Bile acid production in human subjects: rate of oxidation of [24,25-3H]cholesterol compared to fecal bile acid excretion

Bile acid production has been quantitated in seven subjects by methods that compare the results of two independent approaches, namely, quantitation of cholesterol side-chain oxidation and fecal bile acid excretion. Six hypertriglyceridemic (HT) subjects and one normolipidemic control were studied by both techniques. A further control subject was studied by the cholesterol side-chain oxidation method alone. Cholesterol side-chain oxidation was quantitated by measuring the appearance of 3H2O after intravenous administration of [24,25-3H]cholesterol, using multicompartmental analysis of plasma cholesterol and [3H]water specific activity. Body water kinetics were independently defined by use of oral D2O. Two HT subjects were restudied while they were taking cholestyramine, 16 g/day. In all ten studies, multicompartmental analysis closely simulated the observed appearance of 3H2O. Values obtained for bile acid production suggest that cholesterol oxidation, or bile acid input, was significantly greater than fecal bile acid output in the HT subjects (P less than 0.05). Cholesterol side-chain oxidation rates in the two normal subjects were lower than those encountered in HT subjects, being similar to published values for normal subjects both for bile acid synthesis as determined by isotope dilution kinetics and fecal bile acid excretion. Studies conducted with two, synthetically different, preparations of [24,25-3H]cholesterol indicated that, in one of the two preparations, approximately 20% of the tritium label was at positions proximal to C24. In the other preparation examined, all of the tritium was located at, or distal to, C24. Further studies revealed that 0.055-0.24% of the dose was present as labile tritium by virtue of its appearance as 3H2O following in vitro incubation with human plasma. Provided these isotope effects are taken into account, multicompartmental analysis of plasma [24,25-3H]cholesterol and body water appears to be a useful technique for quantitating cholesterol oxidation in human subjects.     

Substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus--a monolayer study.

The substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus was examined in oriented sterol monolayers at the air/water interface. Of the cholesterol analogues with structural alterations in the A- or B-ring that were examined, it was observed that 5 alpha-cholestan-3 beta-ol was oxidized almost as fast as cholesterol itself. When the delta-5 double bond in cholesterol was instead at the delta-4 position, the oxidation rate became 3.2-fold slower. A similar reduction in the average oxidation rate was observed when the delta-5 double bond in cholesterol was instead at the delta-7 position (5 alpha-cholest-7-en-3 beta- ol). 5,7-Cholestadien-3 beta-ol was oxidized 5.1-fold slower compared to cholesterol, whereas 3 beta-hydroxy-5-cholesten-7-one and 5 beta-cholestan-3 beta-ol were not substrates of the enzyme (also verified from the lack of H2O2-production). With C(17) side chain analogues of cholesterol, it was observed that the complete lack of the C(17) side chain (5-androsten-3 beta-ol), or the insertion of an unsaturation at delta-24 (desmosterol), or even an ethyl group at C(24)(24b-ethyl-5,22- cholestadien-3 beta-ol) had no appreciable effects on sterol oxidation rate, implying that the enzyme did not recognize the side chain in oriented sterol monolayers. This study has shown that the sterol monolayer system is a good technique to examine sterol/cholesterol oxidase interactions, since both the orientation of the substrate molecules, and the quality of the interface can be mastered.     

24,25-3H]Cholesterol: presence of tritium at additional sites in the side chain

In order to measure the distribution of radioactivity present in the side chain of [24,25-3H]cholesterol prepared by a sequence involving catalytic tritiation of 3 alpha, 5 alpha-cyclocholest-24-en-6 beta-ol 6-methyl ether, the cholesterol was oxidized to 4-cholesten-3-one, which was then cleaved between C-24 and C-25 to afford the C24 alcohol. Oxidation to the corresponding cholenoic acid, followed by alkali equilibration and esterification completed the sequence. It was found that about 20% of the tritium in the labeled cholesterol is not lost when this tracer is physiologically converted to bile acids. Consequently, measurements of bile acid formation using this tracer must be corrected upward by this amount.     

Effect of 26-oxygenosterols from Ganoderma lucidum and their activity as cholesterol synthesis inhibitors

Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14alpha-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Oxygenation of desmosterol and cholesterol in cell cultures

In order to determine whether hydration of the delta 24 bond of desmosterol contributes to the formation of the regulatory oxysterol, 25-hydroxycholesterol, [3H]desmosterol was incubated with two cultured cell lines and the labeled products were analyzed. Small amounts of 25-hydroxycholesterol were formed with Chinese hamster lung (Dede) cell cultures, but not with mouse fibroblast (L) cell cultures. Apparently, desmosterol was converted into cholesterol, a process that does not occur in L cells, before 25-hydroxycholesterol takes place. No reliable evidence could be obtained for hydration of the delta 24 bond or for the reverse reaction upon incubation of [3H]25-hydroxycholesterol. Oxygenation of desmosterol occurred in both Dede and L cell cultures to give a mixture of 24(R)- and 24(S)-25-epoxy-cholesterol. This reaction, along with the production of 7-oxygenated sterols, may account for low levels of HMG-CoA reductase repressor activity previously found to be associated with delta 24 sterols.     

Biosynthesis of bufadienolides in toads. VI. Experiments with [1,2-3H]cholesterol, [21-14C]coprostanol, and 5 beta-[21-14 C]pregnanolone in the toad Bufo arenarum

[1,2-3H]Cholesterol, 5 beta-[21-14C]cholestan-3 beta-ol (coprostanol), and 3 beta-hydroxy-5 beta-[21-14C]pregnan-20-one were injected into intact Bufo arenarum toads. Arenobufagin, the main bufadienolide present in the venom of the mentioned toad, was isolated and purified by means of chromatographic procedures. The first two compounds, having an intact cholesterol side chain, were incorporated, at comparable levels, into the bufadienolide while the labeled pregnane derivative yielded non-radioactive arenobufagin. The above results support the hypothesis that cholesterol and those steroids having an intact cholesterol-type side chain are able to penetrate to the site of bufadienolide biosynthesis and are converted into bufadienolides by a still-unknown mechanism. On the other hand, those steroid derivatives bearing a degraded side chain, e.g., 20-keto-pregnanes, are not converted into bufadienolides because they are not incorporated into the bufadienolide-producing cells.     

Sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase from the corn earworm, Heliothis zea

The enzymatic activity and sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase (ACAT) were measured in microsomes of cells from Heliothis zea. Under standard assay conditions, the specific enzymatic activity of ACAT was highest in the intestine followed by the fat body and ovary (380.7, 30.7, 8.3 pmol/min per mg, respectively). The structure of the exogenous sterol used in the ACAT assay affected its rate of esterification. The relative rates of esterification of analogs of cholesterol with various modifications of the side chain were: 24-H greater than 24 alpha-CH3 greater than delta 22 greater than delta 24 greater than 24 alpha-C2H5 greater than 24 beta-CH3, delta 22-24 beta-CH3 and delta 22-24 alpha-C2H5. The number and position of double bonds in the B-ring of the sterol nucleus greatly affected the rate of esterification of sterols by ACAT. The average relative rates of esterification of sterols with differences in their B-rings were: delta 7 much greater than delta 8 greater than delta 0 greater than delta 5 greater than delta 5.7. The presence of a 9,14-cyclopropane group and/or methyl groups at the C-4 and 14 positions prevented significant esterification of such sterols. The formation of cholesteryl and lathosteryl esters was partially inhibited in microsomes from the intestine, fat body, and ovary by the addition of the ACAT inhibitor, 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide (Sandoz Compound 58-035).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Biosynthetic studies of marine lipids 36. The origin of common sterol side chains in eleven sponges using [3-3H]-squalene.

1. [3-3H]-Squalene was fed to 11 marine sponges containing a mixture of "common" sterol side chains. All of these sponges possess significant quantities of cholesterol, but their ability to biosynthesize it differs widely. 2. All the sponges possess significant quantities of delta 22 sterols, yet none of them was able to introduce the delta 22 double bond. 22-Dehydro-24-norcholesterol and 24-methyl-22-dehydro-27-norcholesterol side chains also originate from the diet. 3. These sponges biosynthesized between 40 and 80% of their sterols, a typical value being 70%. The remainder is derived from the diet or by modification of dietary sterols.     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

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.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

In vivo evaluation of cholesterol 7 alpha-hydroxylation in humans: effect of disease and drug treatment

7 alpha-Hydroxylation of cholesterol is a stereospecific reaction consisting of the replacement of the 7 alpha-hydrogen with a hydroxyl group. When cholesterol labeled with tritium at the 7 alpha position is administered, the hydroxylation of the substrate will result in the loss of tritium which in turn will label the body water. The rate of tritium enrichment of the body water could thus give a quantitative estimate of the hydroxylation rate. This study describes the validation of the procedure with some 21 studies performed on 15 subjects in different conditions. [7 alpha-3H]cholesterol was administered intravenously in 50 ml of plasma and thereafter blood was sampled at timed intervals for 4 to 5 days. The rate of the hydroxylation of cholesterol was calculated from the time course of the specific activities of plasma cholesterol and body water after tracer administration and was expressed as 7 alpha-hydroxycholesterol formed/24 hr. Calculated values of hydroxylation in three control subjects (493 +/- 206), five patients with hyperlipoproteinemia (539 +/- 168), and seven cirrhotic patients (153 +/- 136) are in good agreement with figures reported for bile acid synthesis determined with other techniques. Cholesterol 7 alpha-hydroxylation rate is reduced in patients with cirrhosis, the impairment being related to the severity of the disease. Cholestyramine administered to one subject for 4 weeks produced a threefold increase of the hydroxylation. Administration of chenodeoxycholic acid resulted in a 50% decrease, whereas that of ursodeoxycholic did not produce consistent changes of the hydroxylation rate. The results support the current view that 7 alpha-hydroxylation of cholesterol is rate-limiting in the synthesis of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of cholesterol in low density and high density lipoproteins by cholesterol oxidase

The cholesterol oxidase-catalyzed oxidation of cholesterol in native low density (LDL) and high density lipoproteins (HDL3) as well as in monolayers prepared from surface lipids of these particles, has been examined. The objective of the study was to compare the oxidizability of cholesterol, and to examine the effects of lipid packing on oxidation rates. When [3H]cholesterol-labeled lipoproteins were exposed to cholesterol oxidase (Streptomyces sp.), it was observed that LDL [3H]cholesterol was oxidized much faster than HDL3 [3H]cholesterol. This was true both at equal cholesterol concentration per enzyme unit, and at equal amounts of lipoprotein particles per enzyme unit. About 95% of lipoprotein [3H]cholesterol was available for oxidation. The complete degradation of lipoprotein sphingomyelin by sphingomyelinase (Staphylococcus aureus) resulted in a 10-fold increase in the rate of LDL [3H]cholesterol oxidation, whereas the effects on rates of HDL3 [3H]cholesterol oxidation were less dramatic. A monolayer study with LDL surface lipids indicated that degradation of sphingomyelin loosened the lipid packing, because the ceramide formed occupied a smaller surface area than the parent sphingomyelin, and since the condensing effect of cholesterol on sphingomyelin packing was lost. The effects of sphingomyelin degradation on lipid packing in monolayers of HDL3-derived surface lipids were difficult to determine from monolayer experiments. Based on the finding that cholesterol oxidases are surface pressure-sensitive with regard to their catalytic activity, these were used to estimate the surface pressure of intact LDL and HDL3. The cut-off surface pressure of a Brevibacterium enzyme was 25 mN/m and 20 mN/m in monolayers of LDL and HDL3-derived surface lipids, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Double bond in the side chain of 1alpha,25-dihydroxy-22-ene-vitamin D(3) is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1alpha,25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] like 1alpha,25(OH)(2)D(3), is also converted into the same final product, calcitroic acid. This finding indicated that 1alpha,25(OH)(2)D(2) also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1alpha,25(OH)(2)D(2) when compared to the side chain of 1alpha,25(OH)(2)D(3), has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1alpha,25(OH)(2)D(2). We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D(2) related compounds. We compared the metabolism of 1alpha,25-dihydroxy-22-ene-vitamin D(3) [1alpha,25(OH)(2)-22-ene-D(3)], a synthetic vitamin D analog whose side chain differs from that of 1alpha,25(OH)(2)D(3) only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1alpha,25(OH)(2)-22-ene-D(3) and 1alpha,25(OH)(2)D(3) are converted into common metabolites namely, 1alpha,24(R),25-trihydroxyvitamin D(3) [1alpha,24(R),25(OH)(3)D(3)], 1alpha,25-dihydroxy-24-oxovitamin D(3) [1alpha,25(OH)(2)-24-oxo-D(3)], 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D(3). This finding indicates that the double bond in the side chain of 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Along with the aforementioned metabolites, 1alpha,25(OH)(2)-22-ene-D(3) is also converted into two additional metabolites namely, 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Furthermore, we did not observe direct conversion of 1alpha,25(OH)(2)-22-ene-D(3) into 1alpha,25(OH)(2)D(3). These findings indicate that 1alpha,25(OH)(2)-22-ene-D(3) is first converted into 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Then the double bonds in the side chains of 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3) undergo reduction to form 1alpha,24(R),25(OH)(3)D(3) and 1alpha,25(OH)(2)-24-oxo-D(3), respectively. Thus, our study indicates that the double bond in 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1alpha,25(OH)(2)-22-ene-D(3) metabolism indicating that prior C-24 hydroxylation of 1alpha,25(OH)(2)-22-ene-D(3) is required for the double bond reduction to occur.     

Side chain functionalization of cholesterol in the biosynthesis of solasodine in Solanum laciniatum

(25R)-26-Amino-cholesterol-[7α-³H], (25R)-26-amino-5-cholestene-3β,16β-diol-[7α-³H] and (25R)-26-acetylamino-5-cholestene-3β,16β-diol-[7α-³H] administered to Solanum laciniatum were converted into solasodine. The results indicate that in the biosynthesis of solasodine the introduction of nitrogen occurs immediately after the hydroxylation at C-26 and before a further oxidation of the side chain of cholesterol. The next step after the amination at C-26 is not hydroxylation at the 16β-position but probably the functionalization of C-22.     

Mechanistic and metabolic studies of sterol 24,25-double bond reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K_m, 3 μM), lanosterol (K_m, 18 μM), and cycloartenol (K_m, 33 μM), to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-²H]lanosterol was transformed by larvae to [24-²H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and ¹H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epimino-lanosterol (IL), was assayed and found to be inhibitory with an I₅₀ of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I₅₀ 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-³H]IL when fed to the larvae. [2-³H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley-Liss, Inc.     

Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.     

Measurement of rates of cholesterol synthesis using tritiated water

Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14C-labeled substrates or [3H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [3H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3H are incorporated into cholesterol from [3H]water in different tissues of several animal species, so that the 3H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [3H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [3H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo.