Worldwide studies find elevated body fluid adenosine deaminase (ADA) levels associated with mycobacterial infections in adjacent tissues. Those results are usually provided by the easily standardized colorimetric procedure of Giusti. However that method cannot be applied to sputa and broncho-alveolar lavage samples because 20-25 per cent of such specimens, both normal and tuberculous, contain large amounts of low molecular weight amines that interfer with the colorimetric procedure. Data presented here support a quantitative fluorometric assay for ADA applicable to sputum and lavage specimens.
The method is based on conversion of residual substrate adenosine (Adn) to the fluorescent 1,N6-ethenoadenosine (e-Adn) on reaction with chloroacetaldehyde (CAA; Barrio et al, Biochem. Biophys. Res. Comm., 46, 597-603 (1972). The assay procedure is achieved with a substrate concentration, 4.0 mM, sufficient to assure maximum velocities of the enzyme levels under test, but low enough to permit accurate measurements of reductions in substrate concentrations. Samples from those reactions mixtures are added to 1,000-mole excesses of CAA in acetate buffer, pH 4.0, and heated 1 hr at 75 OC. The fluorescent e-Adn product is measured at an emission wavelength of 410 nm, with excitation at 313 nm.
While ADA assays by this procedure can be carried out at physiologic pH the reactions described in this presentation are at pH 6.4-6.5, the pH range mandated for the colorimetric assay in order to trap ammonia released from the Adn. Thus, proof of efficacy of the fluorometric procedure is indicated by correlations between results from the two procedures, at the same pH and on the same specimens.
This fluorometric procedure is applicability to quantitative ADA measurements on all sputa.
The technology extends the spectrum of quantitative sputum enzyme assays for diagnostic and therapeutic evaluations.
K.D. Miller, None.