Quantitative analysis of corticosteroids in adrenal cell cultures by capillary column gas chromatography combined with mass spectrometry

Maume, B.F.; Millot, Claire; Mesnier, Danielle; Patouraux, D.; Doumas, J.; Tomori, É.

doi:10.1016/s0021-9673(00)95279-8

Cited by 25 publications

(8 citation statements)

References 18 publications

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“…Culture initiation in a serum supplemented medium. Primary cell cultures of newborn rat adrenal glands were obtained according to Maume and Prost (1973) and Maume et al (1979). Trypsin dissociated adrenal ceils were collected in Ham's F10 medium (Gibco, Grand Island, NY) supplemented with 100 IU/ml penicillin, I00 ~g/ml streptomycin, 10% fetal bovine serum and 10% newborn calf serum and seeded in 20 cm 2 Cooper dishes (Falcon, Oxnard, CA).…”

Section: Hammaml G Maume and B Maumementioning

confidence: 99%

“…The dry unlabeled steroid extract to which cholesteryl butyrate was added as the internal standard was taken up for methoxime-trimethylsilyl (MO-TMS) derivative formation according to a method previously described by Prost and Maume (1974). Quantitative measurements by gas chromatography were done by using the response coefficients calculated by comparing the height of the peak of each adrenal steroid with that of the cholesteryl butyrate according to Maume et al (1979). Gas chromatography was carried out on 25 m long SE30 wall-coated open tubular glass columns (0.21 mm internal diameter) made by Spiral (Dijon, France).…”

Section: Hammaml G Maume and B Maumementioning

confidence: 99%

“…Newborn rat adrenal cells have been shown to retain their differentiated function, i.e., the biosynthesis of corticosteroids, in long-term primary cultures in serum supplemented media (Maume and Prost, 1973;Maume et al, 1976Maume et al, , 1979. Recent progress in culturing established cell lines (Sato et al, 1980) or diploid cell lines initiated from primary cultures in serum-free media provided the opportunity to investigate the steroidogenic functions of adrenal cells in the absence of serum in the culture medium.…”

Section: Introductionmentioning

confidence: 99%

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Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-?-cyclodextrin complex

Hammami¹,

Maume²,

Maume³

1986

Cell Biol Toxicol

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Newborn rat adrenocortical cells were successfully cultured in a serum-free carrier protein-free medium (SPFM) by using alpha-cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol-alpha-cyclodextrin with a molar ratio of almost 1 was obtained for a 5 X 10(-5) mol/1 alpha-cyclodextrin concentration. Cell cultures incubated with [4-14C] cholesterol-alpha-cyclodextrin in SPFM produced, under ACTH stimulation, various 14C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone. As measured by gas chromatography and mass spectrometry, the ratio between corticosteroids (21-hydroxylated steroids) and 20 alpha-reduced steroids produced in SPFM with cholesterol-alpha-cyclodextrin was equal to 1.8. This corresponds to a value of 3.6 times higher than that found in the serum-free medium with cholesterol-albumin. Consequently, the chemically defined SPFM with cholesterol-alpha-cyclodextrin used in this study is more suitable for corticosteroidogenesis by adrenal cells in culture than a serum-free medium with cholesterol-albumin.

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Section: Hammaml G Maume and B Maumementioning

confidence: 99%

Section: Hammaml G Maume and B Maumementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-?-cyclodextrin complex

Hammami¹,

Maume²,

Maume³

1986

Cell Biol Toxicol

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“…The methoxime-trimethylsilyl (MO-TMS) derivatives were prepared according to a previously described method (Maume et al 1979). Excess reagents and polar residues were separated from the steroid MO-TMS deri¬ vatives by chromatography on Lipidex 500 (Packard Instrument Co., Downers Grove, II., USA) following the method of Axelson Se Sjövall (1974 (Maume et al 1979).…”

Section: Incubationsmentioning

confidence: 99%

“…Excess reagents and polar residues were separated from the steroid MO-TMS deri¬ vatives by chromatography on Lipidex 500 (Packard Instrument Co., Downers Grove, II., USA) following the method of Axelson Se Sjövall (1974 (Maume et al 1979). …”

Section: Incubationsmentioning

confidence: 99%

Effect of ACTH on endogenous steroid biosynthesis in long-term primary cultures from newborn rat adrenal cells

Ramirez¹,

Millot²,

Maume³

1984

Acta Endocrinologica

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Normal newborn rat adrenal cells kept in primary culture up to 2\m=1/2\ months respond to ACTH stimulation and produced corticosteroid hormones and smaller amounts of 20\g=a\-reduced progesterone metabolites. Cholesterol from the serum complemented culture medium serves as precursor without further addition of exogenous steroid substrates. A long-term qualitative and quantitative study of individual steroid production under the influence of ACTH was performed. ACTH treatment produced a triphasic effect on steroid production: an induction period (up to 3 days), an acute maximum production period (3rd to 6th day) and a chronic production period (to the end of the treatment). The increase in total steroid production resulted from the increase in the production of corticosteroids only.This indicated an increase of cholesterol side-chain cleavage and of the 21-and 11\g=b\/18-steroidhydroxylations. Removal of ACTH led to a reversible drop in total steroid production. The response to ACTH was dose dependent, so that a 2.2 mU/ml dose elicited lower steroid production than the 6.6 or 22 mU/ml doses. Increasing the lower dose after a week of treatment to a higher dose brought total steroid production and 1 1\ g=b\ / 18\ x=r eq-\ steroid hydroxylation up to the corresponding chronic production levels. The 20\g=a\-steroidreduction system was not affected by ACTH. ACTH changes the importance of the two main steroidogenic pathways. With no ACTH there is approximately a 1:1 ratio between corticosteroid synthesis and progesterone reductive metabolism; a low dose of ACTH increases the total steroid production, but since corticosteroid production and 20\g=a\-reducedmetabolites both increase, the ratio changes little; a high dose of ACTH increases the ratio to more than 30:1.Refractoriness or desensitization to ACTH is postulated to occur through the control of cholesterol availability inside the cell possibly combined with a control of its utilization for steroidogenesis.The function of the adrenal system of various species has been studied using adrenocortical cell cultures as model svstems. Since the original work by Kahri (1968) and O'Hare & Neville (1973a,b) on normal cells from rat adrenal glands, and Buonassisi et al. (1962) and Kowal et al. (1970) on adrenal tumour cells, manv details of corticosteroid synthe¬ sis and metabolism have been studied. Using the method of Maume & Prost (1973) and Maume et al. (1976), newborn rat adrenal cells are maintained in primary culture for 7 -8 weeks in the presence of ACTH. The present article de¬ scribes this cellular system which under the influ¬ ence of ACTH produces all the intermediates and products of the chain of corticosteroid biosynthesis as well as those of the reductive metabolism of progesterone from cholesterol. The maintenance of the activity of the individual steroidogenic en¬zymes and the response of the cells to ACTH, the strongest effector of steroidogenesis in the fasciculata-reticulata zone of the adrenal gland, are good indications of the similarity of the cellular...

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Chromatography as a reference technique for the determination of clinically important steroids

Robards

Towers

1990

Biomedical Chromatography

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Chromatographic methods (paper chromatography, thin layer chromatography, high performance liquid chromatography, gas chromatography and gas chromatography-mass spectrometry) for the determination of clinically important steroids in biological specimens are reviewed. The emphasis is on the use of gas chromatography, gas chromatography-mass spectrometry and high performance liquid chromatography as reference rather than routine techniques. Chromatographic methods are compared with colorimetric, fluorimetric and radioimmunoassay procedures in terms of simplicity of operation, cost and ability to analyse large numbers of specimens. The importance of correct specimen collection and storage are discussed. Sample preparation techniques for the various analytical methods are described. These include extraction of free and conjugated steroids from serum, plasma, urine and saliva by solvent partition, with polymer-based resins such as Amberlite XAD-2, DEAE-Sephadex and Sephadex resins bonded with various other function groups and, more recently, with chemically bonded reversed-phase silicas.

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Quantitative analysis of corticosteroids in adrenal cell cultures by capillary column gas chromatography combined with mass spectrometry

Cited by 25 publications

References 18 publications

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-?-cyclodextrin complex

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-?-cyclodextrin complex

Effect of ACTH on endogenous steroid biosynthesis in long-term primary cultures from newborn rat adrenal cells

Chromatography as a reference technique for the determination of clinically important steroids

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