Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

The stereochemistry of the hydrogen elimination in the biological conversion of cholest-7-en-3β-ol into cholesterol

1. The syntheses of Δ⁷-[4-¹⁴C]cholestenol (XVI, Scheme 3) and Δ⁷-[6α-³H]-cholestenol (XII, Scheme 2) are described. 2. The metabolism of doubly labelled Δ⁷-cholestenol (II, Scheme 1) by rat-liver homogenates was studied. 3. During the enzymic conversion of Δ⁷-cholestenol into cholesterol (IV, Scheme 1) the 6α-hydrogen atom of the former is lost and the overall reaction corresponds to a cis-elimination. 4. In the light of these results various mechanisms for the conversion of Δ⁷-cholestenol into cholesterol are discussed.     

Cerebrotendinous xanthomatosis: An inborn error in bile acid synthesis with defined mutations but still a challenge

Cerebrotendinous xanthomatosis [CTX] is a rare disease characterized by the accumulation of cholesterol and cholestanol in brain and tendons caused by a mutation in the sterol 27-hydroxylase gene [CYP27A1] involved in bile acid synthesis. Disruption of this gene in mice does not give rise to xanthomas. The gene defect leads to reduced bile acid synthesis with a compensatory increase in the activity of the rate-limiting enzyme in bile acid synthesis, cholesterol 7α-hydroxylase. This leads to a marked accumulation of 7α-hydroxylated bile acid precursors, in particular 7α-hydroxy-4-cholesten-3-one. The latter oxysterol passes the blood-brain barrier and is an efficient precursor to cholestanol. The activity of cholesterol 7α-hydroxylase is normalized by treatment with bile acids. Such treatment reduces the xanthomas in CTX patients in parallel with decreased cholestanol levels. The relationship between the accumulation of cholestanol and the development of cholesterol-rich xanthomas has however not been clarified and a suitable animal model is still lacking.     

Aspergillus oryzae produces compounds inhibiting cholesterol biosynthesis downstream of dihydrolanosterol

The formation of cholesterol synthesis inhibiting molecules by five different strains of the koji mold Aspergillus oryzae was studied. After growing these strains on a complex liquid medium we found in crude organic phase extracts and specific fractions there from compounds inhibiting cholesterol synthesis in human hepatic T9A4 cells in vitro at enzyme sites downstream of dihydrolanosterol. This was evidenced by using different radioactively labeled precursors, namely acetate, mevalonate, 24,25-dihydro-[24,25-(3)H2]-lanosterol or [3-(3)H]-lathosterol.     

Cholestanol metabolism in patients with cerebrotendinous xanthomatosis: absorption, turnover, and tissue deposition

To study the metabolism of cholestanol in patients with cerebrotendinous xanthomatosis (CTX), we measured the cholestanol absorption, the cholesterol and cholestanol turnover, and the tissue content of sterols in two patients. Cholestanol absorption was approximately 5.0%. The rapid exchangeable pool of cholestanol was 233 mg, and the total exchangeable pool was 752 mg. The production rate of cholestanol in pool A was 39 mg/day. [4-14C]cholestanol was detected in the xanthomas, but neither [4-14C]cholestanol nor [4-14C]cholesterol was detected in peripheral nerves biopsied at 49 and 97 days after [4-14C]cholesterol given intravenously. Of the 18 tissues analyzed at biopsy and autopsy, the cholestanol content varied from 0.09 mg/g in psoas muscle to 76 mg/g in a cerebellar xanthoma. With the assumption that the cholestanol-to-cholesterol ratio is 1.0, the relative cholestanol-to-cholesterol ratio varied from 1.0 in plasma and liver to 30.0 in the cerebellar xanthoma; cholestanol was especially high in nerve tissue. Our data indicate that CTX patients absorb cholestanol from the diet. They have a higher than normal cholestanol production rate. Cholestanol was derived from cholesterol. In CTX patients, the blood-brain barrier was intact to the passage of [4-14C]cholesterol and [4-14C]cholestanol. The deposition of large amounts of cholestanol (up to 30% of total sterols in cerebellum) in nerve tissues must have an important role in the neurological symptoms in CTX patients. In view of the intact blood-brain barrier, several other explanations for the large amounts of cholestanol in the brain were postulated.     

Synthesis of ubiquinone and cholesterol in human fibroblasts: regulation of a branched pathway.

The current studies demonstrate that cultured human flbroblasts utilize mevalonate for the synthesis of ubiquinone-10 as well as for the synthesis of cholesterol. Study of the regulation of this branched pathway was facilitated by incubating the cells with compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryI coenzyme A reductase, which blocked the formation of mevalonate within the cell. The addition of known amounts of [³H]mevalonate to the culture medium in the presence of compactin permitted the study of the relative rates of mevalonate incorporation into cholesterol and ubiquinone-10 under controlled conditions. When low concentrations of exogenous [³H]mevalonate (10 to 50 μm) were added to cells that were provided with exogenous cholesterol in the form of plasma low density lipoprotein (LDL), the cells incorporated the [³H]mevalonate into ubiquinone-10 at a rate that was two- to threefold faster than the incorporation into cholesterol. When the cells were deprived of exogenous LDL-cholesterol, the incorporation of [³H]mevalonate into ubiquinone-10 decreased and the incorporation of [³H]mevalonate into cholesterol increased. As a result, in the absence of exogenous cholesterol more than 60 times as much [³H]mevalonate was incorporated into cholesterol as into ubiquinone-10. Considered together with previous findings, the current data are compatible with a regulatory mechanism in which LDL inhibits cholesterol synthesis in fibroblasts at two points: (1) at the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, thereby inhibiting mevalonate synthesis, and (2) at one or more points distal to the last intermediate common to the cholesterol and ubiquinone-10 biosynthetic pathways. The latter inhibition allows ubiquinone-10 synthesis to continue in the presence of LDL despite a 98% reduction in mevalonate synthesis.     

Contribution of the shunt pathway of mevalonate metabolism to the regulation of cholesterol synthesis in rat liver

The modulation of the shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in livers from fed, starved, and diabetic rats perfused with a physiological concentration (300 nM) of [5-14C] + [5-3H]mevalonate. Shunt activity was measured by (i) production of 14CO2 (corrected for loss of label by exchange reactions) and (ii) production of 3H2O. Contribution of exogenous mevalonate to total mevalonate production (0.06-0.11%) was assessed in parallel experiments by the incorporation of 3H2O into sterols. Inhibition of non-saponifiable lipid synthesis by starvation and diabetes is not associated with an inhibition of mevalonate production but with a major increase in shunting (7-34%) of sterol-bound mevalonate. The shunt pathway of mevalonate metabolism appears to participate in the regulation of cholesterol synthesis.     

Sterols with 29, 28 and 27 carbon atoms metabolically related to cholesterol, occurring in developing and mature brain

Brain sterols from chick embryos (11 and 18 days of incubation) and mature rats, previously injected with [2-¹⁴C]mevalonate, were analysed. Acetate derivatives of the sterols were chromatographed on Silica Gel:Celite:AgNO₃ columns. Sterol fractions were assayed for radioactivity and the amounts determined by gas chromatography. Sterol structures were elucidated by gas chromatography-mass spectrometry. The method used allowed the identification of some sterols representing no more than 0-01 per cent of the total mixture. The following brain sterols were identified: cholesterol, cholestanol, cholest-5,24-dien-3β-ol (desmosterol); 4,4′-dimethyl-cholest-8-en-3β-ol, 4α-methyl-cholest-8-en-3β-ol, cholest-8-en-3β-ol, 4,4′-dimethyl-choIest-8,24-dien-3β-ol, 4α-methyl-cholest-8,24-dien-3β-ol, cholest-8,24-dien-3β-ol and cholest-7,24-dien-3β-ol. Small amounts of other sterols including polyhydroxy sterols, were also detected. There were no qualitative differences in the sterols detected in developing and mature brain. In the developing chick brain, cholesterol represented approximately 90 per cent of the total sterols. In the mature rat brain, cholesterol accounted for 98 per cent of the sterols. The adult rat brain, as well as the embryonic chick brain, demonstrated the capacity to incorporate mevalonate into cholesterol precursors and cholestanol. The sterols retaining the double bond in the lateral chain, that is, those of the Δ^8,24 series with 29, 28 and 27 carbon atoms and desmosterol, were highly labelled compared with the other identified intermediates. The possibility, supported by our data, that a preferential biosynthetic route for cholesterol exists in brain, is discussed.     

Incorporation of [4-14C] cholesterol into steryl derivatives and saponins of oat (Avena sativa L.) plants

Sterols from both green and etiolated oat plants (Avena sativa) contain sitosterol, stigmasterol, cholesterol, ⁷-stigmastenol, ⁷-cholestenol and campesterol. In the saponin fraction avenacosides A and B and 26-desgluco-avenacosides A and B were detected. Etiolated plants incorporated [4-¹⁴C] cholesterol into steryl derivatives (esters, glycosides and acylated glycosides) and also into all of the 4 saponins. [4-¹⁴C] sitosterol, however, is incorporated only into steryl derivatives, but not in saponins. From this it is concluded that cholesterol, but not sitosterol is the in vivo precursor of oat saponins.Abbreviations GLC gas liquid chromatography - I.D. inner diameter - TMS trimethylsilyl - dpm decompositions per minute     

Isopentenoid synthesis in isolated embryonic Drosophila cells: absolute, basal mevalonate synthesis rate determination.

Embryonic Drosophila cells (Kc cells) and [5-3H]mevalonate (less than or equal to 10 microM) were used to determine the absolute basal in vivo rate of total mevalonic acid synthesis/utilization. An absolute in vivo mevalonic acid synthesis rate of 0.69 nmol/h/mg total cell protein was measured. Absolute mevalonate utilization was obtained by correcting for the extent of endogenous dilution of exogenous [3H]mevalonate at isotopic equilibrium. Cellular [3H]farnesol specific radioactivity was used as representative of a rapidly turning over isopentenoid pool. Although our previous Kc cell study (Havel, C. M., Rector, E. R. II, Watson, J. A., 1986, J. Biol. Chem. 261, 10,150-10,156) demonstrated that greater than or equal to 40% of the metabolized [3H]mevalonate appeared as 3H-labeled media water, this report established that t,t-3,7,11-[3H]trimethyl-2,6,10-dodecatriene-1,12 dioic acid was also secreted. Media accumulation of the C15-alpha,omega-prenyl dioic acid and 3H2O was related directly to [3H]mevalonic acid availability. This is the first mevalonate carbon balance study reported for a eukaryotic organism. It was concluded that (i) Kc cells synthesized more mevalonate than needed for normal growth and essential isopentenoids and (ii) excess mevalonate carbon accumulated intra- and extracellularly as isopentenoid compounds distal to C5 products. Finally, this study emphasized the need to measure total mevalonate utilization and not mevalonate conversion to a single isopentenoid end product in carbon balance investigations.     

Expression of specific high capacity mevalonate transport in a Chinese hamster cell variant

A variant of a low density lipoprotein receptor-negative Chinese hamster ovary (CHO) cell mutant was isolated using a nutritional selection called MeLoCo. The variant, designated met-18b-2, internalized and metabolized mevalonate at rates 10-40 times greater than the progenitor cells from which they were derived. The extent of incorporation of radioactivity from [3H]mevalonate into steroidal and nonsteroidal mevalonate derivatives, including modified proteins, was much greater in met-18b-2 cells than in their progenitors. Much of the internalized [3H]mevalonate was converted to nonpolar lipids. Unlike wild type CHO cells or the receptor-negative progenitors, met-18b-2 cells were killed by high concentrations of mevalonate (greater than 6 mM) in the culture medium. Regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity and cholesterol esterification was dramatically more sensitive to mevalonate in met-18b-2 cells than in progenitor cells. In cell extracts, both the rates of conversion of [3H]mevalonate to cholesterol and mevalonate kinase activities were similar for met-18b-2 and progenitor cells. In contrast to progenitor cells, met-18b-2 cells internalized [3H]mevalonate with high capacity (Km approximately 0.3 mM) kinetics. The increased uptake of [3H]mevalonate was temperature dependent and highly specific. These results suggest that met-18b-2 cells express a mevalonate transport activity that is not normally expressed by CHO cells. This activity may be due to a specific mevalonate transporter that is differentially expressed in specialized tissues. Because intracellular mevalonate in met-18b-2 cells can be labeled to high specific activity, these cells should prove very useful in further characterizing the structures of mevalonate derivatives and their metabolism.     

Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids (Short Communication)

2,4-Dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid inhibited the incorporation of [2-(14)C]mevalonate into cholesterol and non-saponifiable lipids. Both compounds inhibited the conversion of [1-(14)C]isopentenyl pyrophosphate into cholesterol and the synthesis of cholesterol and fatty acids from [2-(14)C]acetate. There was no inhibition of the conversion of [1-(14)C]mevalonate into CO(2). At low concentrations (0.5mm) of the compounds there was a stimulation of acetate incorporation into fatty acids.     

Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Rat liver microsomal cholesterol biosynthesis and drug oxidase activity are affected by chromium ions (Cr3+) in vitro

Chromium ions (Cr3+)evoked a biphasic curve of changes of rat liver microsomal cholesterol biosynthesis using [14C]acetate and/or [14C]mevalonate as precursors. While for the lower range of Cr3+ concentrations the rate of cholesterol biosynthesis rises, at concentrations above 8 X 10(-6) M they evoke a decrease in the cholesterol biosynthesis, up to 50% down on its control value at a concentration of 8 X 10(-4) M. Differences were more pronounced when using [14C]mevalonate instead of [14C]acetate as precursor. The activity of the microsomal enzyme biphenyl-4-hydroxylase showed an equally intense rise to that of cholesterol biosynthesis up to a 8 X 10(-6) M Cr3+ concentration. Above this concentration, however, the activity of the enzyme starts to drop. NADPH-cytochrome c reductase and NADPH-oxidase were decreased at all Cr3+ concentrations used, which cover a 100-fold range. Lineweaver-Burk plots of the cytoplasmic glucose-6-phosphate dehydrogenase demonstrated an uncompetitive mechanism of inhibition by Cr3+ ions. The results are discussed in terms of the possible significance of the Cr3+ concentration-dependent effects on cholesterol biosynthesis, with the observed atherosclerosis in Cr-deficient humans.     

Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin

Abstract: Ubiquinone synthesis has been studied in cultured C-6 glial and neuroblastoma cells by utilizing an inhibitor, 3-β-(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C-6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [¹⁴C]acetate or [³H]mevalonate within minutes. A 95% inhibition was apparent after a 3-h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C-6 glial cells to U18666A caused a marked stimulation of incorporation of [¹⁴C]acetate or [³H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [³H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.     

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.     

The reversibility of the Δ8-cholestenol–Δ7-cholestenol isomerase reaction in cholesterol biosynthesis

The incubation of Delta(7)-cholestenol with a 10000g supernatant or 105000g microsomes in the presence of tritiated water is studied. The reisolated Delta(7)-cholestenol contained up to 0.67g.atom of tritium/mole. This result can best be explained by assuming the reversibility of the reaction Delta(8)-cholestenol right harpoon over left harpoon Delta(7)-cholestenol.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.