[2S] UNIT 6 Total Proteins Flashcards
first rank of importance
“Proteis”
Consists of the elements carbon, oxygen, hydrogen, nitrogen, and sulfur (CHONS)
TOTAL PROTEIN
Synthesized in the liver and secreted by the hepatocyte into the circulation except immunoglobulins
TOTAL PROTEIN
can bear positive and negative charges (amphoteric)
Proteins
pH range of protein
5.5 - 8.0
T/F: Amino acids have no net charge (isoelectric point)
T
pH > pI
Negative charge
pH < pI
Positive charge
Repair body tissues
Functions of Total Proteins
For transport of metabolic substances
Functions of Total Proteins
- Hemostasis
- Acid-base balance
Functions of Total Proteins
- Enzymes (biocatalysts)
- Hormones
- Structure (connective tissue)
Functions of Total Proteins
Most frequently analyzed of all the proteins
PLASMA PROTEINS
- Divided into two groups:
- Albumins
- Globulins
PLASMA PROTEINS
TOTAL PROTEIN METHODS
Reference method. Assume average nitrogen content of
16%
Kjeldahl
TOTAL PROTEIN METHODS
Measurement of refractive index (velocity of light in air and water) due to solutes in serum
Refractometry
TOTAL PROTEIN METHODS
Formation of violet-colored chelate between Cu2+ ions
and peptide bonds (measured at 540 nm)
Biuret
TOTAL PROTEIN METHODS
Protein binds to dye and causes a spectral shift in the absorbance maximum of dye
Dye Binding
Acid precipitation (TCA or tungstic acid) of protein with measurement of
total nitrogen
Total Protein
conversion of nitrogen to ammonia
Kjeldahlization
2 Ammonia measurement
Nessler’s
Berthelot
TOTAL PROTEIN METHODS
breaks the peptide bonds
BIURET: Cupric Ions
TOTAL PROTEIN METHODS
stabilizes cupric ions
BIURET: Potassium iodide
TOTAL PROTEIN METHODS
keeps copper in solution
BIURET: Tartrate Salt
TOTAL PROTEIN METHODS
- Bromphenol blue
- Ponceau S
- Amido black 10B
- Lissamine green
- Coomassie briliant blue
DYE BINDING
- Synthesized in the liver
- General transport protein
ALBUMIN
It is the protein present in highest concentration in plasma
ALBUMIN
Maintains osmotic pressure and is an indicator of nutritional status
ALBUMIN
- Negative acute phase reactant
- Lowest levels are seen in active Nephrotic syndrome
ALBUMIN
ALBUMIN METHOD OF ANALYSIS
Globulins are precipitated
Albumin in supernatant is quantitated by biuret reaction
Salt Precipitation
ALBUMIN METHOD OF ANALYSIS
Nonspecific for Albumin
Methyl orange
ALBUMIN METHOD OF ANALYSIS
Sensitive
Overestimates low albumin levels
Most commonly used dye
BCG (Bromcresol green)
ALBUMIN METHOD OF ANALYSIS
Many Interferences (salicylates, bilirubin)
HABA
ALBUMIN METHOD OF ANALYSIS
Specific, Sensitive and Precise
BCP (Bromcresol purple)
ALBUMIN METHOD OF ANALYSIS
Proteins separated based on electric charge densities
Electrophoresis
ALBUMIN METHOD OF ANALYSIS
- Cellulose acetate/agarose gel (support media)
- After separation, protein fractions are immersed in acid solution then
stained by dyes (e.g. Coomassie blue)
Electrophoresis
ALBUMIN METHOD OF ANALYSIS
The medium is placed in scanning densitometer which compute the area
under the absorbance
Electrophoresis
ALBUMIN METHOD OF ANALYSIS
Monoclonal increase
ELECTROPHORESIS: Monoclonal Gammopathy
ALBUMIN METHOD OF ANALYSIS
Uses higher voltage couple with a cooling system and more concentrated buffer
HIGH RESOLUTION PROTEIN ELECTROPHORESIS
PRE-ANALYTICAL CONSIDERATIONS
❑ Non-fasting test
❑ Sample must be free from hemolysis (↑ increase) and lipemia
Total Proteins
PRE-ANALYTICAL CONSIDERATIONS
❑ Concentration is higher in plasma than in serum
* Presence of fibrinogen
* Increased from 0.2 – 0.4 g/dL (2 – 4 g/L)
Total Proteins
PROCEDURES
Standard of TP
→ Protein: 8 g/dL or 80 g/L,
→ Sodium azide
PROCEDURES
TP Reagent Kit
→ Sodium Hydroxide
→ Potassium sodium tartrate
→ Copper sulfate
→ Potassium iodide
PROCEDURES
Incubation time for TP
10 minutes
PROCEDURES
Measure the absorbance of standard and the reagent blank within __ minutes
30
PROCEDURES
- Wavelength – 520 to 580 nm
- Temperature – 37 degC
- Optical path – 1 cm
- Read against reagent blank
TP
TOTAL PROTEIN: Increased / Decreased
Dehydration
Increased
TOTAL PROTEIN: Increased / Decreased
- Chronic inflammation
- Multiple myeloma
Increased
TOTAL PROTEIN: Increased / Decreased
- Nephrotic syndrome
- Malabsorption
Decreased
TOTAL PROTEIN: Increased / Decreased
- Malnutrition
- Agammaglobulinemia
Decreased
TOTAL PROTEIN: Increased / Decreased
- Overhydration
- Hepatic insufficiency
Decreased
ALBUMIN PROCEDURE
Standard
Albumin 3.5 g/dL or 35 g/L
REFERENCE VALUES (SERUM TOTAL PROTEIN)
6.5 – 8.3 g/dL (65-83 g/L)
ALBUMIN PROCEDURE
Albumin Reagent Kit
→ Succinate buffer (pH 4.2),
→ Bromcresol green
→ Surfactant
→ Sodium azide
ALBUMIN PROCEDURE
Incubation time
5 minutes
ALBUMIN PROCEDURE
Measure the absorbance of standard and the reagent blank within __ minutes
15
PROCEDURE
- Wavelength – 620 nm
- Temperature – 37 degC
- Optical path – 1 cm
- Read against reagent blank
Albumin
ALBUMIN: Increased / Decreased
- Dehydration
- Prolonged tourniquet application
Increased
ALBUMIN: Increased / Decreased
- Malnutrition
- Liver disease
Decreased
ALBUMIN: Increased / Decreased
- Nephrotic syndrome
- Chronic inflammation
Decreased
REFERENCE VALUES (SERUM ALBUMIN)
3.5 – 5.5 g/dL (35-55 g/L)
Computed using Total Protein and Albumin
TP – Albumin =
Normal value of Globulin
1.5 to 4.5 g/dL (15.0 to 45.0 g/L)
GLOBULIN DETERMINATION
A/G ratio
1.3 – 3.0
GLOBULIN DETERMINATION
Increased therapeutic drug in serum
Bisalbuminemia
ODD ONE OUT: GLOBULIN DETERMINATION - Inverted A/G Ratio
Cirrhosis
Multiple Myeloma
Waldenstroms
Bisalbuminemia
Macroglobulinemia
Bisalbuminemia
TP: N/↓
A: ↓
G: ↑
Hepatic Damage & Infections
HEPATIC DAMAGE
TP: N/↓
A: ↓
G: ↑
β-γ bridging
Cirrhosis
HEPATIC DAMAGE
TP: N/↓
A: ↓
G: ↑
↑ γ-globulins
Hepatitis
INFECTIONS
TP: N/↓
A: ↓
G: ↑
- ______ - α1 , α2 globulins
- ______ - ↑ α1, α2 γ, globulins
Acute
Chronic
ODD ONE OUT: Electrophoresis
TP: ↓
A: ↓
G: N
Choices:
Immunodeficiency Syndrome
Inadequate diet
Nephrotic syndrome
↑α2,β-globulins;↓γ-globulins
Immunodeficiency Syndrome
TP: ↓
A: N
G: ↓
Immunodeficiency syndrome
TP: ↑
A: N
G: ↑
Multiple myeloma
Monoclonal & Polyclonal gammopathies
TP: ↑
A: ↑
G: ↑
Dehydration
