MOI UNIVERSITY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF MEDICINE

MSB 103: HEMOPOETIC SYSTEM AND BODY FLUIDS

BIOCHEMISTRY REPORT

MBCH.B/5469/21
 ESTIMATION OF PLASMA/SERUM PROTEINS

INTRODUCTION

Indications for Protein Estimation

i. To asses changes in hydration; Changes in plasma total protein or Albumin concentrations are
certainly due to changes in capillary permeability or changes in hydration.

ii. To investigate oedema; Very low plasma albumin concentrations in the presence of oedema
suggest hypoalbuminemiae. Ginnephritic syndrome and hepatic cirrhosis.

Photometric methods: absorption spectroscopy

When an atom, ion, or molecule absorbs a photon, the added energy results in an alteration of state
and the species is said to be excited. Excitation may involve: Transition of an electron to higher
energy level; a change in the mode of vibration of the molecule’s covalent bonds or alteration of its
mode of rotation about the covalent bonds.

Absorption spectroscopy applies the principle of Beer’s law which states that; the concentration of a
substance is directly proportional to the amount of radiant energy absorbed or inversely proportional
to the logarithm of transmitted radiant energy. If the concentration of a solution is constant and the
path length through the solution that the light must traverse is doubled, the effect on the absorbance is
the same as that caused by doubling the concentration because twice as many absorbing molecules are
now presenting the radiant energy path. Thus the absorbance is also directly proportional to the path
length of the radiant energy through the cell.

Beer Lambert’s law states the mathematical relationship that connects absorbance of radiant energy,
concentration of a solution, and path length. Thus forms the basis of quantitative analysis by
absorption photometry

A= abc

A=Absorbance; a=proportionality constant (absorptivity) ; b=light path in centimetres.

c=concentration of the absorbing compound usually expressed in g/l.

TOTAL PROTEIN CONCENTRATION TEST: BIURET METHOD

Principle

Cupric ions react with protein in alkaline solution to form a purple complex. The absorbance of this
complex is proportional to the protein concentration in the sample.

Normal ranges: Normal born babies 4.6-7.0g/dl or 46-70g/l

Children from 3 years and adults 6.6-8.7g/dl or 66-87g/l

Reagents:

Biurets reagent containing: Sodium hydroxide 200mmol/l

Potassium sodium tartrate 32mmol/l

Copper sulphate 12mmol/l

Potassium iodide 30mmol/l

STD (Protein) 80g/l

Sodium azide 0.095%

Specimen: Serum/plasma

Procedure

1. Arrange three test tubes in a rack

2. Label them as blank, standard and test

3. Add 1000 microliter of Biuret’s reagent to all three test tubes

4. Add 40 microliter of the standard total protein and serum (test) to their respective test tubes

5. Mix each test tube properly

6. Incubate for 10 minutes at 20-25C

7. Use the spectrophotometer to test light absorbance in each test tube

Measure the absorbance of the sample and standard against the reagent blank.
RESULTS

1. Test = 0.324 Optical Density (OD)

2. Standard = 0.3217 Optical Density (OD)

CALCULATION

IF STD of 0.3217 OD 80 g/dl80 g/l

Test of 0.3240 OD 0.324 OD X 80 g/l

0.3217 OD

Total protein concentration = 80.57 g/l

PHOTOMETRIC COLORIMETRIC TEST FOR ALBUMIN: BCG METHOD

Principle

Bromocresol green forms a colored complex with albumin in citrate buffer. The absorbance of this
complex is proportional to the albumin concentration in the sample.
Reference Ranges 3.8-5.1g/dl or 38-51g/l

Reagents:

Citrate Buffer (PH4.2) 30mmol/l

Bromocresol green 260µmol/l

STD: Albumin 4g/dl or 40g/l

Sodium azide 0.095%

Specimen: serum/plasma

Procedure

1. Arrange three test tubes in a rack

2. Label them as blank, standard and test

3. Add 1000 microliter of bromocresol green to each test tube

4. Add 20 microliter of serum (test) and standard albumin in their respective test tubes

5. Mix the contents of each test tube thoroughly

6. Incubate the test tube for 5 minutes

7. Use the spectrophotometer to test light absorbance in each test tube

Measure the absorbance of the sample and standard against the reagent blank.
Results

1. Test = 0.9518 Optical Density (OD)

2. Standard = 1.044 Optical Density (OD)

Calculation

IF STD of 1.044 OD 40 g/l

Test of 0.9518 OD O.9518 OD X 40 g/l

1.044 OD

Albumin concentration = 36.47 g/l

ALBUMIN: GLOBULIN RATIO

Ratio between plasma level of albumin and globulin is called albumin/globulin (A/G) ratio. It is an
important indicator of some diseases involving liver or kidney. Normal A/G ratio is 2 : 1.

Albumin: Globulin ratio = Albumin

Total protein- albumin

= 36.47 g/l

(80.57-36.47) g/l

= 0.8268

CONCLUSIONS

1. The normal range of total protein concentration is 66-87 g/l for adults. The patient’s total protein
concentration was 80.572 g/l and therefore within normal range.

2. The normal range of albumin concentration is 38-51 g/l. The patient’s albumin concentration was
36.4674 g/l and therefore slightly below the normal range.

3. The normal A/G ratio is 2 : 1. The patients A/G ratio was less than normal as a result of lower
plasma albumin concentration.