The fluorimetric determination of ammonia in protein-free filtrates of human blood plasma.
Ammonia has been determined in filtrates of human plasma after precipitation of the proteins by perchloric acid. After restoration of the pH to around 7.5, addition of 2-oxoglutarate, NADH and glutamate dehydrogenase (GDH) convers the ammonia to L-glutamate with oxidation of the NADH to NAD. This latter reaction was utilised in two ways. In the first, reduction of native NADH fluorescence under the conditions of the GDH reaction provided a measure of ammonia concentration. In the second, residual NADH was destroyed by acid treatment, and the fluorescent product generated from NAD under strongly alkaline conditions was assayed. The optimal requirements for both methods were defined, their linearity and precision ascertained, and their relative merits compared. The first method was convenient for "one-off" estimations, and the second for larger batches. Ammonia concentration increased in plasma and in acid protein-free filtrates of plasma irrespective of the conditions of storage; however when the latter were neutralised, storage at -20 degrees C was effective. The distribution of plasma ammonia concentration in healthy subjects was log-normal. The range for males was 21-58 mumol/1 and for females 17-51 mumol/1; this difference was statistically significant (P less than 0.01).
D000641:Ammonia / Q000097:blood* / Q000302:isolation & purification; D001798:Blood Proteins / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D008297:Male; D009243:NAD; D010084:Oxidation-Reduction; D013048:Specimen Handling; D013050:Spectrometry, Fluorescence / Q000379:methods
D001798:Blood Proteins; D009243:NAD; D000641:Ammonia