Simple and Sensitive Determination of Urea in Serum and Urine CLIN.CHEM.38/5, 619-623(1992) CLINICALCHEMISTRY, Vol.38, No. 5, 1992 619 Jean-Luc Orsonneau, Catherine Massoubre, Marie Cabanes, and Patrick Lustenberger In this method for serum and urinary urea determination, the same reagent is used without predilution of urine samples. The method is based on the pH increase result- ing from the ammonia released by urease hydrolysis of urea. oCresolphthalein complexone is used to monitorthe pH change colorimetrically.Urea concentrationand absor- bance at 570 nm are linearly related for concentrationsas great as 600 mmol/L for urine samples and 100 mmol/L for serum. There are no clinicallysignificantinterferencesfrom physiologicalsubstances or drugs, and precision and ac- curacy are excellent (CV -2%, except at very low concen- trations in serum; analytical recovery was 99% in urine, 100% in serum). Results by this method (y) and by the Astra method (x) for urine correlated well (y = 0.991x - 2.87, S = 9.21, r = 0.994), as did the results by this method and by the total enzymatic method (x’) for serum (y = 1.002x’ + 0.192, S’ = 0.598, r = 0.997). This method is applicable to automated as well as manual instruments, and one-reagent or two-reagent formats can be used. Additional Keyphrases: enzymatic methods cresolphtha!eln complexone colonmetr/ Urea was one of the first substances to be determined in biological fluids (1). Early methods for measuring urea involved chemical reagents, of which only o-ph- thalaldehyde (2-4) and diacetylmonoxime (5) are still used. Because of the use of corrosive reagents or incu- bation temperature in these assays, they have been widely replaced by enzymatic methods. All enzymatic methods include hydrolysis of urea by urease (EC 3.5.1.5). The urea concentration can then be measured directly by a differential pH technique (6), an ammonia electrode (7), or a conductivity increase-this last being used in the Astra#{174} and Synchron#{174} systems (Beckman Instruments Inc., Clinical Instrument Divi- sion, Brea, CA 92621). The other enzymatic methods require a second step for the photometric measure of ammonia generated in the urease step. The Berthelot reaction is the only one still being used among the chemical reactions. But again, the reagents are corrosive and the method cannot be processed on numerous auto- mated analyzers. Coupling the urease reaction with L-glutamate dehydrogenase (EC 1.4.1.2) is commonly used and is suitable for all analyzers equipped with an ultraviolet spectrophotometer (8). End point or kinetic Laboratoire de Chimie A, C.H.R. Hotel-Dieu, 44035 Nantea Cedex 01, France. Received May 8, 1991; accepted February 28, 1992. mode can be used, and reading absorbance in the visible range is also possible (9). Two other methods have been described recently-a chemiluminometric method (10) involving L-glutamate dehydrogenase and L-glut.amate oxidase (EC 1.4.3.11), and a method (11) with coupled glutamine synthetase (EC 6.3.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate oxidase (EC 1.2.3.3), and peroxidase (EC 1.11.1.7)-but these have not yet been made com- mercially available. The use of glutamate dehydrogenase makes all the fully enzymatic methods expensive. Moreover, these are not directly suitable for urine samples because of lack of linearity and the presence of large amounts of endogenous ammonia, which might significantly affect the results. We propose here a method based on the pH increase appearing alter urea hydrolysis by urease. The pH variation is monitored by a colorimetric quantification of o-cresolphthalein complexone, a pH indicator dye. The absorption of a pH indicator dye is not usually linearly related to the H’ concentration. However, the use of a buffer and a pH indicator with adequately chosen plC,, and pH range leads to a proportional re- sponse (12), which we used to adapt this approach to urea determination. Materials and Methods Reagents. The color reagent has the following compo- sition: o-cresolphthalein complexone (Merck, Darm- stadt, F.R.G.; no. 7297), 2.5 mmol/L; EDTA, tetrasodium salt (Prolabo, Paris, France; no. 20299.291), 5 mmol/L; Ti-is (Merck; no. 8382), 30 mmol/L; and sodium azide (Merck; no. 6688), 15.4 mmol/L. The pH was adjusted to 7.40 with 1 mol/L HC1 solution. This reagent is stable at least six months at 4#{176}C. The enzyme reagent was prepared by dissolving urease (Boehringer Mannheim France, Meylan, France; no. 791709) in isotonic saline (150 mmol/L NaCl) to give a final concentration 45 kU/L. The reagent is stable for at least six weeks at 4#{176}C. Procedures. A serum (15 pL) or urine (3 ML)sample is mixed with 300 1zLof color reagent and the absorbance at 570 nm (A0) is measured. Then 30 ML of enzyme reagent is added and the absorbance (A5) is measured after a 5-mm incubation at 30#{176}C. The difference (A5 - A0) is then compared with those of the calibrators, to calculate the urea concentration. For this study, we used a Hitachi 717 (Boehringer Mannheim France) at the settings shown in Table 1. Comparison studies were made with the fully enzymatic method (Boehringer Mannheim France; nos. 791695 and