Dietary fat and cholesterol and serum cholesterol in the gerbil

Groups of gerbils were fed purified diets containing either 10 or 20% of safflower, olive, or coconut oil. Each diet was fed without cholesterol and with 0.1 and 0.2% of added cholesterol. The animals were bled after 2, 4, and 8 wk for the determination of the level of serum cholesterol. The major factors affecting the level of serum cholesterol were the kind of dietary oil, the amount of dietary cholesterol, and the length of time the diet was fed. The level of safflower oil had a statistically significant effect but the level of olive or coconut oil had no significant effect. Various other statistically significant interactions were observed which make simple interpretations of the data difficult. The levels of serum cholesterol achieved in the gerbils fed the different oils with no or very low levels of dietary cholesterol were similar to those seen in men fed the same oils. Although the gerbil is apparently resistant to the development of atherosclerosis, it may be a useful model for studying the effect of dietary fats upon cholesterol metabolism.     

Factors influencing the cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay

Factors affecting the esterification rate of cholesterol by lecithin cholesterol acyltransferase (LCAT E.C. 2.3.1.43) in native cold labelled substrates (human, rabbit, rat serum, plasma, VLDL, LDL depleted serum, rabbit intraocular fluids) repaired by use of ready-made 14C-cholesterol discs (Cholesterol kinetics LCAT-test, UVVVR, Czechoslovakia) were investigated. EDTA added to the serum during the cold incubation (18 h, 0 degrees C-4 degrees C) increased the rate of esterification due to elimination of Ca2+ ions. The similar stimulating effect was found in the presence of mercaptoethanol (ME) in the serum, while in the plasma already stimulated by EDTA no additional effect by ME could be noticed. Freezing and thawing did not affect the fractional esterification rate (FER-per cent of total serum unesterified cholesterol esterified per hour) in normolipidaemic sera, whereas in hyperlipidaemic sera, particularly those with high levels of VLDL, FER was stimulated. Esterification partially proceeded during the cold incubation of serum or plasma with 14C-cholesterol ready-to-use discs, attaining the values of about 0.3%/h and 2-6%/h, respectively, in human sera and in rabbit and rat sera. The starting level of esterification did not affect the linearity of LCAT reaction during warm incubation (30 min at 37 degrees C), neither was the absolute value of FER changed as compared with cold labelled sera with those inhibited by DTNB and reactivated by ME. Substantial LCAT activity was also detected in extremely diluted substrates--such as intraocular fluid collected from rabbits with induced uveitis or after preceding paracentesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dietary cholesterol on the regulation of total body cholesterol in man

Studies on the interaction of cholesterol absorption, synthesis, and excretion were carried out in eight patients using sterol balance techniques. Absorption of dietary cholesterol was found to increase with intake; up to 1 g of cholesterol was absorbed in patients fed as much as 3 g per day. In most patients, increased absorption of cholesterol evoked two compensatory mechanisms: (a) increased reexcretion of cholesterol (but not of bile acids), and (b) decrease in total body synthesis. However, the amount of suppression in synthesis was extremely variable from one patient to another; one patient had no decrease in synthesis despite a large increment in absorption of dietary cholesterol, and two patients showed a complete suppression of synthesis. In the majority of cases the accumulation of cholesterol in body pools was small because of adequate compensation by reexcretion plus reduced synthesis, but in a few patients large accumulations occurred on high cholesterol diets when absorption exceeded the compensatory mechanisms. These accumulations were not necessarily reflected in plasma cholesterol levels; these increased only slightly or not at all.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Catalytic properties of cholesterol oxidase in the cholesterol oxidation reaction in an aqueous medium

The effects of organic solvents on cholesterol oxidation by cholesterol oxidase were investigated. It was shown that the increase of isopropanol up to 15% (v/v) in the reaction mixture based on the triethanolamine buffer solution does not influence the value of V, while the Km value for cholesterol shows a bell-shaped dependence and increases more than tenfold within the organic solvent concentration range of about 10%. The changes in the kinetic parameters of the reaction depend not only on the organic solvent concentrations but also on the buffer origin and substrate solution preparation time. This suggests that the composition of the reaction mixture in the first case influences the cholesterol micelle organization which defines its substrate properties in such reactions. Under this stipulation the experimental results can be interpreted within the framework of a new scheme, according to which the specific interaction between cholesterol and delta 4-cholestenon-3 which leads to a decrease of cholesterol mobility and to acceleration of the reaction rate. With a rise in the organic solvent concentration the above-mentioned interactions decelerate, and the enzymatic reaction can be well described by the Michaelis-Menten equation. It is supposed that the cholesterol oxidase-mediated oxidation of cholesterol occurs at the counterface of two phases.     

Automatic gas chromatographic determination of the high-density-lipoprotein cholesterol and total cholesterol in serum

A new analytical method that combines on-line precipitation-filtration, enzymatic hydrolysis, extraction and gas chromatography was developed for the determination of total cholesterol and high-density-lipoprotein cholesterol in human serum. Very-low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein are precipitated with sodium phosphotungstate and magnesium chloride; then, the serum is continuously filtered and unprecipitated high-density-lipoprotein cholesterol is enzymatically hydrolyzed and finally determined as cholesterol by gas chromatography. Total cholesterol is also determined by direct introduction of the serum into the proposed system. The proposed method was validated by analyzing a lipid control serum with certified contents of high-density-lipoprotein cholesterol and total cholesterol. The results obtained were consistent with the certified contents.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

Regulation of gallbladder cholesterol concentration in the hamster. Role of hepatic cholesterol level

The goal of this investigation was to determine how alterations in hepatic cholesterol metabolism influence the cholesterol content of gallbladder bile in hamsters. Although the rate of hepatic cholesterol synthesis was varied over 600-fold, there was no direct relationship between the rate of cholesterol synthesis and the cholesterol content of gallbladder bile. However, expansion of the hepatic cholesterol pool by 42-fold resulted in an 11-fold increase in gallbladder bile cholesterol. Examination of four subfractions of the hepatic cholesterol pool revealed that the cholesterol content of gallbladder bile was most consistently correlated with the free cholesterol level in both hepatic tissue and hepatic microsomes from all experimental groups. In most groups of animals in which gallbladder bile cholesterol was increased, plasma lipoprotein cholesterol levels were also increased. It was concluded that in hamsters, under these experimental conditions, changes in the cholesterol content of gallbladder bile were directly related to alterations in cholesterol content of the liver and most closely related to alterations in the free cholesterol content of that tissue.     

The role of intracellular cholesterol transport in cholesterol homeostasis

How cholesterol is transported among the membranes of the cell is obscure. Similarly, the mechanisms governing the abundance of cell cholesterol are not entirely understood. It may be, however, that a link exists between the intracellular transport of cholesterol and its homeostasis. We propose that cholesterol circulates between the plasma membrane, which contains the bulk of the sterol, and organelle membranes, which contain only traces. A putative sensor translates small fluctuations in plasma membrane cholesterol into relatively large changes in this flux, thereby setting the magnitude of the intracellular pools. The cholesterol concentration in the endoplasmic reticulum and mitochondrial membranes then governs the activities of proteins embedded therein that mediate cholesterol transformations. This arrangement creates a feedback loop through which the intracellular effectors regulate the abundance of plasma membrane cholesterol.     

Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers

The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (CH) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant alpha-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k (p)/(2k (t))(1/2)=R (p)/[LH]R(i)(1/2) by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability. A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals.     

Influence of fasting and cholesterol feeding on the cholesterol synthesis in rats in vivo.

An inhibition of the cholesterol synthesis located between lanosterol and cholesterol could be shown in fasting rats in vivo with glucose, palmitate, acetate, and mevalonate as tracer substances. The same type of inhibition of the cholesterol synthesis was observed after cholesterol feeding. No indication was obtained for any other inhibiting effect located between acetyl-coenzyme A and lanosterol by these in vivo experiments.     

Importance of macrophage cholesterol content on the flux of cholesterol mass

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL₃, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r² = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r² = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [³H]cholesterol efflux and reductions in cholesterol mass (r² = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.