Combination of TLC blotting and gas chromatography-mass spectrometry for analysis of peroxidized cholesterol.
We have established a sensitive and convenient method for analysis of cholesterol hydroperoxides (Chol-OOHs) as trimethylsilyloxyl derivatives using diphenylpyrenylphosphine (DPPP)-thin-layer chromatography (TLC) blotting and gas chromatography-electron ionization-mass spectrometry/selected-ion monitoring (GC-EI-MS/SIM). Chol-OOH standards were prepared by photosensitized oxidation and azo radical-induced peroxidation of cholesterol. Trimethylsilyloxyl derivatives of cholesterol 5alpha-hydroperoxide (Chol 5alpha-OOH), cholesterol 7alpha-hydroperoxide (Chol 7alpha-OOH), and cholesterol 7beta-hydroperoxide (Chol 7beta-OOH) could be separated from one another in the SIM chromatogram using a fragment ion with elimination of trimethylsilanol from the molecular ion. This method was used to characterize peroxidized cholesterol from azo radical-exposed human low-density lipoprotein and UVA-irradiated human keratinocytes in the presence of hematoporphyrin. Finally, we succeeded in the quantification of each Chol-OOH isomer present in hairless mouse skin with and without UVA irradiation by use of beta-sitosterol hydroperoxide as internal standard. The accumulation of Chol 5alpha-OOH with Chol 7alpha/betaOOH in the skin indicates that singlet molecular oxygen ((1)O(2)) participated in the peroxidation of skin cholesterol, because Chol 5alpha-OOH is known to be a (1)O(2) specific cholesterol peroxidation product. We concluded that the combination of DPPP-TLC blotting and GC-EI-MS/SIM is useful for quantifying peroxidized cholesterol in biological samples and confirming the participation of (1)O(2) in oxidative stress.
Minami
Yuko
Y
Department of Food Science, Graduate School of Nutrition and Bioscience, The University of Tokushima, Tokushima, 770-8503, Japan.
Yokoi
Sayuri
S
Setoyama
Mari
M
Bando
Noriko
N
Takeda
Sayaka
S
Kawai
Yoshichika
Y
Terao
Junji
J
D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000032:analysis; D002855:Chromatography, Thin Layer / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006415:Hematoporphyrins / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000528:radiation effects; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000097:blood / Q000737:chemistry; D008297:Male; D051379:Mice; D008812:Mice, Hairless; D012867:Skin / Q000737:chemistry / Q000528:radiation effects; D014466:Ultraviolet Rays
D006415:Hematoporphyrins; D008077:Lipoproteins, LDL; C002843:cholesterol hydroperoxide; D002784:Cholesterol
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