Jaffe’s Method for the Estimation of Creatinine

Creatinine is a non-protein nitrogenous waste product formed from the metabolism of creatine in skeletal muscles. Creatinine is released into the bloodstream at a relatively constant rate and is excreted by the kidneys, leading to stable plasma and urine levels on a day-to-day basis. In individuals with normal renal function, serum creatinine levels remain fairly constant and within the normal range unless there is a significant change in muscle mass. Therefore, estimation of serum creatinine is widely used as a routine and reliable indicator of kidney function in clinical practice.

Methods for Creatinine Estimation

There are several methods available for the estimation of creatinine in blood. Some of the commonly used methods are as follows:

1. Direct Chemical methods:
   • Jaffe’s method
   • Alkaline picrate (end point) or Modified Folin-Wu method
   • Dinitrobenzene method (used in dry chemistry)
2. Indirect Enzymatic methods:
   • Deaminase method (one enzyme step method)
   • Creatininase method (multi-enzymatic method)
3. Other (Gold standard) methods:
   • High-performance liquid chromatography
   • Gas chromatography with mass spectrometry

Jaffe’s method for Creatinine estimation

Jaffe’s method is widely employed in clinical laboratories as a rapid and cost-effective colorimetric technique for measuring creatinine levels in serum and urine. It is highly non-specific. Substances like glucose, protein, ketones, and certain drugs (e.g., cephalosporins) can react with the picrate, causing falsely elevated creatinine readings by 15–25%. It requires deproteinization, typically using tungstic acid (sodium tungstate and sulfuric acid) before the reaction.

Principle:

Creatinine present in a protein-free filtrate of blood or serum reacts with picric acid in an alkaline medium to form an orange-red colored complex known as creatinine picrate (Janovski’s complex or 2,4,6-trinitrophenol). The intensity of the color produced is directly proportional to the concentration of creatinine in the sample. This colored complex is measured spectrophotometrically at a wavelength of approximately 520–540 nm using a green filter.

Requirements

Specimen

1. Serum or heparinized plasma
   Creatinine stability: 24 hours at 2-8°C
2. Urine: dilute sample 1/50 with distilled water.
   Creatinine stability: 1 day at 2-8°C.
   Multiply results by 50 (dilution factor).

Reagents

1. Sodium tungstate (10%)
2. Sulphuric acid (2/3N)
3. Picric acid (0.04 M)
4. Sodium hydroxide (0.75 N)
5. Creatinine standard (4 mg/dl or 0.04 mg/ml)

Instruments

1. Test tubes
2. Pipettes, disposable tips, rack
3. Water bath
4. Colorimeter

Procedure:

A. Preparation of Protein-free filtrate

1. 1. Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
   2. Pipette as follows:

	Blank	Standard	Test
Distilled water	4 ml	3 ml	3 ml
Standard	–	1 ml	–
Serum	–	–	1 ml
Sodium tungstate	2 ml	2 ml	2 ml
Sulphuric acid (2/3 N)	2 ml	2 ml	2 ml

2. Mix the contents after each addition. Keep the tubes at room temperature for 10 minutes.
3. Filter the Test (T) tube and collect the filtrate.

B. Estimation of Creatinine

1. 1. Prepare another set of three tubes and label as Blank (B), Standard (S), and Test (T).
   2. Pipette as follows:

	Blank	Standard	Test
Picric acid	1 ml	1 ml	1 ml
Sodium hydroxide	1 ml	1 ml	1 ml
Distilled water	3 ml	–	–
Standard filtrate	–	1 ml	–
Test filtrate	–	–	2 ml

2. Mix and keep the test tubes at room temperature for 15 minutes.
3. Measure the absorbance of the standard and test sample at 520-540nm (green filter) against blank.

Calculation:

For preparing protein-free filtrate, a 1:4 dilution was done (2 ml of blood in a total volume of 8 ml). However, only 3 ml of the protein-free filtrate was used, which is equivalent to 0.75 ml of the blood sample. Standard contains 0.04 mg/ml, which is diluted to 8 ml; therefore, 3 ml will have 0.015 mg. Hence, we can calculate the concentration of serum creatinine in the specimen using the following formula:

Reference range

Result interpretation and Clinical significance

Increased serum creatinine levels indicate:

• Renal causes
  • Acute and chronic renal failure
  • Glomerulonephritis and nephritis
  • Diabetic and hypertensive nephropathy
  • Drug-induced renal impairment
  • Renal malignancy
  • End-stage renal disease (ESRD)
• Post-renal causes (urinary obstruction)
  • Prostatic hypertrophy
  • Ureteric or bladder stones
  • Urethral stricture
  • Tumors of urinary bladder or ureter
  • Extra-mural urinary obstruction
• Other conditions
  • Severe or vigorous exercise
  • Muscle injury, muscle breakdown, muscular dystrophy
  • Reduced renal blood flow (dehydration, shock, heart failure)
  • Pre-eclampsia and eclampsia
  • Hepatorenal syndrome
  • Drug intake (e.g., aminoglycosides, cephalosporins, cimetidine, trimethoprim)
• Physiological variation
  • Slight increase with advancing age (related to body mass)

Decreased serum creatinine levels are seen in:

• Low muscle mass or small body stature
• Muscle atrophy
• Myasthenia gravis

Advantages and Limitations

Advantages

• Simple, rapid and easy to perform
• Cost-effective and economical
• Suitable for routine clinical laboratory use
• Can be easily automated

Limitations

• Non-specific reaction – other substances also react with alkaline picrate
• Positive interference from glucose, ketone bodies, proteins, ascorbic acid, and bilirubin
• Interference by drugs such as cephalosporins
• Overestimation of serum creatinine values
• Less accurate at low creatinine concentrations
• Affected by hemolysis and lipemia
• Lower specificity compared to enzymatic methods

Modifications

Several modifications of Jaffe’s method have been developed to reduce analytical interference and improve the accuracy of serum creatinine estimation.

References

1. BASU P. BIOCHEMISTRY LABORATORY MANUAL: FOR MBBS, BDS, BHMS, BAMS, BUMS, BNYS AND DMLT STUDENTS. Academic Publishers; 2016.
2. Mohanty B, Basu S. Fundamentals of practical clinical biochemistry.
3. Dandekar SP, Rane SA. Practicals and Viva in Medical Biochemistry. New Delhi: Elsevier; 2004.
4. Vasudevan DM, Das SK. Practical Textbook of Biochemistry for Medical Students. Jaypee Brothers Medical Publishers; 2013.
5. Sood R. Concise book of Medical Laboratory Technology. Jaypee Brothers Pvt. Limited; 2015.