Identification of genetic variants related to metabolic syndrome by next-generation sequencing.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of conditions associated with glucose intolerance, hypertension, abdominal obesity, dyslipidemia, and insulin resistance that increase the risk of cardiovascular diseases (CVD) and type 2 diabetes (T2D). Since MetS is known as a complex symptom with a high incidence of genetic factors, it is important to identify genetic variants for each clinical characteristic of MetS. METHODS: We performed targeted next-generation sequencing (NGS) to identify genetic variants related to obesity, blood glucose, triacylglycerol (TG), and high-density lipoprotein (HDL)-cholesterol level, and hypertension in 48 subjects with MetS and in 48 healthy subjects. RESULTS: NGS analysis revealed that 26 of 48 subjects (54.2%) with MetS had putative non-synonymous variants related to the clinical features of MetS. Of the subjects with MetS, 8 (16.7%) had variants in 4 genes (COL6A2, FTO, SPARC, and MTHFR) related to central obesity, 17 (35.4%) had variants in 6 genes (APOB, SLC2A2, LPA, ABCG5, ABCG8, and GCKR) related to hyperglycemia, 3 (6.3%) had variants in 4 genes (APOA1, APOC2, APOA4, and LMF1) related to hypertriglyceridemia, 8 (16.7%) had variants in 4 genes (ABCA1, CETP, SCARB1, and LDLR) related to low HDL-cholesterolemia, and 5 (10.4%) had variants in ADD1 related to hypertension. CONCLUSIONS: Our findings may contribute to broadening the genetic spectrum of risk variants related to the development of MetS.

Genetic Variants Associated with Elevated Plasma Ceramides in Individuals with Metabolic Syndrome.

Metabolic syndrome (MetS) is a complex condition of metabolic disorders and shows a steady onset globally. Ceramides are known as intracellular signaling molecules that influence key metabolism through various pathways such as MetS and insulin resistance. Therefore, it is important to identify novel genetic factors related to increased plasma ceramides in subjects with MetS. Here we first measured plasma ceramides levels in 37 subjects with MetS and in 38 healthy subjects by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Specifically, levels of C16 ceramide (Cer-16), C18 ceramide (Cer-18), C20 ceramide (Cer-20), C18 dihydroceramide (DhCer-18), C24 dihydroceramide (DhCer-24), and C24:1 dihydroceramide (DhCer-24:1) were significantly increased in MetS group (p < 5.0 × 10−2). We then performed single nucleotide polymorphism (SNP) genotyping to identify variants associated with elevated plasma ceramides in MetS group using Axiom® Korea Biobank Array v1.1 chip. We also performed linear regression analysis on genetic variants involved in ceramide synthesis and significantly elevated plasma ceramides and dihydroceramides. Ten variants (rs75397325, rs4246316, rs80165332, rs62106618, rs12358192, rs11006229, rs10826014, rs149162405, rs6109681, and rs3906631) across six genes (ACER1, CERS3, CERS6, SGMS1, SPTLC2, and SPTLC3) functionally involved in ceramide biosynthesis showed significant associations with the elevated levels of at least one of the ceramide species in MetS group at a statistically significant threshold of false discovery rate (FDR)-adjusted p < 5.0 × 10−2. Our findings suggest that the variants may be genetic determinants associated with increased plasma ceramides in individuals with MetS.

A Rapid and Simple Liquid-Chromatography-Tandem Mass Spectrometry Method for Measuring 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Human Serum: Comparison with Two Automated Immunoassays.

BACKGROUND: Total 25-hydroxyvitamin D (25(OH)D) is well-known to be a reliable biomarker of human vitamin D status, with the recognition of widespread vitamin D insufficiency in general populations. The aims of this study are to validate a fast and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying 25(OH)D2 and 25(OH)D3 in serum and to compare two automated immunoassays with the LC-MS/MS method. METHODS: Samples were prepared by protein precipitation with ethanol including 25(OH)D3-d6, followed by a liquid-liquid extraction with hexane. The analytes were separated within a total run time of 3 min. Accuracy was evaluated with standard reference materials (SRM) 972a. Using 150 samples, the LC-MS/MS method was compared with the LIAISON® assay and ADVIA Centaur® assay. RESULTS: The LC-MS/MS method had a limit of quantitation of 1 ng/mL for the 25(OH)D2 and 25(OH)D3 with linear responses between 1 and 100 ng/mL. Intra- and inter-assay precision were <8.8% and <13.2%, respectively. It also showed a smallest mean difference (+0.9%) for the SRM level 1 to 3, compared to the two immunoassays. Compared to the LC-MS/MS, the mean biases of the RIAISON and ADVIA were +2.4 and +7.9 ng/mL, respectively. Also, the agreement of the LC-MS/MS with the RIAISON was better than that with the ADVIA. CONCLUSION: This study suggests that the LC-MS/MS method traceable to the SRM can be reliably applied in routine quantification of 25(OH)D2 and 25(OH)D3.

Simultaneous determination of cortisol and cortisone from human serum by liquid chromatography-tandem mass spectrometry.

A fast, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and then the levels of cortisol and cortisone from sera of healthy adults were determined by the LC-MS/MS method. One hundred µ L of serum sample was directly extracted by adding 2 mL ethyl acetate, followed by chromatographic separation on a C18 column with a mobile phase consisting of 5 mM ammonium acetate and methanol (25 : 75, v/v). The precision, accuracy, and average recovery of the method were 1.5-5.3%, 95.4-102.5%, and 96.4% for cortisol, and 1.9-6.0%, 89.2-98.8%, and 79.9% for cortisone, respectively. The method was linear from 1.0 to 500.0 ng/mL (r(2) = 0.999) for cortisol and 2.5 to 100.0 ng/mL (r(2) = 0.998) for cortisone. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 1.0 ng/mL for cortisol, and 1.0 and 2.5 ng/mL for cortisone, respectively. The average cortisol concentration (133.9 ± 63.7 ng/mL) of samples collected between 9:00 and 11:00 a.m. was higher approximately 4.4 times than that of cortisone (30.5 ± 10.7 ng/mL) (P < 0.0001). The average cortisone/cortisol ratio was 0.225. Therefore, the LC-MS/MS method may be useful for the diagnosis of some adrenal diseases and the assessment of 11 ß -hydroxysteroid dehydrogenase (11 ß -HSD) activity in clinical laboratories.

Simultaneous analysis and monitoring of 16 UV filters in cosmetics by high-performance liquid chromatography.

Sixteen UV filters were simultaneously analyzed using the high-performance liquid chromatographic method. They were drometrizole (USAN Drometrizole), 4-methylbenzylidene camphor (USAN Enzacamene), menthyl anthranilate (USAN Menthyl anthranilate), benzophenone-3 (USAN Oxybenzone), benzophenone-8 (USAN Dioxybenzone), butyl methoxydibenzoylmethane (USAN Avobenzone), ethylhexyl triazone (USAN Octyl triazone), octocrylene (USAN Octocrylene), ethylhexyl dimethyl p-aminobenzoic acid (USAN Padimate O), ethylhexyl methoxycinnamate (USAN Octinoxate), p-aminobenzoic acid (USAN Aminobenzoic acid), 2-phenylbenzimidazole-5-sulfonic acid (USAN Ensulizole), isoamyl p-methoxycinnamate (USAN Amiloxate), and recent UV filters such as diethylhexyl butamidotriazone (USAN Iscotrizinol), methylene bis-benzotriazolyl tetramethylbutylphenol (USAN Bisoctrizole), and terephthalylidene dicamphor sulfonic acid (USAN Ecamsule). Separation of the UV filters was carried out in a C(18) column with a gradient of methanol-phosphate buffer, and the UV detection was at 300, 320, or 360 nm without any interference. The limits of detection were between 0.08 and 1.94 µg/ml, and the limits of quantitation were between 0.24 and 5.89 µg/ml. The extracting solvent for the UV filters was methanol, except for ethylhexyl triazone and methylene bis-benzotriazolyl tetramethylbutylphenol, which were prepared with tetrahydrofuran. The recoveries from spiked samples were between 94.90% and 116.54%, depending on the matrixes used. The developed method was applied to 23 sunscreens obtained from local markets, and the results were acceptable to their own criteria and to maximum authorized concentrations. Consequently, these results would provide a simple extracting method and a simultaneous determination for various UV filters, which can improve the quality control process as well as the environmental monitoring of sunscreens.

Improved detectability in pharmacokinetic study of tibolone by gas chromatography-high resolution mass spectrometry with selected ion monitoring.

A combination of high resolution mass spectrometry (HRMS) and gas chromatography has been used to improve sensitivity and selectivity in pharmacokinetic study of tibolone. The study was undertaken in 12 healthy volunteers after oral administration of 2.5mg tibolone tablet and plasma concentrations of two major metabolites, 3alpha-hydroxytibolone (3alphaOHT) and 3beta-hydroxytibolone (3betaOHT), were analyzed first by gas chromatography-mass spectrometry (GC-MS). Pharmacokinetic parameters AUC(0-12h) (13.70+/-4.01nghml(-1)), C(max) (3.89+/-1.00ngml(-1)), and T(max) (1.83+/-0.55h) for 3alphaOHT were initially measured by GC-MS. The AUC(0-12h) of 3betaOHT concentration was 5.5-folds higher than that of 3alphaOHT. Improved results in detectability were obtained by GC-HRMS analysis of two metabolites with the same samples. The previously undetected metabolites, 3alphaOHT and 3betaOHT, in samples collected at 15 and 24h in GC-MS system were successfully detected by GC-HRMS analysis and could be calculated pharmacokinetic parameters as well. But, no significant pharmacokinetic parameters were found in two analytical runs. The limit of quantification for HRMS analysis in selected ion monitoring mode of 3alphaOHT and 3betaOHT as trimethylsilyl derivatives down to 0.02ngml(-1), while their recovery rates varied in the range of 82.5-108.0%. This method demonstrated a good overall accuracy and precision as 90.1-102.3% and 1.6-11.4%, respectively. This method could potentially have implications for pharmacokinetic study or clinical trial of rapidly metabolized drugs.