The influence of plasma on basal and ACTH-stimulated in vitro adrenocortical steroidogenesis

Abstract

Early descriptions of in vitro ACTH bioassays all emphasised the need to use extracted plasma samples due to interference by an unidentified component. The aim of these studies was to elucidate the effects of whole plasma on ACTH steroidogenic activity in vitro and to identify the responsible factor. A sensitive in vitro dispersed bovine adrenocortical cell bioassay was established. The addition of 10% ACTH-depleted human pooled plasma to the incubation media resulted in basal steroidogenesis equivalent to that achieved with 10 9 M ACTH 1-24 and potentiated the steroidogenic activity of 10 9 M ACTH 1-24 by 7·8-fold. This potentiation was dependent on the concentration of both ACTH and plasma in the media, but did not result from the mitogenic effect of plasma. A pituitary source was excluded and the potentiating activity was not extractable by Vycor glass. Column chromatography demonstrated two peaks of activity corresponding to molecular weights of 650 and 220 10 3 Da. These peaks did not correspond to the plasma binding of 125 I-ACTH which resulted from non-specific binding to albumin. Lipoprotein-deficient serum had no effect on either basal or ACTH-stimulated steroidogenesis, but both were restored by the addition of purified lipoproteins. However, novel findings demonstrated a differential effect of low (LDL) and high (HDL) density lipoproteins on basal and ACTH-stimulated steroid production; thus, LDL exerted a greater effect on the former, whilst HDL potentiated the steroidogenic activity of added ACTH more than LDL. The addition of the lipoproteins to lipoprotein-deficient serum restored its basal and ACTH potentiating effects, the cholesterol concentrations of the chromatographic fractions exactly paralleling their ACTH potentiating effect. These findings suggest that not only are lipoproteins the plasma factor(s) which potentiates ACTH steroidogenic activity in in vitro bioassays, but also that they exert differential effects on basal and ACTH-stimulated steroid production.

Expression of low and high density lipoprotein receptor genes in human adrenals

“…This is in agreement with a previous report that biosynthesis of corticosteroids from endogenous cholesterol is active in both untreated and cAMP-stimulated human adult adrenal cells (3). Although there is differential accumulation of cholesterol from LDL and HDL according to ACTH stimulation in bovine adrenocortical cells (16), human adult adrenocortical cells may prefer LDL to HDL as a preferential exogenous cholesterol source for steroidogenesis (3). LDL addition increased basal and ACTHinduced cortisol production and it simultaneously slightly inhibited basal expression of LDL receptor mRNA.…”

Use of Glycan Targeting Antibodies To Identify Cancer-Associated Glycoproteins in Plasma of Breast Cancer Patients

“…The presence of albumin and a few other abundant proteins is a major problem in proteomics of plasma. 29 There is concern in any type of chromatography that nonspecific binding of these abundant proteins to columns will later mask the identification of low-abundance proteins. Fortunately, the magnitude of this problem can be assessed by modeling.…”

Determination of Synacthen in human plasma using immunoaffinity purification and liquid chromatography/tandem mass spectrometry

“…Hence, the validation of the presented assay included the analysis of an administration study blood sample. Although no evidence was obtained for the existence of an ACTH‐binding protein,32 non‐specific interaction to other plasma proteins such as albumin might interfere with IAC purification strategies. Hence, an administration study plasma sample was obtained and analyzed according to the developed assay, which provided the proof‐of‐principle of its applicability.…”