Metabolic alterations of short-chain fatty acids and TCA cycle intermediates in human plasma from patients with gastric cancer.

AIMS: Short-chain fatty acids (SCFAs) are produced by gut microbiota from dietary fiber. Since absorbed SCFAs could be introduced into the tricarboxylic acid (TCA) cycle in host cells, the relationships between SCFAs and TCA cycle intermediates might influence to energy metabolism in the human body. For this reason, information on profile changes between SCFAs and TCA cycle intermediates could help unveil pathological mechanisms of gastric cancer. MAIN METHODS: A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to simultaneously determine SCFAs and TCA cycle intermediates in human plasma from patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. We applied a tetra-alkyl ammonium pairing method to prevent loss of volatile SCFAs and base decarboxylation of TCA cycle intermediates during sample preparation. To assess gastric diseases, metabolic alterations of SCFAs and TCA cycle intermediates in human plasma with gastric disorders were analyzed by their plasma levels. KEY FINDINGS: Significantly different metabolic alterations based on the plasma levels of SCFAs and TCA cycle intermediates were investigated in cancer metabolic pathways. Not only propionate and butyrate, mainly produced by gut microbiota, were significantly decreased, but also cis-aconitate, α-ketoglutarate, and fumarate were significantly increased in plasma with IM or gastric cancer, compared to CSG. Further, based on ratios of product to precursor, three metabolic pathways (succinate/propionate, succinate/α-ketoglutarate, and cis-aconitate/citrate) were supposed to be distorted between gastric diseases. SIGNIFICANCE: In conclusion, propionate, cis-aconitate, α-ketoglutarate, and fumarate could be used to assess the progression of gastric cancer.

Efficient Matrix Cleanup of Soft-Gel-Type Dietary Supplements for Rapid Screening of 92 Illegal Adulterants Using EMR-Lipid dSPE and UHPLC-Q/TOF-MS.

An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UHPLC-Q/TOF-MS). As representative green chemistry methods, three sample preparation methods (dispersive liquid-liquid microextraction (DLLME), "quick, easy, cheap, effective, rugged, and safe" dispersive solid-phase extraction (QuEChERS-dSPE), and enhanced matrix removal-lipid (EMR-Lipid) dSPE) were evaluated for matrix removal efficiency, recovery rate, and matrix effect. In this study, EMR-Lipid dSPE was shown to effectively remove complicated matrix contents in soft-gels, compared to DLLME and QuEChERS-dSPE. For the rapid screening of a wide range of adulterants, extracted common ion chromatogram (ECIC) and neutral loss scan (NLS) based on specific common MS/MS fragments were applied to randomly collected soft-gel-type dietary supplement samples using UHPLC-Q/TOF-MS. Both ECICs and NLSs enabled rapid and simple screening of multi-class adulterants and could be an alternative to the multiple reaction monitoring (MRM) method. The developed method was validated in terms of limit of detection (LOD), precision, accuracy, recovery, and matrix effects. The range of LODs was 0.1-16 ng/g. The overall precision values were within 0.09-14.65%. The accuracy ranged from 81.6% to 116.6%. The recoveries and matrix effects of 92 illegal adulterants ranged within 16.9-119.4% and 69.8-114.8%, respectively. The established method was successfully applied to screen and identify 92 illegal adulterants in soft-gels. This method can be a promising tool for the high-throughput screening of various adulterants in dietary supplements and could be used as a more environmentally friendly routine analytical method for screening dietary supplements illegally adulterated with multi-class drug substances.

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.

Histamine receptor 2-mediated growth-differentiation factor-15 expression is involved in histamine-induced melanogenesis.

Vitiligo is a progressive depigmenting disorder. Histamine has been shown to induce melanogenesis via histamine receptor 2, suggesting the possibility of histamine as a repigmenting agent for the treatment of vitiligo. However, the role and signaling mechanism of histamine are still unclear in melanogenesis, especially in relation to growth-differentiation factor-15, which is a protein belonging to transforming growth factor beta and found to be overexpressed in metastatic or malignant melanoma. We found that histamine induces growth-differentiation factor-15 in melanoma cell lines such as SK-MEL-2, B16F10, and melan-a cells. Therefore, in the present study, the role of growth-differentiation factor-15 in histamine-induced melanogenesis was investigated using gene silencing or overexpression of growth-differentiation factor-15 and histamine related compounds such as histamine, amthamine, and cimetidine. Gene silencing of growth-differentiation factor-15 suppressed histamine-induced proliferation, melanin production, tyrosinase activity, and chemotactic migration of SK-MEL-2 cells. Histamine-induced expression of tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 was also suppressed by growth-differentiation factor-15 gene silencing. On the other hand, overexpression of growth-differentiation factor-15 using a plasmid containing growth-differentiation factor-15 in SK-MEL-2 cells increased melanin production and chemotactic migration. Amthamine induced expression of growth-differentiation factor-15 in a time and concentration dependent manner. Amthamine-induced expression of growth-differentiation factor-15 was suppressed by cimetidine. Our results suggest that growth-differentiation factor-15 is a new player in histamine-induced melanogenesis, which can help researchers to extend the knowledge of the role of the transforming growth factor beta family in melanogenesis and in skin pigment disorders such as vitiligo.

Suppression of Transglutaminase-2 is Involved in Anti-Inflammatory Actions of Glucosamine in 12-O-Tetradecanoylphorbol-13-Acetate-Induced Skin Inflammation.

Glucosamine (GS) is well known for the treatment of inflam-mation. However, the mechanism and efficacy of GS for skin inflammation are unclear. The aim of this study was to evaluate the effects and mechanism of GS in the mouse 12-O-tetradecanoyl 13-acetate (TPA)-induced ear edema model. TPA-induced ear edema was evoked in ICR or transglutaminase 2 (Tgase-2) (-/-) mice. GS was administered orally (10-100 mg/kg) or topically (0.5-2.0 w/v %) prior to TPA treatment. Orally administered GS at 10 mg/kg showed a 76 or 57% reduction in ear weight or myeloperoxidase, respectively, and a decreased expression of cyclooxy-genase-2 (COX-2), NF-κB and Tgase-2 in TPA-induced ear edema by western blot and immunohistochemistry. Role of Tgase-2 in TPA ear edema is examined using Tgase-2 (-/-) mice and TPA did not induce COX-2 expression in ear of Tgase-2 (-/-) mice. These observations suggested that Tgase-2 is involved in TPA-induced COX-2 expression in the inflamed ear of mice and anti-inflammatory effects of glucosamine is mediated through suppression of Tgase-2 in TPA ear edema.