A sensitive, specific, and rapid method for quantitating the minor adduct 06-butylguanine (O'BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against 06BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific 06-alkyIguanine. After addition of O'BuG and its deuterium labeled analogue (O'BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/ water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O'BuG recovery from this column were 1.53 nmol 06BuG/column and 62 ± 5%, respectively. The method was applied to evaluate O'BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylam-ine. In the first case the level of modifications present in calf thymus DNA was 104 µmol 06BuG/mol guanine, and in the second case O'BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 Mmol O'BuG/mol guanine) than after N-nitrosodibutylamine (0.34 Mmol O'BuG/mol guanine) treatment. These results indicate that O'BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms. © 1990, American Association for Cancer Research. All rights reserved.

Bonfanti, M., Magagnotti, C., Galli, A., Bagnati, R., Moret, M., Gariboldi, P., et al. (1990). DETERMINATION OF O-6-BUTYLGUANINE IN DNA BY IMMUNOAFFINITY EXTRACTION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY. CANCER RESEARCH, 50(21), 6870-6875.

DETERMINATION OF O-6-BUTYLGUANINE IN DNA BY IMMUNOAFFINITY EXTRACTION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

MORET, MASSIMO;
1990

Abstract

A sensitive, specific, and rapid method for quantitating the minor adduct 06-butylguanine (O'BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against 06BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific 06-alkyIguanine. After addition of O'BuG and its deuterium labeled analogue (O'BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/ water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O'BuG recovery from this column were 1.53 nmol 06BuG/column and 62 ± 5%, respectively. The method was applied to evaluate O'BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylam-ine. In the first case the level of modifications present in calf thymus DNA was 104 µmol 06BuG/mol guanine, and in the second case O'BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 Mmol O'BuG/mol guanine) than after N-nitrosodibutylamine (0.34 Mmol O'BuG/mol guanine) treatment. These results indicate that O'BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms. © 1990, American Association for Cancer Research. All rights reserved.
Articolo in rivista - Articolo scientifico
O-6-BUTYLGUANINE
English
1990
50
21
6870
6875
none
Bonfanti, M., Magagnotti, C., Galli, A., Bagnati, R., Moret, M., Gariboldi, P., et al. (1990). DETERMINATION OF O-6-BUTYLGUANINE IN DNA BY IMMUNOAFFINITY EXTRACTION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY. CANCER RESEARCH, 50(21), 6870-6875.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/32944
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