全血保存过程中血浆中S-亚硝基硫醇的测定

Determination of plasma S-nitrosothiol in preserved whole blood

目的建立荧光法测定血浆中S-亚硝基硫醇(RSNOs)的方法并探讨影响其测定的相关因素。方法建立标准曲线,确定荧光法的准确性与可重复性;以检测过程中某一种影响因素(如样品制备条件、孵育时间等)为变量观察多因素对检测结果的影响;取9份全血(400 ml/份)在保存d1、d7、d14、d21、d28和d35时分别检测血浆中RSNOs含量。结果同一份血浆,相同实验条件下检测10次,变异系数为(CV)=3.5%;不同离心力470、734、1057、1 438 g制备的血浆,RSNOs含量分别为(9.22±0.14)、(9.97±0.11)、(10.40±0.05)、(10.91±0.01)μmol/L;铜离子浓度为100、150、200μmol/L时的RSNOs含量分别为(13.83±0.02)、(14.23±0.03)、(13.22±0.01)μmol/L;在其他条件相同的情况下,孵育1、1.5、2、2.5 h样品的RSNOs含量分别为(10.34±0.04)、(10.75±0.02)、(12.31±0.09)、(10.53±0.06)μmol/L;在其他条件相同的结果下,血浆中加入FHb为20、40、60、80、100μl时,检测结果为(13.58±0.02)、(13.41±0.02)、(13.10±0.01)、(12.60±0.06)、(12.17±0.01)μmol/L;全血保存d1、d7、d14、d21、d28和d35血浆中的RSNO含量分别为(12.94±2.92)、(10.67±2.18)、(6.87±1.24)、(7.13±1.17)、(7.01±1.66)和(5.80±1.55)μmol/L.结论荧光法测定RSNOs具有良好的准确性与可重复性;不同离心力制备的样品、不同的孵育时间、不同的铜离子浓度以及血液样品中的游离血红蛋白对荧光法检测RSNOs的结果均会有影响;全血保存过程中RSNOs含量呈下降趋势。

Objective To establish fluorescent method to determine RSNOs in plasma and to study the influencing factors;to monitor changes in RSNO levels of whole blood during storage.Methods ① Establish the standard curve and determine the accuracy and reproducibility of the fluorescence method.② Change one of the possible influencing factors（sample preparation,incubation time,etc.） to study its influence on the results.③ Nine units of whole blood were tested for RSNOs on the 1 st,7 th,14 th,21 st,28 th and 35 th days of preservation.Results Testing RSNOs in the same plasma sample for 10 times under the same experimental conditions,the coefficient of variation CV% was 3.5%.When whole blood was centrifuged under 470 g,734 g,1 057 g and 1 438 g,the plasma RSNOs were 9.22±0.14,9.97±0.11,10.40±0.05,and 10.91±0.01（μmol/L）,respectively.With copper ion concentrations of 100μmol/L,150μmol/L,and 200μmol/L,plasma RSNOs were 13.83±0.02,14.23±0.03,and 13.22±0.01（μmol/L）,respectively.After 1,1.5,2,and 2.5 h incubation,the RSNOs results were 10.34±0.04,10.75±0.02,12.31±0.09,and 10.53±0.06,（μmol/L） respectively.The plasma RSNO on the 1 st,7 th,14th,21st,28,and 35th days of whole blood preservation were 12.94±2.92,10.67±2.18,6.87±1.24,7.13±1.17,7.01±1.66,and 5.80±1.55（μmol/L）,respectively.Conclusion The determination of RSNO by fluorescence assay is accurate and reproducible.The centrifugal force,the reaction time,the concentration of copper ion and the free hemoglobin in the plasma affect the results.RSNOs in the whole blood decrease gradually during the preservation.