Printer Friendly

Rapid HPLC-electrospray tandem mass spectrometric assay for urinary testosterone and dihydrotestosterone glucuronides from patients with benign prostate hyperplasia.

The development of benign prostatic hyperplasia (BPH) is dependent on androgens, primarily dihydrotestosterone (DHT) (1). To estimate the activity of 5[alpha]-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 [beta]-glucuronic acid, which are excreted mainly (>95%) 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 continuation of studies of steroid profiles in diseases related to steroid enzymes (7, 8), we here demonstrate a sensitive and precise HPLC-tandem MS method for quantification of testosterone and DHT glucuronides by use of a three-column, two-switching valve system and the presence of the indicative biomarker in urine samples from BPH patients.

We obtained 24-h urine samples from 27 patients [mean (SD) age, 46 (5) years; range, 38-55 years] treated at Yonsei Medical Center (Seoul, Korea), including 19 men with BPH and 8 healthy men who underwent digital rectal examination and a prostate-specific antigen blood test. The latter were matched with the BPH patients for age. None of the participants had been treated with chemotherapy before the sampling. Testosterone-17 [beta]-glucuronide (T-G) and DHT-17 [beta]-glucuronide (DHT-G) were purchased from Steraloids. An internal standard (IS), 16,16,17-[[sup.2][H.sub.3]]testosterone-17 [beta]-glucuronide ([d.sub.3] T-G), was obtained from NARL Reference Materials.

[FIGURE 1 OMITTED]

The triple-column system constructed for the HPLC system (NANOSPACE series; Shiseido) included two 2001 inert pumps, a 2003 autosampler, two six-port valves, a CS-300B column oven set at 40 [degrees]C for the mixed functional (MF), and analytical columns (Fig. 1). For sample preparation, 50 [micro]L of urine filtered through a VariSep Nylon Syringe Filter (0.2 [micro]m pore size; 4 mm diameter; Varian) was injected into the sample loop and transferred to the MF column [Capcell Pak Phenyl; 150 x 4.6 mm (i.d.); Shiseido] at a flow rate of 500 [micro]L/min to isolate endogenous interferences, such as proteins and other macromolecules. Most endogenous interferences passed through the column, whereas T-G and DHT-G were retained (Fig. 1A). An isocratic mobile phase consisting of 10 mmol/L phosphate in 100 mL/L acetonitrile was used to prevent precipitation of proteins. Five minutes after injection of a sample, the upper valve was turned to the alternative position so that the MF column and precolumn [Capcell Pak [C.sub.18]; 50 x 2.0 mm (i.d.)] were in line (Fig. 1B). This configuration allowed the T-G and DHT-G on the precolumn to be concentrated. During initial work, all peaks were monitored with ultraviolet detection at 254 nm. After 6.0 min, two switching valves were turned to the alternative positions to disconnect both columns, and the sample was loaded on the analytical column by use of backflush mode[Capcell Pak [C.sub.18]; 150 x 1.0 mm (i.d.)] and eluted with a gradient to complete separation and MS detection (Fig. 1C). Use of the backflush mode allowed the enriched analytes from the pre-column to be loaded on the analytical column with the mobile phase at a flow rate of 100 [micro]L/min. The time required for an analytical run was ~22 min. The retention times of T-G, DHT-G, and the IS were 8.17, 8.48, and 8.21 min, respectively, as measured by a LCQ Advantage ion-trap mass spectrometer (Thermo Finnigan).

All data were recorded on a LCQ ion-trap MS operated in the negative ionization mode (m/z 150-500). The electrospray ionization probe was installed with sheath and auxiliary gasses run at 80 and 20 units, respectively. The heated capillary temperature was maintained at 250 [degrees]C. For the generation of tandem MS data, the deprotonated precursor ions [[M-H][.sup.-] of T-G (m/z 463), DHT-G (m/z 465), and [d.sub.3] T-G (m/z 466) were fragmented by helium gas collisions at the relative collision energy of 55%. The most abundant product ions, m/z 287 for T-G, m/z 289 for DHT-G, and m/z 290 for [d.sub.3] T-G, which were produced by cleavage of glucuronic acid, were chosen for selected-reaction monitoring analysis.

To prepare a calibration curve, we removed T-G and DHT-G from pooled urine by solid-phase extraction (9). Urine samples with increasing concentrations of added analyte (0.2-400 [micro]g/L T-G and 3-200 [micro]g/L DHT-G) and a fixed concentration of added [d.sub.3] T-G (20 [micro]L; 1 mg/L) as an IS were then analyzed according to the procedure described above. The correlation coefficients were >0.962. The recovery of the extraction procedure was measured by comparing the responses obtained from the extracted urine samples to which DHT-G and T-G had been added to those obtained from the corresponding unextracted reference standards prepared at the same concentrations (0.2, 1, 5, 20, and 100 [micro]g/L for T-G; 3, 5, 10, 20, and 100 [micro]g/L for DHT-G). The mean (SD) recoveries were 94% (4%)for T-G and 97% (6%)for DHT-G.

The precision was determined by assaying urine samples in replicate (n = 15) at five different concentrations (0.2, 1, 5, 20, and 100 [micro]g/L for T-G; 3, 5, 10, 20, and 100 [micro]g/L for DHT-G). For within-day imprecision, triplicates of each concentration were analyzed on the same day. For total, day-to-day imprecision, triplicates were analyzed 12 times on 12 separate days. The mean of triplicates was used in calculating total CV. The within-day CV was 4.5-7.5%, and the total CV was 4.9-8.3%. To evaluate recovery of the assay, we added five different concentrations of analyte (0.2,1, 5, 20, and 100 [micro]g/L for T-G; 3, 5, 10, 20, and 100 [micro]g/L for DHT-G), together with 20 [micro]g/L of an IS, to urine. The samples were extracted and analyzed as described above. Recoveries ranged from -2.4% to 6.5%. The limits of detection and quantification were calculated by measuring the analytical background response. The limits of detection were 0.2 [micro]g/L for T-G and 3 [micro]g/L for DHT-G on the basis of a signal-to-noise ratio of 3. The limits of quantification were 1 ~",g/L for T-G and 10 [micro]g/L for DHT-G. The limit of quantification was 10 times the SD of the five urine samples.

The present method was applied to urine from patients with BPH. Mean (SD) urinary T-G was 152 (31) [micro]g/L in control individuals and 170 (42) [micro]g/L in BPH patients (Table 1). No significant difference was found between the two groups, which is similar to the results reported previously for plasma testosterone and urinary T-G (3). In contrast, mean (SD) urinary DHT-G was 15 (8) [micro]g/L in the controls. The mean value was significantly lower in BPH patients [4.9 (2.3) [micro]g/L]. This result is not consistent with previous reports (3,10) of increased DHT in plasma and tissue from patients with BPH, but it provides experimental support for the importance of biologically active free DHT as the main endocrine factor responsible for the etiology of BPH (1,10). Increased DHT may be attributable to decreased glucuronidation in BPH patients.

The metabolite 5[alpha]-androstane-3[alpha],17[beta]-diol (3[alpha]-diol), which is converted by 3[alpha]-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[alpha]-diol is reversibly converted to DHT (12). Urinary DHT-G may be a good index to study both production and metabolism of androgens and a useful tool in assessment of the androgen status of BPH patients. The present method may be useful for differentiating between BPH and other androgen-dependent diseases with clinical features indicative of abnormal 5[alpha]-reductase activity.

We thank Dr. John K. Leach (Massachusetts Institute of Technology, Cambridge, MA) for critical reading of the manuscript and Eun-Koo Yoon (Scinco Corporation) and Ho-Bin Lim (Youngjin Biochrom Corporation) for excellent technical assistance. We thank Dr. Sung Joon Hong (Yonsei Medical Center, Seoul) for the kind donation of urine samples.

References

(1.) Steers WD. 5[alpha]-Reductase activity in the prostate. Urology 2001;58:17-24.

(2.) Horton R, Hsieh P, Barberia J, Pages L, Cosgrove M. Atered blood androgens in elderly men with prostate hyperplasia. J Clin Endocrinol Metab 1975;41: 793-6.

(3.) Wright F, Poizat R, Bongini M, Doukani A, Mauvais-Jarris P. Decreased urinary 5[alpha]-androstane-3[alpha],17 [beta]-diol glucuronide excretion in patients with benign prostate hyperplasia. J Clin Endocrinol Metab 1985;60:294-8.

(4.) Salerno R, Moneti G, Forti G, Magini A, Natali A, Saltutti C, Di Cello V, et al. Simultaneous determination of testosterone, dihydrotestosterone and 5[alpha]-androstan-3[alpha],17[beta]-diol by isotope dilution mass spectrometry in plasma and prostatic tissue of patients affected by benign prostatic hyperplasia. J Androl 1988;9:234-40.

(5.) Graef V, Furuya E, Nishikaze 0. Hydrolysis of steroid glucuronides with [beta]-glucuronidase preparations from bovine liver, Helix pomatia, and E. coli. Clin Chem 1977;23:532-5.

(6.) Tang PW, Crone DL. A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids. Anal Biochem 1989;182:289-94.

(7.) Choi MH, Yoo YS, Chung BC. Biochemical roles of testosterone and epitestosterone to 5[alpha]-reductase as indicators of male-pattern baldness. J Invest Dermatol 2001;116:57-61.

(8.) Choi MH, Hahm JR, Jung BH, Chung BC. Measurement of corticoids in the patients with clinical features indicative of mineralcorticoid excess. Clin Chim Acta 2002;320:95-9.

(9.) Choi MH, Kim KR, Chung BC. Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring. Steroids 2000;65:54-9.

(10.) Wilson JD. The pathogenesis of benign prostatic hyperplasia. Am J Med 1980;68:745-56.

(11.) Walsh PC, Wilson JD. The induction of prostatic hypertrophy in the dog with androstanediol. J Clin Invest 1976;57:1093-7.

(12.) Morimoto I, Edmiston A, Horton R. Alteration in the metabolism of dihydrotestosterone in elderly men with prostate hyperplasia. J Clin Invest 1980;66:612-5.

Man Ho Choi, [1] * Jung Nyun Kim, [2] and Bong Chul Chung [1] ([+]) ([1] Bioanalysis & Biotransformation Research Center, KIST, Seoul 130-650, Korea; [2] -Division of Food Investigation, Korea Advanced Food Research Institute, Seoul 137-060, Korea; * current address: Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, MA 02139; ([+]) address correspondence to this author at: Bioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Korea; fax 82-2-958-5059, e-mail bcc0319@kist.re.kr)
Table 1. Concentrations (a) of urinary T-G and DHT-G in
controls and patients with BPH.

 Mean (range) age, Concentrations,
 years [micro] g/L

Controls (n = 8) 45 (35-59)
 T-G
 Mean (SD) 152 [+ or -] 31
 Median 146
 Range (111-197)
 DHT-G
 Mean (SD) 15 [+ or -] 8
 Median 13
 Range (5-27)
Patients with BPH (n = 19) 46 (38-55)
 T-G
 Mean (SD) 170 [+ or -] 42
 Median 159
 Range (136-230)
 DHT-G
 Mean (SD) 4.9 [+ or -] 2.3 (a)
 Median 4.2
 Range (3.4-8.8)

(a) P <0.001.
COPYRIGHT 2003 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2003 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Technical Briefs
Author:Choi, Man Ho; Kim, Jung Nyun; Chung, Bong Chul
Publication:Clinical Chemistry
Date:Feb 1, 2003
Words:1972
Previous Article:Deletion of the C4-CYP21 repeat module leading to the formation of a chimeric CYP21P/CYP21 gene in a 9.3-kb fragment as a cause of steroid...
Next Article:Simple PCR-based method for synthesis of molecular calibrators and controls.
Topics:


Related Articles
IM testosterone is least costly replacement tx.
Dose-response effect of Red Maca (Lepidium meyenii) on benign prostatic hyperplasia induced by testosterone enanthate.
Evaluation of a deuterium-labeled internal standard for the measurement of sirolimus by high-throughput HPLC electrospray ionization tandem mass...
Testosterone measurement by isotope-dilution liquid chromatography--tandem mass spectrometry: validation of a method for routine clinical practice.
Ion suppression in mass spectrometry.
Liquid chromatographic-tandem mass spectrometric method for the determination of 5-hydroxyindole-3-acetic acid in urine.
Newborn screening by tandem mass spectrometry: gaining experience.
Monitoring of congenital adrenal hyperplasia by microbore HPLC-electrospray ionization tandem mass spectrometry of dried blood spots.
Detection of epitestosterone doping by isotope ratio mass spectrometry.
Analytical advances in detection of performance-enhancing compounds.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters