Plasma ProteinsPlasma Proteins Reading assignment Harper’s Biochemistry: PP 737-745Reading assignment Harper’s Biochemistry: PP 737-745

Dr. Zeyad El-Akawi Jreisat, M.D, M.A, Ph.DDr. Zeyad El-Akawi Jreisat, M.D, M.A, Ph.D

BloodBlood

• Solid elements: Solid elements: – Red cells Red cells

– White cells White cells

– PlateletsPlatelets

• Liquid medium:Liquid medium:

– PlasmaPlasma

BloodBlood

Major Functions of BloodMajor Functions of Blood

- Respiration- Respiration

- Nutrition- Nutrition

- Excretion- Excretion

- Acid-base balance- Acid-base balance

- Water balance- Water balance

- Body temperature regulation- Body temperature regulation

- Defense- Defense

- Hormone transport and regulation of metabolism- Hormone transport and regulation of metabolism

- Metabolite transport- Metabolite transport

- Coagulation- Coagulation

Difference between plasma and serum????

Serum = plasma - Serum = plasma - coagulation factorscoagulation factors

Plasma CompositionPlasma Composition

• Water Water 92% 92%

• Plasma proteins 7% (total 7.0-7.5 g/dL)Plasma proteins 7% (total 7.0-7.5 g/dL)– SimpleSimple

– ConjugatedConjugated• GlycoproteinsGlycoproteins

• LipoproteinsLipoproteins

• Electrolytes (NaElectrolytes (Na++, K, K++, Ca, Ca+2+2, Cl, Cl--, HCO, HCO33--))

• MetabolitesMetabolites

• NutrientsNutrients

• HormonesHormones

• Other solutes Other solutes 1% 1%

Plasma proteinsPlasma proteins

• Most plasma proteins, with the exception of Most plasma proteins, with the exception of immunoglobulins and protein hormones are synthesized in immunoglobulins and protein hormones are synthesized in the liverthe liver

• Plasma proteins are generally synthesized on membrane-Plasma proteins are generally synthesized on membrane-bound polyribosomesbound polyribosomes– Rough endoplasmic membrane → smooth endoplasmic membrane Rough endoplasmic membrane → smooth endoplasmic membrane

→ Golgi apparatus → secretory vesicles → Plasma → Golgi apparatus → secretory vesicles → Plasma

• Almost all plasma proteins are glycoproteinsAlmost all plasma proteins are glycoproteins• Plasma proteins circulate in the blood and between the Plasma proteins circulate in the blood and between the

blood and the extra-cellular tissue spaces. Their movement blood and the extra-cellular tissue spaces. Their movement occurs not only by passive diffusion through junctions occurs not only by passive diffusion through junctions between capillary endothelial cells but by active transport between capillary endothelial cells but by active transport mechanisms and by pinocytosis and exocytosismechanisms and by pinocytosis and exocytosis

Plasma proteinsPlasma proteins

• Because of this movement, most extra-vascular fluids Because of this movement, most extra-vascular fluids normally contain small amount of plasma proteinsnormally contain small amount of plasma proteins

• The concentration of protein in the plasma is important in The concentration of protein in the plasma is important in determining the distribution of fluid between blood and determining the distribution of fluid between blood and tissuestissues

• Many plasma proteins exhibit polymorphismMany plasma proteins exhibit polymorphism

– Alpha1-antitrypsin, haptoglobin, transferrin, Alpha1-antitrypsin, haptoglobin, transferrin, ceruloplasmin and immunoglobulinsceruloplasmin and immunoglobulins

• Each plasma protein has a characteristic half-life in the Each plasma protein has a characteristic half-life in the circulationcirculation

• Most plasma proteins are catabolized in the liver. Most plasma proteins are catabolized in the liver.

Plasma proteinsPlasma proteins

• Alterations in plasma proteins occurs in health and diseaseAlterations in plasma proteins occurs in health and disease• The levels of certain proteins in plasma increase during The levels of certain proteins in plasma increase during

acute inflammatory states or secondary to certain types of acute inflammatory states or secondary to certain types of tissue damagetissue damage

• Some of these alterations have genetic origin, many more Some of these alterations have genetic origin, many more reflect physiological or pathological processesreflect physiological or pathological processes

• Variations in the amount or kinds of protein found in Variations in the amount or kinds of protein found in plasma or extra-vascular fluids depend on many factorsplasma or extra-vascular fluids depend on many factors– GeneticGenetic– PhysiologicalPhysiological– Pathological Pathological

Classification of plasma proteins based on Classification of plasma proteins based on their function their function

• Antiproteases: antichymotrypsin, alpha1-antitrypsin, Antiproteases: antichymotrypsin, alpha1-antitrypsin, alpha2-macroglobulin, antithrombinalpha2-macroglobulin, antithrombin

• Blood clotting: various coagulation factors, fibrinogenBlood clotting: various coagulation factors, fibrinogen• Enzymes: Enzymes:

– Function in blood; coagulation factors, cholinesteraseFunction in blood; coagulation factors, cholinesterase– Leakage from cells or tissues; aminotransferasesLeakage from cells or tissues; aminotransferases

• Hormones: ErythropoietinHormones: Erythropoietin• Immune defense: immunoglobulins, complement proteins, Immune defense: immunoglobulins, complement proteins,

betabeta22-microglobulin-microglobulin• Involvement in inflammatory responses: Acute phase Involvement in inflammatory responses: Acute phase

response proteins (c-reactive proteins, alpha1-acid response proteins (c-reactive proteins, alpha1-acid glycoprotein)glycoprotein)

• Oncofetal: alpha-1 fetoprotein (AFP)Oncofetal: alpha-1 fetoprotein (AFP)

Classification of plasma proteins based on Classification of plasma proteins based on their functiontheir function

• Transport or Binding proteins:Transport or Binding proteins:– Albumin; various ligands, including bilirubin, free fatty acids, ions Albumin; various ligands, including bilirubin, free fatty acids, ions

(Ca(Ca+2+2), metals (Cu), metals (Cu+2+2, Zn, Zn+2+2), metheme, steroids and other hormones, ), metheme, steroids and other hormones, drugs.drugs.

– Ceruloplasmin: contains CuCeruloplasmin: contains Cu+2+2

– Corticosteroid-binding globulin: transcortin (binds cortisol)Corticosteroid-binding globulin: transcortin (binds cortisol)

– Haptoglobin: binds extracorpuscular hemoglobinHaptoglobin: binds extracorpuscular hemoglobin

– Lipoproteins: chylomicrons, VLDL, LDL, HDLLipoproteins: chylomicrons, VLDL, LDL, HDL

– Hemopexin: binds hemeHemopexin: binds heme

– Ritenol-binding protein: binds retinolRitenol-binding protein: binds retinol

– Sex hormone-binding globulin: binds testosterone, estradiolSex hormone-binding globulin: binds testosterone, estradiol

– Thyroid-binding globulin: binds T3, T4Thyroid-binding globulin: binds T3, T4

– Transferrin: transport ironTransferrin: transport iron

– Transthyretin (prealbumin): binds T4 and forms a complex with Transthyretin (prealbumin): binds T4 and forms a complex with retinol-binding protein retinol-binding protein

Plasma proteins separationPlasma proteins separation

• Separation of individual proteins from a complex mixture is Separation of individual proteins from a complex mixture is accomplished by the use of solvents or electrolytes or both to remove accomplished by the use of solvents or electrolytes or both to remove different protein fractions in accordance with their solubility different protein fractions in accordance with their solubility characteristics.characteristics.

• Salting out: a method for separation of plasma proteins using various Salting out: a method for separation of plasma proteins using various

concentrations ofconcentrations of[Sodium or ammonium sulfate][Sodium or ammonium sulfate]

• Plasma proteins can be separated by this method into three groupsPlasma proteins can be separated by this method into three groups– FibrinogenFibrinogen– AlbuminAlbumin– GlobulinsGlobulins

Plasma proteins separationPlasma proteins separation

• Electrophoresis:Electrophoresis:– The most common method of analyzing plasma proteinsThe most common method of analyzing plasma proteins– Using different supporting medium, the most common Using different supporting medium, the most common

in clinical laboratories is cellulose acetate in clinical laboratories is cellulose acetate (electrophoretogram)(electrophoretogram)

– Separated proteins into five bands: albumin, alpha1, Separated proteins into five bands: albumin, alpha1, alpha2, beta, and gamma fractionsalpha2, beta, and gamma fractions

– The amount of bands quantified by densitometric The amount of bands quantified by densitometric scanning machinescanning machine

ElectrophoresisElectrophoresis

Plasma protein electrophoresisPlasma protein electrophoresis

Quantified of plasma proteins by Quantified of plasma proteins by densitometric scanning machinedensitometric scanning machine

Plasma proteins separationPlasma proteins separation

• Antibodies:Antibodies:

– Specific plasma proteins are separated by specific Specific plasma proteins are separated by specific monoclonal antibodies fixed on stationary phasemonoclonal antibodies fixed on stationary phase

(Column Chromatography)(Column Chromatography)– Allowing isolation of pure proteins from the complex Allowing isolation of pure proteins from the complex

mixture present in plasma mixture present in plasma

Acute phase reactant proteinsAcute phase reactant proteins

**Concentration of these proteins rise significantly in acute **Concentration of these proteins rise significantly in acute inflammation, chronic inflammation and cancer** inflammation, chronic inflammation and cancer**

• Alpha1-antitrypsin (AAT): congenital deficiency may be Alpha1-antitrypsin (AAT): congenital deficiency may be associated with emphysema or cirrhosisassociated with emphysema or cirrhosis

• Alpha1-acid glycoprotein (AAG): binds cationic drugs and Alpha1-acid glycoprotein (AAG): binds cationic drugs and hormones hormones

• Haptoglobin (HAP): binds hemoglobin, reduced by Haptoglobin (HAP): binds hemoglobin, reduced by hemolysishemolysis

• Ceruloplasmin (CER): contains copper, antioxidant, Ceruloplasmin (CER): contains copper, antioxidant, decreased in Wilson’s diseasedecreased in Wilson’s disease

• C4: Complement factorC4: Complement factor• C3: Complement factorC3: Complement factor• C-reactive protein (CRP): Nonspecific defense against C-reactive protein (CRP): Nonspecific defense against

infectious agentsinfectious agents• Fibrinogen Fibrinogen **Stimulatory factors, Interleukin-1 (IL-1) and interleukin-6 **Stimulatory factors, Interleukin-1 (IL-1) and interleukin-6

(IL-6) at the gene level** (IL-6) at the gene level**

Plasma proteinsPlasma proteins

• Albumin:Albumin:– is the major protein of human plasma (3.4-4.7 g/dL)is the major protein of human plasma (3.4-4.7 g/dL)– Approximately 40% of albumin is present in plasma and the other Approximately 40% of albumin is present in plasma and the other

60% in the extracellular space60% in the extracellular space– It synthesized in the liver as preproproteinIt synthesized in the liver as preproprotein– The synthesis of albumin is depressed in a variety of diseases, The synthesis of albumin is depressed in a variety of diseases,

particularly those of the liver (decreased albumin/globulin ratio)particularly those of the liver (decreased albumin/globulin ratio)– Responsible for 75-80% of the osmotic pressure of human plasmaResponsible for 75-80% of the osmotic pressure of human plasma– Absence of albumin (analbuminemia) might caused by mutation Absence of albumin (analbuminemia) might caused by mutation

that affect splicingthat affect splicing– It binds many ligands (free fatty acids, calcium, certain steroid It binds many ligands (free fatty acids, calcium, certain steroid

hormones, bilirubin, tryptophan)hormones, bilirubin, tryptophan)– It binds and transport drugs (sulfonamides, penicillin G, It binds and transport drugs (sulfonamides, penicillin G,

dicumarol, aspirin) dicumarol, aspirin) – It transports copper It transports copper

• Haptoglobin:Haptoglobin:– Binds extracorpuscular hemoglobin preventing free hemoglobin Binds extracorpuscular hemoglobin preventing free hemoglobin

from entering the kidneyfrom entering the kidney

– Exist in three polymorphic forms, Hp1-1, Hp2-1, Hp2-2Exist in three polymorphic forms, Hp1-1, Hp2-1, Hp2-2

– Low levels of haptoglobin are found in patients with hemolytic Low levels of haptoglobin are found in patients with hemolytic anemiasanemias

– It is an acute phase protein and its plasma level is elevated in a It is an acute phase protein and its plasma level is elevated in a variety of inflammatory statesvariety of inflammatory states

• Transferrin:Transferrin:– Is a beta-1 globulinIs a beta-1 globulin

– It is a glycoprotein synthesized in the liverIt is a glycoprotein synthesized in the liver

– Shuttles iron to sites where it is neededShuttles iron to sites where it is needed

– Transferrin diminishes the potential toxicity of ironTransferrin diminishes the potential toxicity of iron

– The concentration of transferrin in plasma is approximately 300 The concentration of transferrin in plasma is approximately 300 mg/dL that can bind 300 µg of iron per deciliter (total iron-binding mg/dL that can bind 300 µg of iron per deciliter (total iron-binding capacity) of plasmacapacity) of plasma

• Ferritin:Ferritin:

– Normally there is a little ferritin in human plasmaNormally there is a little ferritin in human plasma

– In patients with excess iron, the amount of ferritin in plasma is In patients with excess iron, the amount of ferritin in plasma is markedly elevatedmarkedly elevated

– Index of body iron storesIndex of body iron stores

– Synthesis of the transferrin receptors and that of ferritin are Synthesis of the transferrin receptors and that of ferritin are reciprocally linked to cellular iron contentreciprocally linked to cellular iron content

– Iron response elementsIron response elements

– Iron-responsive element-binding protein Iron-responsive element-binding protein

• Hemosiderin:Hemosiderin:

– Partly degraded form of ferritin but still containing ironPartly degraded form of ferritin but still containing iron

• Primary hemochromatosis: is a common genetic disorder Primary hemochromatosis: is a common genetic disorder characterized by excessive storage of iron in tissues leading to tissue characterized by excessive storage of iron in tissues leading to tissue damage damage

• Secondary hemochromatosis: can occurs in the result of increased iron Secondary hemochromatosis: can occurs in the result of increased iron levels by transfusion, intake, hemolysis levels by transfusion, intake, hemolysis

• Ceruloplasmin:Ceruloplasmin:– It is an alpha-2 globulinIt is an alpha-2 globulin– Binds copperBinds copper– Low levels of this protein are associated with Wilson diseaseLow levels of this protein are associated with Wilson disease– It exhibits a copper-dependent oxidase activityIt exhibits a copper-dependent oxidase activity

• Copper:Copper:– Is a cofactor for certain enzymes including, amine oxidase, copper-Is a cofactor for certain enzymes including, amine oxidase, copper-

dependent superoxide dismutase, cytochrome oxidase, tyrosinasedependent superoxide dismutase, cytochrome oxidase, tyrosinase– It is excess can cause problems because it can oxidize proteins and It is excess can cause problems because it can oxidize proteins and

lipids, bind to nucleic acids and enhance the production of free lipids, bind to nucleic acids and enhance the production of free radicalsradicals

– Metallothioneins: are a group of small proteins found in the Metallothioneins: are a group of small proteins found in the cytosol of cells particularly of liver, kidney, and intestine they cytosol of cells particularly of liver, kidney, and intestine they control of copper levelscontrol of copper levels

• Menkes Disease: “kinky” or “Steely” hair diseaseMenkes Disease: “kinky” or “Steely” hair disease

– Is due to mutations in the gene for a copper-binding P-type Is due to mutations in the gene for a copper-binding P-type ATPase leads to abnormality in copper metabolismATPase leads to abnormality in copper metabolism

– X-linkedX-linked

– Defect in copper exit from cells leads to its accumulationDefect in copper exit from cells leads to its accumulation

– Involves the nervous system, connective tissue and vasculatureInvolves the nervous system, connective tissue and vasculature

• Wilson Disease:Wilson Disease:

– Is due to mutations in the gene for a copper-binding P-type 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 bileATPase results in the failure of copper to be excreted in the bile

– Copper toxicosesCopper toxicoses

• Alpha-1 antitrypsin deficiency: a serine protease inhibitorAlpha-1 antitrypsin deficiency: a serine protease inhibitor– Is associated with emphysema and liver diseaseIs associated with emphysema and liver disease

– Synthesized by hepatocytes and macrophagesSynthesized by hepatocytes and macrophages