Templar J, Kon S P, Milligan T P, Newman D J, Raftery M J
Department of Renal Medicine and Transplantation, Royal London Hospital, Whitechapel, UK.
Nephrol Dial Transplant. 1999 Apr;14(4):946-51. doi: 10.1093/ndt/14.4.946.
Reactive oxygen species and particularly free radical induced lipid peroxidative tissue damage have been implicated in the pathogenesis of various renal diseases. Lipid peroxidation is assessed indirectly by the measurement of secondary products, such as malondialdehyde (MDA), using the widely employed thiobarbituric acid reactive substances (TBARS) method. However, this method lacks sensitivity and specificity. We have therefore developed and validated an HPLC (high-performance liquid chromatography) method for measurement of MDA and applied this to a variety of plasma samples in renal patients.
The optimized method involves antioxidant treatment of the plasma sample, followed by a protein precipitation step using trichloroacetic acid, acid hydrolysis and formation of an MDA thiobarbituric acid complex. The MDA-(TBA)2 adduct is separated from other interfering compounds by C18 reverse-phase HPLC techniques, with visible detection at 532 nm.
The assay was linear over the ranges 0.25-1.0 microM MDA and the detection limit was 0.06 microM MDA. Within-run precision was <4.5% and between-run precision was <10.0%. MDA plasma concentrations (mean+/-SD) were higher in ESRF diabetic patients (0.32 +/- 0.14 microM, n=20), non-diabetic ESRF patients (0.32 +/- 0.09 microM, n=20), and CRF patients (0.14 +/- 0.06 microM, n=40) compared to healthy controls (0.11 +/- 0.03 microM, n=40), (P < 0.001, P < 0.001 and P = 0.008). Levels were similar in healthy controls with normal renal function and transplanted patients (0.12 +/- 0.03 microM MDA, n=40), (P=NS). No correlation was observed between MDA and creatinine levels (r2 = 0.05, n=80), which suggests that MDA does not correlate with the degree of renal impairment. We matched CRF patients with glomerular and non-glomerular causes of renal failure for creatinine levels and found that MDA levels were higher in patients with glomerulonephritis (0.16 +/- 0.06 microM) than in those with CRF from non-glomerular causes (0.12 +/- 0.04 microM, P = 0.002).
We have introduced a reliable and sensitive HPLC technique to enhance the specificity of MDA-(TBA)2 measurement, with a significant improvement in HPLC column life. Using this method, picomole quantities of MDA can be detected in plasma. We have shown that MDA levels are significantly raised in patients with CRF due to glomerulonephritis, regardless of serum creatinine, which suggests that there is oxidative injury independent of any possible MDA retention due to renal impairment.
活性氧，尤其是自由基诱导的脂质过氧化组织损伤，与多种肾脏疾病的发病机制有关。脂质过氧化通过测量诸如丙二醛（MDA）等次级产物间接评估，采用广泛应用的硫代巴比妥酸反应物质（TBARS）法。然而，该方法缺乏敏感性和特异性。因此，我们开发并验证了一种用于测量MDA的高效液相色谱（HPLC）方法，并将其应用于肾病患者的各种血浆样本。
优化后的方法包括对血浆样本进行抗氧化处理，随后使用三氯乙酸进行蛋白质沉淀步骤、酸水解以及形成MDA硫代巴比妥酸复合物。通过C18反相HPLC技术将MDA -（TBA）2加合物与其他干扰化合物分离，在532nm处进行可见检测。
该测定在0.25 - 1.0微摩尔/升MDA范围内呈线性，检测限为0.06微摩尔/升MDA。批内精密度<4.5%，批间精密度<10.0%。与健康对照组（0.11±0.03微摩尔/升，n = 40）相比，终末期肾衰竭糖尿病患者（0.32±0.14微摩尔/升，n = 20）、非糖尿病终末期肾衰竭患者（0.32±0.09微摩尔/升，n = 20）和慢性肾衰竭患者（0.14±0.06微摩尔/升，n = 40）的血浆MDA浓度（均值±标准差）更高，（P < 0.001，P < 0.001和P = 0.008）。肾功能正常的健康对照组和移植患者（0.12±0.03微摩尔/升MDA，n = 40）的水平相似，（P = 无显著性差异）。未观察到MDA与肌酐水平之间的相关性（r2 = 0.05，n = 80），这表明MDA与肾功能损害程度无关。我们将慢性肾衰竭患者按肾小球和非肾小球性肾衰竭病因进行肌酐水平匹配，发现肾小球肾炎患者（0.16±0.06微摩尔/升）的MDA水平高于非肾小球性病因导致的慢性肾衰竭患者（0.12±0.04微摩尔/升，P = 0.002）。
我们引入了一种可靠且灵敏的HPLC技术，以提高MDA -（TBA）2测量的特异性，同时显著延长HPLC柱的使用寿命。使用该方法，可在血浆中检测到皮摩尔量的MDA。我们已表明，无论血清肌酐如何，因肾小球肾炎导致的慢性肾衰竭患者的MDA水平显著升高，这表明存在独立于肾功能损害可能导致的任何MDA潴留的氧化损伤。
