In principle ammonia may be estimated incubating samples for assay with excess 2-oxoglutarate and reduced nicotinamide adenine dinucleotide (NADH) in the presence of glutamate dehydrogenase (GLDH), observing the total decrease in extinction at 340 nm as NADH is oxidized. Methods on this basis for application to tissue extracts have been reported (l-3)) and the authors have pointed out the advantages over the more conventional rl:icrodiffusion procedures, e.g., of Conway (4) and Archibald (5).
We have employed the GLDH reaction as the basis of ammonia estimation and it has been possible to introduce two noteworthy improvements on previously described eneymic methods.
Commercially available preparations of GLDH 'advertised as 'lammonia-free" do nevertheless contain a little ammonia (1) and with the enzymic procedures hitherto recommended (l-3) this gives a reagent blank value undesirably high when it is required to estimate very small amounts of ammonia, as in freshly obtained tissue extracts. This reagent ammonia value is eliminated in the method to be described. Moreover the method is considerably less time-consuming than those previously reported when a large number of samples require assay.
Further, a reliable and highly specific method for the estimation of L-glutamine in tissue extracts has been developed, measuring the extra ammonia which appears after treatment with glutaminase: glutaminase bglutamine + Hz0 ------+ lrglutamate -+ ammonia GLDH ammonia + P-oxoglutarate + NADH + H+ -L-glutamate + NAD+ 'Most of the work is from the Thesis of P. J. Buttery accepted for the Ph.D. degree,