Nitro-fatty acids formed during virgin olive oil consumption: Dependence on cultivar, fruit ripening, and polyphenols and their protective role in a NAFLD pathophysiology model

Abstract

Extra virgin olive oil (EVOO) consumption is a key contributor to the health benefits of the Mediterranean diet, particularly in mitigating metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). During gastric digestion, the acidic environment promotes the reaction of olive oil-derived unsaturated fatty acids with dietary nitrite (NO₂^–), generating electrophilic nitro-fatty acids (NO₂-FAs), including nitro-oleic acid (NO₂-OA) and nitro-conjugated linoleic acid (NO₂-CLA). These molecules act as potent anti-inflammatory and cytoprotective signaling mediators. However, the influence of olive oil matrix components on gastric NO₂-FA formation and their therapeutic relevance in NAFLD remains incompletely understood. To identify determinants of NO₂-FA formation, two contrasting olive cultivars (Arbequina and Coratina) were evaluated at different ripening stages under in vitro gastric conditions, with particular focus on the roles of endogenous polyphenols and pigments. To assess their protective effects in vivo, a murine NAFLD model induced by a high-fat (HF) diet was supplemented with EVOO and nitrite. Mitochondrial function, tissue lipid accumulation, and cellular respiration were analyzed in fresh liver and heart biopsies. Gastric NO₂-FA formation was strongly influenced by olive cultivar and matrix composition. In Coratina EVOO, NO₂-FA production peaked at the intermediate ripening stage and remained unaffected by polyphenol depletion, likely due to high pigment levels that preserved nitration reactions. In contrast, Arbequina EVOO exhibited relatively constant nitration rates across ripening stages, but polyphenols were essential for directing reactions toward fatty acid nitration rather than lipid oxidation. In the NAFLD model, HF-fed mice exhibited markedly reduced plasma NO₂-FA levels. Supplementation with EVOO plus nitrite restored circulating NO₂-FA concentrations, significantly increased hepatic heme oxygenase-1 expression, and reduced body weight gain and hepatic lipid accumulation. Moreover, NO₂-FA formation rescued mitochondrial dysfunction, improving respiratory parameters, increasing ATPase activity, and enhancing mitochondrial complex II and V activities in both hepatic and cardiac tissues. The generation of bioactive NO₂-FAs from olive oil is a tunable process governed by cultivar selection, fruit maturity, and antioxidant composition. Once formed during digestion, these lipid mediators attenuate NAFLD progression by preserving mitochondrial function and reducing lipotoxicity. These findings identify NO₂-FAs as key molecular mediators underlying the health benefits associated with olive oil consumption.

Extra virgin olive oil (EVOO) consumption is a key contributor to the health benefits of the Mediterranean diet, particularly in mitigating metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). During gastric digestion, the acidic environment promotes the reaction of olive oil-derived unsaturated fatty acids with dietary nitrite (NO₂^–), generating electrophilic nitro-fatty acids (NO₂-FAs), including nitro-oleic acid (NO₂-OA) and nitro-conjugated linoleic acid (NO₂-CLA). These molecules act as potent anti-inflammatory and cytoprotective signaling mediators. However, the influence of olive oil matrix components on gastric NO₂-FA formation and their therapeutic relevance in NAFLD remains incompletely understood. To identify determinants of NO₂-FA formation, two contrasting olive cultivars (Arbequina and Coratina) were evaluated at different ripening stages under in vitro gastric conditions, with particular focus on the roles of endogenous polyphenols and pigments. To assess their protective effects in vivo, a murine NAFLD model induced by a high-fat (HF) diet was supplemented with EVOO and nitrite. Mitochondrial function, tissue lipid accumulation, and cellular respiration were analyzed in fresh liver and heart biopsies. Gastric NO₂-FA formation was strongly influenced by olive cultivar and matrix composition. In Coratina EVOO, NO₂-FA production peaked at the intermediate ripening stage and remained unaffected by polyphenol depletion, likely due to high pigment levels that preserved nitration reactions. In contrast, Arbequina EVOO exhibited relatively constant nitration rates across ripening stages, but polyphenols were essential for directing reactions toward fatty acid nitration rather than lipid oxidation. In the NAFLD model, HF-fed mice exhibited markedly reduced plasma NO₂-FA levels. Supplementation with EVOO plus nitrite restored circulating NO₂-FA concentrations, significantly increased hepatic heme oxygenase-1 expression, and reduced body weight gain and hepatic lipid accumulation. Moreover, NO₂-FA formation rescued mitochondrial dysfunction, improving respiratory parameters, increasing ATPase activity, and enhancing mitochondrial complex II and V activities in both hepatic and cardiac tissues. The generation of bioactive NO₂-FAs from olive oil is a tunable process governed by cultivar selection, fruit maturity, and antioxidant composition. Once formed during digestion, these lipid mediators attenuate NAFLD progression by preserving mitochondrial function and reducing lipotoxicity. These findings identify NO₂-FAs as key molecular mediators underlying the health benefits associated with olive oil consumption.