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Arzneimittelforschung 2008; 58(9): 469-474
DOI: 10.1055/s-0031-1296541
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Editio Cantor Verlag Aulendorf (Germany)

Drug Metabolism Studies with the Incubated Hen’s Egg

Identification of 2,3,5-trihydroxybenzoic acid as a metabolite of gentisic acid
Lutz Kiep
1   Lutz Kiep, Brunow, Germany
,
Jens Burkhardt
2   Department of Organic Chemistry, Faculty of Biology, Chemistry and Earth Sciences, University of Bayreuth, Germany
,
Karlheinz Seifert
2   Department of Organic Chemistry, Faculty of Biology, Chemistry and Earth Sciences, University of Bayreuth, Germany
› Author Affiliations
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Publication History

Publication Date:
19 December 2011 (online)

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Abstract

Previous investigations have shown that an ex vivo model on the basis of the incubated hen’s egg represents a potential alternative to animal experimentation for xenobiotic metabolism studies. This model is characterized by administration of the xenobiotic into the yolk sac and identification of metabolites in the allantoic fluid. In the present study, gentisic acid (2,5-dihydroxybenzoic acid, 2,5-DHBA, CAS 490-79-9) was used as a test substance. The oxidative fate of this salicylate metabolite in human and animal species could not be completely elucidated until now. Inoculation of 2,5-DHBA into the incubated hen’s egg in concentrations up to 8 mg/egg did not affect embryo viability. For the metabolism studies a dose of 2 mg/egg was chosen and 2,5-DHBA was identified as the main metabolite in both the free and conjugated form (glucuronide/sulfate). Two minor metabolites were detected in addition. The first of these could be identified as 2,3,5-trihydroxybenzoic acid (2,3,5-THBA) and the second one as 2-hydroxy-5-methoxybenzoic acid (2-H-5-MBA). 2,3,5-THBA has been characterized as a metabolite of 2,5-DHBA in a vertebrate species for the first time. Since 2,3,5-THBA can be methylated to the guaiacol derivative, 2,5-dihydroxy-3-methoxybenzoic acid (2,5-DH-3-MBA) can not to be excluded as a further possible metabolite. The metabolites were identified by comparative TLC with the authentic reference substances as well as additionally by GC and GC-MS analysis of the trimethylsilyl derivatives. The structures of the synthesized reference substances were confirmed by MS, 1H and 13C NMR spectral data.

Key words

CAS 490-79-9 - Gentisic acid, drug metabolism, ex vivo model - 2-Hydroxy-5-methoxybenzoic acid - Incubated hen’s egg, alternative to animal experimentation - Salicylates - 2,3,5-Trihydroxybenzoic acid
 

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