Estimation of Serum Creatinine by Modified Jaffes’s method

Creatinine:
It is a produced in muscles from creatine by non enzymatic irreversible dehydration. It is
synthesized in liver and kidneys from amino acids , passes into the circulation and is taken up almost
entirely by skeletal muscles for conversion to creatine phosphate and later to creatinine , which serves as
the storage form of energy in skeletal muscles .The concentration of the creatinine in the plasma of the
healthy individual is fairly constant , independent from water intake , exercise and the rate of urine
production .Therefore , increase in plasma creatinine values always indicate decreased excretion , i.e.,
impaired kidney functions.
The creatinine clearance enables quiet good estimation of glomerular filtration rate(GFR),which allows
better detection of kidney diseases and monitoring of renal functions. For this purpose , creatinine is
measured simultaneously in serum and urine, collected over a defined time period.
Aim:
To estimate the creatinine level in the given serum sample by modified Jaffe’s method.
Principle:
Creatinine reacts with picric acid in the presence of an alkali (NaOH) to form orange red color
of creatinine picrate. The intensity of orange red color is equal to the amount of creatinine present in the
serum and is measured by spectrophotometer at 505 nm.
Creatinine + picric acid -----( NaOH )-----> Creatinine picrate
Reagents:
R1: picric acid of pH=1.2, concentration= 40 mmol/l.
R2: sodium hydroxide (NaoH) of pH =13, concentration=0.16mmol/l,
Concentration of the Standard =2mg/dl. Test sample: serum
Working reagent: mix 1000 µL of R1 and 1000 µL of R2, first before use.
Procedure:
1. Take 2 test tubes and label as standard (S) and test (T)
2. Add 50 µL of standard solution in test tube S.
3. Add 50 µL of test sample in test tube T.
4. Add 1000 µL of working reagent in both test tubes i.e., S and T , mix well incubate for 30 sec at
37°C and read the absorbance of 505 nm.
5. The analyzer can select the kidney function test from the main menu and the select the serum
creatinine and select wash and insert probe in distilled water.
6. Wait for few seconds and the absorbance of the standard is displayed.
7. Select wash, insert probe in distilled water, once aspirated, again select test sample then insert
probe in test tube T. Test sample is aspirated ,Wait for few seconds , absorbance of test sample is
displayed. Make a note of absorbance of the test.
TABULAR COLUMN :

Test tubes Standard (s) Test (t)

Standard 50 µL -
Serum sample - 50 µL
Reagent 1000 µL 1000 µL
Mix well incubate for 30 sec at 37°C and read the absorbance of 505 nm.
CALCULATION :
Serum creatinine (mg/dl) = Absorbance of test × concentration of standard
Absorbance of standard

= mg/dl.

Report: The amount of creatinine in the body , in the given serum sample is ……… mg/dl .
NOTES :
Creatinine is present in the tissues (muscle , brain, blood ) as high energy compounds phosphocreatine
and as free creatine .It is produced in the muscle by creatine phosphate. It is an anhydride form of creatine
phosphate.it is not affected by diet , age or exercise. The amount of creatinine produced is directly
proportional to the muscle mass.
Amino acids required for creatinine : Glycine , Arginine and Methionine .

● Creatine , methylguanide acetic acid is synthesized in liver and kidney and carried by the blood to
muscular tissues and brain and converted to creatine phosphate .
● Energy needed for muscular contraction is provided by ATP breakdown to form ADP.
● ATP is regenerated from ADP by the action of creatine kinase. This regeneration of ATP by the
hydrolysis of creatine phosphate is called Lohmann’s reaction.
Normal values of creatinine plasma:
Male adults = 0.7 to 1.4 mg/dl
Female adults = 0.5 to 1.3 mg/dl
Children = 0.4 to 1.2 mg/dl
Methods of estimation of creatinine in serum and urine are:
● Non Enzymatic / Chemical : Jaffe’s method ,
● Enzymatic: Creatinine deaminase , Creatinase.
Clinical Interpretation :
1. Increased serum creatinine is seen in impaired renal function
Causes:
1. Glomerulonephritis
2. Pyelonephritis
3. Shock
4. Urinary tract obstruction
5. Renal failure
6. Congestive cardiac failure.
2. Decreased serum creatinine is seen in muscular atrophy and certain drugs like Thiazide.
* Entire filtered creatinine is excreted, so renal clearance is a measure of GFR’
Clearance :
It is defined as the volume of blood or plasma that is completely cleared off a substance per unit time .
It is expressed as ml of plasma per minute .
Clearance = mg of sub. Excreted / mg of substance per ml plasma
Creatinine clearance :
Creat Clearance is defined as the volume of plasma that is completely cleared creatinine per minute per
unit time ,
It can be calculated by,

Creatinine Clearence ( ml )=U X V X P1.73

XA

where ,
U – Concentration of Creat in urine sample
P– Concentration of Creat in serum or plasma ( in same units as urine conc.)
V – Volume of urine flow in ml/min
A – Body surface area in sq. meters
1.73 – defined standard body surface are in sq .meters
Normal values :
Mal e: 85 - 125 ml /min
Female : 80- 115 ml/ min
Children : 100 ml /min
Interpretation :
1. Decreased creatinine clearance indicates decreased glomerular filtration rate (GFR). This can be due
to conditions such as progressive renal disease, or result from adverse effect on renal hemodynamics
that are often reversible, including drug effects or decreases in effective renal perfusion (eg: volume
depletion, heart failure).

2. Increased creatinine clearance is often referred to as hyperfiltration and is most commonly seen
during pregnancy or in patients with early diabetes mellitus, before diabetic nephropathy has
occurred. It may also occur with large dietary protein intake .

Creatinine Coefficient :
It is defined as the mg of Creatinine excreted in urine per kg of body weight in 24 hours .
Creat. Coefficient = Amount of creat excreted per day / weight of the person
= ………………….. mg /Kg body weight / day
Normal values :
Male : 20 – 28 mg / kg body weight / day
Female : 15 – 21 mg / kg B.W / day
Significance: It is used to assess the muscle mass of individuals and the accuracy of collection of a 24 hrs
urine sample.
Interpretation : The values are decreased in muscular dystrophy .