Zamburlini A, Maiorino M, Barbera P, Roveri A, Ursini F
Department of Biological Chemistry, University of Padua, Italy.
Anal Biochem. 1995 Nov 20;232(1):107-13. doi: 10.1006/abio.1995.9953.
A single photon counting procedure for measuring lipid hydroperoxides in human plasma or LDL-VLDL, escaping from extraction and chromatography, is described. This appears to be a relevant procedure because the recovery of phospholipid hydroperoxides from plasma is a critical point which, in our hands, was limited and poorly reproducible. The sample is added to a reaction mixture containing luminol, hemin, and Triton X-100 in an alkaline buffer, the photon emission is recorded, and the data are processed using the monoexponential decay of the photon emission rate. The measurement is applied to (a) plasma passed through a "desalting" cartridge to eliminate the small water-soluble antioxidants which inhibit the chemiluminescent process or (b) apo-B-containing lipoproteins (LDL-VLDL) isolated by heparin-Sepharose affinity chromatography. The content of lipid hydroperoxides is calculated using an internal calibration with palmitoyllinoleoylphosphatidylcholine hydroperoxide. This procedure, based on a single photon counting technology, was adopted to produce reliable results using samples from which inhibitors of the photon emission process have not been completely eliminated. The specificity of the signal for lipid hydroperoxides was validated by its complete disappearance following incubation of the sample with glutathione and phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12), the sole enzyme specific for all classes of lipid hydroperoxides in lipoproteins. The interassay variability was < 10%. The results indicated that the concentration of lipid hydroperoxides in the plasma of 20 healthy subjects was 353 +/- 78 nM. In different subjects, LDL-VLDL accounted for 40-80% of the lipid hydroperoxides in plasma.
本文描述了一种用于测量人血浆或低密度脂蛋白-极低密度脂蛋白（LDL-VLDL）中脂质氢过氧化物的单光子计数方法，该方法可避免脂质氢过氧化物在提取和色谱过程中的损失。这似乎是一种相关的方法，因为从血浆中回收磷脂氢过氧化物是一个关键点，在我们的实验中，回收率有限且重复性差。将样品加入到含有鲁米诺、血红素和吐温X-100的碱性缓冲液反应混合物中，记录光子发射情况，并使用光子发射率的单指数衰减对数据进行处理。该测量方法应用于：（a）通过“脱盐”柱的血浆，以去除抑制化学发光过程的小分子水溶性抗氧化剂；或（b）通过肝素-琼脂糖亲和色谱法分离的载脂蛋白B的脂蛋白（LDL-VLDL）。脂质氢过氧化物的含量通过棕榈酰油酰磷脂酰胆碱氢过氧化物进行内部校准来计算。该方法基于单光子计数技术，采用未完全去除光子发射过程抑制剂的样品也能产生可靠结果。通过将样品与谷胱甘肽和磷脂氢过氧化物谷胱甘肽过氧化物酶（EC 1.11.1.12，脂蛋白中所有类脂质氢过氧化物唯一特异性的酶）孵育后信号完全消失，验证了脂质氢过氧化物信号的特异性。批间变异小于10%。结果表明，20名健康受试者血浆中脂质氢过氧化物的浓度为353±78 nM。在不同受试者中，LDL-VLDL占血浆中脂质氢过氧化物的40%-80%。
