Practical Analysis Using Spectrophotometer By All Staff Members of Medical Biochemistry Department 2011-2012

Colorimetric estimation of serum creatinine

Aim of practical session The aim of this practical session is to: Estimate the concentration of creatinine in a serum sample using a colorimetric method, the Jaffé reaction. Comment on the provided case. Calculate creatinine clearance.

Introduction Creatinine is a non-protein waste product of creatine phosphate metabolism by skeletal muscle tissue. Creatinine production is continuous and is proportional to muscle mass.

PRINCIPLE The assay is based on the reaction of creatinine with sodium picrate as described by Jaffé. .

Principle of the Jaffé reaction In the Jaffé reaction, creatinine reacts with alkaline picrate solution to yield a deep orange-red complex

Principle of the Jaffé reaction Intensity of the colour formed is directly proportional to the amount of creatinine present in the sample.

For the Standard sample Procedure Step 1:Label 3 dry test tubes B For the Blank T For the test sample S For the Standard sample

B T S Alkaline picrate 1.0 ml Distilled water 2.0 ml …….. Test Step 2- Pipette into the labeled test tubes B T S Alkaline picrate 1.0 ml Distilled water 2.0 ml …….. Test Standard

Step 3: Pipette 2 ml of the given Test sample ,Standard and DW into their corresponding tubes 0.9 2 ml

Step 4: for all ; Pipette 1ml of the alkaline picrate reagent into the previous 3 test tubes.

Step 5: Mix the contents of the tubes

let the tubes to stand for 20 minutes at room temperature.

Record the absorbance of both test & standard against the blank using the Wavelength : 520 nm.

Calculate the test concentration by the following equation: Concentration of serum creatinine (mg/dL) = Absorbance of Test ---------------------------- X Concentration of standard Absorbance of standard

Normal value of serum creatinine Adult males: 0.7 - 1.4 mg/dl: values are slightly higher in males due to larger muscle mass. Adult females: 0.6 - 1.1 mg/dl. Children: 0.2 - 1.0 mg/dl

Impaired renal function. Chronic nephritis. Urinary tract obstruction. Increased serum creatinine levels are seen in: Impaired renal function. Chronic nephritis. Urinary tract obstruction. Muscle diseases such as gigantism, acromegaly, and myasthenia gravis. Congestive heart failure. Shock.

* Persons with small stature. * Decreased muscle mass. Decreased serum creatinine levels are seen in: * Persons with small stature. * Decreased muscle mass. * Inadequate dietary protein. * Muscle atrophy.

Determination of Creatinine clearance Creatinine clearance rate is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for the Glomelular Filteration Rate.

Steps: 1-Advise the patient to avoid eating much protein diet and not to use any type of diuretic drugs or coffee 2- Collect the 24 hours urine ,measure its volume and a blood sample is taken.

3- Estimate the concentration of creatine in serum and in the total urine sample . 4- Calculate creatinine clearance from the following equation

Clearance is measured as milliliters/minute (ml/min) = CALCULATION Clearance is measured as milliliters/minute (ml/min) = Urine creatinine X 24 hs urine volume in ml Serum creatinine X 24 X 60

Normal value of serum Creatinine clearance Adult males: 97 to 137 ml/min. Adult females: 88 to 128 ml/min. Children: 70 to 140 ml/min.

Decreased creatinine clearance levels are seen in: Acute tubular necrosis Bladder outlet obstruction Congestive heart failure Dehydration End-stage kidney disease Glomerulonephritis Kidney failure Renal ischemia (too little blood flow to the kidneys) Renal outflow obstruction (usually must affect both kidneys to reduce the creatinine clearance) Shock

pregnancy, resulting in lower serum levels. Increased creatinine clearance levels are seen in: pregnancy, resulting in lower serum levels.

Thank you