ABSTRACT
O6-(Carboxymethyl)-2'-deoxyguanosine (O6-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by O6-alkylguanine transferases. O6-CMdG has been synthesized by an unambiguous route involving the introduction of a methyl glycolate moiety at C6 of a 3',5'-bis-O-(methoxyacetyl)dGuo derivative by displacement of a quinuclidinium ion. Methanolysis of the methoxyacetyl groups and calcium hydroxide-mediated hydrolysis of the methyl ester afforded the calcium salt of O6-CMdG in good yield. A similar route was used to synthesize O6-(carboxymethyl)guanosine (O6-CMGuo), which was used to prepare protein conjugates for immunization. Rabbit antisera were prepared, and a quantitative competitive ELISA was developed which showed 50% inhibition at 2 pmol of O6-CMdG/ well. O6-CMGuo was 30 times less cross-reactive (50% inhibition at 60 pmol/well), and normal nucleosides and carboxymethylated purines did not cross-react to any significant extent. The antiserum was also used to prepare reusable immunoaffinity columns which retained O6-CMdG. The binding of O6-CMdG was so strong that conditions used to elute the adduct (1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) of the glycosidic bond to give O6-CMguanine which was detected by HPLC with fluorescence detection. DNA treated with azaserine (5 mmol), N-(N'-acetyl-L-prolyl)-N-nitrosoglycine (5 mmol), and potassium diazoacetate (5 mmol) afforded O6-CMdG at levels of 7.3, 393.9, and 496 mumol of O6-CMdG/mol of dG. The antiserum also recognized O6-CMdG in intact DNA.
Subject(s)
Carcinogens/chemical synthesis , DNA Adducts/chemical synthesis , Deoxyguanosine/analogs & derivatives , Glycine/chemistry , Animals , Carcinogens/chemistry , Cattle , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cross Reactions/immunology , DNA Adducts/chemistry , DNA Adducts/immunology , Deoxyguanosine/analysis , Deoxyguanosine/chemical synthesis , Enzyme-Linked Immunosorbent Assay , Glycine/analogs & derivatives , Immunoglobulin G/analysis , Immunoglobulin G/isolation & purification , Nitrosation , RabbitsABSTRACT
Twenty-six patients admitted to hospital for treatment of severe exacerbations of asthma unresponsive to bronchodilators were assigned to high-, medium-, or low-dose corticosteroid treatment regimens. The rates of recovery were assessed by changes in pulse rate, peak expiratory flow rate, and spirometric measurements and were not related to the dose of corticosteroids given. Very high systemic doses of corticosteroids do not offer significant advantages in treating severe exacerbations of asthma.
