Estimation of total creatinine in blood by colorimetric analysis

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BACKGROUND

Creatinine is a byproduct of muscle energy metabolism that is easily extracted by the kidney. It is produced at a constant rate and depends on the muscle mass of the individual. Its level in blood is usually measured to know about the kidney function. High amount of creatinine in blood indicates abnormal functionality of kidney.¹ The objective of this experiment is to estimate the total creatinine in blood by calorimetric analysis. The basic principle of this experiment is

Creatinine + Picric acid=Creatinine pictrate (Red colour compound)

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REQUIREMENTS

Reagent:     2/3 H₂SO₄

10% sodium tungstate solution

Picric acid solution-picric acid (10 gm) +H₂O(1lt)

NaOH solution-NaOH (20 gm) mixed with 200 ml water

Stock solution (1 mg/ml) of creatinine standard- {1 gm creatinine+100 ml of 1N HCL} diluted in 1 litre of water.

Working standard (2 mcg/ml) – Add 2 ml of stock solution in 1litre of water.

Apparatus:  Test tubes

Colorimeter

Conical flask

Filtration unit

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PROCEDURE

In a 100 ml conical flask put 2 ml of sample blood. Then take 14 ml distilled water and 2 ml of sodium tungsten mix and add in the conical flask containing blood. Then slowly add 2 ml of sulphuric acid drop by drop.² While adding sulphuric slowly shake the conical flask. Then keep it in a stationary position for about 5 mins and then filter. Then take three different test tubes and mark it as blank, test and standard.

• Blank– 5 ml distilled water+2 ml picric acid+0.5 ml NaOH
  
• Test– 5 ml blood filtrate+2 ml picric acid+0.5 ml NaOH
  
• Standard– 5 ml standard creatinine+2 ml picric acid+0.5 ml NaOH
  

Mix the contents in test tube very well and allow them to stand for about 15 mins.² Then take measurement of the absorbance of specimen and standard in contrast to reagent blank at 546 nm.

Calculation

Creatinine mg%=Absorbance of specimen /Absorbance of standard×2

Standard values for male and female:

Female: 0.7-1.3 mg/dl

Male: 0.9-1.5 mg/dl

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CONCLUSION

This experiment will give the accurate creatinine content in the blood.

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REFERENCES

1. Goldman, R. Creatinine excretion in renal failure. Proc Soc Exp Biol Med. 1954;85:446–8.
   
2. Owens, CWI, Albuquerque, ZD, Tomlinson, GM. In vitro metabolism of creatinine, methylamine and amino acids by intestinal contents of normal and uraemic subjects. Gut 1979;20:568–74.