Estimation of Blood Creatinine

EXPERIMENT NO - 11 Aim: To estimate creatinine in given sample of blood. Apparatus: Flasks and Graduated pipettes and photoelectric colorimeter. Chemical reagents: Picric acid ... 1% Sodium hydroxide ... 10% Stock creatinine Standard creatinine Method used: Folin method Principle: Creatinine in blood is estimated by folin modified method using photoelectric colorimeter. In this folin Wu i.e. protein free blood filtrate is used. This creatinine (unknown) contain in filtrate is treated with picric acid in alkaline medium to obtain red coloured creatinine picrate. Optical density of this red coloured creatinine thus obtained is compared with that of standard solution, similarly converted by picric acid to creatinine picrate. By using colorimetry principle concentration of creatinine in given blood sample can be calculated. CLINICAL SIGNIFICANCE: Creatinine represents the waste products of creatine metabolism and it arises in the body from the spontaneous breakdown of creatine phosphate. It is a non threshold substance. It is normally filtered by the glomerulli. As its excretion is not related with food protein so its variations in the excretion indicate some of the metabolic disorders. Appearance of urine in urine is known as creatinuria. Its excretion increases in fevers, starvation, on a carbohydrate free diet and in diabetes mellitus. It may increases due to excessive tissue destruction releasing creatine or due to failure of creatine being properly phosphorylated. So creatinine excretion is independent of food proteins and is to be considered as an index of endogenous protein metabolism. Endogenous creatinine clearance is a rough measure of the glomerular filtration rate and is normally 100 - 150 ml/minute an adult of normal size. Values below 90 ml/minute are indicative of diminished glomerular filtration rate. Normal value 0.7 to 2.0 mg/100 ml blood. Procedure: [A] Preparation of unknown sample In a flask labelled as "U", pipette out 5 ml of folin wu filtrate. Add 2 ml of 1 % picric acid mix well. Add 0.5 ml of sodium hydroxide solution 10%. Allow to keep for 15 minutes and obtain the optical density by using green filter (530 M µ). Note it as "Eu" [B] Preparation of standard sample: In a flask labelled as "S", pipette out 5 ml of standard creatinine solution. Add 2 ml of 1 % picric acid. mix well Add 0.5 ml of sodium hydroxide solution 10%. Allow to keep for 15 minutes and obtain the optical density by using photoelectric colorimeter using green filter (530 M µ). Note it as "Es" [C] Preparation of blank sample: In a flask labelled as "B" pipette out 5 ml of distill water. To it add 2 ml of 1 % picric acid and 0.5 ml of 10 % sodium hydroxide solution. Allow to keep for 15 minutes and compare in a calorimeter by using green filter. (530 m µ). Calculations: Note: Optical density of blank must be(minus) subtracted from optical density obtained that of unknown and standard. By using photoelectric calorimetry principle Concentration of creatinine in unknown blood sample CU = (Optical density of (Eu) unknown sample / Optical density of standard sample "Es") × Concentration of standard creatinine (Cs) Standard creatinine solution is prepared by diluting 1 ml of stock solution (1 mg) to 500 ml. i.e. 1 ml of standard creatinine solution = 0.002 mg creatinine. We have used 5 ml of standard creatinine = 0.01 mg creatinine. Now we have used 5 ml of folin wu blood filtrate. 5 ml filtrate = 0.5 ml blood Therefore Concentration of creatinine in unknown blood sample CU = (Optical density of unknown sample "Eu" / Optical density of standard sample "Es") × 0.01 mg creatinine = "x" (suppose) Now "x" mg of creatinine is present in 5 ml of folin wu filtrate. But 1 ml of folin wu filtrate = 0.1 ml of blood ∴ 5 ml of folin wu filtrate = 0.5 ml of blood ∴ "x" mg of creatinine is present in 0.5 ml of blood. To estimate milligram percentage of creatinine If 0.5 ml = 'x' mg creatinine ∴ 100 ml = (x × 100) / 0.5 mg creatinine Substitute value of 'x' We get "cu" = (Eu/Es × 1/100) × (100/1 × 10/5) = (Eu/Es) × 2 mg % creatinine. ∴ CU = (Eu/Es) × 2 mg % creatinine