Evaluation of an Enzymatic Method for Determining Serum Creatinine Assay

The enzymatic method for the determination of serum creatinine is accepted as one of the standard method in a clinical laboratory. The enzymatic method for the determination of serum creatinine was optimized for use with Merilyzer AutoQuant-400 auto analyzer and its performance characteristics were practically compared with Jaffe’s Kinetic. Effects of some interfering substances like Serum bilirubin and plasma glucose on the Jaffe’s kinetic method and the enzymatic method were compared. We measured creatinine in serum samples with the enzymatic method and the Jaffe’s kinetic method in samples divided four groups; Group I-Samples without bilirubin and glucose; Group II-Sample with high level of plasma glucose ; Group III-Samples with high level of bilirubin and Group IV-all the samples. There was an excellent agreement between the two methods in terms of correlation coefficient even in the samples with high levels of glucose or bilirubin. Enzymatic method is having better sensitivity, less interfering effects & having better choice in making decision of critical management of the renal failure patient. KeywORds Creatinine , Enzymatic Assay, Jaffe’s Kinetic Assay, Bilirubin , Glucose Introduction: The creatinine determination in clinical practice is more than 100 years old, there is still much debate regarding its accuracy. Also, numerous methods have been described for determining creatinine in biological fluids. Many of the currently need procedures are based on the Jaffe’s alkaline picrate procedure, which is not specific and is subject to interferences 1. Commonly encountered interfering substances of the Jaffe’s based methods include glucose, acetoacetate, bilirubin and Cefoxitin (Cephalosporins)2. Glucose slowly reduces picric acid to picramate, while bilirubin, under alkaline condition, is oxidized to biliverdin, causing a decrease in absorbance at 520 nm. Acetoacetate and Cefoxitin, conversely react directly with alkaline picrate and cause positive interferences. Acetoacetate , in fact reacts rapidly with picrate than creatinine3. Many investigators have attempted to improve the procedure by minimizing the effect of interfering substances present in the sample. Enzymatic approached have been used, to increase specificity. The enzymatic method exhibits several advantages over Jaffe’s based methods namely, improved specificity, smaller sample volume and hence a rapid sample throughput. Glucose, acetoacetate and Cefoxitin do not interfere with the enzymatic method, although bilirubin causes negative interferences which depends on both creatinine and bilirubin concentrations. The aims of this study was to compare analytical performance and practicability of the enzymatic method and kinetic method for serum creatinine for routine use and compare the effects of some common interfering substances like glucose and bilirubin on the enzymatic method and kinetic Jaffe’s method. Materials and Methods: The Present study was conducted in the clinical biochemistry Laboratory. We assessed 512 consecutive serum samples collected for routine clinical case. Creatinine was analyzed both the Jaffe’s kinetic and the enzymatic method. The Jaffe’s method of serum creatinine determination in based on the principle that picric acid in an alkaline reacts with creatinine to form a yellow-red complex with the alkaline picrate4. Intensity of the colour formed during the fixed time is directly proportional to the amount of creatinine present in the sample. The Enzymatic assay for creatinine involves a series of coupled enzymatic reactions including creatininase enzymatic conversion of creatinine into the product creatinine which is converted to sarcosine by creatine amido hydrolase (creatinase), followed by oxidation of sarcosine by sacrosine oxidase producing hydrogen peroxide. In the presence of peroxidase the hydrogen peroxide is quantified at 550 nm by the formation of Coloured dye2. All Measurements were performed using Merilyzer AutoQuant-400 auto analyzer. We also, estimated serum total bilirubin by Diazo method and plasma glucose by hexokinase method of the respective samples. The two levels (normal and pathological) of quality control, materials used in this study were supplied from BioRad. The data obtained were divided into three groups. Group I comprised 167 samples without interfering substances (samples with plasma glucose <126 mg/dl and serum total bilirubin 1 mg/dl) Group II comprised 206 samples with bilirubin (samples with serum total bilirubin< 1.0 mg/dl and plasma glucose < 126 mg/dl); Group III comprised 139 samples with plasma glucose (Plasma glucose >126 mg/dl and serum total bilirubin <1.0 mg/dl, and Group IV all 512 samples.