Separation and quantitation of peroxidized phospholipids using high-performance thin-layer chromatography with tetramethyl-p-phenylenediamine detection
Separation and quantitation of peroxidized phospholipids using high-performance thin-layer chromatography with tetramethyl-p-phenylenediamine detection
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DOI：
10.1016/j.ab.2003.12.021
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发表时间：
2004-04-01
影响因子：
2.9
通讯作者：
Girotti, AW
中科院分区：
文献类型：
--
作者：
Kriska, T;Girotti, AW
开发了一种选择性测定复杂脂质群中磷脂氢过氧化物 (PLOOH) 家族的简单方法。该方法称为 HPTLC-TPD，基于正相高效薄层色谱法进行 PLOOH 分离，然后使用 N,N,N',N'-四甲基对苯二胺进行喷雾检测，并对紫色谱带进行光密度扫描。亲本磷脂和酒精类似物不发生反应。建立了由磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺制备的氢过氧化物标准品的校准曲线、动态范围和检测限。和心磷脂。对于所有 PLOOH 类别，响应度对于至少 10 nmol 的样品负载呈线性，检测限为 0.1-0.2 nmol。 HPTLC-TPD 数据通过对重复样品进行更复杂的柱色谱和还原模式电化学检测来验证。使用来自两个测试系统的脂质提取物证明了新技术的普遍适用性：(i) 光过氧化脂质体膜和 (ii) 受到呼吸抑制剂抗霉素 A 氧化应激的肿瘤细胞。 HPTLC-TPD 提供了一种方便、特异且高度灵敏的方法，用于定量复杂天然混合物中的各个 PLOOH 家族。 (C) 2004 Elsevier Inc. 保留所有权利。
A simple method for the selective determination of phospholipid hydroperoxide (PLOOH) families in complex lipid populations has been developed. Referred to as HPTLC-TPD, the method is based on PLOOH separation by normal-phase high-performance thin-layer chromatography, followed by spray detection with N, N, N', N'-tetramethyl-p-phenylenediamine and densitometric scanning of the purple bands. Parental phospholipids and alcohol analogues are unreactive. Calibration curves, dynamic ranges, and detection limits were established for hydroperoxide standards prepared from phospatidylcholine, phosphatidylserine, phosphatidylethanolamine. and cardiolipin. For all PLOOH classes, responsiveness was linear out to at least 10 nmol of sample load, the detection limit being 0.1-0.2 nmol. HPTLC-TPD data were validated by subjecting duplicate samples to more complex column chromatography with reductive-mode electrochemical detection. General applicability of the new technique was demonstrated using lipid extracts from two test systems: (i) photoperoxidized liposomal membranes and (ii) tumor cells that had been oxidatively stressed with the respiratory inhibitor antimycin A. HPTLC-TPD provides a convenient, specific, and highly sensitive means for quantifying individual PLOOH families in complex natural mixtures. (C) 2004 Elsevier Inc. All rights reserved.