An improved solvent system for thin-layer chromatography of adrenal steroids

Levin, Scott; Touchstone, Joseph C.; Murawec, Taras

doi:10.1016/s0021-9673(01)80601-4

Cited by 6 publications

(2 citation statements)

References 2 publications

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“…The thin-layer plate was cut at = 0.55, and the lower half (containing the origin and aldosterone, cortisol, 18-hydroxydeoxycorticosterone, and corticosterone standards) was chromatographed in the second dimension in chloroform-ethanol (142.5:7.5) to separate the cortisol and 18-hydroxydeoxycorticosterone standards fully. The upper half (containing the deoxycorticosterone, progesterone, and pregnenolone standards) was chromatographed in the second dimension in ethyl acetatecyclohexane (50:50), The standards were visualized and traced; pregnenolone was visualized with vanillin (Levin et al, 1969). Strips of the thin-layer chromatography plate were cut into 0.5 X 2 cm sections and counted in a liquid scintillation counter.…”

Section: Bioassay Ofmentioning

confidence: 99%

Steroidogenic activity of high molecular weight forms of corticotropin

Gasson¹

1979

Biochemistry

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The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules

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Section: Bioassay Ofmentioning

confidence: 99%

Steroidogenic activity of high molecular weight forms of corticotropin

Gasson¹

1979

Biochemistry

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“…Another review (265) deals with thin-layer chromatography of sterols and steroids. An improved solvent system consisting of 20% acetone-isopropyl ether resolved four adrenal steroids (263). The precise location of steroids on thin-layer plates is determined by the use of marker dyes (449).…”

Section: Applicationsmentioning

confidence: 99%