Colorimetric enzymatic method
This procedure is based upon an improved enzymatic method originally devised to evaluate creatinine in serum and urine. The test is performed in two steps. In the first step creatine is removed during the first minutes of the preincubation stage of the sample with creatinase. In the second step the addition of creatininase, acting also as the reaction starter, hydrolyzes the creatinine of the sample in the presence of sarcosine oxidase (Sar OD) with production of hydrogen peroxide.
Creatinine + H2O Creatine
Sarcosine + H2O + O2 H2O2 + Glicine + HCHO
The hydrogen peroxide produced from the oxidase reaction is cuantified by a Trinder’s type reaction in which the chromogenic derivative HTIB and 4- aminoantipyrine (4-AA) are condensed in the presence of peroxidase (POD) to form a red quinoneimine dye.
4-AA + HTIB Red quinoneimine + H2O
The rate of color development is proportional to the concentration of creatinine in the sample.