Measurement of DHT

The principles and the methods for testosterone assay are basically valid for DHT as well. The main problem in DHT measurement is the elevated cross-reactivity of all polyclonal antisera with testosterone, which renders the direct, accurate quantification of DHT in a male serum sample impossible. As a consequence, DHT has to be separated from testosterone before measurement or, alternatively, testosterone must be chemically modified so that it is not recognized by the antibody. At present there are only two ways of quantifying DHT in serum accurately: after chromatographic separation or after oxidation of testosterone. Both methods involve an extraction step.

Various chromatographic procedures to separate serum steroids were validated in the seventies and in the eighties. The most common methods for chromato-graphic separation of DHT and testosterone are HPLC and celite chromatography. In HPLC the chromatographic medium consists of modified, lipophilic micro-spheres of silica gel onto which the extracted sample is loaded. DHT and testosterone are then sequentially eluted by an aqueous solution with an increasing

012345678 target DHT value or testosterone added (nmol/l)

Fig. 21.3 In-house revalidation of a commercial kit for DHT determination based on oxidation of

012345678 target DHT value or testosterone added (nmol/l)

Fig. 21.3 In-house revalidation of a commercial kit for DHT determination based on oxidation of testosterone. Left panel: DHT values in serum samples measured by HPLC and by the oxidative method. Right panel: recovery of DHT after addition of DHT or testosterone to a serum sample.

concentration of the organic solvent acetonitrile. Samples are then evaporated, reconstituted in assay buffer and assayed by RIA (Lerchl and Nieschlag 1995). Celite is a diatomaceous earth widely used for steroid separation. Minicolumns, prepared fresh by packing a few mg celite, are equilibrated with iso-octane and loaded with the extracted samples. The column is then eluted sequentially with 100% isooctane, 5-10% ethyl acetate in iso-octane (elution of DHT) and 20-50% ethyl acetate in iso-octane (elution of testosterone). Eluates are evaporated and reconstituted in assay buffer for testosterone and DHT determination by RIA (Abraham etal. 1975;Werawatgoompa etal. 1982). Chromatographic methods are still the first choice for accurate quantification of testosterone and DHT in serum. However, they are cumbersome and time-consuming, especially due to the solvent evaporation time.

In non-chromatographic methods, the serum sample is incubated with potassium permanganate. This oxidizing agent converts the double bond at position 4-5 of testosterone to dihydroxyl alcohol and does not affect DHT. The samples are then extractedwith an organic solvent, evaporated, reconstituted in assay buffer and measured by RIA (Werawatgoompa etal. 1982). Since it eliminates the chromatographic step, this type of assay is very convenient when large numbers of samples must be measured, provided that the results have been validated against a chromatographic method (Fig. 21.3). The newest, direct, non-extractive methods for DHT measurement, such as microtitre plate-based ELISA which are commercially available, have not been sufficiently validated. Unless such methods withstand an in-house re-validation against chromatographic methods, they should not be used.

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