Salmon Food-Specific Compounds and Their Metabolites Increase in Human Plasma and Are Associated with Cardiometabolic Health Indicators Following a Mediterranean-Style Diet Intervention.

BACKGROUND: Nutrimetabolomics allows for the comprehensive analysis of foods and human biospecimens to identify biomarkers of intake and begin to probe their associations with health. Salmon contains hundreds of compounds that may provide cardiometabolic benefits. OBJECTIVES: We used untargeted metabolomics to identify salmon food-specific compounds (FSCs) and their predicted metabolites that were found in plasma after a salmon-containing Mediterranean-style (MED) diet intervention. Associations between changes in salmon FSCs and changes in cardiometabolic health indicators (CHIs) were also explored. METHODS: For this secondary analysis of a randomized, crossover, controlled feeding trial, 41 participants consumed MED diets with 2 servings of salmon per week for 2 5-wk periods. CHIs were assessed, and fasting plasma was collected pre- and postintervention. Plasma, salmon, and 99 MED foods were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Compounds were characterized as salmon FSCs if detected in all salmon replicates but none of the other foods. Metabolites of salmon FSCs were predicted using machine learning. For salmon FSCs and metabolites found in plasma, linear mixed-effect models were used to assess change from pre- to postintervention and associations with changes in CHIs. RESULTS: Relative to the other 99 MED foods, there were 508 salmon FSCs with 237 unique metabolites. A total of 143 salmon FSCs and 106 metabolites were detected in plasma. Forty-eight salmon FSCs and 30 metabolites increased after the intervention (false discovery rate <0.05). Increases in 2 annotated salmon FSCs and 2 metabolites were associated with improvements in CHIs, including total cholesterol, low-density lipoprotein cholesterol, triglycerides, and apolipoprotein B. CONCLUSIONS: A data-driven nutrimetabolomics strategy identified salmon FSCs and their predicted metabolites that were detectable in plasma and changed after consumption of a salmon-containing MED diet. Findings support this approach for the discovery of compounds in foods that may serve, upon further validation, as biomarkers or act as bioactive components influential to health. The trials supporting this work were registered at NCT02573129 (Mediterranean-style diet intervention) and NCT05500976 (ongoing clinical trial).

Lipidomics-Based Comparison of Molecular Compositions of Green, Yellow, and Red Bell Peppers.

Identifying and annotating the molecular composition of individual foods will improve scientific understanding of how foods impact human health and how much variation exists in the molecular composition of foods of the same species. The complexity of this task includes distinct varieties and variations in natural occurring pigments of foods. Lipidomics, a sub-field of metabolomics, has emerged as an effective tool to help decipher the molecular composition of foods. For this proof-of-principle research, we determined the lipidomic profiles of green, yellow and red bell peppers (Capsicum annuum) using liquid chromatography mass spectrometry and a novel tool for automated annotation of compounds following database searches. Among 23 samples analyzed from 6 peppers (2 green, 1 yellow, and 3 red), over 8000 lipid compounds were detected with 315 compounds (106 annotated) found in all three colors. Assessments of relationships between these compounds and pepper color, using linear mixed effects regression and false discovery rate (<0.05) statistical adjustment, revealed 11 compounds differing by color. The compound most strongly associated with color was the carotenoid, ß-cryptoxanthin (p-value = 7.4 × 10-5; FDR adjusted p-value = 0.0080). These results support lipidomics as a viable analytical technique to identify molecular compounds that can be used for unique characterization of foods.

Potential Regulation of UGT2B10 and UGT2B7 by miR-485-5p in Human Liver.

The UDP-glucuronosyltransferase (UGT) family of enzymes is important in the metabolic elimination of a variety of endogenous compounds such as bile acids, steroids, and fat-soluble vitamins, as well as exogenous compounds including many pharmaceuticals. The UGT2B subfamily is a major family of UGT enzymes expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contributes to interindividual variability in UGT2B expression and is a crucial component in predicting patient drug response. In the present study, a high-resolution liquid chromatography-tandem mass spectrometry method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples (n = 62). Concurrent in silico analysis identified eight candidate miRNAs as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant inverse correlation between UGT2B10 and UGT2B15 and one of these candidate miRNAs, miR-485-5p. A near-significant correlation was also observed between UGT2B7 and miR-485-5p expression. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the UGT2B10 3'-untranslated region (UTR), and significant reduction in luciferase activity was also observed for a luciferase vector containing the UGT2B7 3'-UTR; however, none was observed for the UBT2B15 3'-UTR. UGT2B10 and UGT2B7 activities were probed using nicotine and 3'-azido-3'-deoxythymidine, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells upon overexpression of miR-485-5p mimic. This is the first study demonstrating a regulatory role of miR-485-5p for multiple UGT2B enzymes. SIGNIFICANCE STATEMENT: The purpose of this study was to identify novel epigenetic miRNA regulators of the UGT2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that miRNA 485-5p is a novel regulator of UGT2B7 and UGT2B10, which play an important role in the metabolism of many commonly prescribed medications, carcinogens, and endogenous compounds. This study identified potential miRNA-UGT2B mRNA interactions using a novel proteomic approach, with in vitro experiments undertaken to validate these interactions.

Regulation of UGT2A1 by miR-196a-5p and miR-196b-5p.

UDP-glucuronosyltransferase (UGT) 2A1 is an important enzyme in the detoxification of polycyclic aromatic hydrocarbons found in cigarette smoke. This enzyme is expressed in aerodigestive tract tissues including lung as both its wild-type and exon 4-deleted splice variant isoforms, with the latter acting as a negative regulator of wild-type UGT2A1 activity. UGT2A1 regulation may also be mediated by microRNA (miRNA). To identify miRNA important in the regulation of UGT2A1, expression analysis in tandem with in silico analysis suggested miR-196a-5p and miR-196b-5p as potential top candidates. Significant reductions in firefly luciferase activity were observed in human embryonic kidney cell line 293 cells cotransfected with the wild-type UGT2A1 3'-untranslated region (UTR)-containing luciferase plasmid and either miR-196a-5p (62%, P = 0.00080) or miR-196b-5p (60%, P = 0.00030) mimics. In pull-down assays, there was a 3.4- and 5.2-fold increase in miR-196a-5p (P = 0.054) and miR-196b-5p (P = 0.035), respectively, using the UGT2A1 3'-UTR biotinylated mRNA probe as compared with the ß-actin coding region control mRNA probe. UGT2A1 mRNA was reduced by 25% (P = 0.058) and 35% (P = 0.023) in H146 and H1944 cells, respectively, after overexpression of the miR196a-5p mimic. A similar 32% (P = 0.030) and 41% (P = 0.016) reduction was observed after over-expression of the miR-196b-5p mimic. In H146 cells transfected with miRNA mimic together with a small interfering RNA (siRNA) specific for the UGT2A1 splice variant, a significant reduction in 3-hydroxy-benzo[a]pyrene-glucuronide formation was observed. The miR-196a-5p- and miR-196b-55p-treated cells exhibited reductions of 35% (P = 0.047) and 44% (P = 0.0063), respectively. These data suggest that miR-196a-5p and miR-196b-5p play an important role in UGT2A1 regulation within the lung and potentially other aerodigestive tract tissues.

Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines.

The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 3' untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P < 0.001) and UGT2B10 (P = 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P < 0.001) and UGT2B10 (P = 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P < 0.001 and P = 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P = 0.021) and Hep3B (P = 0.0068) cells, and increased epirubicin glucuronidation by 85% (P = 0.057) and 50% (P = 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P < 0.001 and P = 0.0048, and P = 0.017 and P = 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.