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Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Practical Analysis Using Spectrophotometer 1 Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Colorimetric estimation of serum creatinine Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Aim of practical session The aim of this practical session is to : 1.Estimate the concentration of creatinine in a serum sample using a colorimetric method, the Jaffé reaction. 2.Comment on the provided case. 3.Calculate creatinine clearance. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department PRINCIPLE The assay is based on the reaction of creatinine with sodium picrate as described by Jaffé. . Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Principle of the Jaffé reaction In the Jaffé reaction, creatinine reacts with alkaline picrate solution to yield a deep orange-red complex Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Principle of the Jaffé reaction Intensity of the colour formed is directly proportional to the amount of creatinine present in the sample. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Procedure Step 1:Label 3 dry test tubes T For the test sample S B For the Standard sample For the Blank Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Step 2- Pipette into the labeled test tubes B T S Alkaline picrate Distilled water Test 1.0 ml 1.0 ml 1.0 ml 2.0 ml …….. …….. …….. 2.0 ml …….. Standard …….. …….. 2.0 ml Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Step 3: Pipette 2 ml of the given Test sample ,Standard and DW into their corresponding tubes o o 2 ml o 2 ml 0.9 2 ml 0.9 0.9 Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Step 4: for all ; Pipette 1ml of the alkaline picrate reagent into the previous 3 test tubes. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department T S B Step 5: Mix the contents of the tubes Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department let the tubes to stand for 20 minutes at room temperature. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Record the absorbance of both test & standard against the blank using the Wavelength : 520 nm. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Calculate the test concentration by the following equation: Concentration of serum creatinine (mg/dL) = Absorbance of Test ---------------------------Absorbance of standard X Concentration of standard Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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 Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Decreased serum creatinine levels are seen in: * * * * Persons with small stature. Decreased muscle mass. Inadequate dietary protein. Muscle atrophy. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 3- Estimate the concentration of creatine in serum and in the total urine sample . 4- Calculate creatinine clearance from the following equation Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department CALCULATION Clearance is measured as milliliters/minute (ml/min) = Urine creatinine X 24 hs urine volume in ml Serum creatinine X 24 X 60 Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department 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. Faculty of Medicine Ain Shams University Decreased creatinine clearance levels are seen Medical Biochemistry & Molecular Biology Department 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 Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Increased creatinine clearance levels are seen in: pregnancy, resulting in lower serum levels. Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Plasma Proteins Normal level of plasma proteins 6-8 gm% Normal level of albumin 3.5 -5 gm% Biuret Method Biuret Method Biuret reagent Biuret reagent+ Protein (peptide bonds) produce Violet color Reagents Test Standard Blank (ml) (ml) (ml) ___________________________________________ Protein solution 1.0 ----Standard solution --1.0 --Distilled Water ----1.0 Biuret reagent 5.0 5.0 5.0 Mix the contents of the tubes by inversion and let stand for 20 minutes at room temperature. Record the absorbance of both test & standard against the blank using the green filter (~550 nm). A patient had chronic renal failure .A check up was done by blood examination. 1- Estimate the level of creatinine in his serum sample. 2-Comment on the result. A pregnant woman has hypertension and edema of lower limbs. Her doctor suspects that she has pre-eclampsia . 1-Estimate the level of albumin in her serum sample 2-The urine sample of this patient may contain A-ketone bodies B- albumin C- bile salts Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Thank you