Profiling of Steroid Metabolic Pathways in Human Plasma by GC-MS/MS Combined with Microwave-Assisted Derivatization for Diagnosis of Gastric Disorders.

Steroid hormones are associated in depth to cellular signaling, inflammatory immune responses, and reproductive functions, and their metabolism alterations incur various diseases. In particular, quantitative profiling of steroids in plasma of patients with gastric cancer can provide a vast information to understand development of gastric cancer, since both sex hormones and glucocorticoids might be correlated with the pathological mechanisms of gastric cancer. Here, we developed a gas chromatography-tandem mass spectrometry-dynamic multiple reaction monitoring (GC-MS/MS-dMRM) method combined with solid-phase extraction (SPE) and microwave-assisted derivatization (MAD) to determine 20 endogenous steroids in human plasma. In this study, MAD conditions were optimized with respect to irradiation power and time. The SPE enabled effective cleanup and extraction for profiling of steroid hormones in human plasma samples. The MAD could improve laborious and time-consuming derivatization procedure, since dielectric heating using microwave directly increase molecular energy of reactants by penetrating through medium. Furthermore, dMRM method provided more sensitive determination of 20 steroids, compared to traditional MRM detection. The limits of quantification of steroids were below 1.125 ng/mL and determination coefficients of calibration curves were higher than 0.9925. Overall precision and accuracy results were below 19.93% and within ±17.04%, respectively. The developed method provided sufficient detection sensitivities and reliable quantification results. The established method was successfully applied to profile steroid metabolism pathways in plasma of patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. Statistical significances of steroid plasma levels between gastric disorder groups were investigated. In conclusion, this method provided comprehensive profiling of 20 steroids in human plasma samples and will be helpful to discover potential biomarkers for the development of gastric cancer and to further understand metabolic syndrome.

Simultaneous Determination of Polyamines and Steroids in Human Serum from Breast Cancer Patients Using Liquid Chromatography-Tandem Mass Spectrometry.

A simultaneous quantitative profiling method for polyamines and steroids using liquid chromatography-tandem mass spectrometry was developed and validated. We applied this method to human serum samples to simultaneously evaluate polyamine and steroid levels. Chemical derivatization was performed using isobutyl chloroformate to increase the sensitivity of polyamines. The method was validated, and the matrix effects were in the range of 78.7-126.3% and recoveries were in the range of 87.8-123.6%. Moreover, the intra-day accuracy and precision were in the ranges of 86.5-116.2% and 0.6-21.8%, respectively, whereas the inter-day accuracy and precision were in the ranges of 82.0-119.3% and 0.3-20.2%, respectively. The linearity was greater than 0.99. The validated method was used to investigate the differences in polyamine and steroid levels between treated breast cancer patients and normal controls. In our results, N-acetyl putrescine, N-acetyl spermidine, cadaverine, 1,3-diaminopropane, and epitestosterone were significantly higher in the breast cancer patient group. Through receiver operating characteristic curve analysis, all metabolites that were significantly increased in patient groups with areas under the curve >0.8 were shown. This mass spectrometry-based quantitative profiling method, used for the investigation of breast cancer, is also applicable to androgen-dependent diseases and polyamine-related diseases.

Serial Hydrolysis for the Simultaneous Analysis of Catecholamines and Steroids in the Urine of Patients with Alopecia Areata.

Catecholamines and steroids are well-known neurotransmitters and hormones that rapidly change the excitability of neurons. Alopecia areata is a disease for which the exact cause is unknown, but it is considered to be associated with stress, and so the simultaneous analysis of catecholamines and steroids is required for the diagnosis of alopecia areata. Thus, we herein report the simultaneous analysis of catecholamines and steroids bearing different functional groups for the first time, during which it was necessary to carry out a serial hydrolysis procedure. Following hydrolysis of the urine samples to produce the free forms from the urinary conjugates, ethyl acetate extractions were carried out, and chemical derivatization was performed using dansyl chloride to increase the sensitivity of the liquid chromatography-tandem mass spectrometry method. The matrix effects and recoveries of this analytical method were validated, giving values of 85.4-122.9% and 88.8-123.0%, respectively. In addition, the method accuracy and precision were assessed, giving values of 0.4-21.5% and 2.0-21.6% for the intra-day and inter-day precisions, respectively. This validated method was then applied to identify differences between patients with and without alopecia areata, wherein the metanephrine content was found to be significantly higher in the alopecia areata patient group. This quantitative profiling method can also be applied to steroid-dependent diseases, as well as catecholamine-related diseases.

Sex-related differences in urinary immune-related metabolic profiling of alopecia areata patients.

INTRODUCTION: Alopecia areata is a well-known autoimmune disease affecting humans. Polyamines are closely associated with proliferation and inflammation, and steroid hormones are involved in immune responses. Additionally, bile acids play roles in immune homeostasis by activating various signaling pathways; however, the roles of these substances and their metabolites in alopecia areata remain unclear. OBJECTIVES: In this study, we aimed to identify differences in metabolite levels in urine samples from patients with alopecia areata and healthy controls. METHODS: To assess polyamine, androgen, and bile acid concentrations, we performed high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Our results showed that spermine and dehydroepiandrosterone levels differed significantly between male patients and controls, whereas ursodeoxycholic acid levels were significantly higher in female patients with alopecia areata than in controls. CONCLUSION: Our findings suggested different urinary polyamine, androgen, and bile acid concentrations between alopecia areata patients and normal controls. Additionally, levels of endogenous substances varied according to sex, and this should be considered when developing appropriate treatments and diagnostic techniques. Our findings improve our understanding of polyamine, androgen, and bile acid profiles in patients with alopecia areata and highlight the need to consider sex-related differences.

Distribution of polyamines may be altered in different scalp regions of patients with hair loss.

Hair loss, from the vertex or front of the head, generally occurs due to increased androgenic steroid levels. Androgenic steroids, particularly testosterone and dihydrotestosterone, are distributed differently across the vertex and occipital regions and are involved in inducing ornithine decarboxylase expression. Therefore, we hypothesized that the distribution of polyamines may be altered in different scalp regions. For the overall metabolic profiling of polyamines in patients with hair loss, a liquid chromatography-mass spectrometry was used. We investigated the differential polyamine levels in different regions of the hair of patients with male pattern baldness and those with female pattern hair loss. The levels of most polyamines were higher in the vertex region than in the occipital region, and N-acetyl polyamine levels differed significantly. We proposed to test our hypothesis by profiling polyamines in human hair fibre to evaluate the distribution of metabolites in various regions of the scalp.

Urinary profiling of tryptophan and its related metabolites in patients with metabolic syndrome by liquid chromatography-electrospray ionization/mass spectrometry.

Tryptophan (Trp) is an essential amino acid that plays an important role in protein synthesis and is a precursor of various substances related to diverse biological functions. An imbalance in Trp metabolites is associated with inflammatory diseases. The accurate and precise measurement of these compounds in biological specimens would provide meaningful information for understanding the biochemical states of various metabolic syndrome-related diseases, such as hyperlipidemia, hypertension, diabetes, and obesity. In this study, we developed a rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry-based method for the simultaneous targeted analysis of Trp and its related metabolites of the kynurenine (Kyn), serotonin, and tryptamine pathways in urine. The application of the developed method was tested using urine samples after protein precipitation. The detection limits of Trp and its metabolites were in the range of 0.01 to 0.1 µg/mL. The method was successfully validated and applied to urine samples from controls and patients with metabolic syndrome. Our results revealed high concentrations of Kyn, kynurenic acid, xanthurenic acid, and quinolinic acid as well as a high Kyn-to-Trp ratio (KTR) in patients with metabolic syndromes. The levels of urine Kyn and KTR were significantly increased in patients under 60 years old. The profiling of urinary Trp metabolites could be a useful indicator for age-related diseases including metabolic syndrome. ᅟ.

Simultaneous Analysis of Free and Sulfated Steroids by Liquid Chromatography/Mass Spectrometry with Selective Mass Spectrometric Scan Modes and Polarity Switching.

Sulfated steroids can act as a latent form of active free steroids, coexisting with them in biological specimens. To evaluate the metabolic significance of free and sulfated steroid species, a simultaneous analysis of eight free steroids [cholesterol, pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone (DHEA), testosterone, and 17ß-estradiol] and four biologically relevant sulfated steroids was developed and validated, using selected-ion and multiple-reaction monitoring modes coupled to polarity-switching liquid chromatography/mass spectrometry (LC/MS). All steroids were separated on a reversed-phase phenyl column (50 mm × 2 mm, 3 µm) at a flow rate of 0.5 mL/min. The limits of quantification ranged from 0.1 to 50 ng/mL at extraction recoveries of 94.1-105.5%, while the precision and accuracy were 2.5-9.3% and 92.4-105.9%, respectively. Quantitative results obtained for samples from obese girls showed that the serum levels of DHEA sulfate were significantly increased (P = 0.004), along with the metabolic ratio representing DHEA sulfotransferase (P < 0.02). The developed novel LC/MS method can quantitatively profile both free and sulfated steroids in a single analytical run.

Bringing GC-MS profiling of steroids into clinical applications.

Abnormalities of steroid biosynthesis and excretion are responsible for the development and prevention of endocrine disorders, such as metabolic syndromes, cancers, and neurodegenerative diseases. Due to their biochemical roles in endocrine system, qualitative and quantitative analysis of steroid hormones in various biological specimens is needed to elucidate their altered expression. Mass spectrometry (MS)-based steroid profiling can reveal the states of metabolites in biological systems and provide comprehensive insights by allowing comparisons between metabolites present in cells, tissues, or organisms. In addition, the activities of many enzymes related to steroid metabolism often lead to hormonal imbalances that have serious consequences, and which are responsible for the progress of hormone-dependent diseases. In contrast to immunoaffinity-based enzyme assays, MS-based methods are more reproducible in quantification. In particular, high-resolution gas chromatographic (GC) separation of steroids with similar chemical structures can be achieved to provide rapid and reproducible results with excellent purification. GC-MS profiling therefore has been widely used for steroid analysis, and offers the basis for techniques that can be applied to large-scale clinical studies. Recent advances in analytical technologies combined with inter-disciplinary strategies, such as physiology and bioinformatics, will help in understanding the biochemical roles of steroid hormones. Therefore, comprehensive analytical protocols in steroid analysis for different research purposes may contribute to the elucidation of complex metabolic processes relevant to steroid function in many endocrine disorders, and in the identification of diagnostic biomarkers.

High-throughput and rapid quantification of lipids by nanoflow UPLC-ESI-MS/MS: application to the hepatic lipids of rabbits with nonalcoholic fatty liver disease.

A rapid and high-throughput quantification method (approximately 300 lipids within 20 min) was established using nanoflow ultrahigh-pressure liquid chromatography-tandem mass spectrometry (nUPLC-ESI-MS/MS) with selective reaction monitoring (SRM) and applied to the quantitative profiling of the hepatic lipids of rabbits with different metabolic conditions that stimulate the development of nonalcoholic fatty liver disease (NAFLD). Among the metabolic conditions of rabbits in this study [inflammation (I), high-cholesterol diet (HC), and high-cholesterol diet combined with inflammation (HCI)], significant perturbation in hepatic lipidome (>3-fold and p < 0.01) was observed in the HC and HCI groups, while no single lipid showed a significant change in group I. In addition, this study revealed a dramatic increase (>2-fold) in relatively high-abundant monohexosylceramides (MHCs), sphingomyelins (SMs), and triacylglycerols (TGs) in both the HC and HCI groups, especially in MHCs as all 11 MHCs increased by larger than 3- to 12-fold. As the levels of the relatively high-abundant lipids in the above classes increased, the total lipidome level of each class increased significantly by approximately 2-fold to 5-fold. Other classes of lipids also generally increased, which was likely induced by the increase in mitogenic and nonapoptotic MHCs and SMs, as they promote cell proliferation. On the other hand, a slight decrease in the level of apoptotic ceramides (Cers) was observed, which agreed with the general increase in total lipid level. As distinct changes in hepatic lipidome were observed from HC groups, this suggests that HC or HCI is highly associated with NAFLD but not inflammation alone itself. Graphical Abstract Schematic of lipidomic analysis from hepatic tissue using nanoflow LC-ESI-MS/MS and nUPLC-ESI-MS/MS.

High-temperature GC-MS-based serum cholesterol signatures may reveal sex differences in vasospastic angina.

Alterations of cholesterol metabolism are responsible for vasospastic angina and atherosclerosis. To comprehensively evaluate cholesterol metabolism, 18 sterols, including cholesterol, 6 cholesteryl esters (CEs), 3 cholesterol precursors, and 8 hydroxycholesterols (OHCs), were simultaneously analyzed using hybrid solid-phase extraction (SPE) purification coupled to high-temperature gas chromatography-mass spectrometry (HTGC-MS). Methanol-based hybrid SPE increased the selective extraction, and HTGC resulted in a good chromatographic resolution for the separation of lipophilic compounds. The limits of quantification of cholesterol and CEs ranged from 0.2 to 10.0 µg/ml, while OHCs and cholesterol precursors ranged from 0.01 to 0.10 µg/ml. Linearity as the correlation coefficient was higher than 0.99 with the exception of cholesteryl laurate, myristate, oleate, and linoleate (r² > 0.98). The precision (% coefficient of variation) and accuracy (% bias) ranged from 1.1 to 9.8% and from 75.9 to 125.1%, respectively. The overall recoveries of CEs ranged from 26.1 to 64.0%, and the recoveries of other sterols ranged from 83.8 to 129.3%. The cholesterol signatures showed sex differences in patients with vasospastic angina and may associate with 24-reductases. This technique can be useful for making clinical diagnoses and for an increased understanding of the pathophysiology of vasospastic angina.

GC-MS-based quantitative signatures of cytochrome P450-mediated steroid oxidation induced by rifampicin.

BACKGROUND: Drug-induced cytochrome P450 (CYP) activity affects endocrine function and drug clearance rates, leading to the development of unpredictable pathologic and toxicologic risks. METHODS: Urinary steroid profiling based on gas chromatography-mass spectrometry (GC-MS) was used for simultaneous quantification of CYP-mediated regioselective hydroxysteroids and their substrates, including 26 androgens, 9 estrogens, 5 progestins, and 7 corticoids. The quantitative data were visualized using a hierarchically clustered heat map to allow identification of CYP-mediated steroid signatures. Twelve healthy subjects were orally administered 600 mg of rifampicin a day for 7 days, and their CYP enzyme activity was evaluated. RESULTS: Using GC-MS, all 47 steroids were well separated with good peak shapes. This assay had good linearity (r > 0.994) in a dynamic range, and the interassay imprecision (% CV) and inaccuracy (% bias) were 3.0%-15.6% and 98.0%-109.2%, respectively. Administration of the CYP3A4 inducer rifampicin produced distinct differences in CYP3A4 and CYP11B1, CYP19A1, HSD11B, and HSD17B, which were indicated by their heat map-visualized steroid signatures. CONCLUSIONS: This CYP-mediated steroid signature profile allows simultaneous assessment of CYP1A, CYP1B, CYP2C, CYP3A, CYP11B, CYP17A, CYP19A, and CYP21A in urine samples. This method could therefore be a useful tool for assessing drug efficacy.

Comparison of metabolic ratios of urinary estrogens between benign and malignant thyroid tumors in postmenopausal women.

BACKGROUND: Estrogen metabolism may be associated with the pathophysiological development of papillary thyroid carcinoma (PTC). METHODS: To evaluate the differential estrogen metabolism between benign and malignant PTCs, estrogen profiling by gas chromatography-mass spectrometry was applied to urine samples from postmenopausal patients with 9 benign tumors and 18 malignant stage I and III/IV PTCs. RESULTS: The urinary concentration of 2-methoxyestradiol was significantly lower in the stage I malignant patients (3.5-fold; P < 0.025) than in the benign group. The metabolic ratios of 16α-OH-estrone/estrone and estriol/estradiol, which are responsible for 16α-hydroxylase activity, were increased more than 2.5-fold in the advanced-stage malignant PTC (P < 0.02 each). The more than 6.2-fold decrease in the urinary 2-/16α-hydroxylase ratio in stage III/IV malignant PTC was consistent with the ratio in postmenopausal patients with endocrine gland cancers. In addition, reductive 17ß-hydroxysteroid dehydrogenase (17ß-HSD; estradiol/estrone or estriol/16α-OH-estrone) was present at significantly higher levels in subjects with stage III/IV malignant PTCs than in benign subjects (>3.5-fold difference; P < 0.002). In particular, the estriol/16α-OH-estrone ratio differentiated between the benign and early-stage malignant patients (P < 0.01). CONCLUSIONS: Increased 16α-hydroxylation and/or a decreased 2-/16α-ratio, as well increased reductive 17ß-HSD, with regard to estrogen metabolism could provide potential biomarkers. The devised profiles could be useful for differentiating malignant thyroid carcinomas from benign adenomas in postmenopausal women.

Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics.

Liver toxicity represents an important healthcare issue because it causes significant morbidity and mortality and can be difficult to predict before symptoms appear owing to drug therapy or exposure to toxicants. Using metabolomic techniques, we discovered common biomarkers for the prediction of hepatotoxicity in rat urine using mass spectrometry. For this purpose, liver toxicity was induced by 5 days of oral administration of carbon tetrachloride (1 ml kg(-1) per day), acetaminophen (1000 mg kg(-1) per day) and methotrexate (50 mg kg(-1) per day). Serum levels of alkaline phosphatase aspartate aminotransferase, alanine aminotransferase and histopathology in liver tissue were then checked to demonstrate liver toxicity. Global metabolic profiling with UPLC-TOF-MS (ultraperformance liquid chromatography-mass spectrometry), multivariate analysis (partial least square-discriminant analysis, hierarchical analysis) and database searching were performed to discover common biomarkers for liver toxicity induced by these three compounds. Urinary concentrations of the newly discovered biomarkers were then quantified to confirm them as biomarkers of hepatotoxicity with targeted metabolic profiling using GC (gas chromatography)-MS and CE (capillary electrophoresis)-MS. In the results, steroids, amino acids and bile acids were metabolically changed between the control and drug-treated groups in global metabolic profiling; 11ß-hydroxyandrosterone, epiandrosterone, estrone, 11-dehydrocorticosterone, glycine, alanine, valine, leucine, dl-ornithine, 3-methylhistidine, cholic acid and lithocholic acid were selected as liver toxicity biomarkers after performing targeted metabolic profiling. In conclusion, we discovered metabolite biomarkers belonging to three different metabolic pathways to check for liver toxicity with mass spectrometry from a metabolomics study that could be used to evaluate hepatotoxicity induced by drugs or other toxic compounds.

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis.

Estrogen metabolites play important roles in the development of female-related disorders and homeostasis of the bone. To improve detectability, a validated gas chromatography-mass spectrometry (GC-MS) method was conducted with two-phase extractive ethoxycarbonlyation (EOC) and subsequent pentafluoropropionyl (PFP) derivatization was introduced. The resulting samples were separated through a high-temperature MXT-1 column within an 8 min run and were detected in the selected ion monitoring (SIM) mode. The optimized analytical conditions led to good separation with a symmetric peak shape for 19 estrogens as their EOC-PFP derivatives. The limit of quantification (LOQ) was from 0.02 to ∼0.1 ng/ml for most estrogens analyzed, except for 2-hydroxyestriol (0.5 ng/ml). The devised method was found to be linear (r² > 0.995) in the range from the LOQ to 40 ng/ml, whereas the precision (% CV) and accuracy (% bias) ranged from 1.4 to 10.5% and from 91.4 to 108.5%, respectively. The good sensitivity and selectivity of this method even allowed quantification of the estrogen metabolites in urine samples obtained from the postmenopausal female patients with osteoporosis. The present technique can be useful for clinical diagnosis as well as to better understand the pathogenesis of estrogen-related disorders in low-level quantification.

Metabolic alteration of urinary steroids in pre- and post-menopausal women, and men with papillary thyroid carcinoma.

BACKGROUND: To evaluate the metabolic changes in urinary steroids in pre- and post-menopausal women and men with papillary thyroid carcinoma (PTC). METHODS: Quantitative steroid profiling combined with gas chromatography-mass spectrometry was used to measure the urinary concentrations of 84 steroids in both pre- (n = 21, age: 36.95 ± 7.19 yr) and post-menopausal female (n = 19, age: 52.79 ± 7.66 yr), and male (n = 16, age: 41.88 ± 8.48 yr) patients with PTC. After comparing the quantitative data of the patients with their corresponding controls (pre-menopause women: n = 24, age: 33.21 ± 10.48 yr, post-menopause women: n = 16, age: 49.67 ± 8.94 yr, male: n = 20, age: 42.75 ± 4.22 yr), the levels of steroids in the patients were normalized to the mean concentration of the controls to exclude gender and menopausal variations. RESULTS: Many urinary steroids were up-regulated in all PTC patients compared to the controls. Among them, the levels of three active androgens, androstenedione, androstenediol and 16α-hydroxy DHEA, were significantly higher in the pre-menopausal women and men with PTC. The corticoid levels were increased slightly in the PTC men, while progestins were not altered in the post-menopausal PTC women. Estrogens were up-regulated in all PTC patients but 2-hydroxyestrone and 2-hydroxy-17ß-estradiol were remarkably changed in both pre-menopausal women and men with PTC. For both menopausal and gender differences, the 2-hydroxylation, 4-hydroxylation, 2-methoxylation, and 4-methoxylation of estrogens and 16α-hydroxylation of DHEA were differentiated between pre- and post-menopausal PTC women (P < 0.001). In particular, the metabolic ratio of 2-hydroxyestrone to 2-hydroxy-17ß-estradiol, which could reveal the enzyme activity of 17ß-hydroxysteroid dehydrogenase, showed gender differences in PTC patients (P < 1 × 10-7). CONCLUSIONS: These results are expected be helpful for better understanding the pathogenic differences in PTC according to gender and menopausal conditions.

Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats.

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC-MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.

Gas chromatography/mass spectrometry based hair steroid profiling may reveal pathogenesis in hair follicles of the scalp.

A method of steroid profiling, including androgens, progestins, corticoids and sterols, was developed to evaluate the concentrations of steroids as well as the activities of the enzymes responsible for steroidogenesis in hair by gas chromatography/mass spectrometry. The extraction efficiencies of steroids from the hair matrix were improved by ultrasonication for 1 h at 50 °C. The overall recoveries ranged from 71 to 132%, with a limit of quantification for all analytes ranging from 1 to 50 ng/g. The devised method was used to identify the metabolic changes for both male-pattern baldness (MPB) and the drug efficiency of dutasteride, which inhibits 5α-reductase. Increased dihydrotestosterone levels and the dihydrotestosterone/testosterone (DHT/T) ratio, which is responsible for the 5α-reductase activity, were observed in the MPB patients. A dutasteride treatment resulted in decreases in the DHT and 5α-androstanedione concentrations and DHT/T ratio in the hair samples. Hair steroid profiling reflects the sebaceous status in the scalp and may be useful for monitoring the metabolic responses to both the disease and drug actions.

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer.

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.

Cellular uptake of ginsenosides in Korean white ginseng and red ginseng and their apoptotic activities in human breast cancer cells.

Panax ginseng has been reported to have cancer-preventive properties and, through anti-inflammatory, antioxidant, and pro-apoptotic mechanisms, to influence gene expression. However, the comparison of Korean white ginseng (WG) and red ginseng (RG) in their apoptotic effects and the identification of the selective cellular uptake of the ginsenosides in human breast cancer cells have not yet been fully understood. In the present study, the relative nonpolar and protopanaxadiol (PPD) class ginsenosides exhibited more cytotoxic and efficient cellular uptake on MCF-7 cells compared with the relative polar and protopanaxatriol (PPT) class compounds. PPD class ginsenosides were present in RG in a 2.5 times higher concentration as compared to WG, while PPT class ginsenosides were only present in WG. Thus, RG exerted more potent cytotoxicity than WG against MCF-7 and MDA-MB231 cells. RG also increased the sub-G1 DNA contents of the cell cycle and Annexin V-positive apoptotic bodies undergoing apoptosis through the caspase-3 activation in MCF-7 cells. In addition, RG downregulated the proliferative and anti-apoptotic gene products and potentiated paclitaxel-induced apoptosis in MCF-7 cells. Overall, RG contained a higher concentration of PPD class ginsenosides as compared to WG; the greater cellular uptake of PPD resulted in more substantial antiproliferative activity in human breast cancer cells.

Alterations in Pattern Baldness According to Sex: Hair Metabolomics Approach.

Pattern baldness has been associated with the male hormone, dihydrotestosterone. In this study, we tried to determine how the overall metabolic pathways of pattern baldness differ in patients and in normal controls. Our study aimed to identify alterations in hair metabolomic profiles in order to identify possible markers of pattern baldness according to sex. Untargeted metabolomics profiling in pattern baldness patients and control subjects was conducted using ultra-performance liquid chromatography-mass spectrometry. To identify significantly altered metabolic pathways, partial least squares discriminant analysis was performed. Our analysis indicated differences in steroid biosynthesis pathway in both males and females. However, there was a remarkable difference in the androgen metabolic pathway in males, and the estrogen metabolic and arachidonic acid pathways in females. For the first time, we were able to confirm the metabolic pathway in pattern baldness patients using hair samples. Our finding improves understanding of pattern baldness and highlights the need to link pattern baldness and sex-related differences.