Radioactive Cholesterol Research Articles

Origin and fate of rat plasma cholesterol in vivo. Modelling of cholesterol movements between plasma and organs

A cholesterol system model was developed in the rat following a single injection of red cells containing free (unesterified) [ 3H]cholesterol. The radioactivity of free and esterified cholesterol in the different parts of the system was measured during the 48 h following tracer introduction. The model consisted of seven compartments (red cell free cholesterol, plasma and liver free and esterified cholesterol, total cholesterol in the rapidly and slowly exchangeable carcass pools). The model was validated by the similarity between simulated and experimental values during the 48 h following tracer introduction. Both the fractional rate of cholesterol esterification in the plasma (0.44 h −1) and liver (0.01 h −) and the fractional exchange rate of free cholesterol from the plasma towards the various organs (particularly 3 h −1 towards the liver for a total of 7 h −1) can be estimated with this model. The results show that cholesterol movements between the plasma and the different organs take place mainly through intense free cholesterol exchanges, resulting in a low net flux.

Cholesterol is not synthesized in membranes bearing 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have shown previously that newly synthesized lanosterol and cholesterol in homogenates of cultured human fibroblasts do not have the same equilibrium buoyant density as the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in the smooth endoplasmic reticulum (SER) (Lange, Y., and Steck, T. L. (1985) J. Biol. Chem. 260, 15592-15597). This finding suggested two alternative and novel hypotheses: (a) that lanosterol and cholesterol might be transported rapidly from the SER to other internal membranes or (b) that synthesis of the sterols is not associated with the SER, or at least not with that portion of this organelle bearing HMG-CoA reductase. We therefore compared the subcellular distribution of HMG-CoA reductase with that of enzymes which convert lanosterol to cholesterol. The two activities studied were the consumption of exogenous [3H]lanosterol and the conversion of exogenous radiolanosterol to radiocholesterol. Differential centrifugation, rate zonal centrifugation, and equilibrium sucrose gradient centrifugation of rat liver homogenates all showed that these enzyme activities did not comigrate with HMG-CoA reductase. The subcellular distribution of newly synthesized sterols also was examined in cultured human fibroblasts. Cells were incubated with radioactive acetate to label endogenous sterols biosynthetically, homogenized, and spun to equilibrium on sucrose gradients. The buoyant density profiles of radioactive cholesterol and lanosterol both had a peak at 1.12 g/cm3. Digitonin treatment shifted both sterols to higher densities, strong evidence that they resided in cholesterol-rich membranes. Pretreatment of intact cells with cholesterol oxidase, which selectively oxidizes plasma membrane cholesterol, abolished the digitonin shift of lanosterol but not of intracellular cholesterol. These findings provide support for the hypothesis that newly synthesized cholesterol and lanosterol are not in the same membrane.

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that 14C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13-14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Turnover of dietary cholesterol and .BETA.-sitosterol in the prawn.

The fate and turnover of cholesterol and β-sitosterol in the prawn Penaeus japonicus were investigated after the feeding of [4-14C] cholesterol and [22, 23-3H] β-sitosterol. Lipids were ex-tracted from the gut, hepatopancreas, hemolymph, muscle, and the remainder for analysis after 1, 24, and 240 hs of feeding of radioactive sterols. In the whole body, the half-lives (T1/2) of cholesterol and β-sitosterol were 5.4 days and 2.8 days, respectively. Cholesterol (T1/2=2.4 days) turned over at a faster rate in the hepatopancreas than β-sitosterol (T1/2=5.7 days), whereas cholesterol (T1/2=12.2 days) was retained in the muscle for a longer tiem than β-sitosterol (T1/2= 5.9 days). The prawn possessed the ability for conversion of β-sitosterol to cholesterol probably via 24-ethylidenecholest-5-enol and 24-methylenecholesterol. Ten days after feeding [3H] β-sitosterol, the whole body of the prawn contained large amounts of radioactive cholesterol (43% of total radioactive sterols).

Opsonization with antimyelin antibody increases the uptake and intracellular metabolism of myelin in inflammatory macrophages.

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.

In vivo flux of plasma cholesterol into human abdominal aorta with advanced atherosclerosis.

To measure the flux of free and esterified cholesterol from plasma into abdominal aortic tissue that had severe atherosclerotic lesions, we intravenously injected two autologous plasma samples containing radioactive cholesterol into patients scheduled for reconstructive arterial surgery. After the injections, blood samples were collected for calculation of the exposure of arterial tissue to labeled free and esterified plasma cholesterol. When tissue specimens were removed a few days after the injection, the aortic influx was determined by the simultaneous use of two differently labeled species of cholesterol. The flux of free and esterified cholesterol into 51 tissue specimens from the abdominal aorta of 12 normocholesterolemic patients was 41 +/- 3 and 45 +/- 3 nmol X cm-2 X day-1 (mean +/- SE), respectively, with 7% to 30% hydrolysis and 8% to 24% esterification of the labeled sterols in the atherosclerotic tissue. The influx was up to 100 times greater than the influx into nonatherosclerotic ascending aorta previously measured in other patients. The cholesterol content of the atherosclerotic tissue corresponded to 16 +/- 3 months (mean +/- SE) of continuous cholesteryl ester influx. Unless counteracted by cholesteryl ester efflux from the plaque, this influx provides enough cholesteryl ester from plasma to cause rapid lesion progression.

Butyrate effects on normal and adapted hepatoma cells: Morphological response and implications for vectoral cholesterol transport

The cell line 4IC6, adapted for growth in 6 m m sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698–7704] , exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 m m sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1- 14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized Cholesterol.

Evidence for different isotopic enrichments of acetyl-CoA used for cholesterol synthesis in the liver and intestine: a study in the rat by mass fragmentography after intravenous infusion of [ 13C] acetate

Wistar rats were killed 4 h after an intravenous infusion of [1,2- 13C]- and [1- 14C]acetic acid sodium salt (39 mg, 12.5 μCi/ml, constant rate: 1.2 ml/h). At this time, labeled free cholesterol movements between the organs are still weak and cholesterol labeling in each tissue mainly originates from the in situ incorporation of the exogenous substrate. In male rats, the specific radioactivity of free cholesterol was found to be higher in the intestine (mucosa and wall) than in the liver and plasma. In female and in cholesryramine-fed male rats, cholesterol 14C labeling was close to that of male rats in the intestine, and was markedly higher in the liver. The same variations of 13C excess, calculated by mass fragmentography, indicated that there was no isotopic effect between 13C and 14C precursors. The advantage of this method consisted in obtaining the proportions of labeled molecules according to their molecular weight ( M + 1- M + 11) for each sample. Then the distribution of 13C atoms in newly synthesized cholesterol was assessed in each sterogenesis site. In the intestine, about 3 4 of the 13C atoms were found in molecules of weight of at least M+4 (after incorporation of at least two labeled acetate units). This proportion was only 1 3 in hepatic and plasma free cholesterol. These distinct 13C-labeling patterns clearly indicate that local variations occurred in the isotopic enrichment of acetyl-CoA used for cholesterol formation. Whatever the experimental conditions of this study, cholesterol was synthesized from an acetyl-CoA more 13C enriched in the intestine than in the liver. Such variations probably result from the different dilutions of exogenous acetyl-CoA by the endogenous pool in the liver and intestine. Consequently, the 14C or 13C incorporations measured in the liver and intestinal sterols do not account for absolute rates of cholesterol production by these organs. This study also indicated that after a few hours of infusion, free cholesterol labeling in the plasma originated mainly from cholesterol newly formed in the liver, even when acetate incorporation into cholesterol was higher in the intestine than in the liver.

Phospholipase A 2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferae reactivity

Human HDL 3 ( d 1.125−1.21 g/ml) were treated by an exogenous phospholipase A 2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. 1. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5–10% haematocrit and HDL 3 (0.6 mM total cholesterol) from 0 to 12–15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 μM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. 2. (2) The lecithin: cholesterol acyltransferase reactivity of HDL 3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A 2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21–1.25 g /ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15–30% lipolysis, the lecithin:cholesterol acyltransferase reactivity of HDL 3 was reduced about 30–40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. 3. (3) The addition of the lecithin:cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL 3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin:cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin:cholesterol acyltransferase-induced removal of cellular cholesterol.

Mode of Action of Probucol in Reducing Serum Cholesterol in Mice

The mode of action of probucol in reducing serum cholesterol was studied in normal and cholesterol-fed mice. Probucol did not affect intestinal absorption of radioactive cholesterol in normal and cholesterol-fed mice. In normal mice, probucol treatment resulted in inhibition of incorporation of [14C]-acetate into cholesterol in the liver, while It stimulated the incorporation in the small intestines. Incorporation of [14C]-mevalonate into cholesterol was not affected by the treatment. These results were consistent with the finding that the HMG-CoA reductase activity was decreased in the liver, but increased in the intestinal tissues of the treated mice. In cholesterol-fed mice, probucol treatment had no effect on cholesterol synthesis in the liver, while It increased the intestinal cholesterol synthesis. The over-all effect of this drug on cholesterol synthesis was not significant, although it tended to be inhibitory in normal mice and stimulatory in cholesterol-fed mice. On the other hand, probucol treatment resulted In acceleration of the clearance of [14C]-cholesterol-derived radioactivity from the circulation and resulted also in a significant increase in fecal excretion of the radioactivity, cholesterol and bile acids without changes in lipid composition of the bile. Cholesterol content in and radioactivity distribution among the tissues were not affected by probucol. Hepatic cholesterol 7α-hydroxylase activity was increased by probucol. These findings indicate that probucol lowers serum cholesterol mainly by increasing catabolic excretion of cholesterol into bile.

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane.

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.

Assays for cholesterol 7α-hydroxylase and 12α-hydroxylase using high performance liquid chromatography

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7 alpha-hydroxylating) and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (enzyme not yet registered) are described. 7 alpha-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12 alpha-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.

Steroid biosynthesis by a sesterterpene pathway in the rat adrenal gland in vitro

Rat adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3β-ol. Steroids were isolated, purified by thin-layer and high performance liquid chromatography and crystallised to constant specific activity. It was found that the rat adrenal gland can utilise 23,24-dinor-5-cholen-3β-ol to produce corticosterone. Also, in contrast to the conversion of cholesterol to corticosterone which occurs in the mitochondrial fraction, the conversion of 23,24-dinor-5-cholen-3β-ol to corticosterone occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat adrenal gland and that the intermediates are converted to steroid hormones in the microsomal fraction.

Dietary cholesterol utilization by the housefly, Musca domestica

The amount of cholesterol and the distribution of its metabolites were studied in various larval tissues of the housefly ( Musca domestica), reared on a high cholesterol diet containing the radioactive cholesterol. The sterol content was also examined in adults derived from larvae reared on increasing dietary cholesterol concentration in separate batches. The results suggest that when the dietary sterol concentration was raised from 0.002 to 0.02% wet weight (an optimal concentration required for a maximal growth and development of insect), the sterol content of six-day old larvae was increased approx. 4-fold. However, a further 10-fold increase of dietary cholesterol (to 0.22% of wet weight) was associated with only a 1.5-fold increase in larval sterol content, and no increase in the sterol content of adult insects derived from such larvae was observed. This increase of sterol content of whole larvae was found to be confined to the larval cuticle and composite gut fractions and may be attributed to unabsorbed cholesterol in the gut, and to a solubilizing effect of the cuticle. These findings suggest that when the cholesterol requirement for the maximal growth of the insect has been reached, larvae are able to regulate the intake of ingested cholesterol, and no more is taken up through the gut even when a high gut concentration of cholesterol is present. The adults contained a higher percentage of esterified sterols than the larvae, approx. 25% of the sterol in females and 14% of sterol in males were esterified.

Origin and fate of cholesterol in rat plasma lipoproteins in vivo. II. Modelling of cholesterol absorption and its release into plasma lipoproteins.

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 lipoproteins (VLDL) and chylomicrons was measured at different times during 2 days. Based on the results, a quantitative model of cholesterol absorption and of its release into the VLDL and chylomicrons has been elaborated. This model takes into account the different processes implied in the turnover of intestinal cholesterol and that of the entire organism. It constitutes a coherent whole (satisfactory simulations for the variables studied, suitable mass balances for each compartment and the absence of major contradictions with preexisting quantitative data). Once again the model demonstrates the important part played by the intestine in rat cholesterol system dynamics. It takes into account the existence of two related exogenous and endogenous cholesterol pools from which the cholesterol released by the intestine into the chylomicrons and VLDL originates. The results suggest the existence of an important esterified cholesterol uptake from other plasma lipoproteins by the chylomicrons.

Origin and fate of cholesterol in rat plasma lipoproteins in vivo. I. Qualitative analysis.

Rats were conditioned to ingest a 15-gram dietary mixture, in a single daily meal, between 6:00 and 9:00 a.m. Labeled cholesterol was introduced into the organism through four different physiological routes: ingestion of 14C-cholesterol, injection of 14C-acetate, injection of red cells or plasma whose cholesterol was tritium-labeled. The specific radioactivities of free and esterified cholesterol, in the different plasma lipoproteins and in the major organs, were measured at various time spans after the introduction. The results revealed a different source of cholesterol in chylomicrons and VLDL discharged by the intestine: the chylomicrons would principally carry esterified cholesterol coming from the intestinal contents, while esterified cholesterol coming from the intestinal wall was found mainly in VLDL. The free cholesterol of these lipoproteins would initially have the same origin as esterified cholesterol but rapid free cholesterol exchanges would introduce precociously cholesterol coming from other structures. The results demonstrated the significant role of the intestine in cholesterol synthesis and revealed that of VLDL in the internal secretion of cholesterol by the intestine. Chylomicrons also appeared to play a significant role in this secretion, by way of exchanges between intestinal cells and chylomicrons in the process of formation. Finally, the results suggested the existence of esterified cholesterol transfers from HDL to lower density lipoproteins (chylomicrons, VLDL and possibly LDL).

Effect of ethanol on lipoprotein synthesis and fecal sterol excretion

The effect of variable doses of ethanol on plasma lipoprotein composition, lipoprotein synthesis and fecal sterol excretion was examined in male, atherosclerosis susceptible squirrel monkeys. Primates were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys given liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. Circulating high density lipoprotein (HDL) free cholesterol and phospholipid, very low density-low density lipoprotein (VLDL-LDL) total cholesterol, and total plasma cholesterol and triglyceride were significantly elevated in High Ethanol primates compared to the other treatments. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups. There were no significant differences in serum glutamate oxalo-acetate transaminase. High Ethanol primates also had significantly greater HDL free cholesterol specific activity following intravenous injection of 3H mevalonolactone compared to the other groups while radioactive VLDL-LDL free cholesterol was elevated in both High and Low Ethanol animals. Although, total fecal bile acid mass was significantly greater in both alcohol treatment groups compared to Controls, fecal neutral sterol specific activity was only higher in monkeys fed the high ethanol diet. This study provides evidence that ethanol at 24% of calories: 1) raises HDL cholesterol levels by enhancing lipoprotein synthesis; 2) increases the fecal output of newly synthesized cholesterol without causing liver dysfunction; and 3) maintains a constant relative distribution of cholesterol among lipoprotein classes.

Effects of dietary fish protein, soybean protein and casein on cholesterol turnover in rats.

The effects of dietary fish, soybean protein and casein on cholesterol turnover were compared in rats. After the injection of [14C]cholesterol into the rats, the specific activities of radioactive cholesterol in feces were followed for 4 weeks. The cholesterol half-lives calculated from the decay curves of the specific activities were 14.7 and 14.6 days in rats fed fish protein and soybean protein, respectively. These were shorter than the half-life (17.4 days) in casein-fed controls. The fish and the soybean protein feedings significantly increased the fecal excretions of cholesterol and coprostanol, respectively, and lowered the plasma cholesterol level, as compared with casein feeding. In addition, both fish and soybean protein feedings also increased the excretion of bile acids. The stimulation of cholesterol metabolism and the increased excretions of cholesterol and its metabolites by feeding fish or soybean protein appear to play important roles in the hypocholesterolemic effects.

In vivo influx of free and esterified plasma cholesterol into human aortic tissue without atherosclerotic lesions.

In order to determine the in vivo influx of plasma cholesterol into human aortic intimamedia tissue, specimens of the ascending aortic wall without visible atherosclerosis were obtained from patients undergoing aortic valve replacement. Before the operation the patients were intravenously injected with autologous plasma in which the lipoproteins were labeled with radioactive cholesterol. The influence of the duration of the exposure time (0.3-114 h) and of the distribution of radioactivity between free and esterified cholesterol in plasma on the amount of radioactivity found in the arterial wall was studied by the simultaneous use of 3H- and 14C-cholesterol. It was shown that the influx of free and esterified cholesterol into the intima-media layer of the tissue could be calculated from a set of linear equations that relate the labeled sterols in the tissue to the average specific activities in plasma. In nine patients between 50 and 70 yr of age with 4.2-5.9 mM total cholesterol in plasma, the influx of free cholesterol and of esterified cholesterol was 1.2-8.8 and 1.0-12.5 nmol X cm-2 X d-1, respectively. Both hydrolysis and esterification of the sterol fractions in the aortic tissue and exchange of free cholesterol between the plasma lipoproteins and the tissue were demonstrated. The cholesterol content of the intima-media layer was 0.6-2.3 mumol X cm-2. This corresponds to the influx of esterified cholesterol during a period of only 0.1-3.5 yr, which is short compared with the lifespan of the patient. Our data thus suggest that removal of esterified cholesterol from aortic tissue without visible atherosclerosis represents a major importance for the cholesterol concentration in the tissue.

Cholesterol Metabolism in Two Strains of Rats with High or Low Response of Serum Cholesterol to a Cholesterol-Rich Diet

Transfer from a low cholesterol commercial diet to a high cholesterol diet, containing 2% (wt/wt) cholesterol and 0.5% cholate, caused an increase in serum cholesterol from about 2.5 mmol/L in two inbred rat strains to 5 mmol/L in the hyporesponsive strain and to 20 mmol/L in the hyperresponsive strain. In both strains the excess of cholesterol in the serum was exclusively located in the very low density lipoproteins. Cholesterol feeding caused a sevenfold increase in the amount of cholesterol in the liver, the increase tending to be greater in the hyporesponders. On the commercial diet, the decay of specific radioactivity of serum cholesterol after the intravenous administration of labeled cholesterol was faster in the hyporesponsive rats. The rate of fecal excretion of radioactive bile acids on this diet was higher in the hyporesponders when compared with the hyperresponders, whereas there was no strain difference with regard to the output of fecal neutral steroids. Sterol balance data showed that whole-body cholesterol synthesis on the low cholesterol diet was about twofold higher in the hypo- than in the hyperresponders. When fed the high cholesterol diet the half-life in the serum of injected radioactive cholesterol was about six times shorter in the hyporesponders. In absolute amounts, the hypo- and hyperresponders excreated similar amounts of endogenous (radioactive) bile acids and fecal steroids with the feces on this diet.