A new enzymatic cycling method for ammonia assay using NAD synthetase.

BACKGROUND: Ammonia is an important marker for liver diseases such as hepatitis and hepatic cirrhosis. Several methods have been developed for ammonia analysis. In particular, the enzymatic assay using glutamate dehydrogenase has been widely used. However, this method is not necessarily high in sensitivity and accuracy due to inhibition by interferences in plasma and instability over long-term storage. METHODS: We developed an ammonia assay using a system consisting of three enzymes, NAD synthetase (NADS; EC 6.3.1.5), glucose dehydrogenase (GlcDH; EC 1.1.1.47), and diaphorase (DI; EC 1.6.99.2). RESULTS: The calibration curve for ammonia with the cycling method was linear (r=0.999) up to 300 micromol/l. The within-run CVs of 10 and 20 micromol/l NH4Cl solutions and 24.1 micromol/l ammonia in human plasma were 2.3%, 1.5%, and 2.8%, respectively. The between-run CVs of them were 4.5%, 3.1%, and 2.8%, respectively. The recovery was between 96.3% and 105%, and the limit of detection was 2.4 micromol/l. No significant interference was observed with addition of the following components: hemoglobin, bilirubin, chyle, EDTA, heparin, and sodium citrate. Due to the high degree of specificity of NAD synthetase to ammonia, no amino compounds exhibited any effect on the ammonia assay. A high correlation was obtained between results of the present method (y) and a conventional glutamate dehydrogenase method in regression analysis; y=0.944x-6.160 with r=0.993 (n=125). However, an addition error was observed from Bland-Altman analysis (the 95% limits of agreement between the two methods; 9.51+/-5.92 micromol/l). CONCLUSION: This new enzymatic method is more sensitive, precise, and accurate than the conventional method. In particular, accurate assay for ammonia can be performed without interference in the presence of various compounds.

Hypoxanthine-guanine phosphoribosyltransferase deficiency and erythrocyte synthesis of pyridine coenzymes.

Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.

Simple and rapid method for determining nicotinamide adenine dinucleotide synthetase activity by high-performance liquid chromatography.

A method is described for the determination of nicotinamide adenine dinucleotide synthetase (NADS) activity in human blood. Using high-performance liquid chromatography (HPLC), the formed NAD is separated from the substrates and the other blood components in less than 13 min. The activity of NADS determined by HPLC is closely correlated with that determined by the conventional spectrophotometric method, which requires two steps of enzyme reaction. The present method is simple and reliable and facilitates the routine analysis of NADS activity.