Establishment Of A Method For Detection Of Redox-Related Small Molecules By LC-MS And Its Application In Myocardial Ischemia Reperfusion

Objective: Oxidative stress plays an important role in the development of myocardial ischemia-reperfusion injury,but there is currently no precise,broad-spectrum method for determining small molecules involved in redox reactions and lipid peroxidation products in plasma or tissue.Based on the technique of ultra performance liquid chromatography tandem mass spectrometry,this study established a method for extracting targeted metabolomics of these small molecules in plasma and tissues.This method can efficiently and accurately screen redox small molecules and lipid peroxidation products related to the development of myocardial ischemia-reperfusion injury.Methods: The left anterior descending artery of the mice was ligated for 30 minutes and then reperfused for 120 minutes.The rats were sacrificed and the heart and plasma were pretreated to extract water-soluble redox molecules and fat-soluble lipid peroxidation products.Through literature review,we have identified redox small molecules and lipid peroxidation products that play important roles in vivo.Among them,water-soluble redox small molecules include tetrahydrobiopterin,Uric Acid glutathione,and oxidized glutathione,nitrosoglutathione,nicotinamide adenine dinucleotide(NAD),reduced nicotinamide adenine dinucleotide(NADH),nicotinamide adenine dinucleotide phosphate(NADP),reduced nicotinamide adenine dinuclear Glucoside phosphate(NADPH),arginine,citrulline,homocysteine,cysteine,vitamin C,vitamin C,etc;Fat-soluble lipid peroxidation products include 4-hydroxy-2-nonenal,4-hydroxy-2-hexenal,crotonaldehyde,Benzaldehyde,hexanal,2.4-nonadienal,2,4-decadienal,etc.In the method establishment,ultra-high performance liquid chromatography(HPLC)was performed on the T3 column for gradient separation,and the mass spectrometer was scanned in the mass spectrometer using an anion multi-reaction monitoring scan mode.Standard curves of redox small molecules and lipid peroxidation products were prepared and analyzed by MultiQuant 2.1.1 software.Results: The method quantitatively determined redox small molecules and lipid peroxidation products in the range of 0.25-2000 ng/ml with a regression coefficient between 0.991 and 0.996.By measuring the ischemia-reperfusion heart of mice by this method,it was found that the reducing small molecules in the dangerous region and the necrotic area of the heart were significantly reduced and the lipid peroxidation products were significantly increased after ischemia-reperfusion.Conclusion: After optimization of the sample extraction method,the compounds detected in cell and heart tissue include 7 lipid peroxidation products and more than 10 redox small molecules.Moreover,it was found that the reductive small molecule with anti-oxidation decreased significantly after ischemia-reperfusion and the lipid peroxidation product increased significantly,suggesting that these small molecules may play an important role in myocardial ischemia-reperfusion injury.Correlation analysis of ejection fraction also determined the potential impact of these small molecules on cardiac function,providing a new idea for future clinical treatment of myocardial ischemia-reperfusion patients.