Highly sensitive fluorescent method for the detection of cholesterol aldehydes formed by ozone and singlet molecular oxygen.
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.
Mansano
Fernando V
FV
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP26077, CEP 05513-970, São Paulo, SP, Brazil.
Kazaoka
Rafaella M A
RM
Ronsein
Graziella E
GE
Prado
Fernanda M
FM
Genaro-Mattos
Thiago C
TC
Uemi
Miriam
M
Di Mascio
Paolo
P
Miyamoto
Sayuri
S
D000447:Aldehydes / Q000032:analysis*; D002784:Cholesterol / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010084:Oxidation-Reduction; D010126:Ozone / Q000737:chemistry*; D026082:Singlet Oxygen / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000447:Aldehydes; D005456:Fluorescent Dyes; D026082:Singlet Oxygen; D010126:Ozone; D002784:Cholesterol