Abstract
Helicobacter pylori diagnosis is fundamental in the management of gastrointestinal pathologies, whose current clinical guidelines support a non-invasive ‘test-and-treat’ strategy. As such, the present work reports the basis of a new, low-cost, specific breath test based on the detection of volatile carboxylic acids resulting from the hydrolysis of short-chain aliphatic amides by H. pylori amidases. Propionamide and butyramide, which are metabolized by amidases to propionic and butyric acids, were elected for this study. Conditions for the extraction of these acids from a vapour phase were optimized concerning the use of solid-phase microextraction (SPME) followed by gas chromatography–quadrupole mass spectrometry (GC–qMS) analysis. SPME–GC–qMS was then used to detect the acids released into a vapour phase upon incubation of a H. pylori reference strain J99 or a clinical specimen with the amides. These experiments have demonstrated that the administration of less than 9 mg of propionamide and/or butyramide to H. pylori cultures, in loads recognized to cause infection (106–109 cells), resulted in the formation of detectable and/or quantifiable amounts of propionic and/or butyric acids after 30 min incubation. As such, propionic and butyric acids can be used as biomarkers for H. pylori upon incubation with the corresponding amides. SPME–GC–qMS was also used to verify the hepatic stability of the acids. These experiments were conducted in mouse liver cells and revealed no signs of metabolization that could compromise their bioavailability in future in vivo assays. Moreover, SPME–GC–qMS permitted the detection of both acids in amounts as low as 0.8 μg in systems mimicking exhaled breath, demonstrating the sensitivity of the method for these compounds.
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Acknowledgments
This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Project POCI/QUI/56393/2004, Ph.D. grant SFRH/BD/19929/2004, Postdoctoral grant SFRH/BPD/66288/2009 and the Research Unit 62/94, QOPNA (project PEst-C/QUI/UI0062/2011). The authors also gratefully thank Professor Maria João Vieira and Dr. Nuno Azevedo from the Centre of Biological Engineering of the University of Minho (Portugal) for, within the scope of project POCI/QUI/56393/2004, providing the Helicobacter pylori strains used in this study and the facilities for bacterial growth.
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Ferreira, J.A., Dias, E., Rocha, S.M. et al. Process for detecting Helicobacter pylori using aliphatic amides. Anal Bioanal Chem 401, 1889–1898 (2011). https://doi.org/10.1007/s00216-011-5259-x
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DOI: https://doi.org/10.1007/s00216-011-5259-x