Rao P S, Rujikarn N, Luber J M
Division of Cardiothoracic Surgery, Long Island Jewish Medical Center, New Hyde Park, NY 11042.
J Chromatogr. 1988 Dec 28;459:269-73. doi: 10.1016/s0021-9673(01)82036-7.
A direct, sensitive (50-200 ng), simple and specific high-performance liquid chromatographic (HPLC) method is described for the quantitation of oxy radicals by means of the consumption of dimethyl sulfoxide (DMSO) by the hydroxy radical and dimethylthiourea (DMTU) by hydrogen peroxide. The specific scavengers catalase and L-methionine were used to quantitate hydrogen peroxide and OH, respectively. The DMSO and DMTU peaks were separated and identified by HPLC on a Waters C18 Resolve 10-microns Radial-Pak column with an isocratic mobile phase (5% aqueous methanol) at 2 ml/min with UV detection (DMSO, 214 nm; DMTU, 240 nm). The OH concentrations were extrapolated by a luminol chemiluminescence technique. A linear relationship was obtained for DMTU consumption by hydrogen peroxide in the range 0.25-0.40 mM with a coefficient of variation (C.V.) of 8.8 +/- 2.1% and for DMSO consumption by hydroxy radicals in the range 0.1-3.2 microM OH, with a C.V. of 9.6 +/- 3.6%. The limits of detection for this method were 50 ng of hydrogen peroxide for DMTU and 200 ng of OH for DMSO. Hydrogen peroxide averaged 10.5 +/- 3.6 nmol/ml in blood and 56.4 +/- 5.3 mumol/g wet weight in left ventricular (LV) tissue. The hydroxy radical concentration was 0.1 microM in blood and 0.3 microM in LV tissue.
描述了一种直接、灵敏（50 - 200 ng）、简单且特异的高效液相色谱（HPLC）方法，用于通过羟基自由基消耗二甲基亚砜（DMSO）以及过氧化氢消耗二甲基硫脲（DMTU）来定量氧自由基。分别使用特异性清除剂过氧化氢酶和L - 甲硫氨酸来定量过氧化氢和OH。在Waters C18 Resolve 10微米径向Pak柱上，采用等度流动相（5%甲醇水溶液），流速2 ml/min，通过紫外检测（DMSO，214 nm；DMTU，240 nm）对DMSO和DMTU峰进行分离和鉴定。OH浓度通过鲁米诺化学发光技术外推得到。过氧化氢消耗DMTU在0.25 - 0.40 mM范围内呈线性关系，变异系数（C.V.）为8.8 ± 2.1%；羟基自由基消耗DMSO在0.1 - 3.2 μM OH范围内呈线性关系，C.V.为9.6 ± 3.6%。该方法对过氧化氢的检测限为DMTU对应的50 ng，对OH为DMSO对应的200 ng。血液中过氧化氢平均为10.5 ± 3.6 nmol/ml，左心室（LV）组织中为56.4 ± 5.3 μmol/g湿重。血液中羟基自由基浓度为0.1 μM，LV组织中为0.3 μM。
