1. Plasma Proteins Reading assignment Harper’s Biochemistry: PP 737-745
Dr. Zeyad El-Akawi Jreisat, M.D, M.A, Ph.D

2. Blood Solid elements: Liquid medium: Red cells White cells Platelets
Plasma

3. Blood

4. Major Functions of Blood
- Respiration
- Nutrition
- Excretion
- Acid-base balance
- Water balance
- Body temperature regulation
- Defense
- Hormone transport and regulation of metabolism
- Metabolite transport
- Coagulation

5. Difference between plasma and serum????
Serum = plasma - coagulation factors

6. Plasma Composition Water 92% Plasma proteins 7% (total 7.0-7.5 g/dL)
Simple
Conjugated
Glycoproteins
Lipoproteins
Electrolytes (Na+, K+, Ca+2, Cl-, HCO3-)
Metabolites
Nutrients
Hormones
Other solutes %

7. Plasma proteins
Most plasma proteins, with the exception of immunoglobulins and protein hormones are synthesized in the liver
Plasma proteins are generally synthesized on membrane-bound polyribosomes
Rough endoplasmic membrane → smooth endoplasmic membrane → Golgi apparatus → secretory vesicles → Plasma
Almost all plasma proteins are glycoproteins
Plasma proteins circulate in the blood and between the blood and the extra-cellular tissue spaces. Their movement occurs not only by passive diffusion through junctions between capillary endothelial cells but by active transport mechanisms and by pinocytosis and exocytosis

8. Plasma proteins
Because of this movement, most extra-vascular fluids normally contain small amount of plasma proteins
The concentration of protein in the plasma is important in determining the distribution of fluid between blood and tissues
Many plasma proteins exhibit polymorphism
Alpha1-antitrypsin, haptoglobin, transferrin, ceruloplasmin and immunoglobulins
Each plasma protein has a characteristic half-life in the circulation
Most plasma proteins are catabolized in the liver.

9. Plasma proteins
Alterations in plasma proteins occurs in health and disease
The levels of certain proteins in plasma increase during acute inflammatory states or secondary to certain types of tissue damage
Some of these alterations have genetic origin, many more reflect physiological or pathological processes
Variations in the amount or kinds of protein found in plasma or extra-vascular fluids depend on many factors
Genetic
Physiological
Pathological

10. Classification of plasma proteins based on their function
Antiproteases: antichymotrypsin, alpha1-antitrypsin, alpha2-macroglobulin, antithrombin
Blood clotting: various coagulation factors, fibrinogen
Enzymes:
Function in blood; coagulation factors, cholinesterase
Leakage from cells or tissues; aminotransferases
Hormones: Erythropoietin
Immune defense: immunoglobulins, complement proteins, beta2-microglobulin
Involvement in inflammatory responses: Acute phase response proteins (c-reactive proteins, alpha1-acid glycoprotein)
Oncofetal: alpha-1 fetoprotein (AFP)

11. Classification of plasma proteins based on their function
Transport or Binding proteins:
Albumin; various ligands, including bilirubin, free fatty acids, ions (Ca+2), metals (Cu+2, Zn+2), metheme, steroids and other hormones, drugs.
Ceruloplasmin: contains Cu+2
Corticosteroid-binding globulin: transcortin (binds cortisol)
Haptoglobin: binds extracorpuscular hemoglobin
Lipoproteins: chylomicrons, VLDL, LDL, HDL
Hemopexin: binds heme
Ritenol-binding protein: binds retinol
Sex hormone-binding globulin: binds testosterone, estradiol
Thyroid-binding globulin: binds T3, T4
Transferrin: transport iron
Transthyretin (prealbumin): binds T4 and forms a complex with retinol-binding protein

12. Plasma proteins separation
Separation of individual proteins from a complex mixture is accomplished by the use of solvents or electrolytes or both to remove different protein fractions in accordance with their solubility characteristics.
Salting out: a method for separation of plasma proteins using various concentrations of
[Sodium or ammonium sulfate]
Plasma proteins can be separated by this method into three groups
Fibrinogen
Albumin
Globulins

13. Plasma proteins separation
Electrophoresis:
The most common method of analyzing plasma proteins
Using different supporting medium, the most common in clinical laboratories is cellulose acetate (electrophoretogram)
Separated proteins into five bands: albumin, alpha1, alpha2, beta, and gamma fractions
The amount of bands quantified by densitometric scanning machine

14. Electrophoresis

15. Plasma protein electrophoresis

16. Quantified of plasma proteins by densitometric scanning machine

18. Plasma proteins separation
Antibodies:
Specific plasma proteins are separated by specific monoclonal antibodies fixed on stationary phase
(Column Chromatography)
Allowing isolation of pure proteins from the complex mixture present in plasma

19. Acute phase reactant proteins
**Concentration of these proteins rise significantly in acute inflammation, chronic inflammation and cancer**
Alpha1-antitrypsin (AAT): congenital deficiency may be associated with emphysema or cirrhosis
Alpha1-acid glycoprotein (AAG): binds cationic drugs and hormones
Haptoglobin (HAP): binds hemoglobin, reduced by hemolysis
Ceruloplasmin (CER): contains copper, antioxidant, decreased in Wilson’s disease
C4: Complement factor
C3: Complement factor
C-reactive protein (CRP): Nonspecific defense against infectious agents
Fibrinogen
**Stimulatory factors, Interleukin-1 (IL-1) and interleukin-6 (IL-6) at the gene level**

20. Plasma proteins Albumin:
is the major protein of human plasma ( g/dL)
Approximately 40% of albumin is present in plasma and the other 60% in the extracellular space
It synthesized in the liver as preproprotein
The synthesis of albumin is depressed in a variety of diseases, particularly those of the liver (decreased albumin/globulin ratio)
Responsible for 75-80% of the osmotic pressure of human plasma
Absence of albumin (analbuminemia) might caused by mutation that affect splicing
It binds many ligands (free fatty acids, calcium, certain steroid hormones, bilirubin, tryptophan)
It binds and transport drugs (sulfonamides, penicillin G, dicumarol, aspirin)
It transports copper

21. Haptoglobin: Transferrin:
Binds extracorpuscular hemoglobin preventing free hemoglobin from entering the kidney
Exist in three polymorphic forms, Hp1-1, Hp2-1, Hp2-2
Low levels of haptoglobin are found in patients with hemolytic anemias
It is an acute phase protein and its plasma level is elevated in a variety of inflammatory states
Transferrin:
Is a beta-1 globulin
It is a glycoprotein synthesized in the liver
Shuttles iron to sites where it is needed
Transferrin diminishes the potential toxicity of iron
The concentration of transferrin in plasma is approximately 300 mg/dL that can bind 300 µg of iron per deciliter (total iron-binding capacity) of plasma

22. Ferritin:
Normally there is a little ferritin in human plasma
In patients with excess iron, the amount of ferritin in plasma is markedly elevated
Index of body iron stores
Synthesis of the transferrin receptors and that of ferritin are reciprocally linked to cellular iron content
Iron response elements
Iron-responsive element-binding protein
Hemosiderin:
Partly degraded form of ferritin but still containing iron
Primary hemochromatosis: is a common genetic disorder characterized by excessive storage of iron in tissues leading to tissue damage
Secondary hemochromatosis: can occurs in the result of increased iron levels by transfusion, intake, hemolysis

23. Ceruloplasmin:
It is an alpha-2 globulin
Binds copper
Low levels of this protein are associated with Wilson disease
It exhibits a copper-dependent oxidase activity
Copper:
Is a cofactor for certain enzymes including, amine oxidase, copper-dependent superoxide dismutase, cytochrome oxidase, tyrosinase
It is excess can cause problems because it can oxidize proteins and lipids, bind to nucleic acids and enhance the production of free radicals
Metallothioneins: are a group of small proteins found in the cytosol of cells particularly of liver, kidney, and intestine they control of copper levels

24. Menkes Disease: “kinky” or “Steely” hair disease
Is due to mutations in the gene for a copper-binding P-type ATPase leads to abnormality in copper metabolism
X-linked
Defect in copper exit from cells leads to its accumulation
Involves the nervous system, connective tissue and vasculature
Wilson Disease:
Is due to mutations in the gene for a copper-binding P-type ATPase results in the failure of copper to be excreted in the bile
Copper toxicoses
Alpha-1 antitrypsin deficiency: a serine protease inhibitor
Is associated with emphysema and liver disease
Synthesized by hepatocytes and macrophages