A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In our study, we used the fat-1 transgenic mouse model as a proxy for long-term omega-3 supplementation to determine, in a well-controlled manner, the molecular phenotype associated with a balanced omega-6/omega-3 ratio. The fat-1 mouse can convert omega-6 to omega-3 PUFAs, which protect against a wide variety of diseases including chronic inflammatory diseases and cancer. Both wild-type (WT) and fat-1 mice were subjected to an identical diet containing 10% corn oil, which has a high omega-6 content similar to that of the Western diet, for a six-month duration. We used a multi-platform lipidomic approach to compare the plasma lipidome between fat-1 and WT mice. In fat-1 mice, an unbiased profiling showed a significant increase in the levels of unesterified eicosapentaenoic acid (EPA), EPA-containing cholesteryl ester, and omega-3 lysophosphospholipids. The increase in omega-3 lipids is accompanied by a significant reduction in omega-6 unesterified docosapentaenoic acid (omega-6 DPA) and DPA-containing cholesteryl ester as well as omega-6 phospholipids and triacylglycerides. Targeted lipidomics profiling highlighted a remarkable increase in EPA-derived diols and epoxides formed via the cytochrome P450 (CYP450) pathway in the plasma of fat-1 mice compared with WT mice. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.

BackgroundCollection of exhaled breath samples for the analysis of inflammatory biomarkers is an important area of research aimed at improving our ability to diagnose, treat and understand the mechanisms of chronic pulmonary disease. Current collection methods based on condensation of water vapor from exhaled breath yield biomarker levels at or near the detection limits of immunoassays contributing to problems with reproducibility and validity of biomarker measurements. In this study, we compare the collection efficiency of two aerosol-to-liquid sampling devices to a filter-based collection method for recovery of dilute laboratory generated aerosols of human cytokines so as to identify potential alternatives to exhaled breath condensate collection.Methodology/Principal FindingsTwo aerosol-to-liquid sampling devices, the SKC® Biosampler and Omni 3000™, as well as Teflon® filters were used to collect aerosols of human cytokines generated using a HEART nebulizer and single-pass aerosol chamber setup in order to compare the collection efficiencies of these sampling methods. Additionally, methods for the use of Teflon® filters to collect and measure cytokines recovered from aerosols were developed and evaluated through use of a high-sensitivity multiplex immunoassay. Our results show successful collection of cytokines from pg/m3 aerosol concentrations using Teflon® filters and measurement of cytokine levels in the sub-picogram/mL concentration range using a multiplex immunoassay with sampling times less than 30 minutes. Significant degradation of cytokines was observed due to storage of cytokines in concentrated filter extract solutions as compared to storage of dry filters.ConclusionsUse of filter collection methods resulted in significantly higher efficiency of collection than the two aerosol-to-liquid samplers evaluated in our study. The results of this study provide the foundation for a potential new technique to evaluate biomarkers of inflammation in exhaled breath samples.

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.