Plasma Proteins/Electrophoresis Flashcards
Immunoglobulins are made from?
B-lymphocytes
Complement system are made by?
Macrophages
Plasma proteins are made from?
Hepatocytes
Measurement of proteins include the measurement of what parameters ?
- Total protein
- Plasma Albumin
- C-reative protein
The amount of proteins in the vascular compartment depends on?
- The balance between the rate of synthesis and catabolism or loss
- Relative distribution between the intravascular and extravascular compartments
Concentration of plasma depends on ?
The relative amounts of proteins and water
Plasma proteins are taken up by what process and into what cells during their catabolism ?
They are taken up by Pinocytosis into the capillary endothelial cells where they are catabolized
Small chain molecular weight proteins are lost passively though what parts of the body?
- The renal glomeruli
- The intestinal wall
**some are reabsorbed with by the Renal tubular cells or after digestion in the intestinal lumen
Functions of plasma proteins?
1) Peptide hormones and blood clotting factors
2 ) control of extracellular fluid distribution
3) Trabdport
4) inflammatory response and control of infections
How does plasma protein function as a transporter?
Albumin and specific binding proteins transport hormones, vitamins, lipids, bilirubin, calcium, trace metals and drugs
How does plasma protein function in inflammatory response and control of infection?
Immunoglobulins and the complement proteins together with CRF and other APRs form part of the immune system and each are involved with inflammatory response
Methods of assessing plasma proteins?
- Plasma proteins may be expressed as either concentration (g/L)
- Plasma proteins may be assessed based on their functions (prothrombin for clotting, or enzyme function )
1) Measurement of Total protein concentration
2) Assay of specific proteins (based on function
3) Quantify serum proteins by serum electrophoresis
In what case does an abnormal form of protein become present at normal concentration ?
- C1 inhibitor of the classical pathway
What can cause raised Total plasma protein?
- Loss of protein free fluid
- Excessive stasis during venepuncture
- Major increase in the constituent of one or more of the immunoglobulins (paraproteins to)
What can cause low total plasma proteins ?
- Dilution
- hypoalbuminemia
- immunoglobulin deficiency
Qualitative methods for studying plasma and urinary proteins include the use of what technique ?
Electrophoresis
What is electrophoresis? ATIBA
Electrophoresis is a technique that separates compounds such as proteins into various components according to their electrical charge
Steps in electrophoresis
- Application of a small amount of serum to a strip of cellulose acetate or agarose gel
- Passage of a current across the supporting medium for a standard time
- serum protein is then separated into a1, a2, beta, gamma globulins, and albumin groups
- Each of the globulins contains several proteins.
- distinguished after protein staining with dye
The most obvious band in electrophoresis includes ?
Albumin - A single protein, makes up the most obvious band
What proteins does alpha 1 globulin band contains ?
- Alpha 1 anti-trypsin
- Alpha 1 anti-chymotrypsin
- Alpha 1 acid-glycoprotein
Alpha 2 globulin consists of what proteins ?
- Alpha 2 - macroglobulin
- *Haptoglobin
Beta globulin contains what proteins ? (B1 & B2)
- Beta 1: Transferrin, LDL
- Beta 2: C3 complement protein
Gamma globulins contain what proteins ?
They are immunoglobulins
Apart from gamma globulin, immunoglobulins can also be found in what other band?
Beta and alpha 2*
What happens if you use plasma for electrophoresis instead of serum?
- Fibrinogen appears as a distinct band in the Beta-gamma region
- May make interpretations difficult
What happens to alpha 2 band In vivo hemolysis and in vitro hemolysis?
Alpha 2 band contains Haptoglobin
- In ViVo hemolysis
Alpha 2 band is reduced**
- in Vitro hemolysis
**Alpha 2 band may split into two*
Electrophoretic pattern of acute phase reaction ? (Acute inflammatory reaction)
- There’s stimulation of synthesis of Acute phase proteins
A rise in alpha 1 globulin and alpha 2 globulin fractions - there’s a rise in ESR and plasma viscosity
Electrophoretic pattern in chronic inflammation?
- An increase in immunoglobulin synthesis hence; (diffuse rise)
Gamma globulin band is INCREASED
Electrophoretic pattern in Liver cirrhosis ?
- Albumin band is REDUCED
- Alpha 1 band is REDUCED
-
gamma globulin is markedly INCREASED
Increased plasma IgA concentrations - FUSION of beta and gamma bands
Electrophoretic pattern in nephrotic syndrome
Depends on severity of the renal lesion
- Early cases:
low plasma albumin concentration may be the only finding
- Established cases;
Albumin band is REDUCED
Alpha 1 band is REDUCED
Gamma globulin band is REDUCED
- increase in HMW alpha 2 macroglobulin;
Alpha 2 band is INCREASED
Electrophoretic patter
In Nephrotic syndrome (SLE )?
Gamma globulin band may be increased for normal
Electrophoretic pattern In alpha 1 anti-trypsin deficiency
* Absence or obvious reduction in alpha 1 band*
- A1 antitrypsin variants;
*split alpha 1’ band
Normal biological Half life and weight of albumin?
HL; About 21 days
Weight : 65Kda
Inherited abnormalities of albumin synthesis ?
1) Bisalbuminaemias
2) Analbuminaemis
Busalbuminaemia ?
Two forms of albumin are present
Analbuminaemia?
Deficient synthesis of protein
Causes of hypoalbuminaemia (5)
1)Dilutional hypoalbuminaemia
2)Redistribution of albumin
3) decreased synthesis of albumin
4) increased loss of albumin from the body
5) increased catabolism of albumin
Dilutional hypoalbuminaemia cases ?
- Result from artefactual changes - Blood sample taken from arm of IV infusion
- Administration of an excess of protein free fluid
- Fluid retention in edematous states or during late pregnancy
Redistribution of albumin from plasma into the interstitial fluid space is caused by?
-
Recumbency
Plasma albumin concentrations may be 5-10g lower in the recumbent position than In the upright position -
increased capillary membrane permeability
Found in most illnesses, in inflammation also or post operatively
Decreased synthesis of albumin may be caused by?
- Liver cirrhosis; impairment of synthesis
- Malabsorption; impaired absorption of dietary peptides and amino acids
- undernutrition; inadequate supply of Dietary nitrogen
How many percent of the body’s albumin is replaced each day?
4 percent
I’m creased loss of albumin from the body can be caused by?
- In increased membrane permeability
- The skin; extensive burns or skin disease (psoriasis)
- **Intestinal wall; protein losing enteropathy
- The glomeruli; nephrotic syndrome
A large part of interstitial fluid is subcutaneous in ?
Increased capillary permeability
Increased catabolism of albumin can be caused by?
Sepsis
Hyperthyroidism
Associated with nitrogen loss
Consequences of Hypoalbuminemia
Fluid distribution; Edema
Binding functions; Albumin binds calcium, bilirubin and free fatty acids. Hence there’s a poor transport of albumin binding hormones
M proteins are also called ?
Paraproteins
What are paraproteins?
Appearance of abnormal, narrow dense band on the electrophoretic strip.
Can be shown to be monoclonal
Paraproteins are most commonly found where ?
Gamma region of the electrophoretic strip
Causes of paraproteinemia ?
- Myelomatosis (most cases)
- Waldenstrom’s macroglobulinaemia
- B cell lymphomas (CLL)
- Cryoglobulinaemia
- SLE, chronic inflammation, infection
Consequences of paraproteins ?
- Increased plasma viscousity
- Pseudohyponatremia (space occupying effect of the protein)
- retinal vein thrombosis
- cerebral thrombosis
- peripheral gangrene
Globin concentration is defined as?
The differences between the total plasma protein concentration and albumin concentration
What are bence jones proteins
Consists of free monoclonal Light chains, found in the urine of patients with B cell malignancy
Causes of hypogammaglobulinaemia
1)Bruton’s or swiss-type agammaglobulinaemia
2) diabetes mellitus
3) Cushing’s syndrome
4) CKD
5) steroids
Consequences of immunoglobulin deficiency?
IgA - recurrent respiratory tract infections
IgG - pyogenic infections in lungs and skin (tissue spaces) from toxin producing organisms (staph and strep)
IgM- septicemia
In chrons disease what Ig is increased?
IgA - it is produced submucosallyv
Define electrophoresis - IDOWU
Electrophoresis is the migration of charged solutes or particles in a liquid medium under the influence of an electric field
Iontophoresis is defined as ?
The migration of small ions
Zone electrophoresis is defined as?
The migration of charged macromolecules in a porous support medium such as paper, cellulose acetate or agarose gel film
The positive electrode is the?
Anode
The negative electrode is the ?
Cathode
Ampholyes (zwitterion) are positive in what solutions?
In solutions more acidic than it’s iso-electric point, and migrates towards the cathode
The result of the zone electrophoresis is called?
Electrophoretogram - consists of sharply separated zones of a macromolecule
The rate of electrolyte mobility is determined by?
1) The net charge of the molecule
2) Size and shape of the molecule
3) Strength of the electric field
4) properties of the supporting medium
5) Temperature of operation
6) Viscosity of the buffer solution
Components of an electrophoresis machine ? Mention 9
- Electrophoretic tank
- The electrodes (anode and cathode)
- The supporting medium
- The buffers
- Power supply
- The specimen applicator
- Stains
- Densitometer
- Comuputer (automated systems)
Types of electrophoresis ?
- Zone electrophoresis
- Isotachophoresis
- Isoelectric focusing
- Two dimensional electrophoresis
- Capillary electrophoresis
Subtypes of zone electrophoresis
- Agarose gel electrophoresis
- Cellulose acetate electrophoresis
- polyacrilamide gel electrophoresis
Dyes used in electrophoresis ? Mention 6
- Ponceau S dye
- Amido black
- Oil red O
- Fat red
- Sudan black
- CBB
stepwise procedure in electrophoresis ?
- Sample is soaked in a hydrated medium for about 5 mins
- supporting medium is put into the electrophoresis chamber (that was previously filled with the buffer)
- A constant voltage is applied to the supporting medium for a specific time
- support is then removed and placed in a fixative/rapidly dried to prevent diffusion of the sample
- this is followed by staining the zones with appropriate dye.
What’s the function of the buffer solution in electrophoresis ?
It must be added to the chamber to maintain contact with the supporting medium
Purpose of putting the supporting medium in a fixative before staining?
To prevent diffusion of the sample
Factors influencing the uptake of dye of the sample provided ?
The uptake is dye of the sample is proportional to the sample concentration
Simplest way to accomplish detection in electrophoresis is ?
Visualization under UV light
The most reliable and common way for quantitation in electrophoresis is through ?
Densitometry
Clinical applications of electrophoresis ?
- Used to establish several clinical conditions
- used to classify lipoproteins: chylomicrons - origin
VLDLP - pre Beta band
IDL - between beta and pre beta bands
LDL - Beta band
HDL - Alpha band - it is a component of other techniques like southern, northern (RNA) and western (protein) blotting
