Decreased Urinary 5a-Androstane-3α,17β-Diol Glucuronide Excretion in Patients with Benign Prostatic Hyperplasia*

Jung

J. Am. Soc. Mass Spectrom.

et al. 2009

Abnormalities in steroid hormones are responsible for the development and prevention of endocrine diseases. Due to their biochemical roles in endocrine system, the quantitative evaluation of steroid hormones is needed to elucidate altered expression of steroids. Gas chromatographic-mass spectrometric (GC-MS) profiling of 70 urinary steroids, containing 22 androgens, 18 estrogens, 15 corticoids, 13 progestins, and 2 sterols, were validated and its quantitative data were visualized using hierarchically clustered heat maps to allow "steroid signatures". The devised method provided a good linearity (r 2 Ͼ 0.994) with the exception of cholesterol (r 2 ϭ 0.983). Precisions (% CV) and accuracies (% bias) ranged from 0.9% to 11.2% and from 92% to 119%, respectively, for most steroids tested. To evaluate metabolic changes, this method was applied to urine samples obtained from 59 patients with benign prostatic hyperplasia (BPH) versus 41 healthy male subjects. Altered concentrations of urinary steroids found and heat maps produced during this 70-compound study showed also differences between the ratios of steroid precursors and their metabolites (representing enzyme activity). Heat maps showed that oxidoreductases clustered (5␣-reductase, 3␣-HSD, 3␤-HSD, and 17␤-HSD, except for 20␣-HSD). These results support that data transformation is valid, since 5␣-reductase is a marker of BPH and 17␤-HSD is positively expressed in prostate cells. Multitargeted profiling analysis of steroids generated quantitative results that help to explain correlations between enzyme activities. The data transformation and visualization described may to be found in the integration with the mining biomarkers of hormone-dependent diseases. Many naturally occurring steroids with similar chemical structures could yield biological information [7]. Endogenous steroids are divided into five groups, namely, androgens, estrogens, corticoids, progestins, and sterols, which are generally synthesized from cholesterol in the adrenal cortex, ovaries, and testes (Scheme 1). In biosynthetic pathways of steroid hormone, two major types of enzymes are involved, cytochrome P450 and steroid oxidoreductase. Abnormalities of these enzymes often lead to hormonal imbalances that have serious consequences, and which are responsible for the development of hormone-dependent diseases (see Supplementary Table 1, which can be found in the electronic version of this article). For example, concentrations of corticoids and their metabolic ratios provide diagnostic evidence of apparent mineralocorticoid excesses caused by 11␤-HSD deficiency [8] and congenital adrenal hyperplasia, which are caused by deficiencies of enzymes like hydroxylase (at C-11, 17, and 21) or 3␤-HSD [9]. In addition, enhanced androgen activity generated by the conversion of testosterone to dihydrotestosterone (DHT) by 5␣-reductase was utilized to allow early therapeutic intervention in young men [10].Enzyme activity profiles can be used to describe the functional diversities of biological systems, which are d...

Section: Quantitative Results Of Benign Prostatic Hyperplasiasupporting

confidence: 92%

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

Jung

J. Am. Soc. Mass Spectrom.

et al. 2009

“…The metabolite 5␣-androstane-3␣,17␤-diol (3␣-diol), which is converted by 3␣-hydroxysteroid dehydrogenase from DHT, has been considered another active androgen and a potential causative agent of BPH (3,4,11 ). In the present study, its glucuronide was not measured because 3␣-diol is reversibly converted to DHT (12 ).…”

mentioning

confidence: 93%

“…The method described here simplifies the procedure to a two-stage PCR amplification and allows the probe sequence to be placed anywhere between the primer binding sites without the need to identify or engineer restriction sites. Similar approaches have been described previously (3)(4)(5), although none of these is as simple or universally applicable as the method described here. As an example, the method has been used to synthesize a molecular construct for use as an internal standard in a quantitative PCR method for measles virus (MV) RNA.…”

mentioning

confidence: 98%

“…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)(3)(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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Choi¹,

Kim

Chung³

2003

Clinical Chemistry

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

Biomedical Chromatography

“…Like the high-temperature GC-MS method, CE-MS/MS method needs no derivatization or sample preparation for solid-phase extraction, and small volumes of sample and solvent can be used. Because the measurement of urinary androgen glucuronides could demonstrate clinical implications with increased DHT in plasma and tissue (Wilson, 1980;Wright et al, 1985), and decreased DHT-17G in urine (Choi et al, 2003) in patients with BPH, determination of urinary androgen glucuronides without deconjugation is very useful technique.…”

Section: Application To the Urine Samplesmentioning

confidence: 99%

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

Cho

Lee²,

Choi³

et al. 2008

Capillary electrophoresis-electrospray tandem mass spectrometry (CE-ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE-MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01-5 microg/mL for glucuronides of androsterone and 11beta-OHAn-3G, and 5-500 ng/mL for glucuronides of 11-ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992-0.998, and the limits-of-detection at a signal-to-noise ratio of 3 were 5-10 ng/mL. The coefficients of variation were in the range of 4.0-9.0% for intra-day assay and 4.1-9.8% for inter-day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis.