Triple-negative breast cancer (TN) is more aggressive than other subtypes of breast cancer and has a lower survival rate. Furthermore, detailed biological information about the disease is lacking. This study investigated characteristics of metabolic pathways in TN. We performed the metabolome analysis of 74 breast cancer tissues and the corresponding normal breast tissues using LC/MS. Furthermore, we classified the breast cancer tissues into ER-positive, PgR-positive, HER2-negative breast cancer (EP+H-) and TN, and then the differences in their metabolic pathways were investigated. The RT-PCR and immunostaining were carried out to examine the expression of ELOVL1, 2, 3, 4, 5, 6, and 7. We identified 142 of hydrophilic metabolites and 278 of hydrophobic lipid metabolites in breast tissues. We found the differences between breast cancer and normal breast tissues in choline metabolism, glutamine metabolism, lipid metabolism, and so on. Most characteristic of comparison between EP+H- and TN were differences in fatty acid metabolism was which were related to the elongation of very long chain fatty acids were detected between TN and EP+H-. Real-time RT-PCR showed that the mRNA expression levels of ELOVL1, 5, and 6 were significantly upregulated by 8.5-, 4.6- and 7.0-fold, respectively, in the TN tumors compared with their levels in the corresponding normal breast tissue samples. Similarly, the mRNA expression levels of ELOVL1, 5, and 6 were also significantly higher in the EP+H- tissues than in the corresponding normal breast tissues (by 4.9-, 3.4-, and 2.1-fold, respectively). The mRNA expression level of ELOVL6 was 2.6-fold higher in the TN tumors than in the EP+H- tumors. During immunostaining, the TN and EP+H- tumors demonstrated stronger ELOVL1 and 6 staining than the corresponding normal breast tissues, but ELOVL5 was not stained strongly in the TN or EP+H- tumors. Furthermore, the TN tumors exhibited stronger ELOVL1 and 6 staining than the EP+H- tumors. Marked differences in fatty acid metabolism pathways, including those related to ELOVL1 and 6, were detected between TN and EP+H-, and it was suggested that ELOVL1 and 6-related fatty acid metabolism pathways may be targets for therapies against TN.

Recently, the use of metabolomic analysis of human serum and plasma for biomarker discovery and disease diagnosis in clinical studies has been increasing. The feasibility of using a metabolite biomarker for disease diagnosis is strongly dependent on the metabolite's stability during pre-analytical blood processing procedures, such as serum or plasma sampling and sample storage prior to centrifugation. However, the influence of blood processing procedures on the stability of metabolites has not been fully characterized. In the present study, we compared the levels of metabolites in matched human serum and plasma samples using gas chromatography coupled with mass spectrometry and liquid chromatography coupled with mass spectrometry. In addition, we evaluated the changes in plasma metabolite levels induced by storage at room temperature or at a cold temperature prior to centrifugation. As a result, it was found that 76 metabolites exhibited significant differences between their serum and plasma levels. Furthermore, the pre-centrifugation storage conditions significantly affected the plasma levels of 45 metabolites. These results highlight the importance of blood processing procedures during metabolome analysis, which should be considered during biomarker discovery and the subsequent use of biomarkers for disease diagnosis.

Helicobacter pylori (H. pylori), which is a spiral-shaped Gram-negative microaerobic bacterium, is a causative pathogen. The entry of H. pylori into gastric epithelial cells involves various host signal transduction events, and its virulence factors can also cause a variety of biological responses. In this study, AGS human gastric carcinoma cells were infected with CagA-positive H. pylori strain ATCC43504, and then the metabolites in the AGS cells after the 2-, 6- and 12-h infections were analyzed by GC/MS-based metabolomic analysis. Among 67 metabolites detected, 11 metabolites were significantly altered by the H. pylori infection. The metabolite profiles of H. pylori-infected AGS cells were evaluated on the basis of metabolite pathways, and it was found that glycolysis, tricarboxylic acid (TCA) cycle, and amino acid metabolism displayed characteristic changes in the H. pylori-infected AGS cells. At 2 h post-infection, the levels of many metabolites related to TCA cycle and amino acid metabolism were lower in H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. On the contrary, after 6-h and 12-h infections the levels of most of these metabolites were higher in the H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. In addition, it was shown that the H. pylori infection might regulate the pathways related to isocitrate dehydrogenase and asparagine synthetase. These metabolite alterations in gastric epithelial cells might be involved in H. pylori-induced biological responses; thus, our findings are important for understanding H. pylori-related gastric diseases.

Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype. Based on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains. Gene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.