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Supplementary data are available at Bioinformatics online.

Qualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student's t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer.

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...

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Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/ mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with singleparameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively. Copyright # 2004 John Wiley & Sons, Ltd.Oxidized nucleosides represent excellent biomarkers for determining the extent of damage in genetic material, which has long been of interest in understanding the mechanism of aging, neurodegenerative diseases, and carcinogenesis. [1][2][3][4][5] Various methods have been developed for quantifying modified nucleosides, based on an enzyme-linked immunosorbent assay (ELISA), 6,7 32 P-post-labelling, 8 capillary electrophoresis, 9,10 HPLC with photodiode array detection 11,12 or ECD, 13 and GC/MS. 14-16 However, GC/MS analysis faced additional problems in the hydrolysis and derivatization steps. 17 HPLC/ECD analysis has limitations in that the requirement for electroactive components meant that the method was mainly applicable to the detection of only 8-oxodeoxyguanosine. 18 Due to their chemical characteristics in HPLC analysis, various kinds of nucleosides could not be analyzed using just one kind of detector.To overcome these problems, in the present work, we developed an analytical method for determining many kinds of nucleosides at one time using LC/MS with ESI (electrospray ionization) detection. We confirmed that mass spectrometric detection with ESI is more specific and sensitive than any other HPLC detection method. We also developed a direct analytical method for nucleosides without extracting urine samples. This approach is both more convenient and more efficient in conducting studies with large sample numbers, e.g., a clinical study, than analytical methods involving complex extrac...

A quantitative analysis of polyamines in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The polyamines were carbamylated with isobutyl chloroformate, extracted with diethyl ether under pH 9.0, and analyzed by LC-MS/MS with single reaction monitoring mode. The limit of quantification was 1 ng/mL based on a signal-to-noise ratio>3, and the correlation coefficient (r2) for the calibration curves was >0.99 for both urine and serum samples. The present method was applied to urine and serum samples from 30 breast cancer patients and 30 normal female controls. There was no significant difference in the urinary polyamine levels between breast cancer patients and controls. However, 1,3-diaminopropane, putrescine, spermine and N-acetylspermidine levels in serum increased in breast cancer patients. These four serum polyamines may be a good index to study both production and metabolism of polyamines, and a useful tool in assessment of the polyamine status of breast cancer patients.

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