1. Practical Analysis Using SpectrophotometerBy
All Staff Members of
Medical Biochemistry Department

2. Colorimetric estimation of serum creatinine

3. 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.

4. 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.

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

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

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

8. 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

9. 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

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

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

12. Step 5: Mix the contents of the tubes

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

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

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

16. Normal value of serum creatinine
Adult males: mg/dl: values are slightly higher in males due to larger muscle mass.
Adult females: mg/dl.
Children: mg/dl

17. 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.

18. * 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.

19. 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.

20. 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.

21. 3- Estimate the concentration of
creatine in serum and in the total
urine sample .
4- Calculate creatinine clearance from
the following equation

22. 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

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

24. 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

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

26. Thank you