Rapid HPLC-Electrospray Tandem Mass Spectrometric Assay for Urinary Testosterone and Dihydrotestosterone Glucuronides from Patients with Benign Prostate Hyperplasia

Abstract

by the locus-specific primer is directly subjected to TaqI digestion and is analyzed on an agarose gel without radioisotopic hybridization. It also appears to be more accurate because all three distinct chimeric CYP21P/ CYP21 genes had 3.2-kb fragments (Fig. 1C, lanes 3 and 4). Simply assuming that the 3.2-kb fragment from conventional Southern analysis (6 ) is a deletion of the CYP21 gene has been shown to be incorrect. The authors of one report have stated that the 3.2-kb fragment analyzed by Southern blotting identified a fused CYP21 gene, but in this study, Southern blotting was performed with radioisotope hybridization and a different endonuclease (8 ).In conclusion, the coexistence of 9.3-and 3.2-kb fragments is consistent with a chimeric CYP21P/CYP21 gene. The method of analysis we have described is a novel tool for identification of such a molecule. These chimeric genes were found to occur frequently in CAH caused by steroid 21-hydroxylase deficiency in a Taiwanese (ethnic Chinese) population. The development of benign prostatic hyperplasia (BPH) is dependent on androgens, primarily dihydrotestosterone (DHT) (1 ). To estimate the activity of 5␣-reductase, which catalyzes the conversion of testosterone to DHT, testosterone and DHT have been quantified in biological samples (2-4 ). Because of their lipophilic properties, they are usually found in the conjugated form, i.e., linked to a hydrophilic sulfuric moiety or ␤-glucuronic acid, which are excreted mainly (Ͼ95%) in human urine. Despite reports of a close association between BPH and testosterone and DHT (1-4 ), to our knowledge, there is no published simultaneous quantitative data for these metabolites as their glucuronides in the urine of patients with BPH.The major problem associated with quantification of total steroids is incomplete hydrolysis, attributable mainly to the matrix effects of urine on the efficiency of enzymatic hydrolysis, which reduces the reproducibility of the assay (5,6 ). Given that it is often necessary to analyze many samples in a short time with good sensitivity and reproducibility, system throughput is a critical issue for many clinical mass spectrometry (MS) groups. In 322 Technical Briefs

Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometry

“…The highest concentration androgen glucuronide was An-3G (2.4 ± 0.5 μg/mL), followed by 11β-OHAn-3G (0.7 ± 0.2 μg/mL), DHEA-3G (41.0 ± 15.7 ng/mL), T-17G (31.7 ± 15.6 ng/mL), Epi-17G (26.2 ± 16.1 ng/mL) and DHT-17G (22.3 ± 6.8 ng/mL). The levels of individual androgen glucuronides measured are in accordance with those of previous reports (Weykamp et al, 1989;Choi et al, 2000Choi et al, , 2003. In our previous report, the levels of urinary androgen glucuronides from subjects well-matched for sex, age and ethnic group with subjects from this study were detected using high-temperature GC-MS (Choi et al, 2000).…”

“…Hence, glucuronidation plays an important role in regulating the levels of androgens, and the measurement of urinary androgen glucuronides is very important in androgen metabolism. Because of changed androgen metabolism, urinary androgen glucuronide levels are altered in various diseases, such as BPH (Choi et al, 2003), acne (Garmina et al, 2002) and alopecia (Legro et al, 1994). As a result, androgen glucuronides can be used as biomedical indicators in these diseases.…”

Heat-map visualization of gas chromatography-mass spectrometry based quantitative signatures on steroid metabolism

“…A dendrogram of ratios revealed that the oxidoreductases formed a cluster (5␣-reductase, 3␣-HSD, 3␤-HSD, and 17␤-HSD, except for 20␣-HSD; see Supplementary Table 1 for full enzyme names), which are differentiated with a cytochrome P450 enzyme, 11␤-hydroxylase. These results support the validity of the data transformation, since 5␣-reductase is a marker of BPH [27][28][29], and 17␤-HSD is positively expressed in prostate cells [40,41]. Other up-regulated enzymes in the map might be biomarkers of BPH.…”

“…The aim of this study was to validate a GC-MS profiling method and a method that allows the quantitative visualization of multiple urinary steroids. To visualize quantitative results, a microarray map (a type of heat map) was designed to present the urine sample results of 59 patients with benign prostatic hyperplasia (BPH) and 41 healthy male subjects; BPH was chosen because steroid metabolism is known to play a role in the progress of prostate diseases [27][28][29]. This study focused on illustrating the usefulness of steroid signatures for explaining both the concentrations of individual steroids and the activities of enzymes correlated global steroidogenesis in BPH, and suggest that enzyme activity profiling may be a useful diagnostic tool and provide a means of identifying mining biomarkers in hormone-dependent diseases.…”

Systematic Error in Gas Chromatography-Mass Spectrometry–Based Quantification of Hydrolyzed Urinary Steroids

“…There are many naturally occurring steroids that are excreted mainly through the urine by their water-soluble conjugates formed by the substitution of 3-or 17-hydroxyl groups with either sulfate or β-glucuronide (1), and their direct measurements have been introduced to clinical studies (2)(3)(4)(5). Gas chromatography-mass spectrometry (GC-MS)-based profiling is a proven technique in steroid analysis, whereas immunoassays have limited applicability due to cross-reactions (6,7).…”