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Arch Oral Biol. 2008 May;53(5):423-8. Epub 2007 Dec 21.

Salivary aldehyde dehydrogenase--reversible oxidation of the enzyme and its inhibition by caffeine, investigated using fluorimetric method.

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1
Department of Biophysics, University of Warmia and Mazury, 4 Oczapowskiego Street, PL-10-719 Olsztyn, Poland. jacek.wie@uwm.edu.pl

Abstract

OBJECTIVE:

We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme.

DESIGN:

Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD(+). Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate.

RESULTS:

Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K(m) of 7.9 microM at pH 7.3. The apparent K(m) for NAD(+) was 1.2 microM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH.

CONCLUSIONS:

Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.

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