With the use of spectophotometer, the estimation of total serum protein is explained,

1. Dr. Almas Arshad
UCMD, UOL

3. METHODS:
1. Kjeldhal's Method
2. Lowrey's Method
3. Biuret Method

4.  KJELDHAL'S METHOD:
In this method protiens are first precipitated to release
nitrogen. Then the amount of protien in the given
volume of a sample is calculated by the amount of
nitrogen component.
LOWREY'S METHOD:
Estimation of Tyrosine in the sample is done by this method.

5.  BIURET METHOD:
The commonly used method for estimation of
total serum protien is the Biuret Method.

6. BIURET METHOD:

7. REAGENT: (BIURET REAGENT)
1. CuSO4 : provides cupric ions
2. NaOH : Gives alkaline medium
3. KI : Indicator
4. Sodium Pottasium Tartarate : Keeps the copper sulphate in
solution
STANDARD SOLUTION:
Protien solution of known concentration: 5 - 6 g/dL

8. REACTION:
Cu2+ Ions + peptide bond
Copper-peptide
complex (Violet
Colour)
Cu2+ Cu2+

9. PRINCIPLE:
Cupric ions bind with the peptide bonds of the protien
in an alkaline medium results in formation of violet
Copper-peptide complex.
The intensity of violet colour is directly proportional to
the number of bonds which in turn is proportional to
the amines of protien in the sample.

10. PROCEDURE:
1. Take 3 cuvettes, label them as Blank (B), Standard (S), Test (T)
B S T

12. B S T
STANDARD
SOLUTION
SAMPLE

13. T S

15. 540 - 546nm
=
Absorbance of
Known.S

16. 540 - 546nm
=
Absorbance of
Unknown.
T

17. Conc. of Sample = Absorbance of Unknown
Absorbance of Known
Conc. of
Standard
Solution
x
RESULT: ...................

18. Normal value: 6-8g/dL
Plasma Protiens: Albumin, Globulin and fibrinogen.

19. • Sources
• Formation
• Absorption
• Excretion

20. HYPOPROTENEMIA: < 6 g / dL
• Chronic liver disease
• Malnutrition
• Malabsorption (celiac disease or IBD)
• Extensive burns
• Nephrotic Syndrome
• Pregnancy
FORMATION x
SOURCE x
ABSORPTION x
LOSS!!!
PHYSIOLOGICAL
PATHOLOGICAL

21. HYPERPROTENEMIA: > 8 g /dL
•Dehydration
•Inflammation or infections, such as viral hepatitis
B or C, or HIV
• Bone Marrow disorders

22. PROCEDURE:
1. Take 3 cuvettes, label them as Blank (B),
Standard (S), Test (T)
2. Add 2ml of Biuret reagent in blank cuvettes.
3. In standard cuvette add 2ml of Biuret reagent
and 40 l of standard solution.

23. 4. In the test cuvette add 2ml of Biuret reagent
and 40 l of serum sample.
5. Mix the content of cuvette and allow them to
stand for 30mins.
6. Observe the absorbance of unknown and standard
solution against blank cuvette at 540 - 546 nm.

24. NORMAL RANGE
Total Protien Concentration 6 - 8 g/dL
Normal range of serum Albumin 3.5 - 5.5 g/ dL
Normal range of serum Globulin 2 - 2.5 g/dL
Normal range of serum Fibrinogen 0.2 - 0.6 g/dL
INTERPRETATION:

25. HYPOPROTIENEMIA: < 6 g / dL
• Chronic liver disease
• Malnutrition
• Malabsorption (celiac disease or IBD)
• Extensive burns
• Nephrotic Syndrome
• Pregnancy
FORMATION x
SOURCE x
ABSORPTION x
LOSS!!!
PHYSIOLOGICAL
PATHOLOGICAL

26. HYPERPROTIENEMIA: > 8 g /dL
•Dehydration
•Acute Inflammation or infections, such as viral
hepatitis B or C, or HIV
• Bone Marrow disorders

27. OBSERVATION AND CALCULATION:
BLANK 'B' STANDARD 'S' TEST 'T'
BIURET REAGENT 2ml 2ml 2ml
STANDARD SOLUTION - 40 l -
SAMPLE SOLUTION - - 40 l

28. Results:
Conc. of Sample = Absorbance of Unknown
Absorbance of Known
Conc. of
Standard
Solution
x