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Estimation of

Creatinine and
residual nitrogen
Dr Tehani Silva

Creatine

Naturally occurring amino acid.

Synthesized in the liver, kidney and


pancreas.

Stored in the skeletal muscle and brain.

Synthesized in the body using L-arginine,


glycine and L-methionine

Creatine and creatine


phosphate/phosphocreatine
Exist

in reversible equilibrium in the skeletal


muscle.

Enzyme

that catalyze this reversible reaction is


creatinine kinase.

In

skeletal muscle 1/4 exist in the free creatine


form, where as 3/4 exist in creatine phosphate
form.

Creatine

phosphate important in energy production.

Creatinine vs Creatine

Creatine and creatinine is not the same.

Creatinine is the break down product of


creatine phosphate and creatine.

Creatinine is excreted via the kidney.

Importance of creatinine

If the Renal function is damaged or impaired,


creatinine excretion is reduced.

Serum creatinine level increase and urine


creatinine decrease.

Creatinine is a reliable indicator of kidney


function.

Serum creatinine level is an important diagnostic


tool in the assessment of renal function.

Importance of creatinine

Blood and urine creatinine levels are used


to calculate creatinine clearance.

This has a direct correlation to glomerular


filtration rate.

Serum creatinine levels

Can be altered by,

Muscle mass
Amount of protein in the diet

Different clearance test used for


the assessment of renal function
Creatinine clearance test
Urine clearance test
Inulin clearance

Causes of high serum creatinine


levels
Renal failure
Starvation and fever
Breaking down of muscle tissue
Diabetes mellitus
Hyperthyroidism

Practical: Renal Function Test

1.
2.

Objectives
Creatinine clearance test
Determination of residual nitrogen in the
blood

Jaffe reaction

Colorimetric method use to determine the


creatinine levels in blood and urine.

Utilizes the properties of creatinine and


picric acid in an alkaline solution.

Colour change is directly proportional to the


creatinine concentration .

Procedure
Creatinine + Picric acid (yellow colour)

NaOH (Alkaline medium)

Creatinine pictrate (orange colour)

Measured colorimetrically at 520mm

Procedure

Standard Test
(ml)
(ml)

Blank
(ml)

Serum

0.5ml

Water

1.5ml

1.5ml

2ml

Standard
for serum

0.5ml

Picric acid

6ml

6ml

6ml

Mix the solutions well


Add 0.4ml of 2.5M NOH
Allow to stand for 20min
Read absorbance against blank

Calculation
Serum creatine = Absorbance of test X conc. of standard
Absorbance of standard

Conc. of standard = 3mg/dL

Residual nitrogen in the blood

Nitrogenic organic compounds, with the exception


of the protein nitrogen and ammonia in the blood.

Residul nitrogen in blood is diagnostic

Normal range 20-40%

Increase amount
Renal failure
Advance liver failure
Tissue damage (e.g: Gangrene, malignant
tumors, compartment syndrome)

Heamodylisis

Determination of residual nitrogen


of blood
1.

Precipitate blood proteins with Trichloroacetic acid and


filter them

2.

Mineralize the filtrate


- To burn down the organic substances with conc.
sulphuric acid and a catalyst
- All nitrogen-containing organic substances are
transformed to the single universal product Ammonia
Sulphate.

Determine the ammonia sulfate using the Nessler Reagent

Determination of residual
nitrogen of blood
The interaction of ammonia sulfate with the
Nessler Reagent, which contains the
mercury salt, leads to the appearance of
the yellow-orange product.
The intensity of its color directly
proportional to the concentration of
residual nitrogen.