CREATININE, Creatinine Clearance Test, Quantitative Estimation of Creatinine, Creatinine test kits

CREATININE

Creatinine Clearance Test

Method Quantitative Estimation of Creatinine

Specimen for Quantitative Estimation

Quantitative Estimation of Creatinine

Calculation of Quantitative Estimation

Creatinine test kits

Reference Values of Creatinine

Normal value of Creatinine clearance

Adult men, Adult women

Interpretation of Creatinine Results 

CREATININE

Creatinine is a nitrogenous product which is produced from the metabolism of creatine in the skeletal muscles. 

It is filtered by the kidneys and excreted in the urine. Unlike urea, creatinine level in the blood is not linked to protein intake. But like urea, the measurement of creatinine level is a test of renal function. 

The formation of creatinine is constant, and has direct relationship to muscle mass. For this reason, it varies with age and sex.

The assay of the creatinine level is a good screening index of renal function. One such useful index relates creatinine excretion to muscle mass or lean body weight. 

This index is known as Creatinine clearance There are several tests available to determine impairment of renal function, but most of them are not routinely performed because of their cumbersome nature. However, the clearance test is in vogue.

Creatinine Clearance Test

The clearance test is done to assess the glomerular filtration capabilities of the kidneys. The creatinine clearance is defined as the millilitres of blood (plasma) that are cleared of creatinine by the kidneys per unit time. The clearance is calculated with this formula:

where

U = concentration of creatinine in urine (mg/dl)

P = concentration of creatinine in plasma (mg/dl)

V = volume in millilitres of urine excreted per minute, i.e., 24 hour urine volume (ml) divided by 1440, the number of minutes in 24 hours.

The standard body surface area is 1.73 square metres. A normogram can be used to determine a patient's body surface area. Most autoanalysers are capable of calculating this value if the patient's height and weight are entered in the system. 

Method It is usual to determine a 24 hour creatinine clearance, but in some cases, it is possible to test for a two to six hour clearance period. 

Specimen For creatinine analysis, serum or plasma and diluted urine can be used. Usually, urine is diluted 1:100 or 1:200; and it is preserved with thymol or toluene only. Urine stored in the refrigerator for up to 5 days is also suitable. Serum or plasma, promptly separated, is suitable for up to I week if refrigerated. 

Fluoride and heparin are good anticoagulants if plasma is to be used. To perform the test, the total volume of urine during the entire 24 hours period is collected and carefully measured. Blood is collected about 12 hours into the urine collection (roughly mid-day) on the day of the test. 

The creatinine levels of the plasma (or serum) and urine are then determined. The clearance is obtained with the formula given above.

Quantitative Estimation of Creatinine

Most of the commonly used methods for the estimation of creatinine level are based on the reaction of creatinine present in the specimen with an alkaline picrate reagent to produce a bright orange-red complex. But unfortunately, a number of other substances give similar reaction, thus giving falsely high values. 

There are various modifications now developed to give a more specific test for creatinine. One of these modifications is the use of aluminium silicate to separate creatinine from nonspecific chromogens. 

This particular modification is now the reference method for "true" creatinine. A popular modification includes an acidification step whereby the impact of acidification of colour development is measured colorimetrically and the difference noted. 

Principle Creatinine reacts with alkaline picrate reagent to form an orange-red colour which is measured in a spectrophotometer.

Reagents

 (i) Saturated picric acid solution Place an excess of picric acid in an amber colored glass bottle containing distilled water. Leave for a few days and the supernatant is the saturated solution.

*Note that dry picric acid is explosive and so, it must always be stored damp. 

(ii) 10% w/v sodium hydroxide solution in distilled water. 

(iii) Creatinine working reagent Mix 10 ml of the saturated picric acid solution and 2.0 ml of the10% sodium hydroxide solution together. This reagent should be prepared fresh for use.

(iv) Stock creatinine standard, 10 mmol/L Weigh 0.113 g of pure creatinine, or 0.602 g of creatinine zinc chloride; dissolve in, and make up the volume to 100 ml with 100 mmol/L (0.1N) hydrochloric acid. This concentration is 10mmol/L (i.e. 10,000 umol/L).

This solution is stable for 2 months when stored at 2-8°C.

(v) Working creatinine standard Dilute the stock standard 1:50 to 200 umol/L. This is stable for about 1 month at 2-8°C. 

Method

(i) Set up two sets of three test tubes and label each as follows:

(ii) Add reagents as shown

	blank	Standard	test
Blank Working reagent	2.0 ml	2.0 ml	2.0 ml
2.0 ml Distilled water	0.2 ml
Standard (200 umol/L)		0.2 ml
0.2 ml - Test sample			0.2 ml

(iii) Mix well and leave at room temperature for 90 minutes.

(iv) First reading Read absorbance of the first set of tubes with blue-green filter, or at 490 nm. Zero the instrument with the blank, (first set of tubes) 

(v) Second reading Into the second set of tubes, add 50 ul (0.05 ml) acetic acid, 60% v/v, to all the tubes. Leave at room temperature for 6-10 minutes. Read absorbance of the second set of tubes with blue-green filter; or at 490 nm. Zero the instrument with the blank, (second set of tubes) 

(vi) Final reading Subtract second reading from the first reading to eliminate the colour developed due to non-specific chromogens.

Note

Alternatively, a calibration curve for creatinine can be prepared and from it the value of the creatinine can be obtained, if the reading of the 200 umol/L standard agrees with the calibration.

When the creatinine result is over 500 umol/L, the assay should be repeated with a l in 3 dilution of specimen in distilled water. The result is then multiplied by 3. 

Creatinine test kits There are various brands of test kits for creatinine in the market. Majority of them are based on the principle of the alkaline picrate method before and after adding acid (to give 'true' creatinine value). The important rule of using any test kit is to follow the manufacturer's instructions and the creatinine test kit is no exception.

Reference Values

Creatinine Serum/Plasma

Adult men 88-135 umol/L (0.9-1.5 mg/dl)

Adult women 62-115 umol/L (0.7-1.3 mg/dl) mg/dl of creatinine = umol/L < 0.011

Creatinine clearance

Adult men 97-137 ml/min 

Adult women 88-128 ml/min

Interpretation of Creatinine Results

High serum or plasma creatinine levels are all attributed to renal causes of uraemia. But while the urea level will continue to rise in cases of severe, permanent renal dysfunction, the creatinine level tends to plateau.