GC/MS analysis of catecholamines (CAs) in biological sample may produce poor reproducible quantitaion when chemical derivatization is used as the technique to form a volatile derivative. Significant quantities of the side products can be formed from CAs with primary amine during the derivatization reaction under un-optimized conditions. We have tested various chemical derivatization techniques in an attempt to find an optimum derivatization method that will reduce side product formation, enable to separate several catecholamine derivatives in GC chromatogram, and obtain significant improvement of detection sensitivity in GC/MS analysis. Whereas several derivatization techniques such as trimethylsilylation (TMS), trifluoroacylation (TFA), and two step derivatization methods were active, selective derivatization to form O-TMS, N-heptafluorobutylacyl (HFBA) derivative using N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) and N-methyl-bis(heptafluorobutyramide) (MBHFBA) reagents was found to be the most effective method. Moreover, this derivative formed by selective derivatization could provide sufficient sensitivity and peak separation as well as produce higher mass ion as base peak to use selected ion in SIM mode. Calibration curves based on the use of an isotopically labeled internal standard show good linearity over the range assayed, 1 ~ 5000 ng/mL, with correlation coefficients of > 0.996. The detection limits of the method ranged from 0.2 to 5.0 ppb for the different CAs studied. The developed method will be applied to the analysis of various CAs in biological sample, combined with appropriate sample pretreatment.
RAGE (Receptor for Advanced Glycation end Products) plays a pivotal role in the atherosclerotic process. The cytoplasmic binding domain of RAGE interacts with mDia‐1, a member of the formin family involved in actin and microtubule reorganization. Here we report that mDia‐1 is required for RAGE effects on vascular stress. Immunostaining of human and mice atherosclerotic tissue showed mDia‐1 expression in the atherosclerotic plaque, particularly in smooth muscle cells (SMCs). We established primary cultures of murine aortic SMCs from wild type (WT) and mDia1 null mice. Recombinant S100B was used as a prototypic RAGE ligand. We found that compared to control cells, SMCs devoid of mDia‐1 (KO or mdia1 siRNA transfected) showed significant and specific inhibition of S100B‐stimulated superoxide production, lamellopodia formation and cell migration. Interestingly, RAGE induced Rac activity and phosphorylation of AKT and GsK3beta were also greatly inhibited in these cells. In WT cells, blockade of RAGE‐induced ROS production with Diphenyiodinium or dominant negative Rac construct, significantly prevented RAGE‐induced AKT/Gsk3beta phosphorylation and SMCs migration. We concluded that in SMCs, mDia‐1 is essential for RAGE‐induced Rac1 activation and ROS production. ROS may then activate PI3Kinase and induce AKT dependent phosphorylation of GSK3beta, leading to cell migration.
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