Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Reduced synthesis and rapid esterification of cholesterol in the liver of hamsters with spontaneous hypercholesterolemia

The rates of in vitro incorporation of [2-14C]mevalonate and of [2-14C]acetate were determined in the liver and in the ileum obtained from two groups of hamsters. The first (N-H) were conventional hamsters with normal cholesterolemia, the second (FEC-H) were hamsters which develop high levels of cholesterol in plasma and in the liver with age. In FEC-H, the levels of cholesterol reached 130 to 220 mg/100 ml in plasma and 600-2400 mg/100 g fresh tissue in the liver in contrast to about 100 mg and 350 mg respectively in the N-H group. The accumulation of cholesterol in the tissues of FEC-H was found to be associated with a strong decrease in the incorporation rate of the precursors into cholesterol, but above all, the distribution of the radioactivity derived from [2-14C]mevalonate between free cholesterol and esterified cholesterol was markedly different in the two groups of hamsters. In FEC-H, 78% of the radioactivity was found in the esters, as opposed to 10% found in the N-H group. Thus, the development of hypercholesterolemia with age in FEC-H might be related to alterations in the enzyme systems (ACAT, CEH) which modulate the level of cholesterol esters in the liver. No significant difference was observed between the two groups of hamsters either in the concentration of cholesterol or in the rate of cholesterogenesis in the ileum.     

Pantethine inhibits cholesterol and fatty acid syntheses and stimulates carbon dioxide formation in isolated rat hepatocytes

The effects of pantethine on cholesterol and fatty acid metabolism were investigated in isolated rat hepatocytes. Preincubation of the cells with pantethine induced a concentration-dependent decrease of the radioactivity incorporated into carbon dioxide and lipids in incubations with [2-14C]acetate. When pantethine and the labeled substrate were simultaneously added to the cell suspension, there was an enhancement of carbon dioxide radioactivity at short incubation time (5 min) whereas, at longer incubation time, values were comparable to those of controls; lipid radioactivity, instead, was dramatically reduced by pantethine even at short incubation time and decreased further during the incubation, being 23% of that of controls at 60 min. Analysis of the incubation medium showed that pantethine induced a concentration- and time-dependent release of acetate into the medium. Results of the effect of the acetate concentration on the incorporation of [2-14C]acetate radioactivity into CO2 and lipids in control hepatocytes allowed the conclusion that the above-described modifications induced by pantethine are only partially attributable to the dilution of the labeled substrate, and that catabolism of acetate to carbon dioxide is stimulated by the disulphide pantethine, whereas cholesterol and fatty acid syntheses are inhibited.     

Biosynthesis of cholesterol in rabbit reticulocytes and its exchange with plasma

When rabbit reticulocytes were incubated in normal blood plasma containing mevalonic acid-2-(14)C, radioactivity was incorporated into cholesterol, cholesteryl esters, and squalene in the cells. The squalene reached a steady level of radioactivity much more rapidly than did cholesterol. Rabbit reticulocytes which were labeled as a result of previous incubation with mevalonic acid-2-(14)C were incubated with normal autologous blood plasma. The specific activity of the cholesterol in the plasma rapidly became higher than that of the cells. This suggests that there is compartmentation of cholesterol in the reticulocyte and that a pool involved in exchange with plasma cholesterol has a specific activity which is much higher than the average for the whole cell.     

Reverse cholesterol transport in the isolated perfused rat spleen.

1. A method has been developed which enables the rat spleen to be loaded in vivo with [3H]cholesterol to a high specific radioactivity using cholesterol-labelled erythrocytes. The erythrocytes were shown to be rapidly degraded by the spleen and not released intact during subsequent perfusion. 2. When labelled spleens were perfused with whole blood or serum, lipoproteins in the high-density lipoprotein (HDL) range were shown to be the principal lipoprotein vehicles for the removal of cholesterol, the specific radioactivity of cholesterol being much greater in the HDL fractions than in other lipoproteins, particularly in the d 1.175-1.210 fraction. 3. The formation of [3H]cholesteryl ester was restricted to the major HDL fractions. 4. Experiments utilizing individual HDL fractions added to a basal perfusate indicated that HDL1 (d 1.050-1.085) was of less importance in the removal of cholesterol from the spleen than HDL subfractions of higher density. Also, a decrease in density of the lipoproteins was observed during perfusion, concurrent with uptake of cholesterol, especially in the d 1.085-1.125 subfraction. 5. When [3H]cholesterol-labelled spleens were perfused with whole blood, about half of the radioactivity released was detected in erythrocytes, indicating a rapid exchange or transport of cholesterol. Thus erythrocytes could play an important role in the transfer of unesterified cholesterol when the chemical potential gradient is favourable.     

The mechanism and regulation of dolichyl phosphate biosynthesis in rat liver

Rat liver slices were pulse labeled for 6 min with [3H]mevalonolactone and then chased for 90 min with unlabeled mevalonolactone in order to study the mechanism of dolichyl phosphate biosynthesis. The cholesterol pathway was also monitored and served to verify the pulse-chase. Under conditions in which radioactivity in the methyl sterol fraction chased to cholesterol, radioactivity in alpha-unsaturated polyprenyl (pyro)-phosphate chased almost exclusively into dolichyl (pyro)phosphate. Lesser amounts of radioactivity appeared in alpha-unsaturated polyprenol and dolichol, and neither exhibited significant decline after 90 min of incubation. The relative rates of cholesterol versus dolichyl phosphate biosynthesis were studied in rat liver under four different nutritional conditions using labeled acetate, while the absolute rates of cholesterol synthesis were determined using 3H2O. From these determinations, the absolute rates of dolichyl phosphate synthesis were calculated. The absolute rates of cholesterol synthesis were found to vary 42-fold while the absolute rates of dolichyl phosphate synthesis were unchanged. To determine the basis for this effect, the rates of synthesis of cholesterol and dolichyl phosphate were quantitated as a function of [3H]mevalonolactone concentration. Plots of nanomoles incorporated into the two lipids were nearly parallel, yielding Km values on the order of 1 mM. In addition, increasing concentrations of mevinolin yielded parallel inhibition of incorporation of [3H]acetate into cholesterol and dolichyl phosphate. The specific activity of squalene synthase in liver microsomes from rats having the highest rate of cholesterol synthesis was only 2-fold greater than in microsomes from rats having the lowest rate. Taken together, the results suggest that the maintenance of constant dolichyl phosphate synthesis under conditions of enhanced cholesterogenesis is not due to saturation of the dolichyl phosphate pathway by either farnesyl pyrophosphate or isopentenyl pyrophosphate but coordinate regulation of hydroxymethylglutaryl-CoA reductase and a reaction on the pathway from farnesyl pyrophosphate to cholesterol.     

Evidence for reverse cholesterol transport in vivo from liver endothelial cells to parenchymal cells and bile by high-density lipoprotein.

Acetylated low-density lipoprotein (acetyl-LDL), biologically labelled in the cholesterol moiety of cholesteryl oleate, was injected into control and oestrogen-treated rats. The serum clearance, the distribution among the various lipoproteins, the hepatic localization and the biliary secretion of the [3H]cholesterol moiety were determined at various times after injection. In order to monitor the intrahepatic metabolism of the cholesterol esters of acetyl-LDL in vivo, the liver was subdivided into parenchymal, endothelial and Kupffer cells by a low-temperature cell-isolation procedure. In both control and oestrogen-treated rats, acetyl-LDL is rapidly cleared from the circulation, mainly by the liver endothelial cells. Subsequently, the cholesterol esters are hydrolysed, and within 1 h after injection, about 60% of the cell- associated cholesterol is released. The [3H]cholesterol is mainly recovered in the high-density lipoprotein (HDL) range of the serum of control rats, while low levels of radioactivity are detected in serum of oestrogen-treated rats. In control rats cholesterol is transported from endothelial cells to parenchymal cells (reverse cholesterol transport), where it is converted into bile acids and secreted into bile. The data thus provide evidence that HDL can serve as acceptors for cholesterol from endothelial cells in vivo, whereby efficient delivery to the parenchymal cells and bile is assured. In oestrogen-treated rats the radioactivity from the endothelial cells is released with similar kinetics as in control rats. However, only a small percentage of radioactivity is found in the HDL fraction and an increased uptake of radioactivity in Kupffer cells is observed. The secretion of radioactivity into bile is greatly delayed in oestrogen-treated rats. It is concluded that, in the absence of extracellular lipoproteins, endothelial cells can still release cholesterol, although for efficient transport to liver parenchymal cells and bile, HDL is indispensable.     

The formation of cholest-5-ene-3β,26-diol as an intermediate in the conversion of cholesterol into bile acids by liver mitochondria

1. When [(14)C]cholesterol was incubated with rat liver mitochondria, radioactive 26-hydroxycholesterol, 3beta-hydroxychol-5-enoic acid and other bile acids were isolated from the incubation mixture. 2. In the absence of added 26-hydroxycholesterol, the specific radioactivity of the 26-hydroxycholesterol formed from [(14)C]cholesterol during the incubation was higher than that of the 3beta-hydroxychol-5-enoic acid. Addition of increasing amounts of 26-hydroxycholesterol led to a progressive fall in the specific radioactivity, and to a progressive increase in the mass, of the 3beta-hydroxychol-5-enoic acid recovered at the end of the incubation. 3. It is concluded that 26-hydroxycholesterol is an intermediate in the formation of 3beta-hydroxychol-5-enoic acid from cholesterol. 4. Comparison of the specific radioactivity of the 26-hydroxycholesterol formed in the incubation mixture with that of the added [(14)C]cholesterol indicates that endogenous cholesterol in mitochondria is accessible to cholesterol 26-hydroxylase.     

Effect of diosgenin on lipid metabolism in rats.

The purpose of this study was to determine whether diosgenin suppresses cholesterol absorption in rats, and to examine relevant changes in cholesterol and bile acid metabolism. Diosgenin fed with the diet for 1 week inhibited cholesterol absorption as determined by the serum isotope ratio technique, as well as by measuring in the feces the amount of unabsorbed radioactivity from orally administered [3H]cholesterol. In addition, diosgenin suppressed the serum and liver uptake of radioactivity from co-administered [3H]cholesterol as well as the accumulation of liver cholesterol in the cholesterol-fed rat; diosgenin was substantially more active than cholestyramine or beta-sitosterol. In vitro, diosgenin had no effect on the activity of rat pancreatic esterase. Diosgenin decreased the elevated cholesterol in serum LDL and elevated cholesterol in the HDL fraction of cholesterol-fed rats; diosgenin had no effect on serum cholesterol in normocholesterolemic rats. In contrast to cholestyramine, diosgenin markedly increased neutral sterol excretion without altering bile acid excretion; in vitro, diosgenin had no effect on bile acid binding. Diosgenin treatment increased hepatic and intestinal cholesterol synthesis as well as the activity of hepatic HMG CoA reductase. This was accompanied by increased biliary concentration of cholesterol, but not of bile acids. Diosgenin had no effect on cholesterol synthesis when added to normal rat liver homogenates. It was concluded that diosgenin interferes with the absorption of cholesterol of both exogenous and endogenous origin; such interference is accompanied by derepressed, i.e., increased, rates of hepatic and intestinal cholesterol synthesis. The increased unabsorbed cholesterol together with enhanced secretion of cholesterol into bile resulted in increased excretion of neutral sterols without affecting the biliary and fecal excretion of bile acids.     

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.     

Exogenous cholesterol transport in rabbit plasma lipoproteins

1. The appearance of exogenous cholesterol in free cholesterol and ester cholesterol of plasma chylomicra, very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins was studied in unanaesthetized rabbits after ingestion of a meal containing 5% fat and 0.08% [³H]cholesterol. 2. The specific radioactivity of ester cholesterol of VLD lipoproteins reached the highest value of any lipoprotein fraction and for each lipoprotein it increased at a faster rate and reached a higher maximum than that of free cholesterol; the maximum in VLD lipoproteins occurred later than in chylomicra. 3. The pattern of appearance of exogenous cholesterol in chylomicra and VLD lipoproteins of plasma was similar to the pattern previously observed in lymph. The specific radioactivity of ester cholesterol in plasma VLD lipoproteins was higher than that in chylomicra in spite of a larger pool size and dilution of cholesteryl esters from VLD lipoproteins produced by the liver. These results support the concept that during absorption the major portion of exogenous cholesterol is transported in VLD lipoproteins as ester cholesterol. 4. The specific radioactivity of ester cholesterol of chylomicra and VLD lipoproteins increased at a faster rate than that of LD and HD lipoproteins. However, the rate of increase and the absolute values of the specific radioactivity in LD and HD lipoproteins were identical. Since cholesteryl esters are thought not to exchange between lipoproteins, this observation supports the hypothesis that a result of VLD lipoprotein and chylomicron metabolism is the formation of LD and HD lipoproteins. 5. Results in vivo showed that the free cholesterol of individual plasma lipoproteins does not equilibrate within a period of 24h.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Cholesterol exchange in platelets, erythrocytes and megakaryocytes

Cholesterol exchange between plasma and human platelets and erythrocytes and guinea pig platelets, erythrocytes and megakaryocytes was studied. The characteristics of exchange of cholesterol between [3H]cholesterol-labeled plasma and human platelets and erythrocytes were similar: exchange per cell was independent of cell concentration in whole plasma, decreased only 2-fold over a wide range of cell concentrations in low concentrations of plasma and approached a plateau at 1/3 normal plasma cholesterol concentration, and there was no net change in the cholesterol content of either cell. The activation energy for exchange for both cells was 47 kJ/mol. In all experiments, erythrocyte cholesterol was labeled to approximately twice the specific activity of platelet cholesterol. Guinea pig megakaryocyte cholesterol exchanged at 25-33% of the rate of guinea pig platelet cholesterol in vitro. Similarly, when guinea pigs were fed [3H]cholesterol, erythrocyte cholesterol specific activity after 24 h was 90%, platelet 50-65%, and megakaryocyte 20-26% that of plasma. Guinea pig platelets incubated with plasma radiolabeled in free and esterified cholesterol incorporated radioactivity from free but not esterified cholesterol. The similarity of free cholesterol exchange in platelets and erythrocytes in vitro and in vivo and the apparent inability of platelets to take up cholesterol esters from lipoproteins suggest that the interaction between normal platelets and normocholesterolemic plasma is limited to cholesterol exchange.     

Enzyme blockade: a nonradioactive method to determine the absolute rate of cholesterol synthesis in the brain

The standard in vivo method to determine rates of brain cholesterol synthesis involves systemic injection of (3)H(2)O and measurement of incorporated radioactivity in sterols. Herein, we describe an alternative method ("enzyme blockade") that obviates the use of radioactivity. The method relies on the ability of AY9944, a potent and relatively selective inhibitor of cholesterol synthesis, to cause the time-dependent accumulation of 7-dehydrocholesterol (DHC), a cholesterol precursor detected with sensitivity and specificity by reverse-phase HPLC-coupled spectrophotometry at 282 nm. To validate the method, adult AY9944-treated and control mice were injected with [(3)H]acetate. After 24 h, most of the radioactivity in brain sterols from treated mice accumulated in DHC, without significantly perturbing overall sterol pathway activity, compared with controls (where cholesterol was the dominant radiolabeled sterol, with no label found in DHC). When adult mice were treated continuously with AY9944, the time-dependent accumulation of DHC in brain was linear (after approximately 8 h) for 3 days. The rate of brain cholesterol synthesis determined by this method ( approximately 30 microg/g/day) closely agrees with that determined by the radioactive method. We also determined the cholesterol synthesis rate in different regions of adult mouse brain, with frontal cortex having the highest rate and cerebellum having the lowest rate.     

High density lipoprotein metabolism in normolipidemic and cholesterol-fed rabbits.

New Zealand white rabbits were used to determine the compositional and metabolic changes induced in high density lipoproteins (HDL, rho = 1.063--1.21 g/ml) in response to cholesterol feeding. There was no change in total HDL cholesterol in plasma due to cholesterol feeding (12 weeks), but the triglyceride level was decreased to 29% of pretreatment values. Total protein content of HDL was decreased in response to cholesterol feeding, resulting in a significant increase in the cholesterol/protein ratio. There was a decrease in some isomer of the major apolipoproteins (A-I2) of HDL. The decay of radioactivity in HDL or its apolipoproteins was biphasic in both normolipidemic and hypercholesterolemic rabbits. The first phase was much more rapid than the second. The decay rates for the radioactivity in HDL depended upon the dietary status of the recipient animal.     

Acyl-coenzyme A: cholesterol acyltransferase. Transfer of cholesterol to its substrate pool and modulation of activity

The preincubation at 37 degrees C of rat liver microsomal fraction, followed by re-isolation of the treated vesicles, results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The presence of cholesterol-phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration-dependent. The preincubation of the microsomal fraction in the presence of [3H]cholesterol-phospholipid liposomes results in transfer of [3H]cholesterol to the re-isolated microsomal vesicles and this transfer follows first-order kinetics in respect to the donor concentration. These preincubations result also in a time-dependent and liposomal cholesterol concentration-dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half-time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltransferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time-dependent and temperature-dependent increase in cholesterol acyltransferase activity is due to the transfer of non-esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.