C. Lutton

Vitesse des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat: III. Modifications et étude critique de la méthode d'équilibre isotopique

Abstract Since the isotopic equilibrium method as applied to rat cholesterol was described, some practical modifications have been made which are reported here. Sometimes the use of a subcutaneous implantation of capsules containing tritiated cholesterol leads to an over-estimate of the rate of fecal cholesterol excretion. For the questionable cases, it is necessary to use the initial method of daily subcutaneous injections of radioactive cholesterol. In another connection, 17 experiments using the isotopic equilibrium method have shown great variability of the weekly radioactive balance. The radioactivity of cholesterol and its transformation products released from the organism of the rat and that of absorbed cholesterol are rarely equal as would be expected. To define the origins of these losses an experiment using the isotopic equilibrium method was carried out on rats which were killed after 4, 8 or 12 weeks. Bacterial degradation cannot explain the deficit in the weekly radioactive balances, for the total radioactive balance which includes the radioactive cholesterol of the rat is always nil. In fact, these deficits can be explained on the basis that isotopic equilibrium is not really obtained or that the organism is not in a steady state but in an expansion state. In both cases the application of suitable relations permits the calculation of all the rates involved in the dynamics of cholesterol with certainty. But for an organism in an expansion state it is necessary to know the rate of cholesterol increase. After these corrections have been made, the percentage of error on each rate is defined. In the particular case of this critical experiment, animals were fed with a basal diet to which, 0.2% taurocholate was added. Although this results in an expansion state, the rates of synthesis, excretion and absorption are constant. The increase of mobile cholesterol in the organism is associated with a decrease of its transformation to bile acids.

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol.

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.

Biodynamics of cholesterol and bile acids in the lithiasic hamster

By using the isotopic equilibrium method in the young male Syrian hamster, the rates of cholesterol turnover processes, i.e. dietary cholesterol absorption, cholesterol synthesis, cholesterol excretion in the faeces and urine and cholesterol transformation into bile acids, were determined in the hamster receiving a control (C) or a lithogenic diet (L) for 7 weeks. At the end of this period the gall bladder of all animals in group L contained cholesterol gallstones. The coefficient of dietary cholesterol absorption was reduced by 26%, cholesterol synthesis and cholesterol faecal excretion were twofold higher in group L than in group C. Bile acid content in the small intestine was diminished in group L, but bile acid composition was similar in the two groups. The increase in cholesterogenesis in lithiasic animals essentially took place in the liver. Bile acid biosynthesis did not significantly differ in the two groups, but represented only 35% of total cholesterol input (dietary absorption + internal secretion) in group L v. 52% in group C. Thus, in the lithiasic hamster, hepatic synthesis of cholesterol and bile acids are not coupled. The molar percentage of cholesterol in bile was twofold higher in group L than in group C but those of bile acids and of phospholipids were not modified. In the lithiasic hamster the specific activity of biliary cholesterol was similar to that in plasma and liver. Consequently, biliary cholesterol does not derive directly from cholesterol newly synthesized in the liver but from hepatic cholesterol rapidly exchangeable with plasma cholesterol.

Inducing cholesterol precipitation from pig bile with β-cyclodextrin and cholesterol dietary supplementation

BACKGROUND/METHODS In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile. RESULTS Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0<<10<5% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0<5<10% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet. CONCLUSIONS We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.

Cholesterol metabolism in the genetically hypercholesterolemic rat (RICO) I. Measurement of turnover processes

The rates of mobile cholesterol turnover processes were measured by the isotopic equilibrium method in normocholesterolemic (SW) and hypercholesterolemic homozygote (RICO) rats fed a semi-synthetic base diet containing 0.05% cholesterol. When the absorption rate is similar in SW and RICO rats, the internal secretion rate is 60% higher in RICO (25.3 mg/day) than in SW (16.2 mg/day). This increase is compensated by an increase in fecal excretion (RICO: 5 mg/day; SW: 3.8 mg/day), urinary excretion (RICO: 1.7 mg/day; SW: 1.1 mg/day) and above all the transformation of cholesterol into bile acids (RICO: 24.2 mg/day; SW: 15.3 mg/day). The fact that 70 minutes after [14C]acetate administration, the only variations obtained in RICO compared to SW rats are a doubled sterol radioactivity in the small intestine and a tripled one in the liver suggests that the increase in internal secretion of the RICO rat has both an intestinal and hepatic origin. This cholesterogenic stimulation in RICO rats takes place in the jejunum as well as in the ileum and in the crypt cells as well as in the villosities. It is concomitant with a doubled cholesterolemia, a doubled intestinal, caecal and colon bile acid pool and a 20% increase in the enterocyte protein content.

Induction of long-lasting hypercholesterolemia in the rat fed a cystine-enriched diet

The influence of dietary excess (5%) of L-cystine on rat plasma lipoproteins was examined. After only one week of cystine feeding, an increase in the plasma cholesterol level and a decrease in triglyceride levels were observed. The increase in cholesterol level became greater when the duration of cystine-enriched diet increased until eight weeks (+131% after eight weeks), but no further increase occurred between 8 and 20 weeks. This change was essentially due to the progressive increase in cholesterol levels in high density lipoproteins (HDL) and in lipoproteins isolated between 1.040 and 1.063 g/ml, i.e., certain low density lipoproteins (HDL2), and containing mainly apoE-rich lipoproteins (HDL1). The decrease in plasma triglycerides resulted from that of chylomicrons and very low density lipoproteins (VLDL). The effects observed after four or eight weeks of cystine feeding were maintained for eight weeks after replacing the cystine diet by the standard diet. Ingestion of the standard diet containing either cholestyramine (2%) or probucol (0.25%) following eight weeks of cystine feeding significantly decreased plasma cholesterol levels. It is concluded that cystine-fed rats are a useful tool of investigation for understanding mechanisms leading to increased plasma cholesterol level and for hypocholesterolemic drug trials.

Biliary cholesterol absorption in normal and L-thyroxin-fed rats.

Infusion of bile containing labeled cholesterol into bile fistula rats has permitted an in vivo study of the movements and of the absorption of biliary cholesterol in the digestive tract. The specific activities of cholesterol were similar in the micelles and the sediment of the luminal content after a 6 hr infusion, indicating rapid exchange of cholesterol between these fractions. In animals fed a basal diet, the biliary cholesterol absorption was higher (83%) than that of dietary cholesterol (70%). Bile cholesterol is essentially absorbed in the jejunum while the absorption of cholesterol from the diet takes place all along the small intestine but preferentially in its second and third quarters. Both alimentary cholesterol and bile cholesterol enter the top cells of the villi in preference to those of the crypts. In L-thyroxin-fed rats, a parallel decrease in biliary and dietary cholesterol absorption was observed. The increase in the intestinal transit of cholesterol and epithelium cell renewal of the jejunum accounted for this observation.

Origin and Fate of Cholesterol in Rat Plasma Lipoproteins in vivo

After a single ingestion of a diet containing 14C-cholesterol, cholesterol radioactivity in the stomachal and intestinal contents, in the different organs and in the very low density lipopr

Cholesterol crystallization in gall-bladder bile of pigs given cholesterol-β-cyclodextrin-enriched diets with either casein or soyabean concentrate as protein sources

Cholesterol precipitation from supersaturated bile is the earliest and determinant step in the formation of cholesterol gallstones, which is thought to be diet-dependent. Bile composition, appearance and growth of cholesterol crystals were studied in fresh gall-bladder biles from pigs adapted to four different protein-containing diets over 3 weeks: 160 g dietary protein/kg as casein (C16; n 6), or as soyabean-protein concentrate (S16; n 6), or a mixture of both protein sources (casein-soyabean protein, 70:30, w/w) (CS16; n 6), or 320 g of the mixed protein/kg (CS32; n 6). Moreover, all four diets contained 3 g cholesterol/kg and 50 g beta-cyclodextrin/kg as modifiers of bile composition towards cholesterol pro-crystallization. Cholesterol precipitation was most active after the high-protein diet, CS32, and the casein diet, C16, and lowest after the soyabean-protein diet, S16. It was intermediate after the mixed diet, CS16, but still much lower than in the former two groups. These diet-induced variations were suggested to be mediated through modifications in the biliary profile of bile acids, whereas all other biliary constituents studied were essentially unchanged. The fasting level of plasma cholesterol was lowest in both 160 g protein/kg diets containing soyabean protein (S16 and CS16), highest for the high-protein diet CS32, and intermediate for the C16 diet. These results should encourage clinical studies on the effect of soyabean protein, or other vegetable proteins, for primary or recurrence prevention of cholelithiasis at its earliest stage.

Diet and sterol biohydrogenation in the rat: Occurrence of epicoprostanol

The fecal sterols from rats fed several types of semipurified or commercial diets were analyzed by a combination of thin layer and gas liquid chromatography. In rats fed semipurified diets with lard, sucrose, and casein, increasing proportions of lard (0, 8, 20, 65%) enhanced the fecal coprostanol/coprostanol + cholesterol ratio (from 0.50 to 0.85). This ratio was reduced by replacing lard with triolein or a mixture of calcium oleate and linoleate (1∶1) and did not change when trierucin was substituted. No coprostanol formation was observed in rats fed a diet with tripalmitin or tristearin. The addition of sodium hyodeoxycholate (0.5%) or cholestyramine (2%) to the basal diet was without effect on the coprostanol/coprostanol + cholesterol ratio in the feces. The addition of sodium taurocholate (0.2, 0.75, and 4%) strongly reduced coprostanol formation, while a chronic bile duct ligation led to an enhancement. Cholesterol feeding (0.05, 0.2, and 0.5% in the diet) slightly increased (from 51 to 66%) coprostanol formation. Trace amounts of epicoprostanol were generally found in the feces. However, in some cases a very high proportion (up to 60%) of this sterol was observed. Possible relationships between the presence of epicoprostanol and the nature of the diet are discussed.