氧化应激,其特征是抗氧化剂和自由基之间的不平衡,在冠心病的发病机制中起着举足轻重的作用,一种常见而严重的心血管疾病,并对其发展和进步做出了重大贡献。无血清硫醇是人体抗氧化防御系统的重要组成部分。血清游离硫醇水平的准确测定为了解机体状态和监测冠心病发生、进展的危险因素提供了参考依据。在这项研究中,开发了一种基于2,2'-二硫代二吡啶衍生化反应的高效液相色谱(HPLC)方法,以同时获得总游离硫醇(Total-SH)的浓度,低分子量游离硫醇(LMM-SH),和人血清中的无蛋白质硫醇(P-SH)。AgilentEclipseXDB-C18色谱柱(150mm×4.6mm,5μm)用于分析,以1mL/min的流速进行梯度洗脱。0.1%甲酸水溶液用作流动相A,和0.1%甲酸乙腈溶液用作流动相B。梯度洗脱程序如下:0-0.1分钟,12%B-30%B;0.1-2分钟,30%B;2-2.1分钟,30%B-100%B;2.1-6分钟,100%B;6-6.1分钟,100%B-12%B;6.1-7分钟,12%B.在5分钟的运行时间后出现分离良好的峰。2-硫代吡啶酮的峰代表样品的总SH含量,吡啶基二硫代衍生物的峰代表LMM-SH含量。这两个峰之间的差异表明P-SH含量。优化了衍生化反应条件,并对该方法进行了验证。该方法表现出良好的线性,相关系数≥0.9994,浓度范围为31.25-1000μmol/L。Total-SH和LMM-SH的检出限分别为2.61和0.50μmol/L,Total-SH和LMM-SH的定量限分别为8.71和1.67μmol/L,分别。Total-SH和LMM-SH的回收率为91.1%~106.0%。日内和日间精确度从0.4%到9.1%不等。所开发的方法用于分析714名志愿者的血清样品。总SH浓度范围为376.60至781.12μmol/L,平均浓度为555.62μmol/LLMM-SH浓度从36.37到231.65μmol/L不等,平均为82.34μmol/L。P-SH浓度范围为288.36~687.74μmol/L,平均值为473.27μmol/L。Spearman相关检验显示,血清硫醇水平与冠状动脉病变严重程度及临床常见生化指标相关。该研究为检测血清游离硫醇及其与冠心病的关系提供了一种简单可靠的HPLC方法。为冠心病风险相关标志物的研究提供了新的参考。
Oxidative stress, which is characterized by an imbalance between antioxidants and free radicals, plays a pivotal role in the pathogenesis of coronary heart disease, a common and serious cardiovascular condition, and contributes significantly to its development and progression. Serum free thiols are crucial components of the body\'s antioxidant defense system. The accurate determination of serum free thiol levels provides a reference basis for understanding the body\'s status and monitoring the risk factors associated with the occurrence and progression of coronary heart disease. In this study, a high performance liquid chromatographic (HPLC) method based on the derivatization reaction of 2,2\'-dithiodipyridine was developed to simultaneously obtain the concentrations of total free thiols (Total-SH), low-molecular-mass free thiols (LMM-SH), and protein-free thiols (P-SH) in human serum. An Agilent Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μm) was used for the analysis, and gradient elution was performed at a flow rate of 1 mL/min. A 0.1% formic acid aqueous solution was used as mobile phase A, and a 0.1% formic acid acetonitrile solution was used as mobile phase B. The gradient elution program was as follows: 0-0.1 min, 12%B-30%B; 0.1-2 min, 30%B; 2-2.1 min, 30%B-100%B; 2.1-6 min, 100%B; 6-6.1 min, 100%B-12%B; 6.1-7 min, 12%B. Well-separated peaks appeared after a run time of 5 min. The peak of 2-thiopyridone represented the Total-SH content of the samples, and the peak of the pyridyldithio derivative represented the LMM-SH content. The difference between these two peaks indicated the P-SH content. The derivatization reaction conditions were optimized, and the method was validated. The method demonstrated good linearity, with a correlation coefficient ≥0.9994, over the concentration range of 31.25-1000 μmol/L. The limits of detection for Total-SH and LMM-SH were 2.61 and 0.50 μmol/L, and the limits of quantification for Total-SH and LMM-SH were 8.71 and 1.67 μmol/L, respectively. The recoveries of Total-SH and LMM-SH were in the range of 91.1%-106.0%. The intra- and inter-day precisions ranged from 0.4% to 9.1%. The developed method was used to analyze serum samples from 714 volunteers. The Total-SH concentrations ranged from 376.60 to 781.12 μmol/L, with an average concentration of 555.62 μmol/L. The LMM-SH concentrations varied from 36.37 to 231.65 μmol/L,with an average of 82.34 μmol/L. The P-SH concentrations ranged from 288.36 to 687.74 μmol/L, with an average of 473.27 μmol/L. Spearman\'s correlation test showed that serum thiol levels were correlated with the severity of coronary artery disease and common clinical biochemical indicators. The proposed study provides a simple and reliable HPLC method for detecting serum free thiols and exploring their relationship with coronary heart disease, offering a new reference for the study of markers related to the risk of coronary heart disease.