Combining First and Second-Tier Newborn Screening in a Single Assay Using High-Throughput Chip-Based Capillary Electrophoresis Coupled to High-Resolution Mass Spectrometry.

BACKGROUND: Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up. METHODS: A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS). RESULTS: The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity. CONCLUSIONS: Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.

Arachidonic acid-derived hydroxyeicosatetraenoic acids are positively associated with colon polyps in adult males: a cross-sectional study.

Oxylipids are potent lipid mediators associated with inflammation-induced colon carcinomas and colon tumor survival. Therefore, oxylipid profiles may be useful as novel biomarkers of colon polyp presence. The aim of this study was to investigate the relationship between plasma non-esterified oxylipids and the presence of colon polyps. A total of 123 Caucasian men, ages 48 to 65, were categorized into three groups: those with no polyps, those with one or more hyperplastic polyps, and those with one or more adenomas. Plasma non-esterified oxylipids were analyzed using solid phase extraction and quantified using a targeted HPLC tandem mass spectrometric analysis. Statistical analyses included Kruskal-Wallis one-way ANOVA with Dunn's test for multiple comparison and generalized linear models to adjust for confounding factors such as age, anthropometrics, and smoking status. In general, monohydroxy omega-6-derived oxylipids were significantly increased in those with polyps. Concentrations of 5-hydroxyeicosatetraenoic acid (HETE) and 11-HETE were significantly higher in those with hyperplastic polyps and adenomas compared to those with no polyps. Arachidonic acid-derived HETEs were significantly associated with colon polyp types, even after adjusting for age, smoking, and body mass index or waist circumference in regression models. Since many of these oxylipids are formed through oxygenation by lipoxygenases (i.e., 5-, 12-, and 15-HETE, and 15- hydroxyeicosatrienoic acid [HETrE]) or auto-oxidative reactions (i.e., 11-HETE), this may indicate that lipoxygenase activity and lipid peroxidation are increased in those with colon polyps. In addition, since oxylipids such as 5-, 12-, and 15-HETE are signaling molecules involved in inflammation regulation, these oxylipids may have important functions in inflammation-associated polyp presence. Future studies should be performed in a larger cohorts to investigate if these oxylipids are useful as potential biomarkers of colon polyps.

The role of the lipidome in obesity-mediated colon cancer risk.

Obesity is a state of chronic inflammation influenced by lipids such as fatty acids and their secondary oxygenated metabolites deemed oxylipids. Many such lipid mediators serve as potent signaling molecules of inflammation, which can further alter lipid metabolism and lead to carcinogenesis. For example, sphingosine-1-phosphate activates cyclooxygenase-2 in endothelial cells resulting in the conversion of arachidonic acid (AA) to prostaglandin E2 (PGE2). PGE2 promotes colon cancer cell growth. In contrast, the less studied path of AA oxygenation via cytochrome p450 enzymes produces epoxyeicosatetraenoic acids (EETs), whose anti-inflammatory properties cause shrinking of enlarged adipocytes, a characteristic of obesity, through the liberation of fatty acids. It is now thought that EET depletion occurs in obesity and may contribute to colon cell carcinogenesis. Meanwhile, gangliosides, a type of sphingolipid, are cell surface signaling molecules that contribute to the apoptosis of colon tumor cells. Many of these discoveries have been made recently and the mechanisms are still not fully understood, leading to an exciting new chapter of lipidomic research. In this review, mechanisms behind obesity-associated colon cancer are discussed with a focus on the role of small lipid signaling molecules in the process. Specifically, changes in lipid metabolite levels during obesity and the development of colon cancer, as well as novel biomarkers and targets for therapy, are discussed.