Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP26077, CEP 05513-970, São Paulo, SP, Brazil.
Anal Chem. 2010 Aug 15;82(16):6775-81. doi: 10.1021/ac1006427.
Cholesterol oxidation gives rise to a mixture of oxidized products. Different types of products are generated according to the reactive species being involved. Recently, attention has been focused on two cholesterol aldehydes, 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxyaldehyde (1a) and 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (1b). These aldehydes can be generated by ozone-, as well as by singlet molecular oxygen-mediated cholesterol oxidation. It has been suggested that 1b is preferentially formed by ozone and 1a is preferentially formed by singlet molecular oxygen. In this study we describe the use of 1-pyrenebutyric hydrazine (PBH) as a fluorescent probe for the detection of cholesterol aldehydes. The formation of the fluorescent adduct between 1a with PBH was confirmed by HPLC-MS/MS. The fluorescence spectra of PBH did not change upon binding to the aldehyde. Moreover, the derivatization was also effective in the absence of an acidified medium, which is critical to avoid the formation of cholesterol aldehydes through Hock cleavage of 5alpha-hydroperoxycholesterol. In conclusion, PBH can be used as an efficient fluorescent probe for the detection/quantification of cholesterol aldehydes in biological samples. Its analysis by HPLC coupled to a fluorescent detector provides a sensitive and specific way to quantify cholesterol aldehydes in the low femtomol range.
胆固醇氧化会产生氧化产物的混合物。不同类型的产物是根据参与的反应性物质生成的。最近，人们关注的焦点是两种胆固醇醛，3β-羟基-5β-羟基-B-降胆甾烷-6β-羧醛（1a）和 3β-羟基-5-氧代-5,6-降胆甾烷-6-醛（1b）。这些醛可以通过臭氧以及单线态氧介导的胆固醇氧化生成。有人认为 1b 优先由臭氧生成，1a 优先由单线态氧生成。在这项研究中，我们描述了使用 1-蒽丁酸肼（PBH）作为检测胆固醇醛的荧光探针。通过 HPLC-MS/MS 证实了 1a 与 PBH 之间形成了荧光加合物。PBH 的荧光光谱在与醛结合时没有变化。此外，衍生化在没有酸化介质的情况下也很有效，这对于避免通过 5α-羟过氧胆固醇的 Hock 裂解形成胆固醇醛至关重要。总之，PBH 可以用作检测/定量生物样品中胆固醇醛的有效荧光探针。通过与荧光检测器耦合的 HPLC 进行分析，可以提供一种在低 femtomol 范围内定量胆固醇醛的灵敏和特异的方法。
