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Combination of TLC Blotting and Gas Chromatography–Mass Spectrometry for Analysis of Peroxidized Cholesterol

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Lipids

Abstract

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 5α-hydroperoxide (Chol 5α-OOH), cholesterol 7α-hydroperoxide (Chol 7α-OOH), and cholesterol 7β-hydroperoxide (Chol 7β-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 β-sitosterol hydroperoxide as internal standard. The accumulation of Chol 5α-OOH with Chol 7α/βOOH in the skin indicates that singlet molecular oxygen (1O2) participated in the peroxidation of skin cholesterol, because Chol 5α-OOH is known to be a 1O2 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 1O2 in oxidative stress.

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Abbreviations

AAPH:

2,2′-Azobis(2-amidinopropane)dihydrochloride

AMVN :

2,4-Dimethylvaleronitrile

Chol-OH:

Cholesterol hydroxide

Chol 7α-OH:

7α-Hydroxycholesterol

Chol 7β-OH:

7β-Hydroxycholesterol

Chol-OOH:

Cholesterol hydroperoxide

Chol 7α-OOH:

Cholesterol 7α-hydroperoxide

Chol 7β-OOH:

Cholesterol 7β-hydroperoxide

Chol 5α-OOH:

Cholesterol 5α-hydroperoxide

DPPP:

Diphenyl-1-pyrenylphosphine

GC–EI–MS:

Gas chromatography–electron ionization–mass spectrometry

HPLC:

High-performance liquid chromatography

LAOOH:

Linoleic acid hydroperoxide

LDL:

Low-density lipoprotein

LOOH:

Lipid hydroperoxide

LOH:

Lipid hydroxide

1O2 :

Singlet molecular oxygen

PCOOH :

Phosphatidylcholine hydroperoxide

PEOOH :

Phosphatidylethanolamine hydroperoxide

PVDF :

Polyvinylidenedifluoride

PTFE :

Polytrifluoroethylene

SIM :

Selected-ion monitoring

St-OOH :

β-Sitosterol hydroperoxide

TLC :

Thin-layer chromatography

TLOOH :

Trilinoleoylglycerol hydroperoxide

UVA :

Ultraviolet A

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Acknowledgments

This study was partly supported by the program of the twenty-first century Center of Excellence “Human nutritional science on stress control” and a grant-in aid for scientific research of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Department of Food Science, Graduate School of Nutrition and Bioscience, The University of Tokushima, Tokushima, 770-8503, Japan

    Yuko Minami, Sayuri Yokoi, Mari Setoyama, Noriko Bando, Sayaka Takeda, Yoshichika Kawai & Junji Terao

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  1. Yuko Minami
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  2. Sayuri Yokoi
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Correspondence to Junji Terao.

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Minami, Y., Yokoi, S., Setoyama, M. et al. Combination of TLC Blotting and Gas Chromatography–Mass Spectrometry for Analysis of Peroxidized Cholesterol. Lipids 42, 1055–1063 (2007). https://doi.org/10.1007/s11745-007-3099-4

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  • DOI: https://doi.org/10.1007/s11745-007-3099-4

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