Determination of Dehydroepiandrosterone in Human Blood Plasma

The first method for the determination of dehydroepiandrosterone (DHEA) in blood plasma was published by Migeon (1).To follow variations of DHEA in the blood within a rather short period, it seemed desirable to reduce not only the time necessary for one analysis, but also the volume of plasma needed for this procedure.Furthermore, by using the method of Migeon, our recoveries of added Na DHEA sulfate did not exceed 65 per cent.The losses might be attributed in part to variations in the activity of Florisil (2).Also, as found during our experiments, the precipitation of proteins by means of ethanol leads to losses as high as 25 per cent of DHEA due to coprecipitation of the steroid conjugates.Hence, to avoid these difficulties, another procedure was developed which comprised the following steps: (1) precipitation of protein with ethanol (EtOH) ; ( 2) acid hydrolysis of EtOH extract and protein precipitate; (3) extraction; (4) distribution between 80 per cent MeOH and 10 per cent ethyl acetate (EtOAc) in hexane; (5) concentration of extract on paper strips; (6) descending chromatography; (7) elution of DHEA; (8) Zimmermanii reaction. MethodsPrecipitation of Protein-10 ml. of plasma are shaken with 20 ml. of EtOH in a 50 ml.centrifuge tube.After centrifugation, the extract is transferred into a second centrifuge tube and the residue submitted to two similar extractions with 5 ml. of EtOH each.The combined extracts are kept at -15" overnight, centrifuged in the cold, and the supernatant solution is transferred into a third centrifuge tube (3).The fatty residue is mashed with 5 ml. of ice-cold EtOH and added to the precipitated material, 5 ml. of a 0.1 N acetate buffer being used (pH 5.5).The EtOH extract and the wash are combined and evaporated to dryness in vacua in a water bath of 45".Acid Hydrol@ of EtOH Extract and Protein Precipitate-To the residue