is a new technique for the measurement of cholesterol absorption, as well as a ligated-loop method (in situ). The results obtained were as follows: 1. The absorption of beta-sitosterol itself was significantly less than cholesterol. Cholesterol was selectively absorbed from rat intestine. 2. When 100 to 1,000 microgram of beta-sitosterol were added to the dose solution containing 10 microgram of cholesterol, cholesterol absorption by the in vivo experiment decreased with the increae of additional beta-sitosterol. 3. A similar inhibitory effect of beta-sitosterol was observed by the in situ ligated-loop method. These results suggest that beta-sitosterol actually inhibits cholesterol absorption in the physiological state of an animal.

Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats

Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol ((14)C or (3)H) and an intravenous dose of colloidal labeled cholesterol ((3)H or (14)C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-hr period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method.