Download creatinine+ protein 5

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