A patented kinetic uricase method was evaluated for serum uric acid assay. Initial absorbance of the reaction mixture before uricase action (A0) was obtained by correcting the absorbance at 293 nm measured before the addition of uricase solution, and background absorbance (Ab) was predicted by an integrated method. Uric acid concentration in reaction solution was calcu-lated from ?A, the difference between A0 and Ab, using the absorptivity preset for uric acid. This kinetic uricase method exhibited CV<4.3% and recovery of 100%. Lipids, bilirubin, hemoglobin, ascorbic acid, reduced glutathione and xanthine <0.32 mmol/L in serum had no significant effects. ?A linearly responded to 1.2 to 37.5 μmol/L uric acid in reaction solution containing 15 μl serum. The slope of linear response was consistent with the absorptivity preset for uric acid while the intercept was consistent with that for serum alone. Uric acid concentrations in clinic sera by different uricase methods positively correlated to each other. By Bland-Altman analysis, this kinetic uricase method accorded with that by quantifying the total change of UV absorbance on the completion of uricase reaction. These results demonstrated that this kinetic uricase method is reliable for serum uric acid assay with enhanced resistance to both xanthine and other common errors, wider range of linear response and much lower cost.
LIAO FeiZHAO Yun-shengZHAO Li-naTAO JiaZHU Xiao-yunLIU Lan
At 0.12 mmol/L γ-glutamyl p-nitroaniline(GGPNA),an improved integrated method was developed for kinetic analysis of γ-glutamyltransferase(GGT) reaction process and the integration with the classical initial rate method to measure serum GGT.For the improved integrated method,an integrated rate equation,which used the predictor variable of reaction time and considered inhibitions by both GGPNA and products,was nonlinearly fit to GGT reaction processes.For the integration strategy,classical initial rates were estimated when GGPNA consumption percentages were below 50%;otherwise,maximal reaction rates of GGT were estimated by the improved integrated method and converted into initial rates according to the differential rate equation at 0.11 mmol/L GGPNA.The inte-gration strategy was validated using optimized GGT kinetic parameters and 10-s intervals to record reaction curves within 8.0 min.By the integration strategy,there was a linear response from 0.9 to 32.0 U/L GGT,coefficients of variation were below 3.5%for GGT from 8.0 to 32.0 U/L(n=5) ,and GGT activities in clinical sera responded linearly to their classical initial rates at 2.00 mmol/L GGPNA with an expected slope.Therefore,the integration strategy was successful in measuring GGT at 0.12 mmol/L GGPNA.
The correlation of serum arylesterase(PON1) activity on phenylacetate determined by an integrated method to clas-sical biochemical indexes of liver damage was investigated for the use of PON1 activity to evaluate liver damage.PON1 reaction curve as absorbance at 270 nm for 0.20 mmol/L phenylacetate hydrolysis was analyzed by the integrated method to determine maximal PON1 reaction rate.Classical biochemical indexes of liver damage were determined routinely.The 95% confidence threshold of PON1 activity in sera from healthy individuals was 2.12 mkat/L [(4.73±1.31) mkat/L,n=105].PON1 activity in clinical sera was closely correlated to serum albumin,total protein and the ratio of albumin to globulins,but was weakly correlated to both direct and total bilirubin in serum.There were no correlations of PON1 activity to γ-glutamyltransferase,alkaline phos-phatase,alanine aminotransferase and aspartate aminotransferase.Among 127 clinical sera with PON1 activity>2.12 mkat/L,there were 92% healthy individuals examined by albumin,90% healthy individuals examined by total protein,88% healthy individuals examined by total bilirubin,86% healthy individuals examined by direct bilirubin and 64% healthy individuals examined by the ratio of albumin to globulins,respectively.In each group of healthy individuals judged by classical biochemical indexes of close correlation to PON1 activity,percentage of healthy individuals examined by PON1 activity was always >80%.These results suggested PON1 activity on phenylacetate estimated by the integrated method was also suitable for the evaluation of liver damage.
Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transferase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the others and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation ln [Am/(Am-Ai)]+Ai/(ε×Km)=(Vm/Km)×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the concentration of one substrate twenty-fold above the others, this integrated method was reliable to measure the activity of enzyme on two substrates, and substrate concentration of the lower one close to its apparent Km was able to be used.
