Abstract
During this research a simple, accurate, and environmentally friendly method to determine lipoyllysine and lipoic acid in meat was developed and validated. The presented approach was based on the hydrolysis of the proteins containing lipoic acid, reduction of disulfide bonds with tris(hydroxymethyl)phosphine, and precolumn derivatization of free thiol groups with 1-benzyl-2-chloropyridinium bromide long-term followed by HPLC separation with a diode-array detector. The method has been validated in accordance with the U.S. FDA guidelines and was linear in the range of 0.1–10 μmol/L in concentration with R2 values ≥0.9997 for both analytes. For lipoyllysine and lipoic acid, intra- and interday precision values were lower than 10%. The intraday accuracy values ranged from 91.0% to 99.4% for lipoyllysine and from 99.1% to 107.3% for lipoic acid, whereas the interday accuracy values for lipoyllysine and lipoic acid were 92.0–95.6% and 93.5–98.8%, respectively. Additionally, in this research the antioxidant activity of lipoyllysine and reduced lipoyllysine compound using spectrophotometric method with 1,1-diphenyl-2-picrylhydrazyl was examined for the first time. The data showed that dihydrolipoyllysine exhibits stronger antioxidant capacity than lipoyllysine based on a lower value of concentration required to achieve a 50% antioxidant effect in 1,1-diphenyl-2-picrylhydrazyl radical scavenging test.
Highlights
For the past few years, medical research has shown that healthy diets and long-term consumption of foods rich in antioxidants may lower oxidative stress and the occurrence of chronic diseases, such as cancer and diabetes, as well as neurodegenerative and cardiovascular diseases.[1,2]In regular and healthy people, there is an equilibrium between endogenous antioxidant defenses and the generation of reactive oxygen species (ROS) or free radicals
Our studies show that when the reaction was carried out at a physiological temperature of 37 °C and under mild alkaline conditions, the reaction reached a plateau after 22 h using the appropriate amounts of proteases, which were recommended for the selected animal tissue (SI Table S1)
The reduced LLys was fluorescently labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate at 60 °C for 1 h, whereas the results of the proposed method indicated total derivatization occurred within 15 min at room temperature
Summary
In regular and healthy people, there is an equilibrium between endogenous antioxidant defenses and the generation of reactive oxygen species (ROS) or free radicals. If this balance is disturbed, it can lead to oxidative stress and related damage. Oxidative stress damages proteins, lipids, and nucleic acids, compromising cell viability.[3,4] To minimize the harmful effects of free radicals, organisms are endued with a very effective antioxidant defense system. Natural antioxidant enzymes (catalase, superoxide dismutase, horseradish peroxidase) and nonenzymatic antioxidant compounds, such as vitamins E and C, α-lipoic acid (LA), and glutathione, inhibit the oxidative mechanisms that lead to the development of degenerative diseases and aging.[3,5] Because oxidative damage of human cells increases with age, the intensified ingesting of exogenous antioxidants derived from fruits, vegetables, and meat may assist the endogenous antioxidant defense system and reduce the risk of chronic diseases.[6]
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