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).


CONCLUSIONS
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.
Yamaguchi
Fumihiko
F
Planova Technology Development, Asahi Kasei Pharma Corporation, 6-4158 Asahimachi, Nobeoka, Miyazaki 882-0847, Japan.Miyazaki, Japan. yamaguchi.fb@om.asahi-kasei.co.jp
Etoh
Takashi
T
Takahashi
Mamoru
M
Misaki
Hideo
H
Sakuraba
Haruhiko
H
Ohshima
Toshihisa
T
D019732:Amide Synthases / Q000097:blood*; D000641:Ammonia / Q000097:blood*; D004796:Clinical Enzyme Tests / Q000379:methods* / Q000592:standards; D004795:Enzyme Stability; D005260:Female; D042843:Glucose 1-Dehydrogenase / Q000097:blood; D006801:Humans; D008297:Male; D009252:NADPH Dehydrogenase / Q000097:blood; D012680:Sensitivity and Specificity
D000641:Ammonia; D042843:Glucose 1-Dehydrogenase; D009252:NADPH Dehydrogenase; D019732:Amide Synthases; C038479:NAD+ synthase