Studies towards the development of a multi-purpose home self-test kit for the detection of urinary tetrahydrocortisone and testosterone metabolites : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University

Abstract

The development of homogeneous enzyme immunoassays (HEIA) for testosterone glucuronide (TG) and tetrahydrocortisone glucuronide (THEG) in urine are described. The proposed test system is based on the Ovarian Monitor homogeneous immunoassay system, established by J.B Brown and L.F. Blackwell et al Brown JB, Blackwell LF, Cox RI, Holmes JM and Smith MA (1988). Chemical and homogenous enzyme immunoassay methods for the measurement of estrogens and pregnanediol and their glucuronides in urine. Progress in Clinical and Biological Research 285:119-38. as a simple, laboratory accurate, monitoring device for the measurement of estrone glucuronide (E1G) and pregnanediol glucuronide (PdG) as markers of the fertile phase during a womans menstrual cycle. This information can be used readily by women to identify their cyclical periods of fertility and infertility. The major testosterone metabolite in the urine of males, testosterone β-glucuronide, was synthesised by firstly preparing the glycosyl donor α-bromosugar and conjugating this with testosterone under standard Koenigs-Knorr conditions. ¹H nmr studies confirmed that the synthetic steroid glucuronide had the same stereochemistry as the naturally occurring urinary testosterone glucuronide. Testosterone glucuronide and tetrahydrocortisone glucuronide conjugates of hen egg white lysozyme were prepared using the active ester coupling method in good yield. Unreacted lysozyme was successfully removed from the reaction mixture by a combination of cation-exchange chromatography in 7 M urea and hydrophobic-interaction chromatography. S- Sepharose chromatography allowed two of the major conjugation products in each steroid- glucuronide reaction mixture to be isolated and characterised by MALDI mass spectrometry. All of the selected conjugates were found to be mono-acylated species, with the exception of one of the testosterone glucuronide conjugates, which was found to contain a mixture of mono-and di-substituted hen egg white lysozyme conjugates. Conjugates prepared in this way were tested for their suitability as signal generators in a homogenous immunoassay system for measurement of testosterone glucuronide and tetrahydrocortisone glucuronide in urine samples based on the already established Ovarian Monitor home fertility assay. Immunogens required for raising anti-steroid antibodies were also prepared using the active ester method to conjugate testosterone glucuronide and tetrahydrocortisone glucuronide to the carrier protein, bovine thyroglobulin. These immunogens were then used to raise anti- testosterone glucuronide and anti-tetrahydrocortisone glucuronide antibodies in four New Zealand White rabbits. The antisera obtained over a number of months from each rabbit were screened for their ability to inhibit the lytic activity of the corresponding steroid glucuronide- lysozyme conjugate. Although all the antisera showed the immunogens had stimulated the production of anti-steroid glucuronide antibodies, the antiserum titre was low, and meant that the volume of antiserum required to inhibit the lytic activity was high. Nevertheless, despite the low antiserum titres, the selected antisera could be used to produce good standard curves for testosterone with sensitivity close to that required for TG and an excellent standard curve for THEG. Potential applications of home assays for urinary testosterone glucuronide are in self-monitoring of testosterone levels in men undergoing long term testosterone supplementation for a diagnosed androgen deficiency or steroidogenic abnormality. Tetrahydrocortisone glucuronide is a major metabolite of cortisol, the main glucocorticoid produced by the body in response to stress and disease, and has potential as a biomarker for assessing therapies designed to reduce stress and for individuals suffering from stress related conditions such as hypertension and heart disease.