Association Study between Phospholipid Profile and SNPs Related to Fatty Acid Metabolism in Hanwoo Beef

Abstract

Many positive physiological effects of omega-3 fatty acids have been demonstrated for over three decades. Lowering the attack rate of CVD, cancer, rheumatoid arthritis, auto-immune diseases and recurrence of myocardial infarction are representative effects of omega-3 fatty acids. Additionally, eicosanoids, the signaling molecules have contrast immune and inflammatory effects by their origin, omega-3 or omega-6. Therefore, omega-6 to omega-3 ratio is critical to maintaining immune homeostasis, normal development, and mental health. However, the modern western diets are very imbalanced in omega fatty acids ratio, and most people all over the world take much more omega-6 fatty acids than omega-3 fatty acids. Agriculture and livestock industry focused on productivity are the one of the reasons of omega fatty acid imbalance. Hanwoo industry is also focused on productivity and marbling score using maize as a main feed grain which is high in omega-6 fatty acids, and thus resulting in imbalanced omega fatty acids ratio in Hanwoo beef. In this study, methods for beef fatty acid analysis was established and associations were studied between omega fatty acid composition in fifty-six Hanwoo beef samples and SNPs related to fatty acid metabolism. To study the associations between fatty acid composition and SNPs, instead of neutral fat, fatty acids of phospholipid was chosen since it is mainly under control of the genes related to fatty acid metabolism. Six SNPs in five genes related to fatty acid composition were selected as candidate SNPs

FASN (rs41919985), SCD (rs41255693), FABP4 (rs41729173), FADS1 (rs136261927 and rs42187261) and FADS2 (rs109772589). 1. Total lipid was extracted using Folch method, and phospholipid was separated from total lipid using thin-layer chromatography. Phospholipid fraction was scraped from TLC plate and directly methylated using O'Fallons direct methylation method. This preparation method was validated by comparing with O'Fallons direct methylation method and analyzing gas chromatography data of empty silica through the preparation method. The same amount of phosphatidylcholine from egg yolk was treated by above preparation method or direct methylation without pretreatment, and GC data from each method were compared. Total areas through the preparation method or direct methylation were comparable having 407 and 470, respectively. 2. Fatty acid composition in phospholipid of the samples were analyzed with GC. Genomic DNA was extracted from the fifty-six samples and genotyped for the candidate SNPs using restriction fragment length polymorphism or Sanger sequencing. In the case of rs109772589 in FADS2, all the samples showed the same hetero genotype, and thus it was omitted from the association analysis. On the other hand, in the case of rs41919985 in FASN, there were only two genotypes except AA type. 3. Associations between omega fatty acid composition and genotypes were analyzed by ANOVA. There were four SNPs showing statistically significant associations.  GA type of rs41919985 with the highest C20:5 n-3 (p=0.027)  CC type of rs41729173 with the lowest C22:2n-6 (p=0.049)  AG type of rs42187261 with the lowest C20:4 n-6 (p=0.044)  GA type of rs136261927 with the lowest C20:3n-6 (p=0.027) The ratio of total n-3/n-6 of the individual which have GA, CC, AG and GA types in rs41919985, rs41729173, rs42187261 and rs136261927, respectively, was around 28:1. This value was the third highest grade in total n-3/n-6 ratio of the samples and much higher than the mean value of total n-3/n-6 ratio. Consequently, these four SNPs could be applied as potential genetic markers to select Hanwoo steers in the aspect of improvement of n-3/n-6 balance in the future.