Serum level of 7 alpha-hydroxycholesterol in hypercholesterolemic patients treated with cholestyramine.

Serum levels of 7 alpha-hydroxycholesterol (7 alpha (OH)C) were found to increase considerably in hypercholesterolemic patients treated with cholestyramine. After 3-5 days, the normal level, which is in the range of 0.04 mg/l, rose to 1 mg/l. When the treatment was stopped it decreased rapidly with a half-life analogous to that of LDLs. This phenomenon was also observed in non-hypercholesterolemic humans. More than 75% of 7 alpha (OH)C is carried in blood by the LDLs.

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. II. Effects of sterol biosynthesis inhibitors on cell division.

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity--25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9-16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G2M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.

Serum-induced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells.

Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.

Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle.

A cell-free system assay involving cell freeze-thawing and protein fractionation by ammonium sulfate precipitation was developed to characterize a cytosol binding protein specific for oxysterols in rat embryo fibroblasts. This protein shared common characteristics with the oxysterol-binding protein described in L cells and in normal human lymphocytes: 8 S sedimentation coefficient, sterol-protein complex of Mr 160 600, saturability, high affinity (Kd in the range of 10(-9) M) and specificity for cholesterol derivatives oxidized on the side chain. These compounds were better inhibitors of DNA synthesis than the compounds oxidized on the nucleus, whereas no difference was found between sterols oxygenated either on the side chain or on the nucleus, as far as inhibition of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) was concerned. Macromolecular components capable of specifically binding 25-hydroxycholesterol were also detected in the fibroblast nucleus. The cytosol oxysterol-binding protein showed a sharp increase (5-fold) in the G2M phase of the cell cycle and in exponentially growing cells (maximal binding site number/cell: 43 500, versus 8850 in confluent cells). Neither the affinity nor the sedimentation coefficient of the protein changed in either situation. The quantitative (but not qualitative) variations of oxysterol-binding protein could be related to the inhibitory effect of 25-hydroxycholesterol on DNA synthesis, which becomes critical when this sterol is added in the G2M phase of the cell cycle.

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. I. Temporal relationships between HMG-CoA reductase activity, sterol biosynthesis and thymidine incorporation into DNA.

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C27 sterols, and [3H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C27 sterols occurred, with two maxima in S and G2M phases; the relative shortness of the G1 phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C27 sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C27 sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-14C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.

Evidence that liver microsomes of human neonates desaturate essential fatty acids.

delta 6- and delta 5-Desaturation of essential fatty acids of n-6 and n-3 series are required for the biosynthesis of polyunsaturated fatty acids (PUFAs), which are precursors of eicosanoids and constituents of membrane phospholipids. This pathway could be of special importance during the perinatal period, when PUFAs accretion in the central nervous system is very active. However, experimental evidence of delta 6- and delta 5-desaturase activities in man is very scarce, and no data are available for newborns. We report the delta 6- and delta 5-desaturase activities detected in human liver microsomes from three neonates who died from associated malformations. Radiochemical assays of delta 6- and delta 5-desaturase activities performed with reverse phase HPLC analysis of the products in the n-6 series ranged from 4.8-13.6 to 3.2-16.4 pmol substrate converted.min-1.mg-1 microsomal proteins, respectively. In the n-3 series delta 6-desaturase activity ranged from 5.3 to 12.8 pmol.min-1.mg-1. The relationships between enzyme activities and substrate concentrations suggest excess substrate inhibition for n-6 and not for n-3 fatty acids. These results demonstrate significant delta 6- and delta 5-desaturase activities in human liver of neonates, but this activity was lower than previously reported in adult humans and in mammals, especially rodents.

Oxysterol activation of arachidonic acid release and prostaglandin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase C activity [corrected].

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca2+ level. Since both Ca2+ variations and protein kinase C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein kinase C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxysterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC50 (4 x 10(-9) M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE2 synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3 beta, 25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca2+ ion fluxes, therefore oxysterols could affect earlier events triggered by serum growth factor binding to their cell membrane receptors.

Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: role of calcium.

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [14C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E2 biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A2 activity is Ca2(+)-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca2+ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca2+ concentration; when external Ca2+ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca2+ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca2+ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 micrograms/ml), oxysterols are not able to increase, immediately or after a short time of contact (90 min) the concentration of intracellular free Ca2+ ([Ca2+])i measured by fluorescence of Quin-2; at very high concentration of oxysterol (25 micrograms/ml), [Ca2+]i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca2+ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect of oxysterols on cell Ca2+ level.

Phosphoinositide breakdown and superoxide anion release in formyl-peptide-stimulated human alveolar macrophages. Comparison between quiescent and activated cells.

Human alveolar macrophages (AMs) from allergic asthmatics (AAs) showed continuous Li+-sensitive production of IP1, indicating that the cells were continuously activated. Furthermore, whereas the accumulation of IP1, IP2 and IP3 rapidly increased by as much as 125-175% in chemotactic-factor-stimulated AMs from healthy subjects, stimulation of cells from AAs increased these inositol phosphates only slightly. This moderate production could be due to a permanent state of activation leading to a depleted pool of polyphosphoinositides, corroborating the greater capacity of these cells to generate superoxide anion after stimulation by a chemoattractant. The activation state could be due to the action of priming agents, which are known to be released into the inflammatory sites.

Rat liver cytosol oxysterol-binding protein. Characterization and comparison with the HTC cell protein.

A cytosol protein that specifically binds cholesterol derivatives oxygenated on the side chain has been demonstrated in rat liver and transformed HTC cells. This protein, of which the sedimentation coefficient is about 8 S, was saturable and showed a high binding affinity (Kd about 5 X 10(-9) M) for 25-hydroxycholesterol. Its molecular mass is about 160 kDa. The physicochemical characteristics of this protein were identical whether the model was normal or transformed. This oxysterol-binding protein differs from the well-known sterol carrier proteins.

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol. Effects of high salt concentrations and molybdate.

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to 'activate' the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KCl) but the resulting 4.3 S complex dissociates easily whereas the 'native' 8.3 S form does not. Moreover, molybdate did not prevent the 8.3----4.3 S transformation induced by KCl and neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors.

Differential incorporation of fish-oil eicosapentaenoate and docosahexaenoate into lipids of lipoprotein fractions as related to their glyceryl esterification: a short-term (postprandial) and long-term study in healthy humans.

We investigated how the distribution of eicosapentaenoate (EPA, 20:5n-3) and docosahexaenoate (DHA, 22:6n-3) in the sn-2 and sn-1(3) positions of fish-oil triacylglycerols influenced their respective incorporation into triacylglycerol, cholesterol esters, and phospholipids of two lipoprotein fractions: low- and very-low-density lipoprotein (VL/LDL) and high-density lipoprotein (HDL). Nine healthy volunteers were studied over both a short-term (0-8 h) and a long-term (30 d) postprandial period of daily supplementation with 2 g EPA and 1.3 g DHA given as 11 g fish-oil triacylglycerol in which DHA was predominantly situated in the sn-2 position. Our results strongly suggest that the higher triacylglycerol incorporation of DHA and the higher metabolic availability of EPA compared with DHA for phospholipid accumulation (particularly in the short-term study) depend on their respective preferential sn-2/sn-1(3) positions in fish-oil triacylglycerol, emphasizing the important role of the triacylglycerol structure and its potential manipulation for modulating availability of either or both fatty acids.

Human placental glutamate dehydrogenase. Purification--kinetic and regulatory properties--physicochemical studies.

Glutamate dehydrogenase (EC. 1.4.1.3) has been purified more than 9,000 times from human placental alcoholic subfractions as a homogenous protein of 55,155 daltons (subunit molecular weight). Kinetic constants for the reverse reaction (reductive amination of alpha-ketoglutarate) have been shown to be similar to those of the bovine liver enzyme, while the kinetic constants for the forward reaction were markedly different as well as some regulatory properties (lack of activation by ADP in the reverse reaction). The amino acid composition differs from the bovine liver enzyme composition. Furthermore, the tryptic peptide patterns of the placental enzyme and the human liver enzyme have been compared. Besides the low specific activity of this enzyme, the results indicate that human placental glutamate dehydrogenase is closely related to other mammalian glutamate dehydrogenases.

[Structure of adducts prepared from nicotinamide adenine dinucleotide and their oximes]. / Structure des composés d'addition formés par le nicotinamide adénine dinucléotide et les oximes.

Preparation of adducts from nicotinamide adenine dinucleotide and a number of oximes is described; these include acetoxime, pyruvatoxime, cyclohexanoxime, cyclopentanoxime. These adducts are closely related to the corresponding NAD-ketone adducts in their spectra properties, but they are stable in acid solutions (pH 5).

Characterisation of an associate 17-beta-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-alpha-hydroxysteroid dehydrogenase of Pseudomonas testosteroni.

The 3-alpha-hydroxysteroid dehydrogenase and the 3-beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyaerylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration. The pure 3-alpha-hydroxysteroid dehydrogenase was completely devoid of 3-beta-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-beta at an appreciable rate. This activity accounts for about 40 per cent of the total 17-beta-estradiol dehydrogenase of the crude bacterial extract. Affinity labelling of pure 3-alpha-hydroxysteroid dehydrogenase was carried out using 5-beta-pregnane 3,20-dione-12-alpha-iodoacetate and 5-alpha-androstane 3-one-17-beta-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.

A specific hydroxysterol binding protein in human lymphocyte cytosol.

A discriminating system capable of recognizing the oxygenated sterols was investigated in human lymphocytes. After labelling entire cells with 25-hydroxy [3H] cholesterol (10 nM) the cytosol was ultracentrifuged on a linear sucrose density gradient. Bound 25-hydroxy [3H] cholesterol was located in a single peak with a sedimentation coefficient of 8.3 S. Pronase treatment abolished the radioactive peak. This 8.3 S protein had a low binding capacity for 25-hydroxy [3H] cholesterol and probably a high affinity. This last parameter was not determined on account of some difficulties encountered in a cell-free system relating to the physico-chemical properties of 25-hydroxycholesterol. Only the hydroxylated sterols closely related to 25-hydroxycholesterol were capable of specifically binding to the 8.3 S protein, in contrast with cholesterol. This protein differed from the binding proteins of oxygenated derivatives of vitamin D3 and glucocorticoids. With the human lymphocyte as a model and under our experimental conditions, this hydroxylated sterol-binding protein seems to be involved rather in the cell division control than in the regulation of HMG-CoA reductase activity: indeed, the hydroxysterols able to inhibit thymidine [3H] incorporation into DNA are recognized by this protein whereas the hydroxysterols active on HMG-CoA reductase activity without affecting thymidine [3H] incorporation into DNA are not.

Studies of covalent adducts of NAD(P) and enolizable ketones as specific glutamate dehydrogenase inhibitors.

Structural analogues of the reduced coenzymes, NADH or NADPH, of dehydrogenases are prepared by addition of carbonyl compounds including: pyruvate, alpha ketoglutarate, oxaloacetate, butyraldehyde, acetaldehyde and acetone, to the oxidized coenzymes NAD(P). Some of the adducts obtained are specific inhibitors of the glutamate dehydrogenase. The specificity is related to the carbonyl compound used. The high selectivity of the dehydrogenases for adducts is evidenced by inhibition studies of NAD(P)-pyruvate and NAD(P)-alpha ketoglutarate adducts on both activities of glutamate dehydrogenase. The inhibitions are competitive with the reduced coenzymes and the oxidized substrates: adducts could be considered as structures closely related to the ternary complexes of the dehydrogenase.

Comparative effects of simvastatin and pravastatin on cholesterol synthesis in patients with primary hypercholesterolemia.

The effects of simvastatin and pravastatin on cholesterol biosynthesis were compared in 26 hypercholesterolemic patients who were randomly allocated to either simvastatin or pravastatin treatment (20 mg once daily) for 6 weeks in a crossover trial. Serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) lathosterol (latho) concentrations and lathosterol/cholesterol (latho/chol) ratios (the latter two are considered as reliable indices of whole body cholesterol synthesis) were evaluated at the beginning and end of each therapeutic sequence. Reductions in TC and LDL-C were more pronounced (P < 0.001) with simvastatin (TC = -28.0%, LDL-C = -35.6%) than with pravastatin (TC = -19.6%, LDL-C = -25.2%). These results were associated with concomitant decreases in both latho concentrations (-59.0% with simvastatin and -37.0% with pravastatin) and latho/chol ratios (-43.0% with simvastatin and -20.3% with pravastatin). Simvastatin resulted in more marked diminutions of latho concentrations (P < 0.01) and latho/chol ratios (P < 0.05) than pravastatin. These results suggest that the better efficacy of simvastatin on serum cholesterol and LDL cholesterol might result in part from a greater inhibitory action of simvastatin on cholesterol synthesis compared with that of pravastatin.

Coupling of delta 5,3-ketosteroid isomerase to human placental lactogen with intermolecular disulfide bond formation. Use of this conjugate for a sensitive enzyme immunoassay.

An immunoassay for human placental lactogen (HPL) has been developed with the use of hormone labelled with delta 5,3-ketosteroid isomerase. The enzyme is coupled to the antigen via an intermolecular disulfide interchange reaction. HPL-enzyme conjugates are prepared in high yield and purified by affinity chromatography. An enzyme immunoassay using these hormone-enzyme derivatives is compared with radioimmunoassay for determination of HPL in serum.

Affinity chromatography: a tool for enzyme immunoassay.

Affinity chromatography has been widely used for enzyme purification and for the preparation of antigen-enzyme conjugates. Most enzyme immunoassay use immunoadsorbent (specific or double insolubilized antibodies) which makes automation difficult as centrifugation and washing steps are necessary. We propose affinity chromatography for rapid separation of antigen-enzyme bound to antibodies from free conjugate and its application in enzyme immunoassay. We describe enzyme immunoassays for human placental lactogen and progesterone which use delta 5,3-ketosteroid isomerase as marker and depend upon retention by specific absorbent of enzyme conjugate not bound to antibodies.