Tritiated Cholesterol Research Articles

Functional differences in behavior of monocyte/macrophage from HIV+ patients versus healthy subjects

Abstract Increased risk of atherosclerotic cardiovascular diseases is common in HIV+ adults on stable antiretroviral treatment (ART). A key step in the development of the atherosclerotic plaque is the transmigration of monocytes to the plaque area and their differentiation into macrophages and eventually foam cells. The objective of this study was to determine if the propensity of monocytes to transmigrate as well as their ability to take up and efflux cholesterol is affected by HIV infection. Monocytes (transmigration) and differentiated macrophages (lipid metabolism) from HIV+ ART treated patients were compared against those from matched controls as a single point cross-sectional study. Monocyte transmigration was assessed by transwell assay using MCP1 as a chemoattractant, whereas lipid uptake was measured by flow cytometry analysis of internalized acetylated LDL and cholesterol efflux by tracking tritiated cholesterol excreted from the cell after exposure to HDL. Interestingly, isolation of monocytes via negative selection with magnetic beads revealed that the yield of monocyte was significantly lower from HIV+ patients compared to controls. While no significant differences were found in cholesterol uptake or efflux parameter, the ability of monocyte to transmigrate was slightly increased in HIV patients compared to controls suggesting that the chemotactic function of monocytes may be partly responsible for the differences in the atherosclerosis risk in patients with HIV.

A high‐throughput, fluorescence‐based cholesterol efflux assay (LB44)

Numerous studies have established a negative correlation between cellular cholesterol efflux and cardiovascular disease.Cholesterol efflux from peripheral tissues and cells by a process called Reverse Cholesterol Transport (RCT). RCT allows extrahepatic cells, including macrophage‐derived foam cells in arterial atherosclerotic plaque, to transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Standard protocols for quantitation of cholesterol efflux involve labeling cells with tritiated cholesterol and measuring the release of labeled sterol in a 12 or 24 well plate. This protocol, however is not ideal for high‐throughput screening of large numbers of serum samples or screening compounds/small molecules for their effect on cholesterol efflux.We report here a simple, convenient and high‐throughput cell‐based assay using fluorescently‐labeled cholesterol in a 96‐well plate format. Our results demonstrate that the percentage cholesterol efflux elicited by 2% ApoB‐depleted normal human serum in J774 macrophage cells is comparable to those published in literature. Our assay method utilizes a positive control for this assay which induces efflux independent of any receptor, that is used as a measure of the reliability of the assay. This assay is highly reproducible with low inter‐assay variability (蠄 10%). We demonstrate that this is an efficient method of measuring cholesterol efflux in a cell‐based assay. The increased sensitivity of the assay coupled with its high‐throughput screening capability enables scientists to screen large libraries of reagents and serum samples and provides a valuable tool for drug discovery in the field of cardiovascular disease research.

Abstract 439: Niacin Does Not Accelerate Reverse Cholesterol Transport in Man

Background HDL may be atheroprotective by accelerating reverse cholesterol transport (RCT). Niacin raises HDL cholesterol (HDL-c) and niacin monotherapy up to 3g/d prevented coronary events, possibly by accelerating RCT. However, up to 2g/d niacin did not prevent outcomes beyond aggressive LDL lowering including statins, arguing against RCT-mediated atheroprotection. We developed a novel method to assess macrophage-to-lipoprotein RCT in vivo. Preliminary studies in mice demonstrate that albumin-bound tritiated cholesterol (3H-Chol) is rapidly removed ( 95% in 30min) by resident macrophages. We monitored subsequent reappearance over 8d by macrophage-to-lipoprotein RCT as 3H-Chol rate of appearance (RA pre-post therapy, %3H-Chol injected/mol cholesterol/h) and tracer fecal excretion (%3H-Chol injected/g feces), and further analyzed RCT by multi-compartmental modeling. Results Niacin raised HDL-c 44% vs 17% on placebo (p=0.02) and dropped non HDL-c 40% (p<0.0001). Niacin and placebo did not differ by HDL 3H-Chol RA (placebo: 4.16-3.72, niacin: 3.24-2.61, p=0.8), total plasma 3H-Chol RA (placebo: 2.70-2.42, niacin: 2.35-2.14, p=0.9), macrophage-to-HDL transfer rate (placebo: 4.8-4.2, niacin: 5.1-4.7 fractional transfer/h, p=0.7), ex vivo cholesterol efflux capacity ratio (placebo: 0.73-0.82, niacin: 0.91-0.95, p=0.2), or tracer fecal excretion (placebo: 34.1-37.4, niacin: 21.2-28.0, p=0.6). Conclusion Despite potently raising HDL-c, high-dose niacin had no benefit on in vivo or ex vivo macrophage-to-lipoprotein RCT or fecal excretion in man, suggesting niacin’s atheroprotective potential lies elsewhere.

Photochemical molecular imprinting of cholesterol

Cinnamoylated photocrosslinkable cyclodextrin derivatives (BCC) were synthesized by the substitution of β-cyclodextrin (β-CD) with cinnamoyl chloride (CC) and crosslinked with either hexamethylenediisocyanate (HMDI) or toluenediisocyanate (TDI). Cyclodextrin rings were substituted with one or two cinnamoyl moieties, as found from mass spectrometry. The polymeric matrix with cholesterol molecular imprint was obtained on irradiation of molecular assembly formed by the cinnamoyl-functionalized β-cyclodextrin-cholesterol with light at 275 nm, absorbed exclusively by the cinnamoyl chromophores. Irradiation induced crosslinking due to the photodimerization of the cinnamoyl moieties. To determine the adsorption properties of the produced material imprinting was performed in the presence of tritiated cholesterol and the intensity of β radiation from the material was measured. The materials obtained by the adsorption of tritiated cholesterol by nonirradiated polymer were used as controls. It was found that the polymer photocrosslinked in the presence of cholesterol have shown a considerable higher adsorption capacity for cholesterol than the control materials. This confirmed successful formation of molecularly imprinted polymer (MIP) by photochemical crosslinking. The selectivity of imprinting was also confirmed using compounds of similar structures, i.e. ergosterol, dehydroergosterol, and Vitamin D.

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay

A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes. Absorption of tritium beta particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium beta particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of approximately 600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.

Mechanism of platelet-derived growth factor-dependent caveolin-1 phosphorylation: relationship to sterol binding and the role of serine-80.

In human vascular smooth muscle cells, inhibitors of protein kinase C activity reduced serine phosphorylation of caveolin-1 and increased sterol binding by this protein. This was measured after immunoprecipitation of caveolin-1 from cells labeled with tritiated cholesterol or the photoactivable cholesterol analogue FCBP [Fielding et al. (2002) Biochemistry 41, 4929-4937]. At the same time cellular sterol efflux was inhibited. Mutagenesis within a caveolin-1 central domain (residues 80-104) suggested a major role for serine-80 in mediating both of these effects. To perturb sterol binding, platelet-derived growth factor was added to the cells, leading to a transient loss of caveolin-1-associated sterol. Under these conditions, sterol efflux was stimulated, and caveolin-1 phosphorylation at tyrosine(14), assayed with a selective antibody, was substantially increased above baseline levels. These changes were also blocked by inhibitors of protein kinase C activity. Selective inhibitors of the platelet-derived growth factor receptor and downstream kinases were used to show that loss of sterol from caveolin-1 preceded tyrosine phosphorylation, but relipidation was dependent on phosphotyrosine hydrolysis.

Fractional esterification rate of HDL particles in patients with type 2 diabetes. Relation to coronary heart disease risk factors.

To study the fractional esterification rate of cholesterol on HDL particles (FERHDL) in adults with type 2 diabetes and assess its correlation with serum lipids and other coronary heart disease (CHD) risk factors. FERHDL was measured in 90 adult (57 men, 33 women) patients by an isotopic assay method involving several steps, including preparation of VLDL- and LDL-depleted plasma, labeling of the sample with a trace amount of tritiated cholesterol, separation of free and esterified cholesterol fractions by chromatography post incubation, and subsequent counting of radioactivity in the individual fractions. Male patients have higher FERHDL values than their female counterparts. When HDL cholesterol was controlled for in a multivariate regression analysis, the sex factor was not significant. There was a significant positive correlation between FERHDL and plasma total cholesterol (r = 0.32), triglycerides (r = 0.82), apolipoprotein B (apo B; r = 0.48), insulin (r = 0.46), BMI (r = 0.31), and waist-to-hip ratio (WHR; r = 0.50). There was a negative correlation between FERHDL and HDL cholesterol (r = -0.76) and apolipoprotein AI (r = -0.60). When both HDL cholesterol and triglycerides were controlled for, the only significant correlation was between FERHDL and BMI. Non-insulin-requiring type 2 diabetic patients have FERHDL, which correlated positively with triglycerides and negatively with HDL cholesterol. The positive correlation of FERHDL with serum insulin, WHR, total cholesterol, and apo B, but not that with BMI, loses its significance when HDL cholesterol and triglycerides are controlled. The sex difference between men and women in FERHDL also loses its significance when HDL cholesterol is controlled.

Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation

Studies in vitro revealed that intact ring glands of Drosophila melanogaster convert tritiated cholesterol (C) and 25-hydroxycholesterol (25C) via 7-dehydrocholesterol (7dC) and 7-dehydro-25-hydroxycholesterol (7d25C), respectively, to ecdysone (E) and 2-deoxyecdysone (2dE), while both intact and homogenized ovaries synthesize only 2dE from these precursors. Emulsified 7d25C was incorporated directly into ecdysteroids by these tissue preparations at a much greater rate than was 7d25C made in situ from 25C. To probe the basis of the biochemical defect in the ecdysteroid deficient conditional mutant ecdysoneless (ecd 1), the differential incorporation into ecdysteroids of C (via 7dC), and particularly of 25C (via 7d25C), was measured relative to that observed after the incubation of 7d25C directly with both wild type and mutant tissues in vitro at 30°C, the restrictive temperature. Both C and 25C were equally 7,8-dehydrogenated in situ to 7dC or 7d25C, respectively, by both wild type and mutant tissues at 30°C. However, the rate of subsequent conversion of either of these Δ 5,7-sterol intermediates synthesized in situ to ecdysteroids was reduced an average of 50% in the mutant tissues relative to the wild type. Yet, when emulsified 7d25C was incubated directly with either the wild type or mutant tissues at the restrictive temperature, the amplified rate of conversion of the freely available 7d25C to ecdysteroid by these tissues was identical. These data suggest that the defect in ecd 1 tissue-mediated ecdysteroidogenesis does not involve a “hit” on any of the enzymes involved in either the 7,8-dehydrogenation of C or 25C or in the subsequent oxidation of 7d25C or 7dC to ecdysteroid. Rather, the mutation appears to affect the expression of a gene governing the translocation of Δ 5,7-sterol intermediates from the subcellular compartment where they are synthesized and/or stored to the site of subsequent oxidation to ecdysteroid.

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH 4) 2SO 4 to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 m M KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex ( M r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.

Endothelin-1-induced incorporation of cholesterol into rat adrenals

The effect of endothelin-1 (ET-1) on cholesterol uptake by adrenal cortex was evaluated through several experimental approaches: infusion of ET-1 followed by measurement of endogenous cholesterol in excised adrenals; infusion of ET-1 followed by tritiated cholesterol incorporation into adrenal quarters in vitro; coinfusion of ET-1 with tritiated cholesterol-enriched serum and determination of adrenal-associated radioactivity; and tritiated cholesterol incorporation in incubations of adrenal cells. In all cases ET-1 increased cholesterol uptake. Subcellular fractionation showed an ET-1-mediated augmentation in mitochondrial fraction. This increase was mediated by the subpopulation B of adrenal receptors for ET-1. In addition, ET-1 also increased cytochrome P450-SCC (side-chain cleavage) activity.

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-GA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.

Metabolism of plasma lipoproteins in the genetically hypercholesterolemic rat (RICO)

Experiments were performed to determine the turnover processes of plasma cholesterol in genetically hypercholesterolemic rats (RICO). Specific activity of plasma cholesterol was monitored during 4 months following an intravenous injection of tritiated cholesterol. The results were subjected to two-pool model analysis. Cholesterol production in the RICO rat was significantly higher (28.9 ± 1.7 mg/d) than in the SW control (18.5 ± 0.7, P < .01). The study also revealed a 30% decrease in the rate constant for cholesterol movement from the plasma toward the majority of organs in the RICO rat versus the SW control. Very—low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) turnover were investigated following injection of labeled lipoproteins (on cholesteryl ester or apolipoproteins). Results from these experiments showed that the higher HDL cholesterol concentration in the RICO rat as compared with the control is due to the greater production rate of esterified cholesterol in these lipoproteins (1.3 ± 0.05 mg/h v 0.8 ± 0.03 in the control, P < .001). The fractional catabolic rate (FCR) or production rate for VLDL were not significantly different between the two groups (3.4 ± 0.01 and 3.6 ± 0.01 h −1 and 2.6 ± 0.4 and 3.3 ± 0.1 mg/h, respectively). However, radioactivity of VLDL recovered in LDL at death was considerably higher in RICO rats (14% ± 1% v 6% ± 1%, P < .01). The greater concentration of LDL cholesterol in RICO rats is due to a higher LDL production (0.40 ± 0.05 v 0.19 ± 0.03 mg/h, P < .01) together with a lower catabolism (FCR, 5.5 ± 0.6 v 7.9 ± 0.8%/h, P < .05). Cross-injection experiments showed that this lower catabolism of LDL is partly due to the nature of the lipoprotein particle. Taken together, these data are consistent with the hypothesis of a reduced uptake of apolipoprotein (apo)E-containing lipoproteins (VLDL and LDL), which results in a higher LDL cholesterol concentration in RICO rats.

On the control of ecdysone biosynthesis by the central nervous system of blowfly larvae.

Ecdysone was found to be the major secreted steroid of ring glands dissected from blowfly larvae and incubated in vitro. Other secretory products such as 3-dehydroecdysone and 20-deoxy-makisterone A could not be detected when the glands were labelled with tritiated cholesterol. Ecdysone synthesis and secretion were found to be tightly coupled. The highest rate of secretion was observed a few hours before pupariation. In vitro, the rate of ecdysone secretion by ring glands was affected significantly by coincubation with the central nervous system (CNS). Modulating effects from the CNS to the gland were mediated both by culture medium and by nerve connections. Distinct parts of the CNS revealed multiple and partially opposite effects on ecdysone secretion, suggesting a more complex control than had been anticipated. Multiple neural control systems appear to be involved. Moreover, the observed effects changed with development during the second half of the third instar, reflecting a significant plasticity of neural control.

Use of a human microvascular endothelial cell line as a model system to evaluate cholesterol uptake.

CDC/EU.HMEC-1 (HMEC-1) cells provide a reliable source of human microvascular endothelial cells free of mycoplasma and viral infection. This cell line has potential for use in the further study of the endothelial cell modification of low-density lipoproteins and for anticholesterol drug evaluation assays. HMEC-1 cells will fill a gap that is present for in vitro investigations of cholesterol metabolism in conjunction with previously established hepatic, monocytic, or macrophage cell lines. This paper presents a simple assay that demonstrates a linear uptake of tritiated cholesterol by he HMEC-1 cells and shows that the cellular cholesterol load can be regulated using anticholesterol drugs.

Erythrocyte and plasma cholesterol exchange in sickle cell anemia

The effects of sickeling on cholesterol exchange between red cell membranes and serum lipoproteins were studied by following the movement of tritiated cholesterol incorporated into erythrocytes. The initial rate of this exchange was greater in sickled cells than in normal cells. One quarter of the cholesterol in the sickled cells is quickly exchanged with plasma lipoproteins. After 15 minutes, the rate becomes identical for these two types of cells, reaching similar equilibrium at end. The sickling of red cells would explain the observed differences, although conditions of hypoxia and the saturation of the incubation medium with oxygen tend respectively to accentuate and to cancel this phenomenon.

Conversion of ecdysone and 20-hydroxyecdysone into 3-dehydroecdysteroids is a major pathway in third instar Drosophila melanogaster larvae

Ecdysone and 20-hydroxyecdysone metabolism was investigated in third instar Drosophila larvae both in vivo by injecting radiolabelled ecdysteroids and in vitro by incubating various tissues with labelled ecdysteroids. Ecdysone metabolism proceeds through different pathways: (1) C-20 hydroxylation; (2) C-26 hydroxylation and C-26 oxidation leading to the formation of 26-hydroxyecdysteroids (26-hydroxyecdysone and 20,26-dihydroxyecdysone) and acidic compounds (ecdysonoic acid and 20-hydroxyecdysonoic acid); C-3 oxidation and C-3 epimerization then conjugation leading to the formation of 3-dehydrocompounds (3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone), 3-epimers (3-epiecdysone and 3-epi-20-hydroxyecdysone) and conjugates (only one conjugate was tentatively characterized as 3-epi-20-hydroxyecdysone-3-phosphate). 3-Dehydrocompounds are the major metabolites formed in third instar Drosophila larvae and C-3 oxidation occurs in various tissues. Experiments using tritiated cholesterol provided evidence that 3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone are true endogenous ecdysteroids in Drosophila larvae.

Specific binding of cholesterol to chromatin prepared from mouse spleen cells.

Mouse spleen cell suspensions were incubated with tritiated cholesterol for various time intervals. The chromatin of these cells was then isolated, washed with Triton X-100, and fractionated on Sephadex columns. It was found that cholesterol binds specifically to the chromatin. The binding was saturated after 45 min of incubation and also displayed characteristics typical of dose-dependent binding. The number of cholesterol molecules bound per nucleus was estimated to be on the order of 10 000. Oxygenated sterols, such as 25-hydroxycholesterol and 7-ketocholesterol, did not compete for the binding with [3H]cholesterol if added in 20-fold molar excess to the incubation medium of the cells. Chromatographic analyses on Sephadex columns displayed a distinct peak of radioactivity. The protein-sterol complex had an apparent molecular weight of 180 000 +/- 27 000. Using extensive digestion with DNase I (EC 3.1.21.1) it could be concluded that DNA, binding to the complex, did not influence the estimate of the molecular weight, whereas digestion with pronase or treatment with sodium dodecyl sulfate destroyed the complex. Additional experiments using sucrose density gradients (5-20%) showed also, that [3H]cholesterol was bound to chromatin by one or several proteins.

Fate of maternal conjugated ecdysteroids during embryonic development in Locusta migratoria

Newly laid eggs of Locusta migratoria contain impressively high concentrations of conjugated 2-deoxyecdysone and conjugated ecdysone of maternal origin. These molecules are metabolized during embryonic development, the changes concerning not only the ecdysteroid genins but also the conjugating moieties. In the present paper the fates of the maternal conjugates were followed during embryogenesis in the eggs. The conjugates were separated both by silica gel TLC and reverse-phase HPLC and measured, before and after hydrolysis, by RIA. Fluctuations of radioactive ecdysteroid conjugates were also investigated in eggs laid by females subjected to massive injections of tritiated cholesterol. The results are discussed in relation to recent data on identification of ecdysteroid conjugates in Locusta and a model for the sequences of metabolic events leading from maternal ecdysteroid conjugates to the embryonic ecdysteroids is proposed.

In vivo study of cholesterol turnover in tissues of adult sows

The in vivo study of free and esterified cholesterol turnover was carried out in 15 tissues of adult Large White sows maintained at a constant weight for 10–12 weeks. They received a single intravenous injection either of [1- 14C] acetate, or of an autologous red cell suspension or of plasma, previously labelled in vitro (for red cells) or in vivo (for plasma) with tritiated cholesterol. The tissues can be separated into four groups according to their relative rate of free cholesterol exchange between plasma and tissues. The liver and the lungs have a very fast exchange rate whereas the brain and the spinal cord have a very slow one. The whole lipoprotein particle transfer — an exclusive model for the esterified cholesterol transport from plasma to tissues — has been found in all sow tissues. When [1- 14C] acetate is used as a substrate for cholesterol synthesis, lungs, adrenal glands and heart do not seem — or at an extremely low rate — to convert acetate into cholesterol whereas an intense cholesterol synthesis takes place in the small intestine. Its contribution to cholesterol synthesis in sows — taking into account the cholesterol transfer processes — reaches 70 per cent.

Unreliability of tritiated cholesterol: studies with [1,2-3H]-cholesterol and [24,25-3H]cholesterol in humans.

Over the past 18 months different lots of [1,2-3H]cholesterol and [24,25-3H]cholesterol were found to be radiochemically acceptable by conventional chemical and other in vitro tests, yet, when co-administered with [4-14C]cholesterol to human subjects, an abrupt fall in the 3H/14C specific activity ratio in plasma cholesterol was discovered in every case. We have concluded that all batches of [3H]cholesterol should be regarded as radiochemically unreliable unless they are shown to behave identically in all respects to [4-14C]cholesterol in an appropriate in vivo assay system.