A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.

Objective To assess the diagnostic value of saline infusion test ( SIT) and its optimal cutoff in the diagnosis of primary aldosteronism ( PA ), and to analyze whether the dietary salt intake affects the SIT accuracy. Methods This is a prospective study. All 236 patients with a high risk for PA underwent the screening test, SIT and the fludrocortisone suppression test (FST) in separate days. The diagnosis of PA was established according to the FST criteria. According to the 24 h urinary sodium level, the patients were divided into low salt, normal salt, and high salt groups respectively, and the effect of salt intake on SIT was analyzed. Receiver operating characteristic (ROC) analysis was performed to compare the diagnostic accuracies. Results Finally, in 236 patients with high risk for PA, 134 patients with PA and 102 patients with essential hypertension ( EH) were diagnosed. Using post-test plasma aldosterone concentration (PAC) for diagnosis, the area under the ROC curve (AUCROC) of the SIT was 0.974 (0.957, 0.991), which was significantly higher than that of the post-test plasma aldosterone to renin ratio (ARR) [0.900 ( 0. 862, 0. 938)] and that of the PAC suppression percentage [ 0. 752 ( 0. 690, 0. 813)] ( both P<0.01). Considering both sensitivity and specificity, an optimal cutoff of PAC post-SIT was set at 8 ng/dl, resulting in a sensitivity of 88. 1% and a specificity of 95. 1%. The PAC post-SIT, whether in PA or EH patients, had no statistically significant differences among low salt, normal salt, and high salt groups (P>0.05). Conclusion SIT is reliable for the diagnosis of PA. PAC post-SIT more than 8.0 ng/dl is recommended to confirm PA.

Objective : Based on metabolomics and transcriptomics analysis , to explore the molecular mechanism of spleen inflammation induced by high altitude hypoxia in mice through NOD⁃like receptor signaling pathway . Methods : C57BL/6 mice were raised at an altitude of 400 m and 4 200 m respectively , with five mice in each group , and spleen tissues were collected after 30 days . Differential metabolites and differentially expressed genes in key pathways were screened by metabolomics and transcriptome analysis and correlation KEGG enrichment analysis , and the related network interaction diagram of differential metabolites and differentially expressed genes in key pathways was constructed and verified by RT⁃qPCR . Results : Metabolomics analysis showed that 133 differential metabolites were screened from in the plain spleen control group (PSC group) and the plateau spleen test group (HST group) , 95 of which were up⁃regulated while 38 of which were down⁃regulated . KEGG enrichment analysis showed that they were mainly involved in NOD⁃like receptor signaling pathway , HIF⁃1 signaling pathway , cholesterol metabolism and other metabolic pathways . The results of transcriptome analysis showed that a total of 4213 differentially expressed genes were identified in PSC group and HST group , including 1947 up⁃regulated genes and 2266 down⁃regulated genes . KEGG was enriched in 173 signaling pathways , including NOD⁃like receptor signaling pathway , MAPK signaling pathway , NF⁃κB signaling pathway and other pathways . Comprehensive analysis showed that the differential metabolites and differentially expressed genes were obviously enriched in NOD⁃like receptor signaling pathway . Therefore , the correlation network interaction map was constructed for the differential metabolites ATP and differentially expressed genes in NOD⁃like receptor signaling pathway . RT⁃qPCR results showed that compared with PSC group , the expression levels of DEGs related to NOD1 and NOD2 (CHUK , TAB3 , MAPK8) in the signaling pathway of NOD⁃like receptor and NLRP1 ⁃CASP1 pathway (NLRP1b , CASP1) in HST group were significantly enhanced . The mRNA expression levels of downstream inflammatory factors IL⁃6 , IL⁃1 β , IL⁃18 , INF⁃γ and TNF⁃α were up⁃regulated and differentially expressed . Conclusion Based on the combined analysis of metabolomics and transcriptomics , it was found that hypoxia stimulation at high altitude may affect the NOD⁃like receptor signaling pathway in vivo , and the differential metabolite ATP is positively correlated with the differential key genes in the pathway . ATP mediates the release of downstream inflammatory factors by activating NOD1 , NOD2 pathways and NLRP1 inflammable⁃CASP1 pathways . Inflammatory response occurred in spleen tissue of mice.

ObjectiveTo explore the regulatory mechanism of hypoxia exposure on carbon metabolism pathway in spleen of mice. MethodsC57BL/6 mice were raised at altitudes of 400 m and 4 200 m， with 5 mice in each group. After 30 days， spleen tissues were aseptically removed for analysis of differentially expressed genes， proteins， and metabolites using transcriptome sequencing， proteomics， and non-targeted metabolomics. GO and KEGG enrichment analysis were conducted to explore key pathways. The key genes and protein in the pathway were validated by RT-qPCR and Western blot. ResultsTranscriptome sequencing revealed a significant difference in the expression of 4 213 genes in hypoxic exposure， of which 1 947 were up-regulated and 2 266 were down-regulated. The analysis of differentially expressed proteins showed that 166 proteins were up-regulated and 39 proteins were down-regulated. The results of non-targeted metabolomics showed that 133 different metabolites were screened under high altitude hypoxia condition， of which 95 were up-regulated and 38 were down-regulated. KEGG enrichment analysis showed that differentially expressed genes， differentially expressed proteins and differentially expressed metabolites were enriched into the carbon metabolic pathway. Therefore， the key genes and proteins in the carbon metabolic pathway were verified. The mRNA and protein expressions of PGAM2、ENO3、PRPS2、PGLS、RPE、IDH3A、SUCLA2 and MDH2 were significantly down-regulated in the carbon metabolism pathway. ConclusionLow oxygen environment at high altitude weakens glycolysis， tricarboxylic acid cycle and pentose phosphate pathway by inhibiting the carbon metabolism pathway of the body， resulting in oxidative stress and energy metabolism imbalance.