J. Férézou

A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine.

The present study deals with an attempt to describe how the plasma cholesterol level is related to input into the plasma of cholesterol synthesized in the liver and in the intestine. It has previously been shown in our laboratory that, for a given absorption of alimentary cholesterol, the rat plasma cholesterol level decreases when internal secretion of cholesterol (cholesterol synthesized in the organs and poured into the plasma) increases. This relationship was established using rats in which the major source of cholesterol synthesis was the intestine. We used rats fed a cystine-enriched diet (5%) which was previously shown to increase cholesterolemia and internal secretion of cholesterol. It was first demonstrated that a significant positive linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol. Secondly, using [14C]acetate as the cholesterol precursor it was shown that ingestion of the cystine-enriched diet increased hepatic but not intestinal cholesterogenesis. Individual values of cholesterolemia were linearly correlated to those of [14C]acetate incorporation into the hepatic sterols. Results obtained by this method were validated by determining the 13C-labeling pattern of cholesterol synthesized de novo by the liver and the intestine after [13C]acetate infusion. Indeed, this labelling indicated that the dilution of exogenous acetyl-CoA in the liver was not changed by cystine feeding, whereas that in the intestine was enhanced. It is concluded that the plasma cholesterol level varies with internal cholesterol secretion, depending on the organ which determines the variations of this secretion: it decreases when intestinal cholesterogenesis increases, whereas it increases when hepatic cholesterogenesis increases. Finally, the use of [14C]acetate coupled with lipoprotein analysis in rats fed the cystine-enriched diet, in control rats and in rats fed a cholesterol-enriched diet, allowed a new linear correlation to be demonstrated: between cholesterol concentration in LDL2 (lipoproteins of density 1.040-1.063 g/ml) and [14C]acetate incorporation into liver sterols. Our results suggest that LDL2 are produced by the liver in relation to cholesterogenesis in this organ.

Origins of Neutral Sterols in Human Feces Studied by Stable Isotope Labeling (D and 13C)

An experimental procedure using stable isotope-labeled cholesterol (13C and D) was carried out on 15 healthy subjects to distinguish the different origins of neutral fecal sterols in man: nonabsorption of dietary cholesterol, fecal excretion by transfer of plasmatic cholesterol and external secretion of cholesterol biosynthetized in digestive tract and directly eliminated. For a mean daily mass of 652 mg of fecal cholesterol, unabsorbed dietary cholesterol is 20% (133 mg), excreted cholesterol 67% (434 mg) and cholesterol from external secretion 13% (85 mg). A short treatment (4 days) with cholestyramine or different bile acids was then administered to each subject to study the possible variations in their fecal elimination of cholesterol. The more evident effect was the large stimulation of external secretion of cholesterol (234 mg/day) observed after chenodeoxycholic acid feeding (1 g/day). This treatment tends also to decrease dietary cholesterol absorption and to enhance excretion of cholesterol.

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.

Squalene isolation by HPLC and quantitative comparison by HPLC and GLC

A new procedure is described for isolating and measuring squalene in plasma and in several organs of the rat. The unsaponifiable material was fractionated by normal phase HPLC on a silica gel column using a mobile phase consisting of hexane/propanol-2/water. The eluate was monitored at 215 nm. The squalene in the hydrocarbon fraction thus collected was than quantified on an analytical column eluted with hexane. Squalene concentrations ranging from 3 to 200 μg per ml of plasma or per g of fresh tissue were accurately measured. The results obtained agree with those of the squalene assays carried out by gas chromatography on a packed or capillary column.

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.

Plasma lipids, lipoproteins and apolipoproteins in two kindreds of hypobetalipoproteinemia

Plasma lipids and apolipoproteins were quantified in two kindreds of hypobetalipoproteinemia. All affected members were asymptomatic but showed a decrease of 75% in apolipoprotein B and of 69% in LDL-cholesterol. There were no major changes in apo A-I and A-II but all affected family members had reduced levels of apo C-II (by 58%) and C-III (by 59%) without significant decrease in apo C-I and no specific decrease of apo C-III1. Apolipoprotein E is decreased in SDS-PAGE. The plasma level and phenotype of Lp(a) are not affected by HBL, suggesting that a catabolic rather than a synthetic mechanism is responsible for the disease. As shown by density gradient ultracentrifugation, HDL2 particles that contain essentially apolipoprotein A-I, cholesterol and phospholipids represent in affected subjects the major part of HDL. Due to the net reduction of apolipoprotein B-containing particles (VLDL and LDL) as acceptors of lipids in HBL, there is an accumulation of large particles rich in cholesteryl esters.

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.

Hepatic apolipoprotein and LDL receptor gene expression in the genetically hypercholesterolemic (RICO) rat

The present study was designed to examine apolipoprotein and LDL receptor gene expression in genetically hypercholesterolemic RICO rats. In the plasma of RICO rats as compared to SW (control) rats, the hypercholesterolemia (+41%) was associated with a significant increase in plasma apo B (+23%) and apo E (+68%) concentrations. Study of apolipoprotein synthesis in the liver has shown that this increase in plasma apo B and apo E concentrations was not associated with modification in their synthesis and mRNA levels. Study of apo E mRNA level in various tissues has shown only the modification in adrenals in RICO as compared to SW rats (2.7-fold increase). Study of LDL binding, LDL receptor mass and LDL receptor mRNA level in the liver of RICO and SW rats has shown no significant differences between these two strains. EDTA-resistant binding of rat LDL was lower in RICO than in SW rats suggesting that binding sites others than the LDL receptor are present in lesser amount in this hypercholesterolemic strain.

Comparative methods of quantifying fecal neutral sterols in rats and humans after intravenous [14C]-, [3H] or [2H] cholesterol labeling

Several methods used to estimate the fecal elimination of neutral sterols and of cholesterol having a plasmatic origin (called excreted cholesterol) were compared in rats and humans according to the tracer intravenously administered: 14-14C], [1,2-3H]- or octadeuterated cholesterol. In both species, octadeuterated cholesterol had no isotopic effect and the chance occurrence of epicoprostanol in fecal sterols induced an error in the calculation of the fecal excretion of cholesterol. In humans, the use of [1,2-3H]cholesterol appeared to be inaccurate in measuring the fecal flows of cholesterol, because of a loss of 3H radioactivity during the bacterial transformation of cholesterol in the digestive tract. Consequently, the reference method needed to calculate the proportion of excreted cholesterol in fecal cholesterol consisted in dividing the isotopic concentration measured in purified fecal cholesterol by that measured in the appropriate plasma cholesterol sample.

Receptor-dependent and -independent catabolism of low-density lipoprotein in a kindred with familial hypobetalipoproteinemia

Three affected members of a kindred with asymptomatic hypobetalipoproteinemia (HBL) were injected intravenously with 125I-labeled native low-density lipoproteins (LDL) and 131I-labeled cyclohexanedione (CHD)-treated LDL. Plasma and urine radioactivity data were collected for 15 days at regular intervals. A compartmental model using the SAAM program was built to fit simultaneously 125I and 131I plasma radioactivity decay and urine excretion data. This model allows precise calculation of the kinetic parameters of both receptor-independent (NR) and receptor-dependent (R) pathways. Compared with normal subjects, HBL patients show a 90% increased fractional catabolic rate (FCR) of LDL by both routes, more marked for the R pathway (215% increase), and an approximately 50% reduced production rate (PR). Structural analysis did not show significant abnormalities of apolipoprotein (apo) B in HBL patients compared with normal. These data suggest that the very reduced, LDL-apo B plasma levels result from a combination of two processes: (1) an increased activity of all catabolic routes, and (2) a reduced synthesis rate. The latter may result from a decreased conversion of very-low-density lipoprotein (VLDL) to LDL secondary to an increased direct removal of large VLDL, suggested by apo C-II and C-III turnover studies previously reported.