AS described in our previous study [16 (link)], the venous blood was placed at room temperature for three hours, then it was centrifuged by 3000× g for 20 min at 4 °C to obtain serum samples. The serum samples were sent to Servicebio (Wuhan, China). A biochemical analyzer (Chemray 240, Rayto Life and Analytical Sciences Co., Ltd. Shenzhen, China) with the corresponding reagent (Huili Biology, Changchun, China) was used to measure the serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).
In the presence of cholesterol hydrolase, cholesterol is hydrolyzed into fatty acids, which are then further oxidized to produce hydrogen peroxide. The Trinder reaction was used to detect hydrogen peroxide concentrations. Calculated using the formula, the TC concentration was determined by comparing the hydrogen peroxide concentration in the cholesterol standard solution to the hydrogen peroxide concentration in the serum. To remove free glycerol from the serum, glycerol kinase was applied. Hydrogen peroxide was produced by breaking down TG with lipoprotein lipase. Follow-up tests were consistent with cholesterol testing methods. Phosphotungstic acid-magnesium reagents were needed to precipitate apoB-containing lipoproteins, and HDL-C was measured from the supernatant. The detection principle was the same as cholesterol. Sulfated polyethylene was used to precipitate and disperse LDL-C, then it was calculated by subtracting the cholesterol in the supernatant without LDL-C from the total cholesterol.
Serum NEFAs were determined using commercial assay kits according to the instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Acetyl-CoA synthase catalyzed the production of hydrogen peroxide from NEFA. It was consistent with the above method of detecting hydrogen peroxide. All the detection methods were recommended by the Chinese Medical Association.
In the presence of cholesterol hydrolase, cholesterol is hydrolyzed into fatty acids, which are then further oxidized to produce hydrogen peroxide. The Trinder reaction was used to detect hydrogen peroxide concentrations. Calculated using the formula, the TC concentration was determined by comparing the hydrogen peroxide concentration in the cholesterol standard solution to the hydrogen peroxide concentration in the serum. To remove free glycerol from the serum, glycerol kinase was applied. Hydrogen peroxide was produced by breaking down TG with lipoprotein lipase. Follow-up tests were consistent with cholesterol testing methods. Phosphotungstic acid-magnesium reagents were needed to precipitate apoB-containing lipoproteins, and HDL-C was measured from the supernatant. The detection principle was the same as cholesterol. Sulfated polyethylene was used to precipitate and disperse LDL-C, then it was calculated by subtracting the cholesterol in the supernatant without LDL-C from the total cholesterol.
Serum NEFAs were determined using commercial assay kits according to the instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Acetyl-CoA synthase catalyzed the production of hydrogen peroxide from NEFA. It was consistent with the above method of detecting hydrogen peroxide. All the detection methods were recommended by the Chinese Medical Association.
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