Luminometric single step urea assay using ATP-hydrolyzing urease.
An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 micromol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96-103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) were 3.6-8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02-1.03 and intercepts of 0.08-0.23 mmol/L.
Näslund
B
B
Clinical Research Centre and Division of Medicine, Karolinska Institute, Huddinge University Hospital, Sweden. birgitta.naslund@roche.com
Ståhle
L
L
Lundin
A
A
Anderstam
B
B
Arner
P
P
Bergström
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000737:chemistry; D000438:Alcohols / Q000737:chemistry; D000581:Amidohydrolases / Q000378:metabolism*; D049454:Firefly Luciferin; D006801:Humans; D007674:Kidney Diseases / Q000097:blood; D008156:Luciferases; D008163:Luminescent Measurements; D017551:Microdialysis; D015999:Multivariate Analysis; D006435:Renal Dialysis; D012680:Sensitivity and Specificity; D014508:Urea / Q000032:analysis* / Q000097:blood
D000438:Alcohols; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D014508:Urea; D008156:Luciferases; D000581:Amidohydrolases; C023271:urease (ATP-hydrolyzing)