The Interference of Free and Esterified Cholesterol in the Spectrofluorimetric Measurement of Plasma 11-Hydroxysteroids

Abstract

Cholesterol and cholesterol esters have been identified on the basis of thin-layer and gas\p=m-\liquid chromatographic data and fluorimetric characteristics as major interfering fluorogens in a simple fluorimetric method for determining plasma corticosteroids.In a small series ofnormal subjects the average concentration of fluorogen, which was not 11-hydroxysteroid, was 3\m=.\4 \g=m\g.apparent cortisol/100 ml. plasma, equivalent to 22\m=.\4 % of the total fluorogen present.

Fluorometric analysis

“…Much work has been done on the fluorometric determination of corticosteriods (646) such as cortisol and corticosterone (185, 288, 585,806) usually by means of a fluorogenic reagent of HiSCh-ethanol in varying proportions (115,186,218,805). There have been many papers on the fluorometry of the 11-hydroxycorticosteroids cortisol and corticosterone (318, 836, 520, 921, 1042) including some on corrections for nonspecific fluorescence (346,889,898,989) and the separate determination of the two compounds by TLC (87). In the fluorometric procedure for cortisol with a 7:3 H2SC>4-ethanol reagent, various improvements have been proposed (497,758,880, 902) such as correction for interferences (563,1001), the use of phase-separating filter paper (490), and metaperiodate oxidation to achieve increased specificity (173, 174)• Other papers described the fluorometry of 11deoxycortisol (629, 630), of cortisol and transcortin (876), and of cortisol and 20-dihydrocortisol (988).…”

“…Aldosterone was assayed by its fluorescence after ELSCL-methanol treatment (928), or after periodic acid oxidation to formaldehyde which was then quantitated by condensation with acetylacetone and ammonia (381). Individual bile acids were determined fluorometrically after TLC separation and H2SO4 treatment (694); total bile acids which are also 3-hydroxysteroids were assayed by NADH fluorometry after enzymic reaction with 3-hydroxysteroid dehydrogenase (684); and fluorometry was evaluated in relation to other quantitative methods for bile acids (898).…”

“…quinacrine (909). Luminescence techniques have been used for studies of: microcirculation in the skin (884) and brain (784); changes in the human intervertebral disk with age (707); lipofuscin pigments in the heart (177) and as a result of lipid peroxidation of subcellular organelles (167); orange fluorescence in the acoustic nerve (786); the changes caused by electrical stimulation of nerve stained with various fluorochromes (982); the physiological state of lymphocytes in the peripheral blood of schizophrenic patients (546); the fluorescence properties of visual pigments (366) and their relationship to ultraviolet sensitivity in insect eyes (471); and the evaluation of pollen viability by intracellular enzymic hydrolysis of fluorescein diacetate (898).…”