Two-point kinetic determination of uric acid in serum with uricase, catalase and aldehyde dehydrogenase on the KONE CD analyser

Two methods are commonly used for the determination of uric acid--the first is according to Praetorius and Poulsen [1] and the second was devised by Kageyama I-2]. Neither method is readily applicable to he KONE CD analyser (manufactured by KONE OY, Finland). The new aldehyde dehydrogenase (ALDH) method, proposed by Haeckel [3] and improved by Bartl et al. 1-4-], overcomes many of the disadvantages in Praetorius’s and Kageyama’s methods. This paper presents the results of tests on two commerically available reagents for the ALDH method for their applicability to the KONE CD. One of these reagents is produced by Boehringer Mannheim and is formulated according to Bartl. The other is manufactured by Human and is formulated according to the original Haeckel prescription= Bartl’s method avoids the disturbing effect of ethanolconverting enzymes by the addition of oxalic acid and pyrazole, and also has a large linear range due to the use of xanthine, which is a competitive inhibitor of uricase. Moss [5] described in detail the enzymatic determination of uric acid by the equilibrium procedure, measurement of two-point kinetics, and measurement of the initial rate of reaction. Moss showed that two-point kinetic measurement is the best method available. Due to the long lag time of the KONE CD’s initial reading, a blank-corrected equilibrium method cannot be used. Normal end-point determination appears to be slow and reagent consuming. It was therefore decided to optimize the two-point kinetic method for use on the KONE CD.


Introduction
Two methods are commonly used for the determination of uric acid--the first is according to Praetorius and Poulsen [1] and the second was devised by Kageyama I-2]. Neither method is readily applicable to he KONE CD analyser (manufactured by KONE OY, Finland).
The new aldehyde dehydrogenase (ALDH) method, proposed by Haeckel [3] and improved by Bartl  Kageyama's methods. This paper presents the results of tests on two commerically available reagents for the ALDH method for their applicability to the KONE CD. One of these reagents is produced by Boehringer Mannheim and is formulated according to Bartl. The other is manufactured by Human and is formulated according to the original Haeckel prescription= Bartl's method avoids the disturbing effect of ethanolconverting enzymes by the addition of oxalic acid and pyrazole, and also has a large linear range due to the use of xanthine, which is a competitive inhibitor of uricase. Moss [5] described in detail the enzymatic determination of uric acid by the equilibrium procedure, measurement of two-point kinetics, and measurement of the initial rate of reaction. Moss showed that two-point kinetic measurement is the best method available.
Due to the long lag time of the KONE CD's initial reading, a blank-corrected equilibrium method cannot be used. Normal end-point determination appears to be slow and reagent consuming. It was therefore decided to optimize the two-point kinetic method for use on the KONE CD. An=absorbance after completion of the reaction, and At= the absorbance at time after starting the reaction). The reagent solution was prepared by dissolving a bottle of lyophilisate in 20ml of buffer and adding 100 ul ofuricase (see curve in figure  1). To test whether this solution can be diluted further, a 20ml aliquot was diluted with 14 ml of demineralized water (dilution factor 1.7; see curve II in figure 1) and another 20ml aliquot was diluted with 28 ml ofdemineralized water (dilution factor 2.4; see curve II! in figure 1). Under all three reaction conditions, the concentration of uric acid in the cuvette is 60 #mol/1.

Reafents
As figure illustrates, the reaction does follow pseudo-firstorder kinetics over the measurement interval both for diluted and undiluted reagents. Table  presents the regression equations from the pseudo-first-order kinetics at various uric acid concentrations (40-120 #mol/1) with a reagent dilution factor of 1.7. All the regression equations obtained for the different uric acid concentrations are linear. The variation in slope (rate constant) is significant. This variation is similar to the slope obtained with undiluted reagent and with reagent diluted 2.4 times with demineralized water. However, despite this variation in slope at reagent dilution factor 1.7, a good linear uric acid dilution curve is obtained.  This sensitivity, however, decreases if the first measurement is taken at 50s. The KONE CD is connected electronically to the KONE C. As a result, it is possible to determine accurately the time lag between the addition of the reagent and the measurement. The instrument has a minimum lag time between the addition of reagent to three rows ofcuvettes in the cuvette block and the first measurement. The shortest possible lag time varies between the several program versions on the tape which is used to program the KONE C. The best lag time is produced by KONE C's program version 3.2, which has a minimum lag time of 40 at a measuring time of 2 min. The newest program, program 4.0, has a lag time of 50 s at a measuring time of 2 min.
In view of practical constraints, experiments with the Boehringer Mannheim kit will, in future, be run using a lag time of 50s.

Interferences
Interference from xanthine oxidase The Boehringer Mannheim kit contains xanthine in order to increase the Michaelis constant; a test was made to discover whether xanthine oxidase interferes in the determination of uric acid. Shamma'a et al. [6] investigated the level of the enzyme activity of xanthine oxidase in serum from patients with liver diseases. They found marked increases in xanthine oxidase in the serum from patients with infectious hepatitis. The values given by Shamma'a et al. ranged from 1.9 to 1412 International mU/1 throughout the course of the disease. Only slight increases of serum xanthine oxidase were found in patients suffering from other diseases of the liver. Several concentrations of xanthine oxidase were investigated. When 1500 ImU/1 was added to sera, using routine assay conditions, an apparent increase in uric acid (about 18/mol/l) was found. Thus only slight increases in uric acid can be expected at high serum xanthine oxidase levels.

Interference from xanthine
Stepatschuk [7] found, at pathologic serum xanthine concentrations, an apparent decrease in urate with a uricase-based method (Uricaquant, cat. No. 124753, supplied by Boehringer Mannheim). His method uses low uricase concentrations during kinetic measurement.
Since Hande et al. [8] noted serum xanthine concentrations as high as 148 mg/l after a patient with Burkitt's lymphoma was treated with allopurinol, sera containing added xanthine up to mmol/1 were analysed. At mmol/1 of xanthine and at a uric acid concentration of 200/mol/1 an increase of 25/mol/1 was found, at at a uric acid concentration of600 #mol/1 an increase of 40/mol/1 in uric acid was found.

Human kit
The Human kit was also tested to see if the reaction follows pseudo-first-order kinetics. The experiments were carried out with the following refigents: buffer volumes of 12, 15, 18, 25, 52 and 79 ml; uricase concentrations in the cuvette of 5, 10, 20 and 40 U/l; and uric acid concentrations in the cuvette of 12, 20, 30, 40, 50 and 60/mol. Pseudo-first-order kinetics was not obtained under any of these conditions.

Linearity
To find out whether the uric acid dilution curve is linear, dilution curves were set up for all of the above reaction conditions. Nonlinearity was found at low uric acid concentrations. At higher uric acid concentrations the curve is linear in relation to the dilution. Non-linearity is more extreme the later the first measurement is taken from starting the reaction. At the shortest possible lag time for 24 cuvettes (40 using KONE C's program 3.2) all the curves displayed poor linearity. When an LKB 8600 reaction-rate analyser was used a linear dilution curve was obtained (lag time was 10s), although the reaction still did not follow pseudo-first-order kinetics. A drawback of the KONE CD is that it does not allow a short lag time.

Routine uric acid determination
For routine determination of uric acid with the KONE CD, it was decided to use the Boehringer Mannheim ALDH method. The KONE dispenser is programmed for 30C, for a 50 /1 sample and for 200 y1 of physiologic saline. It is unnecessary to incubate the thermostatted cuvette, so 300 y1 ofstarter reagent is added straight away. This starter reagent is prepared by adding 20 ml of buffer, 15 ml of demineralized water, and 100/A of uricase to a bottle oflyophilized reagent. Since the linear range extends to a concentration of 120/mol/1 in the cuvette (see figure 3), the linear range for the serum is 1350 /mol/1. Program supplied by Ortho Diagnostics. Two S.D. limits were applied to these sera for quality control of the results obtained with serum samples from patients.

Day-to-day precision
To test day-to-day precision, 30 separate days' lyophilized bovine control sera were reconstructed with low, medium and high concentrations. These three controls were analysed as unknowns between the patients' sera. The precision for Autonorm was 4.3% (mean concentration 170#tool/l), for General Diagnostics serum it was 3"5% (mean concentration 452 #tool/l) and for Wellcome serum 2.5% (mean concentration 737 ymol/1).

Correlation
Since the machine used for routine uric acid determination at the Academic Hospital was changed from a Technicon SMA 6/60 to a KONE CD system a correlation study was made between the two instruments. The phosphotungstate reduction method was used for the SMA 6/60. 100 patients' samples were used in the correlation experiment. The regression equation was calculated using Deming's method, which is recommended by Cornbleet and Gochman [9]. Taking x for the SMA 6/60 and y for the KONE CD, the regression equation was y= 1.042 x-43.1.

Conclusions
The Human H1004 kit cannot be adapted for two-point kinetic determination of uric acid on the KONE CD. The procedure used with the Boehringer Mannheim kit No. 242616 is quick (allowing 300 determinations/h) and provides good reproducibility of routine use.
In comparison with the phosphotoungstate reduction method, the procedure followed with the Boehringer Mannheim kit gives excellent linearity. Finally, only a minor interference is to be expected in patients' sera from xanthine oxidase and from xanthine.
Symposium" New technology allowing pathology near the patient (clinical chemistry, haematology and microbiology), chaired by A. H. Free (USA).
The congress languages are English, Spanish and French and simultaneous translation will be available during the plenary sessions and symposia. Participants will be able to attend poster sessions, workshops and a commercial exhibition during the three days of the congress.
Papers presented at the congress will be published in Pure and Applied Chemistry, abstracts of free communications in this Journal.
The registration fee for people over 28 is now 12 000 pesetas, under 28s can register for 6000 pesetas. This does not include travel, hotels or any meals.