An enzymatic cycling method for the determination of ammonia

An enzymatic cycling method for the determination of ammonia has been developed by using glutamate dehydrogenase(GLDH) and glutamate oxidase(GLOD). The measurement was carried out by observing the decrease in light absorbance of NADPH continuously at the wavelength of 340 nm. The sample volume necessary for one assay is only 50 µl. The calibration curve and the senitivity were dependent on the reaction time. In the case of 1.5U/assay for GLDH and 21 U/assay for GLOD with the reaction time of 30 min for example, there was linearity up to 300µg/l00ml. Compared with the other method without any addition of GLOD, this method was 13 times more sensitive. Under the conditions of this method, a change of 0.001 in light absorbance was equivalent to an ammonia volume of 1.7 µg/l00ml, and the number of cycles was two per minute. Since the GLOD used in this method is specific for L-glutamate, its application to this cycling method was carried out by inhibiting glutamate decarboxylase (GLDC) with aminooxyacetic acid after eliminating L-glutamate with GLDC, when any L-glutamate was suspected to exist in the samples. L-Glutamate at 80 mM could be eliminated by reacting with GLDC at 6.0 U/ml for 10 min at 37 °C. Due to the high substrate specificity of GLOD, this method is strongly affected by the presence of large amounts of L-aspartate and L-glutamine in the samples.