Protein – from greek word proteis meaning first rank of importance

- Synthesized by the liver and excreted by the hepatocyte into the circulation
- Most abundant macromolecule that are constructed from common amino
acids joined by peptide bonds
- Product of genes, and considered as an active in all cells
- Are amphoteric
Functions of Proteins
1. As an enzyme
2. Transportation and storage
3. Cell and tissue growth
4. Mechanical support
5. Coordination and motion
6. Immune protection
7. Nerve generation and impulses
8. Fluid balance / buffer
9. Hormones
Clinical significance of PROTEIN
INCREASED TP DECREASED TP
Malignancy Hapatic cirrhosis
Multiple myeloma Glomerulonephritis
Waldenstrom’s macroglobulinemia Nephrotic syndrome
Starvation
Reference value: Adult – 6.5 – 8.3 g/d L (65 – 83 g/L)
Major groups of PROTEIN
ALBUMIN GLOBULIN
 Comprise approximately 60% of  Globulins comprise the 40% of
total serum proteins total serum protein
 Are made in the liver and are  The globulins are a heterogeneous
homogenous in structure group of molecules
 Soluble in water, insoluble in  Composed of antibodies, blood
saturated salt solution and coagulation proteins & enzymes
hydrocarbons solvents  Soluble in weak salt solution &
hydrocarbon solvents
 Insoluble water & saturated salt
solution
Functions of ALBUMIN
o Regulation of oncotic pressure
o Act as mobile repository of amino acid
o Acts as carrier protein molecule
o Molecules transported by albumin; thyroxine, penicillin, estrogen, bilirubin,
cortisol, fatty acid
Reference range of serum albumin : 3.8 to 5.3 g /d L (35-53 g/L)
Conversion factor : 10
Clinical significance of ALBUMIN
INCREASED ALBUMIN DECREASED ALBUMIN
 Dehydration  Chronic liver disease
 Prolonged tourniquet application  Inflammation of the GI tract
 Albuminuria
 Severe burns
 Acute disease states
 Mutation
- Analbuminuria
- bisalbuminuria
Functions of GLOBULIN
 as immune system it will help fight infection
 transport nutrients
 it consists of α1, α2, β, µ fractions, each fraction consists of a
number of different proteins with different functions
reference value: 2.3 – 3.5 g/dL (23-35 g/L)
clinical significance of GLOBULIN
INCREASED IN GLOBULIN DECREASED IN GLOBULIN
 chronic inflammation  liver disease
 multiple myeloma  Kidney disease
 waldenstrom’s macroglobulinemia
 immune disorders

Specimen considerations for PROTEIN:

 Proteins concentrations are 3% higher in the morning than in samples
collected during the afternoon
 Changing the patient position from standing to lying down, induce a
passage of liquid from extravascular to intra-vascular compartment that
may reduce to relative concentrations of protein and of the analytes
related to the proteins up to 10%.
 During the pregnancy, due to the rise of intra-vascular liquid, the total
protein decreases significantly up to 14%.
Specimen considerations for PROTEIN / ALBUMIN TEST
 Tourniquet use for more than 3 minutes increases total protein concentration in
blood up to 5%.
 Physical exercises increases the proteins concentrations.
 Triglycerides values between 500 and 1100 mg/d L provide positive
interferences which can be minimized by using the blank of the sample using
0.85% NaCl as diluent
 Hemoglobin values over 130 mg/d L provide positive interferences which can
not be minimized by using the blank of the sample
 Fasting sample is not required
 Serum is preferred over plasma
 Hemolyze sample should not be used
Methods of TOTAL PROTEIN
1. Kjeldahl method:
- Reference method but routinely used
- It is based on measurement of nitrogen content
- Acid digestion with H2SO4 in the presence of catalyst releasing ammonium
ions from nitrogen containing compounds. The ammonium ions are then
quantified by:
 titration w/ alkali
 nesslerization
 berthelot reaction
2. Colorimetric Methods
 Biuret method:
- Most widely used method
- It requires 2 peptide bonds and alkaline medium
- End product: violet – colored chelate
 Folin Ciocalteu Reagent
- Involves oxidation of phenolic compound to give a deep blue color
using phenol &biuret reagent
 Coomasie Brilliant Blue Dye
- Sensitive for detection down to 1µg of protein
 Ninhydrin
- Produces violet color by reacting to primary amines
 Greenberg
- Measures TP and Albumin using the Folin – Ciocalteu reagent
which oxidizes phenolic compouns such as; tyrosine, tryptophan,
phenylalanine, and histidine to give deep blue color.
 Looney-Walsch
- Measures TP and globulin in urine and CSF which precipitates
protein using SAA and TCA.
3. Refractometry
 Accurate for measuring serum protein concentration based on RI due to
solutes in serum for levels above 2.5 g /d L
4. Specific gravity
 Simple, used widely in urine specimen as a screening test for protein
5. Turbidimetric and Nephelometric methods
 Measures protein in CSF and urine with addition of SSA and TCA the
measurement depends on the formation of uniform fine precipitates which
scatter incident light in suspension or block the light
6. UV absorption Method
 Proteins absorb UV light at 280 nm the absorbance readings obtained
may be converted to concentration of CHON in the sample using the
molar absorptivity values.
7. Chromatographic technique
 Separate protein fractions
8. Electrophoresis
 Movements of protein fraction in an electric field
TOTAL PROTEIN USING BIURET METHOD
Principle:
Cupric ions complex the groups involved in the peptide bond forming a violet
colored chelates which is a proportional to the number of the peptife bonds present
and reflects the total protein level at 545 nmn.
 It requires at least 2 peptide bonds and an alkaline medium
 Reagents
 Rochelle salts (potassium sodium tartrate)
 Alkaline Copper Sulfate
 NaOH
 Potassium (K) iodide
Reference value: Adult: 6.5 – 8.3 g/dL (65-83 g/L)
Conversion factor: 10

BIURET METHOD FOR TP

 Bring reagents to room temperature
 Prepare equipment and sample
 Switch on machine to warm for 15 minutes
BLANK STANDARD CONTROL SAMPLE
Blank 20µL
Standard 20µL
Control 20µL
Sample/Unknown
Reagent 1000µL 1000µL 1000µL 1000µL
Mix and incubate for 5minutes at 25 degree Celsius and read absorbance at 540-545
nm within 60 minutes.

Special consideration for Protein test

Linearity : ranging from 1.0 to 14.0 g/dL
If total proteins concentrations exceeds 14.0 g/dL the sample must be diluted with
0.85% NaCl.
Multiply the result by the appropriate dilution factor.
Calculation:
Total Proteins = Abs unknown / Abs. standard x 4 (g/d L)

SERUM ELECTROPHORESIS
 The migration and separation of charged particles under the influence of an
electric field
 Uses an electric field to separate the proteins in the blood serum into groups of
similar size, shape, and charge.
 Each of the five protein groups moves at a different rate in an electric field and
together form a specific pattern. This pattern helps identify some diseases.
Normal step pattern

Normal SEP:
1. Albumin = 1st and fastest band (53-65%)
2. Alpha – 1 – globulin = 2nd fastest band (2.5-5%)
3. Alpha – 2 – globulin = 3rd fastest band (7-13%)
4. Beta – globulin = 4th band (8-14%)
5. Gamma globulin = 5th, slowest band (12-22%)

Abnormal SPEP Pattern

 1. Gamma Spike Multiple Myeloma
2. Beta – Gamma Bridging Hepatic Cirrhosis
3. Alpha 2 – Globulin Band Spike Nephrotic Syndrome
4. Alpha – 1 Globulin Flat Curve Juvenile Cirrhosis
5. Spikes of Alpha1, Alpha2 and Beta Inflammation
Globulin Bnad

METHODS FOR ALBUMIN

Salt precipitation method
 Globulins are precipitated in high salt concentrations; albumin is supernatant is
quantified by biuret reaction
Dye binding method
 Albumin can be measured by direct method based on its dye-binding property.
1. Bromcresol green (most commonly used dye ; used extensively in automatic
analyzers)
2. Bromcresol purple (most specific)
3. Methyl orange
4. HABA (2,4’ – hydroxyazobenzene – benzoic acid)

Bromcresol green dye method for ALBUMIN

Principle
Albumin + bromcresol green (BCG) green blue colored complex

 Bring reagents to room temperature

 Prepare equipment and sample
 Switch on machine to warm for 15 mins
BLANK STANDARD CONTROL SAMPLE
Blank 10µL
Standard 10µL
Control 10µL
Sample/Unknown 10µL
Reagent 1000µL 1000µL 1000µL 1000µL
Mix and incubate for 10minutes at 25 degree Celsius and read absorbance at 540-545
nm within 60 minutes.
GLOBULIN
GLOBULIN = Total Protein – Albumin
Albumi / Globulin ratio is determined to validate if globulin is higher than albumin.
 The ratio is used to try to identify causes of change in total serum protein
 If globulin is greater than albumin it is known as inverted A/G ratio seen in
some diseases.
A/G Ratio = Albumin ÷ Globulin
Reference value : 1.1 – 2.4 g/L

Albumin / Globulin Ratio Clinical Significance

High level of A/G ratio:

 Infection
 Inflammatory disease
 Immune disorders
Low level of A/G ratio:
 Overproduction of Globulins
 Underproduction of albumin
 Selective loss of albumin from the circulation