Flow monitoring of NADH consumption in bioassays based on packed-bed reactors bearing NAD+-dependent dehydrogenases: Determination of acetaldehyde using alcohol dehydrogenase

An enzymatic method for the individual or simultaneous determination of pyruvic acid and acetaldehyde is described. Alcohol dehydrogenase (ADH) was immobilized onto aminopropyl-modified controlled pore glass, which was then used for the construction of packed-bed (PB) reactors. ADH catalyses the reduction of acetaldehyde to ethanol, in the presence of the coenzyme NADH, which is oxidized to NAD+. Photometric measurements in a fully automated flow injection (FI) manifold are used to monitor the decrease of NADH absorbance at 340 nm. The possibility of pyruvate measurements, by combining the above mentioned system with soluble pyruvate decarboxylase (PyDC) is also demonstrated. PyDC catalyses the decarboxylation of pyruvate to acetaldehyde. Analytical parameters such as the buffering system, working pH, flow rate, sample size, and NADH concentration were studied. The interference of various compounds present in real samples was also investigated. Linear calibration graphs over the ranges 0.08–1.25 and 0.04–0.4 mM acetaldehyde were constructed in the presence of 50 mM succinate pH 7.5 and 50 mM phosphate pH 7.0 buffering systems, respectively. A linear calibration graph over the range 0.08–1.25 mM pyruvate was also constructed in the presence of 50 mM succinate pH 7.5 buffer solution. The reactors remain active for more than 6 months under specified storage conditions. The maximal sample throughput is 30 h−1 and the R.S.D. of the method is 0.9% for 0.2 mM acetaldehyde (<F>n=6</F>). The suitability of the proposed method for real samples was tested by recovery studies. [Copyright &y& Elsevier]