Creatinine: Principle of measurement

This is a non protein nitrogenous compound. Measurement of excretion rate of creatinine is used as diagnostic indicators of kidney function. Creatinine is synthesized in kidney liver and pancreas. A proportion of free creatine in muscle (1-2%/day) spontaneously and irreversible converts to its anhydride creatinine. So creatinine production is fairly constant and related to muscle mass.

Measurement:

Chemical method:

Measured by Jaffe’s reaction of alkaline picrate method. Here creatinine reacts with picrate ion in an alkaline medium to yield an orange red complex. Although many other compounds like protein, glucose, ascorbic acid, ketone bodies, bilirubin (negative interferent), and cephalosporins can produce positive interfere with the test.

True creatinine can be measured by using hydrated aluminum silicate (Fuller’s earth, Lloyd’s reagent). Here creatinine will only get adsorbed and precipitated. The precipitate is eluted and the creatinine is measured by alkaline picrate method. Jaffes method overestimates true plasma creatinine 20% due to non creatinine interferences.

Kinetic method: These are more sensitive, specific and lack of interferences. Early studies of interferences into the kinetic methods identified two kinds of noncreatinine chromogens, those whose rate of adduct formation were very rapid in first 20 seconds after mixing reagent and sample (e.g. acetoacetate) and those whose rates did not become rapid until 80 to 100 s after mixing (e.g. protein). The window between 20 and 80 therefore was a period of signal produced by creatinine-picrate reaction. These methods are now widely used. This is kinetic chemical method.

Enzymatic methods:

This is more popular method. Addition of potassium ferricyanide or bilirubin oxidase is done to remove bilirubin interference. Interference by ascorbic acid is removed by adding ascorbate oxidase. This system is also included in POCT device using polarographic detection.

Dry chemistry system:

Multilayer dry reagent methods are available for creatinine measurement using enzyme reactions in POCT. A two slide approach employs creatinine deaminase, which deaminates creatinine with the ammonia diffusing through a semipermeable and optically opaque layer to react with bromophenol blue to give an increase in absorbance at 600 nm. A 2^nd multilayer film lacking the enzyme was used to quantitate endogenous ammonia, enabling blank correction. A later single-slide method used the creatininase creatinase reaction sequence. Here the color produced in the film is quantified by reflectance.

Reference value

Serum/plasma creatinine = 0.7 – 1.5 mg/dl in men and 0.6 – 1.1 in women.

Urine = 0.7 – 1.8 gm/day

In ESRD creatinine may exceed 11 mg/dl.