Characterization of fatty acid esters of hydroxy fatty acids, a novel class of bioactive lipids, in milk fat of cows supplemented with stearic and palmitic acid

Document Type


Publication Date



American Dairy Science Association


Journal of Dairy Science




Suppl. 1






fatty acid esters of hydroxy fatty acids (FAHFA), functional lipids, milk fat


Fatty acid (FA) esters of hydroxy FA (FAHFA) are classified as estolides and have been characterized as potential anti-inflammatory and antidiabetic bioactive FA. FAHFA are classified into families according to the FA and hydroxy FA makeup [i.e., palmitic acid (PA) esters of hydroxystearic acid are PAHSA] and within each family multiple regioisomers exist depending on the location of the hydroxy group in the hydroxy FA (i.e., 9-PAHSA). The objective of this study was to characterize 5 FAHFA families made of PA, stearic (SA), palmitoleic (PO), or oleic (OA) acid (PAHSA, SAHSA, POHSA, PAHPA, OAHSA) in a retrospective analysis of milk fat samples from a fat supplement experiment. Briefly, 12 multiparous Holstein cows were arranged in a 4 × 4 Latin square. Treatments were a no supplement control (CON) or a 2% DM inclusion of either a highly enriched PA or SA or a combination of both (PA/SA). FAHFA families were detected by LC-TQMS using multiple reaction monitoring and quantified using a standard curve for each FAHFA regioisomer. Data were analyzed with a mixed model with cow and period as random effects and treatment as a fixed effect and a regression analysis was done between FAHFA and production traits and plasma metabolites. PA increased 9- and 12-PAHPA concentration 2.1- and 5.5-fold (P ≤ 0.01 for both) and tended to decrease 10-PAHSA (P = 0.07), when compared with CON. There was no effect of treatment on 9-SAHSA, 9-OAHSA, 9-POHSA, 9-PAHSA, 12-PAHSA. Interestingly, the combination of PA/ SA abolished the effect of PA on PAHPA levels. Notably, 12-PAHPA was positively related to milk 16 carbon FA (R2 = 0.12, P = 0.02) and plasma glucose (R2 = 0.10, P = 0.04) and negatively related to milk de novo FA (R2 = 0.10, P = 0.03). Additionally, 10-PAHSA was positively related to plasma nonesterified FA (R2 = 0.26, P < 0.001). The results, to the best of our knowledge, are the first characterization of FAHFA in bovine milk, suggest a role of dietary FA on FAHFA concentrations, and show that some FAHFA are correlated with production traits and plasma metabolites in Holstein dairy cows.