Bong Chul Chung

GC-MS determination of steroids related to androgen biosynthesis in human hair with pentafluorophenyldimethylsilyl–trimethylsilyl derivatisation

An efficient method for the simultaneous determination of eight steroids, androstenedione, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androsterone, etiocholanolone, progesterone and pregnenolone, in human hair by gas chromatography-mass spectrometry (GC-MS) using d3-testosterone as internal standard is described. The method involves alkaline digestion, liquid-liquid extraction and subsequent conversion to mixed pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected ion monitoring (SIM) mode. This method showed good overall repeatability and reproducibility of 4.88-11.24 and 3.19-9.58%, respectively. For the first time, the quantification of DHT, DHEA and pregnenolone in human hair has been achieved by GC-MS, testosterone was also quantified. The detection of four steroids in hair samples was possible in the concentration range 0.12-8.45 ng g-1. The other four steroids, androstenedione, androsterone, etiocholanolone and progesterone, were not detected. The detection limits for SIM of the steroids varied in the range 0.02-0.5 ng g-1, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.996 for most of the steroids studied. The concentrations of the four steroids detected were different in male and female hair samples.

Multivariate classification of urine metabolome profiles for breast cancer diagnosis

BackgroundDiagnosis techniques using urine are non-invasive, inexpensive, and easy to perform in clinical settings. The metabolites in urine, as the end products of cellular processes, are closely linked to phenotypes. Therefore, urine metabolome is very useful in marker discoveries and clinical applications. However, only univariate methods have been used in classification studies using urine metabolome. Since multiple genes or proteins would be involved in developments of complex diseases such as breast cancer, multiple compounds including metabolites would be related with the complex diseases, and multivariate methods would be needed to identify those multiple metabolite markers. Moreover, because combinatorial effects among the markers can seriously affect disease developments and there also exist individual differences in genetic makeup or heterogeneity in cancer progressions, single marker is not enough to identify cancers.ResultsWe proposed classification models using multivariate classification techniques and developed an analysis procedure for classification studies using metabolome data. Through this strategy, we identified five potential urinary biomarkers for breast cancer with high accuracy, among which the four biomarker candidates were not identifiable by only univariate methods. We also proposed potential diagnosis rules to help in clinical decision making. Besides, we showed that combinatorial effects among multiple biomarkers can enhance discriminative power for breast cancer.ConclusionsIn this study, we successfully showed that multivariate classifications are needed to precisely diagnose breast cancer. After further validation with independent cohorts and experimental confirmation, these marker candidates will likely lead to clinically applicable assays for earlier diagnoses of breast cancer.

Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor in Bladder Cancer Epithelia and GnRH Effect on Bladder Cancer Cell Proliferation

Introduction: Gonadotropin-releasing hormone (GnRH) is the pivotal hormone in the hypothalamopituitary gonadal axis. Recently, localization of extrahypothalamic GnRHs and GnRH receptors has been made in several organs but not in the bladder. We studied the extrahypothalamic GnRH and GnRH receptor in bladder cancer epithelia, and the role of GnRH on bladder cancer cell proliferation. Material and Methods: Normal human bladder mucosa, human transitional bladder cancer tissue, and 4 bladder cancer cell lines were used. For culture media, normal or charcoal-stripped serum with exogenous GnRH of different concentrations (0, 10–3, 10–5, and 10–7M) were supplemented. For detection of GnRH and the GnRH receptor and its mRNAs, RNase protection assay, in situ hybridization, and immunocytochemistry were performed. Cellular proliferation was measured by hemocytometer and the cell cycle by flow cytometer. Results: GnRH and GnRH receptor mRNA expression were detected in all cancer cell lines and human bladder cancer tissues. GnRH and GnRH receptors were localized in bladder cancer epithelial cells, but the density was not even. There were no significant differences (p > 0.05) in proliferation, and no significant change (p > 0.05) in cell cycle with GnRH supplements compared to controls. Conclusion: The bladder epithelia produce GnRH and the GnRH receptor in both normal and malignant tissues, but GnRH does not induce proliferation or cell cycle change of bladder cancer cells.

Gas chromatographic–mass spectrometric determination of plasma saturated fatty acids using pentafluorophenyldimethylsilyl derivatization

An improved method for the detection of 11 saturated fatty acids (SFAs) including C12:0-C26:0 (even numbers only), C17:0, C19:0 and C23:0 in human plasma by gas chromatography-mass spectrometry (GC-MS) with a stable isotope internal standard as d3-stearic acid is described. This procedure was based on acidic treatment, liquid-liquid extraction, and chemical derivatization prior to instrumental analysis. Eleven pentafluorophenyldimethylsilyl-SFA derivatives were well separated without any interfering peaks in plasma samples. The characteristic ions at M-15, constituting the base peaks in the electron impact mass spectra for 11 SFAs, permitted their sensitive detection by GC-MS in the selected ion monitoring (SIM) mode. The SIM responses were linear with correlation coefficients varying from 0.993 to 0.999 in the concentration range of 0.05 to approximately 50 microg/ml for the 11 SFAs. The detection limits for SIM of the SFAs varied in the range of 0.05 to approximately 10.0 pg. When applied to the plasma samples of normal subjects and patients with X-linked adenoleukodystrophy, which is one of the hereditary peroxisomal disorders, the present method enabled us to determine the SFAs with good sensitivity and good overall precision and accuracy within the concentration ranges of 0.14 to approximately 82.35 micromol/l.

Determination of testosterone : epitestosterone ratio after pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation using gas chromatography-mass spectrometry in equine urine

A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of steroids: the selective exchange of the alcoholic flophemesyl ether for the trimethylsilyl ether. The sensitivity and specificity of the mixed 3-TMS-17-flophemesyl derivatives allow adequate determinations of testosterone and epitestosterone, even in urine from mares, in 5 ml samples. The repeatability of testosterone and epitestosterone was 6.2 and 5.7%, respectively, and their reproducibility was in the range of 6.4-8.7%.

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

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.