MEASUREMENT OF FREE CORTICOSTEROIDS IN RAT PLASMA: PHYSIOLOGICAL VALIDATION OF A METHOD¹

Abstract

Levels of free plasma corticosteroids (expressed as jug. Compound B/100 ml. plasma) have been measured routinely in 2 ml. of rat plasma using a method based on fluorescence of Compound B in H 2 SO.i. Resting levels are 9 to 15 Mg./lOO ml. After stress, levels increase up to 500%. After total adrenalectomy or hypophysectomy readings correspond to 3.5-6.5 jUg./100 ml. There is no change upon exposure to stress of the adrenalectomized or hypophysectomized animal. Intra-venous injection of 0.1 to 1.0 milliunit of ACTH U.S.P. Standard, 24 hours after hypophysectomy increases the concentrations of corticoids in a linear log-dose response. After adrenalectomy ACTH is without effect. In the hypophysectomized animals, large doses of highly purified FSH, LH, TSH, STH, a-MSH and arginine vasopressin have no effect on the corticoid levels. Considerable stimulation of corticoidogenesis can be observed in the absence of detectable changes in the adrenal ascorbic acid concentrations when the two variables are measured simultaneously in the same hypophysectomized animals.A SIMPLE method based on the fluorescence of corticosterone in sulfuric acid was recently devised by Silber to measure levels of "free" corticosteroids in small volumes of plasma (1). This report is a summary of experiments performed to assess the validity of this method in various experimental conditions. Two preliminary notes on some of these data have been published elsewhere (2, 3). MATERIALS AND METHODS 1. Animals. Approximately 700 animals were used in the experiments reported here; they were male rats, weighing 160-210 gm. obtained from Holtzman Farms in Houston and Madison. Females were used in a series of experiments devoted to a study of the relationships between the ovarian cycle and the resting levels of plasma corticosteroids; stages of the cycle (proestrus, estrus, diestrus) were assessed for 7 days before measurement of corticosteroid levels by daily vaginal smears stained with 1% toluidine blue.

Adrenal Involvement in Determining the Time of Onset of Puberty in the Rat1

“…The sensitivity of the assay was such that values down to 4.0 µg/ JOO ml could be routinely measured and at times values as low as 2.5 µg/ 100 ml could be detected. This compares well with values in the literature (85,157) and is well below the background fluorescence found in place of adrenalectomized animals in other studies (6.0 ! .07 µg/100 ml) 52 and in our own (6.5 ± 0.9 µg/100 ml).…”

Effect of single sub-lethal doses of barbital sodium on the adrenal glands of the garden lizard, Calotes versicolor: Histomorphic and histochemical investigations.

“…Pentobarbital inhibits release of ACTH in varying degrees in rats (Munson and Briggs 1955, Ohler and Sevy 1956, Sayers 1957, Royce and Sayers 1958, Rerup and Hedner 1962, Oliver and Troop 1963, Leonard 1966, dogs (Suzuki et al 1962), monkey (Harwood and Mason 1957) and man (Siker et al 1956), and hexobarbital does the same in dogs (Suzuki et al 1962). On the other hand, it is on record that in rats, pentobarbital (Guillemin et al 1958, Holzbauer and Vogt 1958, Barrett and Stockham 1963), hexobarbital (Lauber et al 1937) and phenobarbital (Ashford and Shapero 1962) stimulate ACTH secretion by acting on the nervous system. However, the prominent effect of pentobarbital appears to be inhibitory.…”

TOLERANCE TO THE EFFECT OF Δ1‐TETRAHYDROCANNABINOL ON CORTICOSTERONE LEVELS IN MOUSE PLASMA PRODUCED BY REPEATED ADMINISTRATION OF CANNABIS EXTRACT OR Δ1‐TETRAHYDROCANNABINOL

“…Both In the experiments concerning measurement of plasma corticosterone levels, all injections were made between 10 h 00 min and 11 h 00 minutes. The corticosterone was assayed fluorometrically by a method based on procedures described by Guillemin, Clayton, Smith & Lipscomb (1958) and by Silber (1966). Each sample of plasma analysed was derived from the blood of two adult male mice.…”