plasma proteins in disease diagnosis (4)

8/13/2019 Plasma Proteins in Disease Diagnosis (4)

1/81

Blood (Plasma, Serum)

Proteins


2/81

Objectives

Know the major plasma proteins and where they areproduced.

Describe the main properties and functions of some

key plasma proteins.

Give a detailed explanation of the role of at least twoplasma proteins in disease and clinical diagnosis


3/81


4/81

Blood

When cells are removed

Plasma

Water (90%)

blood proteins (8%)

inorganic electrolytes salts, lipids glucose

remove fibrinogens/clotting factors (clot)

serum


5/81

All bodily fluids have some proteins:

Plasma (3-8g/dL)

lymphatic fluids

cerebrospinal fluid (45mg/dL).

Urine (trace amounts)


6/81

Examples of plasma proteins:

Albumin,

Clotting factors,

Haptoglobin

Transferrin,

Ceruloplasmin

Lipoproteins

-antitrypsin

-macroglobulin,

Immunoglobulins

The complement

system.


7/81

Plasma 2DE proteomics with andwithout depletion of high

abundance plasma proteins byaffinit removal


8/81

How are plasma proteins categorised?

Simple proteins:

Albumin


9/81

Conjugated Proteins:

Glycoproteins,

Lipoproteins


10/81

Size small globulins (40kda)

macroglobulins (1Mda)

lipoproteins (20-40 Mda


11/81

The salting-out method

produces three groups: Fibrinogen

Albumin

Globulins


12/81

Separat ion

Cellulose acetate electrophoresis

Gels (agarose)

five bands

Albumin, 1, 2, , and

quantified by densitometry


13/81

F igure 1.

The image on the lef t is pure serum.

The image on the seperation of serum into dif ferent categori es

when the serum goes through electrophoresis


14/81


15/81

http://www.youtube.com/watch?v=z2D7ZkT3aOo


16/81

Five groups on cel lu lose acetate or

agarose:

Albumin (quantitatively the largest protein).

1-globulins (mainly 1-antitrypsin).

2-globulins (2-macroglobulin and haptoglobin,

immunoglobulins).


17/81

-globulins ( two types 1; transferrin & LDL, 2theC3component of complement, immunoglobulins).

a fibrinogen band will be seen if plasma is used.

-globulins (immunoglobulins).


18/81

The Electrophoresis pattern

of plasma proteins in disease


19/81


20/81


21/81


22/81


23/81

The Electrophoresis pattern

of plasma proteins in disease


24/81

Reduction of all proteins in malnutrition

(maladsorption)

Acute phase pattern (increases in acute phase

proteins associated with inflammation; 1- and 2-

globulins and an increase in plasma viscosity)

Chronic inflammation increased immunoglobulins

especially -globulins, but the pattern is complicateddepending on cause.


25/81

Cirrhosis of the liver (raised -globulins and reduced

albumin and 1

-globulins)

Nephrotic syndrome (reduced albumin and -

globulins, there may be increases in 2-

macroglobulin

1-antitrypsin deficiency decreased 1-antitrypsin


26/81

Paraproteinaemia (a dense abnormal band onelectrophoresis strip, can be suggestive of

malignancy)

hypogammaglobulinaemia


27/81

Classification by function Immunoglobulins

Clotting factors

Enzymes

Transporters


28/81

What determines the level of

Plasma Proteins ?

Synthesis

Catabolism (excretion)

Tissue distribution


29/81

Most major plasma proteins are

synthesised by a few cell types:

Hepatocytes, (albumin, clotting factors, transferrin)

Macrophages

B cells (-globulins)

Low abundance proteins?


30/81

Other important characteristics

Plasma Proteins exhibit polymorphism

Almost all are glycosylated (N- or O-linked

oligosaccharide chains).

Removal of the sugars alters function andhalf-life

Excessive glycosylation seen in whichdisease?


31/81

The circulation half-life is important ,

why?Albumin 20 days

Haptoglobin 5 days

altered by certain diseases.


32/81

In Crohns disease (regional ileitis)

Inflamed intestinal mucosa

loss of plasma proteins into the bowel

albumin half-life to as little as 1 day.


33/81

Plasma proteins levels may

also inc rease fol low ing : Cancer

Chronic inflammation

In acute Inflammation


34/81

Several Plasma proteins increase in response to

trauma.

"acute phase proteins:

Levels increase in the plasma following acute

inflammation, trauma


35/81

Examples of acute phase proteins C-reactive protein

alpha-1 anti-chymotrypsin

alpha-1 anti-trypsin haptoglobins

caeruloplasmin

serum amyloid A

fibrinogen

ferritin

complement components C3, C4


36/81

The level of some plasma proteins may decrease :

pre-albumin

albumin transferrin


37/81

synthesis of acute phase proteins by hepatocytes

triggered by (cytokines) lnterleukins (IL-l, IL-6),released from mononuclear phagocytic cells

C-reactive protein ( reacts with the C polysaccharideof pneumococci),


38/81

1-antitrypsin

haptoglobin,

1-acid glycoprotein

Fibrinogen.

The increase may range from a 2-l000-fold.


39/81

Why elevated ?

They mediate the response to inflammation.

Stimulation of complement pathway (CRP),

neutralize proteases released during the acuteinflammatory state (-AT).


40/81

Acute-phase proteins

trapping of microorganism and their products,

activating the complement system, binding cellular remnants (nuclear fractions)

neutralizing enzymes,

scavenging free hemoglobin and radicals

modulating the hosts immune response.


41/81

The functions of plasma

proteins

Functionally plasmaproteins can be placed in 7categories


42/81

1 Th di t ib ti f fl id b t bl d d ti


43/81

1. The distribution of fluid between blood and tissues.

They exert osmotic pressure (oncotic pressure),

approximately 25 mm Hg.

[Plasma proteins] (severe protein malnutrition, liver

disease),

oncotic pressure of plasma

fluid accumulates in the extravascular tissue spaces,a condition known as oedema.


44/81

2. Carriers for cations and insoluble compounds,fatty acids, billirubin, steroids, lipids, vitamins.Drugs

3. The immune system:

Antibodies (gamma globulins), alsocomplement, acute phase proteins.

4. Hormones (quantitatively small, - fetoprotein)


45/81

5.Clotting factors (quantitatively small)

6. Source of energy and amino acids

7. Enzymes (intracellular from

breakdown of cells,acetylcholinesterase)


46/81

THE PLASMA PROTEINS


47/81

Albumin The major plasma protein

Albumin (69 kDa) is the major plasma protein (3.4-

4.7 g/dL)

> 60% of the total plasma proteins.

40% in the plasma,

60% in the extracellular space.


48/81

Produced by the liver (12g/day) 25% of total hepatic protein synthesis

Synthesised as a preproprotein

In some diseases the synthesis of albumin is

reduced

decreased albumin/globulin ratio


49/81

Decreased albumin synthesis is an

early indicator of protein malnutrition(kwashiorkor).

Analbuminia: individuals lack albumin(mutation),


50/81

Other proteins may be increased to

compensate

Albumin is responsible for 80% of the osmotic

pressure of plasma

Analbuminia may result in mild oedema


51/81

Changes in plasma proteins may

Affect binding of various ions in the blood

pharmacokinetics of certain drugs


52/81

Structure:

one polypeptide chain of 585 amino acids and

contains 17 disulphide bonds.

3 domains may be identified using proteases,


53/81

Despite its high concentration it does notincrease the viscosity of the plasma.

A significant part of the transport roleof plasma is carried out by albumin.


54/81

Binding

fatty acids

calcium,

amino acids,

bilirubin

steroid hormones

drugs.


55/81

Transferrin

A glycoprotein

-globulin

Molecular mass 76 kDa.

Synthesized in the liver.

20 polymorphic forms


56/81

What does transferrin do?

A central role in iron transport (2 mol of Fe3+per moleof transferrin)

Iron (Fe2+) ingested at the intestinal mucosa is tightlyregulated.


57/81

Plasma transferrin concentration is approximately

300 mg/dL

Capable of binding 300g/dL

But normally only one-third saturated

Very little iron is lost in healthy adult (1 mg/d) more

in adult females


58/81

RBC destruction

25mg/day released (potentially toxic)

Binding to transferrin ( toxicity)


59/81

What happens to iron-bound transferrin?

Transferrin cell surface receptor binds the protein

The receptor is internalized by endocytosis

The iron released (to ferritin)

Receptor returns to the surface


60/81

Problems of Iron Metabolism:

Particularly important for two groups:

Women

Older people


61/81

Iron deficiency (anaemia) due to:

inadequate intake

inadequate utilisation,

excessive loss


62/81

In terms of diagnosis transferrin is used

for?

Low iron intake


63/81

Ferritinbinds and stores 23% iron in the body

in tissues such as:

liver

spleen.


64/81

Ceruloplasmin

2-globulin

Copper binding protein (90% of plasma copper, eachmolecule binds six atoms of copper very tightly.)

About 160 kDa.

Plasma levels are reduced in liver disease.


65/81

Low levels in:

Wilson disease

abnormal metabolism of copper.


66/81

1-Antiproteinase (1-Antitrypsin)

The principal serine protease inhibitor of humanplasma (serpin).

As name suggests first discovered as 1-Antitrypsin

Controls the proteolytic action of lysosomal enzymes.


67/81

Synthesized in hepatocytes and macrophages

But aslo intestinal and bronchial epithelial cells

About 52 kDa

A single-chain protein of 394 amino acids,

Contains three oligosaccharide chains,

The major component (> 90% ) of the 1fraction of humanplasma.

Levels increase after infection/trauma


68/81

How does it work?

Complexes with proteins causing inhibition of:

Trypsin

Elastase (other proteases)

Highly polymorphic ( > 70 forms) identified byelectrophoresis.


69/81

The basic clinical problem is due to the mutations in

the 1-Antitrypsin gene

Highly polymorphic ( > 70 forms) identified byelectrophoresis. More by PCR


70/81

Autosomal codominant

Major genotype is MM (phenotypic; PiM) onchromosome 14

There is also the Z allele and the S allele

Most individuals have two copies of the M allele (MM)

Any deviation from this may result in either liverdisease or lung disease (emphysema).


71/81

With the ZZ (PiZ) genotype

Less protein is secreted from cells this can lead to tissuedamage.

PiZZ results from a single point mutation at position 342on the gene encoding the substitution of lysine forglutamate (Glu342Lys).


72/81

The PiZ mutant is responsible for more than

95% of cases of pulmonary and hepatic diseaseassociated antititrypsin deficiency,

Another common mutant is the PiS,

Individuals may have two of these abnormal geneslabelled PiSS or PiZZ, or one of each PiSZ


73/81

Accumulation of the PiZ protein in the ER ofhepatocytes is seen in some liver diseases

The PiZ mutations produce a peptide which formspolymers in the rough endoplasmic reticulum ofhepatocytes,these aggregate and are retained in theliver.

This may lead to hepatitis or cirrhosis (accumulation

of massive amounts of collagen, resulting in fibrosis)of the liver.


74/81

Lung tissue damage and 1-antitrypsin

deficiency

How does 1-antitrypsin deficiency result in tissuedamage ?


75/81

Normal 1-ant i t rypsin exp ression and level:

Phagocyte in lung

Active elastase Tissue damage

+1-AT

Inactive elastase:1-AT complex No proteolysis oflung

(no tissue damage)


76/81

Abnormal 1-ant it ryps in expression and level :

Active elastase (no 1-antitrypsin)

Proteolysis of lung Tissuedamage


77/81

Smoking results in the oxidation of a methionine

residues in the protein decreasing binding.

In patients with the PiZZ phenotype

The effect is exaggerated accelerating the

the emphysema

l b li


78/81

2-Macroglobulin

a large glycoprotein (720 kDa)

four identical subunits of 180kDa

10% of the plasma proteins

transportation zinc in plasma (10%)


79/81

The major member of a group of plasma proteinscalled:

thiol ester plasma protein family(complement proteins C3 and C4)

they contain a unique internal cyclic thiol ester bondand a proteolytic cleavage region

These helps attract and deactivate protineases


80/81


81/81

What is the function of 2-macroglobulin

To neutralize a range of Proteinases

To bind and targets certain cytokines to tissues.

plasma proteins in disease diagnosis (4)

Documents