Chan Seo

Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been studied for their therapeutic potential. However, evaluating the quality of hBM-MSCs before transplantation remains a challenge. We addressed this issue in the present study by investigating deformation, the expression of genes related to reactive oxygen species (ROS) generation, changes in amino acid profiles, and membrane fluidity in hBM-MSCs. Deformability and cell size were decreased after storage for 6 and 12 h, respectively, in phosphate-buffered saline. Intracellular ROS levels also increased over time, which was associated with altered expression of genes related to ROS generation and amino acid metabolism. Membrane fluidity measurements revealed higher Laurdan generalized polarization values at 6 and 12 h; however, this effect was reversed by N-acetyl-l-cysteine-treatment. These findings indicate that the quality and freshness of hBM-MSCs is lost over time after dissociation from the culture dish for transplantation, highlighting the importance of using freshly trypsinized cells in clinical applications.

Metabolomic study of aging in mouse plasma by gas chromatography-mass spectrometry.

Metabolomic analysis of aging was performed in plasma samples of young (8 weeks) and old (72 weeks) mice as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry (GC-MS). As new approaches, study of altered metabolism from aging was attempted by simultaneous profiling analysis of amino acids (AAs), organic acids (OAs) and fatty acids (FAs) by GC-MS in a single run combined with pattern analysis. As a result, 27 amino acids (AAs), 17 organic acids (OAs) and 24 fatty acids (FAs) were positively screened with large variations in plasma samples. Among altered metabolites, levels of six AAs (proline, methionine, 4-hydroxyproline, pipecolic acid, glutamic acid, α-aminoadipic acid) as neurotransmetters and nutrients, five OAs (2-hydroxybutyric acid, 2-hydroxyglutaric acid, cis-aconitic acid citric acid, isocitric acid) including intermediate metabolites in the TCA cycle, and three n-3 polyunsaturated FAs (PUFAs) of α-octadecatrienoic acid, eicosapentaenoic acid and docosahexaenoic acid as potential biomarkers were significantly different between young and old groups. Their levels were normalized to the corresponding mean values of the young group and then plotted into star symbol patterns, which were clearly distinct compared with numerical data and readily distinguishable for young and old groups. Thus, the present metabolomic screening and the star pattern recognition method might be useful for understanding the complexity of biochemical events in aging.

Simultaneous analysis of seven 2-hydroxy fatty acids as tert-butyldimethylsilyl derivatives in plasma by gas chromatography–mass spectrometry

An efficient method for the simultaneous analysis of seven 2-hydroxy fatty acids (2-HFAs) as tert-butyldimethylsilyl (TBDMS) derivative was developed by gas chromatography-mass spectrometry in selected ion monitoring mode. New mass spectral data on 2-hydroxycapric, 2-hydroxypalmitic, 2-hydroxystearic and 2-hydroxybehenic acids as di-TBDMS derivatives for hydroxyl and carboxyl groups were built. Under the optimal conditions, the present method showed a good correlation coefficient (r ≥ 0.999) in the range of 0.01-0.5 µg. The precision showed low relative standard deviation of <10%, and the accuracy (percentage relative error) varied from -5.2 to 0.3 for the seven 2-HFAs studied. Recovery rates of all 2-HFAs were  ≥ 93.2% with good precision. When applied to normal human plasma, seven 2-HFAs were positively identified. Therefore, the present efficient method will be useful for simultaneous analysis of 2-HFAs in plasma.

Monitoring of altered amino acid metabolic pattern in rat urine following intraperitoneal injection with γ-hydroxybutyric acid

Introductionγ-Hydroxybutyric acid is a well-known prescription medicine that is used for the clinical treatment of alcohol dependence and narcolepsy. However, the biochemical mechanism underlying γ-hydroxybutyric acid intoxication remains unclear, and metabolomic amino acid profiling and pattern analyses have not been attempted following treatment with γ-hydroxybutyric acid.ObjectivesWe carried out urinary amino acid profiling and pattern analyses in rats to determine the biochemical events associated with altered amino acid metabolism and biomarker detection of intoxication following treatment with γ-hydroxybutyric acid.MethodsMetabolic profiling analysis of amino acids in rat urine samples was performed as ethoxycarbonyl/tert-butyldimethylsilyl derivatives by gas chromatography–mass spectrometry following intraperitoneal administration of γ-hydroxybutyric acid once per day for 1 and 10 consecutive days.ResultsA total of 28 amino acids were positively identified in urine samples from the control, single and multiple groups treated with γ-hydroxybutyric acid. Their levels from the single and multiple treated groups were normalized to the corresponding mean control values. The star graphic pattern of the amino acids was characteristic and readily distinguishable for each group owing to its distorted nonacosagonal shape. In the principle component analysis, we monitored phenylalanine, glutamic acid, aspartic acid, asparagine, and methionine as contributing factors that discriminated the three groups.ConclusionThe present metabolomic study may explain the altered metabolism of amino acids following administration, and intoxication with γ-hydroxybutyric acid.

Metabolic Profiling of Aliphatic, hydroxy, and Methyl-Branched Fatty Acids in Human Plasma by Gas Chromatography–Mass Spectrometry

ABSTRACT An efficient method for the simultaneous profiling analysis of 44 fatty acids including 24 aliphatic, 12 hydroxyl, and eight methyl-branched fatty acids as tert-butyldimethylsilyl (TBDMS) derivatives was developed by gas chromatography–mass spectrometry. In this study, new mass spectral databases of isolauric, 11-methyllauric, isomyristic, isopentadecylic, isopalmitic, isostearic, 17-methylstearic, and 19-methylarachidic acids were constructed as TBDMS derivatives. Under the optimal conditions, linearity in the range of 0.1–10.0 µg/mL (24 aliphatic fatty acids) and 0.01–1.0 µg/mL (eight methyl-branched and 12 hydroxy fatty acids) showed good correlation coefficients (r ≥ 0.997). The repeatability showed relative standard deviation of less than 11.2% and accuracy (percent relative error) varied from −11.0 to 7.8 for the 44 fatty acids studied. The recoveries ranged from 61.7 to 90.1% with good repeatability. When applied to human plasma sample, 18 aliphatic, three 2-hydroxy, three 3-hydroxy, and five methyl-branched fatty acids were positively identified and quantified. Therefore, the present method will be useful for profiling analysis of various fatty acids in biological samples including plasma.

Metabolomic study for monitoring of biomarkers in mouse plasma with asthma by gas chromatography–mass spectrometry

Asthma is a multifaceted chronic disease caused by an alteration of various genetic and environmental factors that is increasing in incidence worldwide. However, the biochemical mechanisms regarding asthma are not completely understood. Thus, we performed of metabolomic study for understanding of the biochemical events by monitoring of altered metabolism and biomarkers in asthma. In mice plasma, 27 amino acids(AAs), 24 fatty acids(FAs) and 17 organic acids(OAs) were determined by ethoxycarbonyl(EOC)/methoxime(MO)/tert-butyldimethylsilyl(TBDMS) derivatives with GC-MS. Their percentage composition normalized to the corresponding mean levels of control group. They then plotted as star symbol patterns for visual monitoring of altered metabolism, which were characteristic and readily distinguishable in control and asthma groups. The Mann-Whitney test revealed 25 metabolites, including eight AAs, nine FAs and eight OAs, which were significantly different (p<0.05), and orthogonal partial least-squares-discriminant analysis revealed a clear separation of the two groups. In classification analysis, palmitic acid and methionine were the main metabolites for discrimination between asthma and the control followed by pipecolic, lactic, α-ketoglutaric, and linoleic acids for high classification accuracy as potential biomarkers. These explain the metabolic disturbance in asthma for AAs and FAs including intermediate OAs related to the energy metabolism in the TCA cycle.

Metabolomic Study on Fatty Acids in Placenta of Preeclamptic Pregnancies by Gas Chromatography-Mass Spectrometry

Preeclampsia is a serious pregnancy-associated complication and can critically affect the health of the mother and fetus. However, the pathogenesis of preeclampsia remains unclear. One of the characteristics of preeclampsia is abnormal lipid metabolism. Thus, profiling analysis of 23 fatty acids (FAs) as tert-butyldimethylsilyl derivative was performed in the placenta of preeclamptic term pregnancies and uncomplicated term pregnancies by gas chromatography-mass spectrometry. The compositions of saturated, monounsaturated and n-3 polyunsaturated FAs were significantly reduced, whereas those of oleic acid among monounsaturated FAs and arachidonic acid among n-6 polyunsaturated FAs were significantly increased in preeclampsia group compared to the normal group. The distorted star pattern of the preeclamptic pregnancy group was different from the tricosagonal shape of the normal group. Thus, the present FA profiling analysis combined with the star symbol plotting method will be useful for the biochemical monitoring of placental abnormalities and visual discrimination between preeclamptic and normal pregnancies.