PLASMA PROTEINS

By Dr J Onubi

MBBS, M.Sc, FMC Path

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DEFINITION

Plasma Proteins

They are proteins found in the plasma ( intravascular) compartment.

Many proteins are found in the plasma – over hundred. The functions of many are unknown. Many diseases are associated with changes in the concentration, structure or function.

PLASMA PROTEINS

Albumin & globulins

1. Pre-albumin (Transthyretin)
2. Albumin
3. Globulins

- α1-globulin e.g α1-antitrypsin

- α2-globulin e.g haptoglobulin

- β-globulin: β1 e.g fibrinogen, β2 e.g glycoproteins

- γ-globulins –immunoglobulins

4. Complements

Synthesis

Major plasma proteins are synthesized and secreted into the blood stream by the liver.

The exceptions are the immunoglobulins secreted by the lymphoreticular system (Plasma cells).

They are affected by:

• Hormones – T4 and corticosteroids increase the rate of albumin synthesis.
• Nutritional Status- in protein malnutrition, kwashiorkor, protein synthesis decrease.
• Environment – heat decrease protein synthesis.
• General state of health- decrease in malignancy and neoplastic disease.

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Functions

1. Transport- serves as transporter when bound to this agents- makes them stable.

- Metabolites e.g. lipoproteins

- Hormones- T4 Thyroxine binding Albumin (TBA),cortisol- cortisol binding globulin(CBG),Thyroxine-binding pre-albumin(TBPA).

-Metals- iron transferrin

-Calcium- albumin

-Excretory products- bilirubin –albumin

-Drugs and other toxic substances

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2. Colloidal – oncotic pressure maintenance , mainly by albumin (approximately 80% of colloid osmotic pressure of plasma ). In hypoalbumianaemia- oedema is likely to develop.

3. Inflammatory response & control of infections- the immunoglobulins and complement systems form parts of the immune systems, the complement proteins & the acute phase proteins or reactants are also involved in inflammatory response.

4. Nutritional monitoring- Pre albumin,RBP,CRP,Fibronectin,Interleukins

5. Buffer – Hb (Haemoglobin)

6. Coagulation & fibrinolysis

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Pre-albumin

• Also known as thyroxine-binding pre albumin(Transthyretin)
• Produced in the liver and choroid plexus in the brain
• Moves ahead of albumin
• Ref value 15-36mg/dl (150-360mg/l)
• Half life 2days
• Transport T4 and vitamin A
• Nutritional marker
• Albumin precursor

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Albumin

• Synthesized in the liver
• Concentration influenced by synthesis, degredation and distribution
• Half life 2-3 weeks (20 days)
• Molecular weight- 65,000 daltons
• Ref .value -3.5 to 5.2g/dl, if less than 2.5 (oedema formation) -35 to 52g/l
• Highest protein in the plasma
• Major contributor to colloid osmotic pressure
• Capacity to bind substances in the blood

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• Binds various substances in the blood. It has 4 binding sites.

thyroid hormones

• sex hormones- estrogen and progesterone
• iron
• Unconjugated bilirubin
• Salicylic acid (aspirin)
• Fatty acids
• Calcium (ca²⁺) & Mg²⁺ ions.
• Many drugs – serum alb level can affect the half-life of drugs
• Some dyes.
• Glycated albumin – short term hyperglycemia control more sensitive than glycosylated hemoglobin

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Globulins

Globulins –consists of α₁, α₂ β and λ fractions.- They are enzymes, proteins and antibodies.

• α_1-fraction – consists of number of different proteins with different functions.
• - α₁ – Antitrypsin (AAT) – Syntesized in the liver most important function by inhibiting protease neutrophil elastase ( this can destroy alveoli and can lead to emphysema if not controlled by α₁ – antitrypsin)
• If the abnormal form of α_{1 –} antitrypsin accumulate in the liver leading to cirrhosis

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• It is a acute phase reactant i.e

It is increased in inflammatory reactions, pregnancy , contraceptives

• Note : Abnormal α₁ antitrypsin (AAT) levels are most often found in the laboratory by the lack of α_1- globulin band on protein electrophoresis because it is the major component of the fraction of serum proteins that migrates immediately following albumin.

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b) α₁ – fetoprotein (AFP) – synthesized in the developing embryo & fetus and then by the parenchymal cells of the liver. AFP has no known function in normal adults.

• Elevated AFP – Spina bifida, neural tube defects, abdominal wall defects ,anencephaly, general fetal distress.
• Low levels of maternal AFP indicate an increased risk for down syndrome and trisomy 18. It is increased in the presence of twins. AFP screening is done between 14-20 weeks gestational age.

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c) α ₁ – Acid Glycoprotein (AAG)

• Synthesized in the liver
• It is acute –phase reactant
• Elevated in stress,
• Inflammation and tissue damage
• AMI, trauma, pregnancy, cancer , pneumonia , surgery

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d) α₁ – Antichymotrypsin – synthesized in the liver. It is an acute-phase reactant. Defficiency is associated with liver disease.

α₂ Fraction

• Haptoglobin (Hp) – α ₂ – glycoprotein
• synthesized in the liver
• acute phase reactants. Increased in inflammation, e.g ulcerative colitis, heart attack and severe infection.
• used to evaluate rheumatic diseases. Increased in burns and nephrotic syndrome.
• The function of haptoglobin is to bind free hemoglobin to prevent the loss of hemoglobin and its constituent, iron, into the urine

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b) Ceruloplasmin – α ₂ glycoproteins enzymes synthesized in the liver

- Acute – phase reactant – increased in inflammation , severe infection, tissue damage, pregnancy and use of oral contraceptives

• Transport 90% copper
• Wilson disease – An autosomal recessive inherited disorder associated with decreased levels of cerulosplasmin and excessive storage of copper in the liver , brain and other organs resulting in hepatic cirrhosin and neurologic damage
• Low cerulosplasmin is seen in malnutrition, malabsorption, severe liver disease, nephrotic syndrome.
• C) α ₂ – macroglobulin -
• Synthesized in the liver, major component of the α ₂ band in protein electrophoresis

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• β- globubin Fraction –
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I. Transferrin ( siderophilin) – Negative acute- phase protein synthesized in the liver. Binds iron for transport.

• It is the major component of the β – globulin fraction. Appears as distinct band on SPE
• Prevents iron loss through kidney
• Prevents iron deposition in the tissue
• Transport iron to storage sites
• Low transferrin can inpair hemoglobin production and leads to anemia
• Low transferrin can lead to iron deposits in tissues

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2. Hemopexin- β – globulin region – acute –phase reactants. It scavenges the heme released or lost by the turnovers of heme proteins such as hemoglobin which protects the body from oxidative damage that free heme can cause.
3. Liproproteins – Appears at β – globulin band. complexes of proteins and lipids that transports cholesterol, triglycerides and phopholipds in the blood. Classified as chylomicrons, VLDL,IDL, LDL and Lipoproteins (a) and HDL

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IV. β_{2 –} Microglobulin- Light chain components of the major histocompatibility complex (HLA). Filtered in the glomerulus and ˃99% reabsorbed in the PT. Elevated serum levels are the result of impaired clearance by the kidney or overproduction of the protein that occurs in a number of inflammatory diseases – rheumatoid arthritis and systemic lupus erythematosus (SLE) .

• A high β 2M level in HIV in the absence of renal failure indicates a large lymphocyte turnover rate which suggests the virus is killing lypmphocytes.

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Immunoglobulins

• There are glycoproteins produced by B-lymphocytes
• Contains two identical heavy chains (H) and two identical light chains (L) linked by two disulfide bonds
• The five classes are

IgG-75%, IgM, IgA-15%, IgE, IgD

Each heavy chain has two regions, the constant region and the variable region. The constant region are involved in complements binding, placental passage and binding to cell membrane.

• The two light chains are

Kappa(κ)

Lamda(λ),

• IgG :Most abundant class of antibodies in blood plasma and lymph (75%). Act on bacteria, fungi, viruses, and foreign particles by agglutination, opsonization( theprocess by which a pathogen is marked for ingestion and destruction by a phagocyte). Increased in liver disease, infection, myeloma, parasitic disease.

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• IgA: Main one found in mucous secretin – tears, saliva, colostrum, vaginal fluid, secretions from the respiratory and GIT mucosa. Serum IgA and secretary IgA.

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• IgM : The first antibody that appears in response to antigenic stimulation. It is the naturally occurring antibody to red cell antigens.

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• IgM : The first antibody that appears in response to antigenic stimulation. It is the naturally occurring antibody to red cell antigens.
• IgD : Helps regulates B cell function. Its function is largely unknown.
• IgE : Produced by B cells and plasma cells and is the immunoglobinassociated with allergic and anaphylactic reactions. Its concentration in circulation is very low.

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Complement

• 30 plasma proteins in active form. They are synthesized by macrophages and hepatocytes.
• The natural defense mechanism that protects the human body from infections
• In classic pathway- c1 binds to antigen-antibody complex, c2-c9 are activated, to cell membrane and then lysis of the cell.
• Complement is increased in inflammatory state and decreased in mal-nutrition

Changes of plasma proteins in diseases

Hypoalbuminaemia:

Causes

A. Dilutional

• Artefactural – taken blood from infusion hand.
• Fluid retention
• Late pregnancy

B. Redistribution of albumin from plasma to interstitial as a result of increase capillary permeability

C. Decreased synthesis of albumin.

D. Increased breakdown of albumin.

E. Increased loss from the body

• Nephrotic syndrome
• Burns
• Skin disease- psoriasis
• Protein loosing enteropathy- malabsorption

F. Liver disorders that results in:

• Bisalbuminaemia- presence of albumin that has unusual molecular characteristics usually demonstrated by the presence of two albumin bands instead of single. no oedema
• Analbuminaemia- absence of albumin in the serum. there is oedema 

Inflammation:

The immunoglobulins and complement systems form parts of the immune systems, the complement proteins & the acute phase proteins or reactants are also involved in inflammatory response.

Paraproteinaemia

This is the condition in which a single clone of the antibody producing cells produces an excessive amount of a single type and class of immunoglobins. These immunoglobins are called paraproteins.

Two types of paraproteins

1. The Benign

• Rheumatoid Arthritis
• Multiple Sclerosis
• Systemic lupus erythematosis
• Myasthemia gravis

2. Malignant

• Multiple myloma
• Macroglobulinaemia e.g. Waldenstrom’s macroglobulinaemia 

Disease Patterns as shown on serum electrophoresis

1. Monoclonal immunoglobin disease. Myelomatosis – multiple myeloma, a spike in γ- region. There may also be spike in the α2 and β-region.*if there is deficiency, the γ- region will be pale (i.e.in IgG which is the predominant immunoglobulin).

2. Nephrotic syndrome- Patient loses serum albumin and low molecular weight proteins in the urine. Some IgG is also lost. At the same time, an increase in α2 macroglobulin, β- lipoprotein , complement components and haptoglobulin. These two events leads to a dramatic decrease in the relative amount of albumin and a marked increase in the relative amounts of α2-globulin fractions.

3. α-1 antitrypsin deficiency- decrease in α1 anti trypsin

4. Inflammatory pattern- Decrease in albumin, increase in α2 globulin (celuloplasmin and haptoglobin), increase in α1 globulin (α1- antitrypsin), increased β-globulin band(C- reactive protein)

5. Liver disease- e.g. Liver Cirrhosis

β-γ bridge or fusion characteristic of cirrhosis. It is due to some fast moving γ-globulins that prevent resolution of the β and γ-globulins bands.

Other conditions

Infectious hepatitis- γ-globulin rises with increasing hepatocellular damage.

Obstructive Jaundice- increase in α2 and β -globulins.

Protein electrophoresis

Separation of proteins on the basis of their electric charge densities.

Movement of the charged protein depends on the following

• PH of the surrounding buffer and on the charge carried on the protein Cations (+ ve charge) moves towards cathode (-ve terminal)and Anions (-ve charge) towards Anode (+ve terminal)
• Speed of migration depends on degree of ionization and the protein of the PH of the buffer.
• The more the PH of the buffer differs from the PI, the greater the magnitude of the net charge of that protein and the faster it will move in the electric field.
• Size of the molecule
• Electric field
• Shape of the molecule
• Temperature

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• Serum Protein Electrophoresis

Anode Cathode

+ve _-ve

+ve Alkaline buffer

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Support medium (agarose gel or Cellulose acetate paper)

The PH of the alkaline buffer is set at 8.6. At the PH all major proteins carry net negative charge as such migrate towards the anode.

The following 5 BANDS are noted

– Albumin travels farthest to the anode then

-α1- globulins

-α2-globuluns

-β globulins

-γ globulins (immunoglobulins)

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Fixing

Support strip is inserted in an acid solution (e.g. acetic acid) to denature the protein and immobilize them on the support medium. The proteins are then stained.

Staining

Ponceau S, amido black or coomassie blue. The protein appears as bands on the support medium.

Cellulose acetate electrophoretic patterns are shown.

Visual inspection of the membrane

Scanning densitometer

Concentration is calculated as a percentage of the total protein that was determined by one of the proteins methods such as the Biuret procedure.

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Electrophoretic patterns:

Inadvertent use of plasma will result in a narrow band in the β2-globulin region because of the presence of fibrinogen.

The electrophoretic pattern can give information about the relative increase and decrease in the protein population as well as information about the homogeneity of a fraction.

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