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led steps. After optimizing the applied tension, the application of substances was delayed until a steady contraction force was observed. Substances were directly added to the bath solution. At the beginning of the measurements, 5 9 10-8 M isoprenaline, a beta-receptor agonist, was applied to the organ bath to further elevate the contraction force of the muscle. Following this prestimulation, increasing concentrations of the OR51E1 ligand, nonanoic acid, were applied to the bath solution. The administered concentration started at 1 lM, with the concentration increased fivefold every 510 min until 5 mM was reached. Meanwhile, changes in the contraction force were closely monitored. To exclude possible toxic effects of nonanoic acid, stimulation with 1 mM isoprenaline was performed after each experiment. Determination of the fatty acid pattern in the triglyceride PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19796427 and non-esterified fatty acid fraction from epicardial adipose biopsies and from plasma Approximately 25 mg of human epicardial adipose tissue was homogenized using a TissueLyser MM 300 in 0.25 ml 1% Triton X-100 in PBS. Then, 1.25 ml 2-propanol, n-heptane and 2 mol/l phosphoric acid were added to the tissue extract or to 0.25 ml plasma samples and mixed by vortexing. After 10 min, 0.5 ml toluene/methanol and 0.75 ml water were added and mixed by vortexing, and after centrifugation at 4000 rpm, the upper phase was dried under stream of nitrogen. The lipids were dissolved in 75 ll CHCl3/CH3OH and applied to a silica gel chromatography plate. The lipid fractions were separated using a mixture of n-hexane, diethylether and acetic acid as a solvent. The lipid fractions were identified using a pooled control plasma and were separated on each plate; the lipid fractions were then visualized by 2,7dichlor-fluoresceine under ultraviolet light. The fractions were scraped off the TLC plate, transferred to screw-capped vials and dissolved in a 2 ml methanol/toluene mixture containing cis-13,16,19-docosatrienoic acid as an internal standard. Trans-esterification was performed by incubation with acetyl chloride at 100 C for 60 min. The cold sample was neutralized with 5 ml 6% K2CO3, shaken for 2 min, and centrifuged, and the upper phase was concentrated to 100 ll under nitrogen. The fatty acid methyl esters were measured by gas chromatography 7890A with a flame ionization detector and MedChemExpress DM-1 quantified using the corresponding fatty acids standards. All data analysis were performed using the JMP 11.0 software package. 123 13 Page 6 of 20 Basic Res Cardiol 112:13 Results Olfactory receptor OR51E1 is expressed in the human heart and in stem cell-derived cardiomyocytes Comparative transcriptome analysis of OR expression identified OR51E1 in various human tissues, including the heart. In the human adult and fetal heart, OR51E1 is the highest expressed OR, with an expression level similar to that of the beta- 2 adrenergic receptor and the muscarinic acetylcholine receptor M2. On a rough scale, 1 FPKM corresponds to a weak expression level, 10 FPKM represents a moderate expression level and 100 FPKM indicates a high expression level. Moreover, OR51E1 is one of the few ORs for which the ligand has been identified. Therefore, in this study, we focused on OR51E1 for functional characterization of ORs in the human heart. First, we validated the results of the transcriptome analysis using reverse transcription PCR and could detect transcripts of OR51E1 in the Fig. 1 Expression of OR51E1 in the human heart tissue