Topic 4: Plasma Proteins; Pathological Markers in Blood

What is the difference between plasma and serum

plasma = complete straw coloured fluid without cells

serum = the straw coloured fluid left after clotting without clotting factors or cells

What are some functions of plasma proteins

• transport and storage

• balance of electrolytes and water

• acid/base regulation

• acute phase response

• enzymes

• hormones

• clotting

What are some cation binding proteins and their ligands

albumin - divalent and trivalent cations: Cu2+ and Fe3+

ceruloplasmin - Cu2+

transferrin - Fe3+

What are some hormone binding proteins and their ligands

thyroid binding globulin - thyroxine (T4), tri-iodothyrine (T3)

cortisol binding globulin - cortisol

sex hormone binding globulin - androgens, estrogens

What are some haemoglobin/protoporphyrin binding proteins and their ligands

albumin - haem, bilirubin, biliverdin

haptoglobin - Hb dimers

What are some FFA binding proteins and their ligands

albumin - FFA, steroids

Why are proteins measured?

A change in plasma level of a protein can be indicative of disease affecting circulation or the tissue that synthesises the protein

• proteins with physiological function in circulation

A change in level of plasma can be indicative of disease affecting tissue that synthesises protein

• proteins that leak from cells/tissues

Clinical Biochemistry labs measure:

• total protein

• albumin

• Ig’s

• serum protein electrophoresis

• protein and peptide hormones

• enzymes, fibrinogen, clotting factors

• CRP - inflam marker

• transferrin - transport

• complement - immune

• alpha-1-antitrypsin - protease inhibitor

• ceruloplasmin - transport

• haptoglobin - binding protein

What are conditions that alter plasma protein levels/activity

• changes in cellular prolif, cell turnover or damage, protein synth

• inherited protein variants with altered activity

• altered protein conformation

How are enzymes used as disease biomarkers?

cellular damage triggers release of certain enzymes from affected tissues

• elevated enzyme levels can be measured in serum, saliva, urine

measurable changes in enzyme activity can serve as disease biomarkers, often before clinical symptoms appear

What is proteomics

the systematic, high-throughput approach to protein expression analysis of a cell or organism

• cells respond to internal and external changes by regulating activity and level of proteins therefore can be used to understand structure, function and interactions of the entire protein content in specific organism

What are the types of protein assays used in clinical biochemistry labs

• spectrophotmetric

  • total protein

  • albumin

• electrophoresis

  • protein

• immunochemical

  • nephelometry

  • rate nephelometry

  • turbidimetric immunoassay

• immunoassay

  • immunoassay

  • ELISA

  • western blotting

  • protein hormones

How is total protein measured?

Biuret assay

• Cu2+ ions and peptide bonds in alkaline medium form violet coloured complex and is measured at 540nm

How can serum albumin be measured?

Bromocresol green

• albumin binds BCG

• absorbance at 628nm: increases in proportion to protein concentration

• some non specific A changes over time

Bromocresol purple

• albumin binds BCP

• absorbance at 603nm: increases in proportion to protein concentration

• more specific for albumin

• does not bind animal albumin

Bradford assay

• red form of coomassie blue donates free e- to ionisable groups on protein

• causes disruption of proteins exposing hydrophobic pockets

• negatively charged coomassie blue binds to protein and forms stabilised blue form in proportion to amount of protein

• absorbance at 595nm

Lowry assay

• divalent copper ions in folin-ciocalteu reagent form complex with peptide bonds at alkalkine pH and reduced to monovalent copper ions

• monovalent copper and radical groups of tyrosine, tryptophan and cystein react to folin reagent to produce unstable product that becames reduces to tungsten blue

• blue = proportional to amount of tyrosine, trytophan and cystein in protein

• absorbance at 650nm

what is the clinical significant of measuring total protein

RR = 60-80g./L

• limited clinical value, concentration reflects protein and fluid content

• low levels

  • dilution

  • hypoalbuminaemia

  • profound Ig deficiency

• high levels

  • loss of protein free fluid, or stasis during venipuncture

  • major increase in Ig

what is the clinical significant of measuring albumin?

RR = 32-50g/L

assesses:

• Ca2+ concentration (binds to albumin)

• severity of liver disease, myeloma

• contribution to oedema formation

• dehydration resulting in haemoconcentration (increased Hct)

What is the principle of protein electrophoresis

migration of charged particles in support medium due to electric field

• proteins = zwitterionic = can be negatively or positively charged depending on pH of solution

migration depends on:

• electric charge

• size and shape

• electric field strength

• properties of support material

• temp

How is the velocity of a molecule moving in an electric field calculated?

v = Eq/d6 pi r n

What are the uses of protein analysis

• determine MW

• analyse protein purity

• protein ID

• western blotting

What is the difference between Native PAGE and SDS PAGE

Native

• proteins separated based on native conformation and charge

• used to analyse serum proteins

SDS

• uses SDS to bread disulfide bonds and denature proteins

• SDS unfolds proteins into their linear shape and masks charged groups

• separate based on MW

What are types of solid support gels for electrophoresis

polyacrylamide

• for proteins

• chemically inert and electrically neutral

• hydrophilic

• does not interact with solutes

agarose

• melted and set polysaccharide

• sieving effect based on MW

• uncharged support

What are the PAGE protein stains and their sensitivities?

Coomassie blue

• sensitivity = 30ug protein

  • normal/common

Amido black

• sensitivity = 75ug protein

  • used when lots of protein and not concerned about sensitivity

Silver

• sensitivity = 0.2ug protein

What are the functions of acute-phase proteins?

• mediators: CRP

• modulators: fibrinogen

• inhibitors: alpha-1-antitrypsin

• scavengers: haptoglobin

• immunomodulators: alpha-acid glycoprotein

• repair: alpha-1-AT

What is the acute phase response?

• physiological events that follow tissue damage and inflammation

• systemic effects = fever, muscle proteolysis, leukocytosis

• acute phase proteins increase by >25% in first 7 days following tissue damage

How do some plasma proteins increase in APR

CRP = 1000x increase in 6-10 hours

alpha-1-AT, haptoglobin and ferritin = 2-4x increase in 10-24 hours

ceruloplasmin and C3 and C4 = 0.5x increase in 48-72 hours

What is CRP (and its normal concentration) and what are some conditions with elevated CRP?

pentraxin family of proteins that bind phosphocoline, microbial cell walls and microbial and host chromatin

• 0.8mg/L

inflammatory = systemic vasculitis, rheumatoid arthritis

infections = bacterial and restricted viral

malignant = carcinoma, sarcoma

ischaemic necrosis = MI

trauma = surgery, burns

What are complement proteins and their functions?

removal or destroy antigen by opsonisation and lysis of microorganisms

peak 5 days after APR

classic: Ag-Ab complexes, C1q, C4, C2

alternate: bacterial cell wall components, endotoxins

both activate C3 → C5-9 MAC

What is haptoglobin and its functions?

binds free Hb released from RBC during intravascular haemolysis

• 1 HAP binds 2 Hb

rapid endocytosis and recovery of iron

Significance:

• decreased in intravascular haemolysis and haem turnover

  • used up

• increased in APR and burns + nephrotic syndrome

What is alpha-1 antitrypsin and its functions?

a major inhibitor of neutrophil elastase which is released from WBC to fight infection, and inhibits trypsin

• changes conformation and tightly binds to inactivate protease

• neutrophil elastase may attack normal tissues if not controlled by a1AT

• uncontrolled → emphysema

• smoking causes oxidation of a1AT → prevents inhibition

Z mutation (PiZZ)

• poorly secreted a1AT and it aggregates in liver

• homozygotes develop emphysema and liver disease at early age

• dyspnoea

• cirrhosis

• infection and dehydration

• defining symptoms:

  • cholestasis in infants

  • cirrhosis of unproven etiology

  • emphysema

• diagnosis:

  • electrophoresis: decrease in a1 band

  • functional decrease in ability of serum to inhibit trypsin

What is ceruloplasmin and its functions?

multifunctional copper binding protein that has transport and enzymatic activities

• binds 95% of serum Cu - 6-8 molecules at a time

• oxidises Fe2+ to Fe3+ for incorporation into apotransferrin

• antioxidant activity

• low levels = wilsons disease (excess Cu) and menkes disease

  • results in increased total body Cu and liver Cu

What is a serum paraprotein

a monoclonal Ig or complement light or heavy chain

produced by clonal prolif of plasma cells or other B-cell lineage cells

What is monoclonal gammopathy and its characteristics?

an abnormal overproduction of specific clone of B cells/plasma cells producing Ig’s

monoclonal gammopathy of undetermined significant (MGUS)

• paraprotein production with no associated organ damage

• benign phenotype

  • serum paraprotein conc <30ug/L

  • <10% affected BM plasma cells

  • no anaemia, renal failure or hypercalcaemia

What is Multiple Myeloma and its characteristics?

proliferation of malignant plasma cells in the BM → producing abnormal monoclonal paraprotein and evidence of end-organ damage

• symptoms: hypercalcaemia, anaemia, renal impairment and or bone pain

• serum paraprotein > 30g/L or BM plasmacytosis of >10%

How can paraproteins be detected?

immunofixation - serum protein electrophoresis

1. serum separated on native PAGE

2. lanes with seperated proteins incubated with specific anti-Ig antisera

3. monoclonal Ig detected as bands

   • anti-IgG

   • anti-IgA

   • anti-IgM

   • anti-kappa light

   • anti-lambda light

What are bence jones proteins?

free monoclonal light chains of kappa or lambda

• ligh chains are filtered at glomerulus due to low MW to give heavy BJP proteinuria

• presence of light chains implies de-differentiation due to their synthesis in excess of heavy chains (G, A, M)