Bioconversion of 16 alpha-hydroxyprogesterone to 16 alpha-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture.

Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups. Minor metabolites resulted from 18-hydroxylation and 6 beta-hydroxylation of the substrate. The metabolism of 16 alpha-hydroxyprogesterone is similar to that of progesterone in the same cell-culture system; however, there are two exceptions. The 21-hydroxylation of 16 alpha-hydroxyprogesterone occurs at a rate similar to that of its 11 beta-hydroxylation, whereas the 21-hydroxylation of progesterone is faster than its 11 beta-hydroxylation. The ratio of 11 beta- to 18-hydroxylation of 16 alpha-hydroxyprogesterone is about 3, whereas the ratio of 11 beta- to 18-hydroxylation of progesterone, 20 alpha-dihydroxyprogesterone and DOC is between 1./ and 2. It is most likely the rate of 18-hydroxylation which is decreased by the hydroxyl group at C-16. The use of adrenal cell cultures is a practical, simple method for the preparation of a variety of 16 alpha-hydroxylated steroids from a single substrate. Its adaptation to the production of important amounts of 16 alpha-hydroxylated corticosteroids will permit the study of their biological activity.

Bioconversion of 2 alpha-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture.

The bioconversion of 2 alpha-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11 beta-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6 beta-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2 alpha-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2 beta-epimers of the different metabolites arose principally from the transformation of 2 beta-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11 beta-hydroxylation where the reaction appears stereospecific for the 2 beta-epimer. The 2 alpha-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3 beta-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium.

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4-14C]cholesterol-HDL in serum-free medium compared to those in medium with lipoprotein-deficient serum, in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20 alpha-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7-150 micrograms cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.

[Study fo sterols from brown seaweeds of the genus Cytoseira. Identification by gas-liquid chromatography coupled with mass spectrometry (author's transl)]. / Etude des sterols d'algues brunes du genre Cystoseira. Identification par chromatographie gaz-liquide couplée à la spectrométrie de masse

All the samples of brown seaweeds (Cystoseria) that we have studied present the same deltas sterols fucosterol, 22 trans-dehydrocholesterol, brassicasterol, 24-methylene cholesterol as well as cystosterol, a new C27 sterol. This sterol has been submitted to gas-liquid chromatographic-mass spectrometric analysis.

Effect of rat plasma high density lipoprotein with or without apolipoprotein E on the cholesterol uptake and on the induction of the corticosteroid biosynthetic pathway in newborn rat adrenocortical cell cultures.

High density lipoprotein (HDL) has been shown to induce the cellular accumulation of cholesterol esters and the biosynthetis of 21-hydroxysteroids (corticosteroids) newborn rat adrenocortical cells cultivated in serum-free medium. In order to identify the component(s) of HDL responsible for these effects, we investigated the ability of rat HDL subfractions and HDL with or without apolipoprotein E to deliver cholesterol to cells and to stimulate the steroid biosynthetic pathways in adrenal cultured cells. The total cholesterol uptake from HDL2 was greater than that observed with HDL rich in apolipoprotein E (HDL1 and HDLc). Furthermore, the increase of the ratio between 21-hydroxysteroids and reductive metabolites of progesterone was higher with HDL2 than with HDL1 or HDLc. The results of competitive studies between LDL and HDL subfractions indicate that adrenal cells take up cholesterol from HDL2 and LDL by separate mechanisms but that LDL and HDL containing apolipoprotein E share the same uptake processes. In experiments with various concentrations of HDLc or HDL without apolipoprotein E, the adrenal cells displayed a higher affinity for rat HDLc than for rat HDL without apolipoprotein E. However, HDL without apolipoprotein E produced a higher enhancement of the cholesterol cell content and was 3-fold more effective in stimulating 21-hydroxylated steroid production than rat HDLc. Although these findings suggest a participation of HDL with apolipoprotein E in the HDL interaction with rat adrenal cells, the predominant effect on these cells is devoluted to HDL containing mainly apolipoprotein A.

Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst.

Suspension-cultured cells of Nicotiana tabacum generated active oxygen species (AOS) when they were treated with the proteinaceous elicitor, cryptogein. This response was blocked by diphenylene iodonium, an inhibitor of the neutrophil NADPH oxidase. When microsomal extracts of tobacco cells were probed with an antibody directed against the human small G protein Rac2, two immunoreactive proteins were detected at 18.5 and 20.5 kDa. The same experiment performed with cytosolic extracts of tobacco cells led to the observation of a strong immunoreactive protein at 21.5 kDa only in the cryptogein-treated cells. The appearance of this cytosolic protein was related to the production of AOS by the elicited cells. These results provide evidence for the possible involvement of small G proteins, homologous to the neutrophil Rac2 protein, in the regulation of the elicitor-induced oxidative burst in plant.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid-cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

Combination of the novel farnesyltransferase inhibitor RPR130401 and the geranylgeranyltransferase-1 inhibitor GGTI-298 disrupts MAP kinase activation and G(1)-S transition in Ki-Ras-overexpressing transformed adrenocortical cells.

To test the Kirsten-Ras (Ki-Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl-pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase-1 inhibitor GGTI-298. In Ki-Ras-overexpressing transformed adrenocortical cells, RPR130401 at 1-10 microM inhibited very efficiently the [(3)H]farnesyl but not [(3)H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC(50)=30 microM). GGTI-298 inhibited the growth of these cells with an IC(50) of 11 microM but cell lysis was observed at 15 microM. The combination of 10 microM RPR130401 and 10 microM GGTI-298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G(0)/G(1) and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non-cytotoxic concentrations, acting on the farnesyl-pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase-1 respectively is an efficient strategy for disrupting Ki-Ras tumorigenic cell proliferation.

A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

Adrenal and liver metabolites of 18-hydroxy-11-deoxycorticosterone in the rat. Identification of reduced compounds (18-OH-TH-DOC).

The presence of reduced metabolites of 18-hydroxy-11-deoxycorticosterone has been investigated in the adrenals of 23 day-old and adult rats and in the liver of adult rats. By thin-layer chromatography a fraction of the adrenal steroid extract migrating like tetrahydrocorticosterone has been isolated. By gas chromatography-mass spectrometry several isomers of 3,18,21-trihydroxy-pregnan-20-one (18-OH-TH-DOC) have been separated in this fraction and identified by comparison with authentic samples which have been chemically and enzymatically synthesized. The major tetrahydrogenated metabolite in the adult and prepuberal rat adrenals is 3beta,18,21-trihydroxy-5alpha-pregnan-20-one (18-OH-TH-DOC II). The 3alpha,18,21-trihydroxy-5beta-pregnan-20-one has been found only in the prepuberal rat adrenal. A third tetrahydrogenated isomer has been tentatively indentified as 3alpha,18,21-trihydroxy-5alpha-pregnan-20-one. Quantitative measurements by mass fragmentography show that adrenal reductase activity on 18-hydroxy-11-deoxycorticosterone is higher than on corticosterone. The 18-OH-TH-DOC II has been identified in the liver of adult male rat.

Synthesis and gas chromatographic-mass spectrometric analysis of reduced compounds derived from 6 alpha- and 6 beta-hydroxy-11-deoxycorticosterone.

A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4-5 double bond, the 3-oxo group, and/or the 20-oxo group of 6 alpha- and 6 beta-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given. The gas chromatographic separation of the syn- and anti-isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6 alpha-series than for the 6 beta-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a saturated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.

Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

[Enzymatic activity assays in the hepatic cell by mass fragmentography associated with gas-liquid chromatography]. / Mesure d'activités enzymatiques dans la cellule hépatique par la fragmentographie de masse couplée à la chromatographie gaz-liquide

The most generalized methods in enzymology are based on the quantitative assay of compounds, substrate or coenzyme, by spectrophotometry without any separation. Such a method is ruled out if the colorimetric reaction is not specific of the compound. In liver enzymology, aside the classical metabolic pathways, such assays are difficult to apply, especially when several metabolic steps are investigated. It is therefore necessary to use separative methods to isolate the metabolized substrate(s). For instance, the reductive catabolism of corticosterone leads to fourteen isomers (two dihydrocompounds, four tetrahydrocompounds and eight hexahydrocompounds) in which their respective productions are sex and age-linked. A position isomer of corticosterone, the 18-hydroxy-11-deoxy-corticosterone, follows the same reductive route. In adrenals some reduced metabolites arise from these two steroid hormones and are age dependent. When such metabolites are amenable to volatilization for gas chromatography, the interfacing of the gas chromatograph to the mass spectrometer allows to identify each compound introduced in the spectrometer. Among the ions produced by fragmentation of a compound or of a family of compounds, several specific fragments can be selected to be monitored along the chromatographic run leading to mass peaks which are quantitatively proportional to the amount of compounds, as far as other foreign molecules do not contribute fo fragment productions. These methods called mass fragmentography or multiple ion detection, or selected ion monitoring, allow with the help of all the resources of gas chromatography such the derivatization of studied molecules with heavy isotope labeled reagents to use the same unlabeled derivatized molecules as carriers and internal standards at once. This method allows to quantitate at the level of the picomolecule or less. Examples will be given with the study of the metabolism hormone steroids and xenobiotic compounds by the liver and adrenals in the animal and by isolated liver and adrenal cell cultures.

A method for the analysis of catecholamines by selected ion monitoring and 14C isotope dilution in adrenal medullary cell culture.

Extracts have been made from culture medium of rat medullar adrenal cells developed in tissue culture in this laboratory. After pentafluorobenzylimine-trimethylsilyl ether formation the catecholamine derivatives were characterized by gas-liquid chromatography chemical ionization mass spectrometry. In order to assess the catecholamine production capabilities of the cells in culture, a mass spectrometric method with isotope dilution has been devised. Chemical ionization selected ion monitoring allows specific detection at the nanogram level in a higher mass range (400-600 amu) than in the electron impact mode. The isotopic dilution method with 14C catecholamines gives rise to accurate measurements and linear response in the picomole range. The use of the [M-15]+ ion for monitoring m/z values minimizes errors in selected ion monitoring analysis. The results obtained are computerized and treated by the data system for fine background subtraction when high sensitivity and accuracy are required.

[Metabolism of exogenous steroids in adrenal cortex rat cells in culture: establishment of hydroxylation in position 11-beta or 18]. / Métabolisme de stéroïdes exogènes par les cellules corticosurrénaliennes de rat en culture: place de l'hydroxylation en position 11 beta ou 18.

Newborn rat adrenocortical cells in primary culture have allowed us to establish the place of 11 beta/18-hydroxylation in the biosynthesis and metabolism of corticosteroid hormones. The affinity of the 11 beta/18-hydroxylation system has been determined with respect to known or potential intermediates. It has equally been examined in relation to 2 alpha-, 6 alpha-, 6 beta-, 16 alpha or 17 alpha-hydroxyprogesterone and 6 alpha-, 6 beta- or 17 alpha-hydroxy-11-deoxycorticosterone.