Plasma Proteins

Mark GuyDept Clinical Biochemistry

Hope HospitalSalford M6 8HD0161 206 4968

mark.guy@srft.nhs.uk

Clinical use of plasma protein measurements

• Total Protein

• Albumin

• Protein electrophoresis

• Specific protein measurement– Acute phase proteins, immunoglobulins– Genetic variants / inherited defects– Proteins in nutritional assessment

Specific protein measurement

• Immune systemDeficiency, allergy, malignancy, immune response IgG, A, M, E

• Inflammation /acute phase responseCRP, acute phase proteins

• Complement activationC3, C4, complement activation products

• Nutritional assessmentPrealbumin, transferrin

Specific protein measurement

• Protein losing states

eg. nephrotic syndrome, protein losing enteropathy

? albumin

• Intravascular haemolysis

haptoglobin

• Genetic variants/hereditary diseases

α1- antitrypsin, ceruloplasmin, haptoglobin

Acute phase proteins

• Acute phase response

Following inflammation cellular changes occur to facilitate phagocytosis of microorganisms or cell debris and initiate repair

Complex control of processes at cell level mediated by many different molecules,

eg. cytokines

From Evans and Whicher, Adv Clin Chem 1993;30:1-88

Acute phase proteins• Inflammatory mediators

– Complement opsonization, chemotaxis, mast cell degranulation

– CRP phosphorylcholine binding, complement activation, opsonization

– Factor VIII fibrin matrix formation– Kallikrein vascular permeability, dilatation– Plasminogen complement activation, clotting, fibrinolysis

• Proteinase inhibitors– Antithrombin III, C1-INH Control of mediator pathways– α1-antitrypsin Elastase, collagenase– α1-antichymotrypsin Cathepsin G– Haptoglobin Cathepsins B, H, L– Thiol proteinase inhibitors Cysteine proteinases

• Scavengers– Ceruloplasmin ?free oxygen radicals– CRP ?DNA– Haptoglobin Haemoglobin– Serum amyloid A Cholesterol

Acute phase proteins

• Immune regulation– α1-acid glycoprotein Monocyte production of IL-1ra

– CRP T and B cell interaction

• Repair and Resolution– α1-acid glycoprotein Fibroblast growth

– α1-antitrypsin Binds to elastic fibres

– α1-antichymotrypsin Inhibits remodeling by leukocyte proteases

From Evans and Whicher, Adv Clin Chem 1993;30:1-88

From Thompson et al. Ann Clin Biochem 1992; 29: 123

From Thompson et al. Ann Clin Biochem 1992; 29: 123

Acute phase proteins

• Accurate, precise, sensitive measurement

• Low cost

• Specific for inflammation

• Short half-life

• Large fold increase over baseline

• (Use in differential diagnosis)

CRP

• Pentraxin family of proteins• 5 covalently linked subunits – pentameric, MW 105 kDa• Ca dependent binding to lipids enhancing agglutination,

lattice formation. Bound CRP activates complement• Detectable within 6 hours, half-life 19 hours• “Normal” <1 mg/L. 99% individuals <10 mg/L

10-40 mg/L mild inflammation

40-200 mg/L significant acute inflammation or bacterial infection

>300 mg/L burns, serious bacterial infection

Proteins in nutritional assessment

• Patients who are malnourished with protein energy malnutrition require source of amino acids for synthesis and turnover of proteins

• When intake does not meet demand – breakdown of eg. skeletal muscle

• Protein synthesis in liver prioritised – synthesis of non essential proteins reduced, reflected in serum concentration

• PEM leads to eg. increased infections, mortality

Protein markers – nutritional assessment

• Short half-life

• Protein status reflected by serum concentrations

• Small whole body pool

• Rapid synthetic rate

• Constant catabolic rate

• Responds only to protein or energy restriction

Protein markers – nutritional assessment

MW (kDa) Half lifeConc

Albumin 65 20 d 33-48 g/L

Fibronectin250 15 h 220-400 mg/L

Prealbumin 55 48 h 160-350 mg/L

Retinol binding pr. 21 24 h 30-60 mg/L

IGF-1 7.65 2 h 0.1-0.4 mg/L

Transferrin76 10 d 1.6-3.6 g/L

Genetic variants / inherited disease

• Haptoglobin

• α1- antitrypsin

• Ceruloplasmin

Haptoglobin

• Complex group of allotypically related glycoproteins

• α2 globulin, acute phase protein• Possesses two α and two β chains• Exhibits genetic polymorphism

– 3 common phenotypes

Hp 1-1 15% 86 kDa

Hp 2-1 50% 86 kDa + polymers

Hp 2-2 35% >200 kDa (polymers)

Haptoglobin

• Function – binds free Hb, serves to conserve Fe released by red cell lysis

Binds oxyHb, metHb, α chains, αβ dimers but not deoxyHb or free haem.

Half-life of free Hp = 3-4 d

Hp/Hb complex = 8 min

Large size therefore not filtered by glomerulus, catabolism by reticuloendothelail system, Fe recycled

Haptoglobin

• Decreased levels:– Congenital deficiency (rare)– Intravascular haemolysis

– Thalassaemia, sickle cell, G6PDH deficiency

– Acute and chronic liver disease– Malabsorption

• Increased levels– Acute phase response– Cholestasis

α1-antitrypsin

• Glycoprotein, 54 kDa• Major α1 globulin• 90% of tryptic inhibitory activity in serum• Slow acute phase reactant• Increased in pregnancy, oestrogens, OCP

α1-antitrypsin

• Genetics

Approx. 60 genetic variants described

Nomenclature – PI system, based on mobility on IEF

PI Alleles α1-antitrypsin variants Anodal M region CathodalB M (1-5) N S

Bsaskatoon Mduarte Nadelaide T

C Mchapelhill Nhampton Wsalerno

D Msalla Nyerville Wgazak

E NgrossoeuvreWfinneytown

Elemberg MmaltonWcolumbus

Ematsue Nletrait X

Efranklin Pbudapest Xalben

Etokyo Pstlouis Ybrighton

Ecincinnati R Yhagi

Ecler Peuskurchen Ytoronto

G Pkyoto Z

I Pcastoria

Jhouyao P

L Pleverkusen

F Pclifton

Lbeijing V

α1-antitrypsin

• Deficiency alleles– S – P– W– Z – mmalton– mduarte– null

α1-antitrypsin deficiencyVariants with clinical relevance:

PI S (moderate deficiency)

PI Z (severe deficiency)

(Caucasian UK )Gene frequencies Genotypes:M 92% MM 85%S 5% MS 10%Z 2% MZ 3.5%

SZ 0.2%ZZ 0.04%

Inheritance : autosomal codominant

From Protein Reference Units Handbook of Clinical Immunochemistry

From Brantly Chest 1991;100:703

α1-antitrypsin deficiency

• Approx 1:2000• Consequences

– Liver disease adults + neonates– Lung disease adults

chronic obstructive airways disease, emphysema

From Protein Reference Units Handbook of Clinical Immunochemistry

StressedMetastable

RelaxedHyperstable

α1-antitrypsin deficiency

• Treatment

– Counselling

– Synthetic AAT

• Measurement

– Phenotyping below 1.2 g/L

– ? screening

Ceruloplasmin

• α2 globulin, MW 150 kDa• Reversibly binds 8 Cu atoms per molecule,

carries 90% of all Cu in plasma• Ferroxidase, superoxide dismutase and amine

oxidase activities• Role in Fe incorporation in transferrin, free

radical scavenging• Plasma levels 0.2-0.6 g/L• Slow acute phase reactant

Ceruloplasmin

• Increased levels– Acute phase response– Pregnancy, OCP, oestrogens

• Decreased levels– Primary deficiency - aceruloplasminaemia

(v.rare)– Secondary deficiency – Wilsons disease

Menkes diseaseCu deficiency

Ceruloplasmin

• Cu + apoceruloplasmin → (holo)ceruloplasminhalf life apocer = few hours, cer =4 days

• Wilsons disease (hepatolenticular degeneration)– Autosomal recessive 1:50000 to

1:100000carriers 1:2000

– Gene product, copper transport protein (ATPase) resulting in defective biliary excretion of Cu and incorporation of Cu into apoceruloplasmin

– 27 mutations in gene

Wilsons disease

• Manifests 6-50 years old, usually early adult• Free Cu deposits in liver, brain, cornea, kidney• Presents liver disease (42%), neurological

(34%), renal tubular damage, haemolytic anaemia

• Cu deposits in cornea – Kayser Fleischer rings, almost pathognomonic

Wilsons disease

• Diagnosis– Kayser Fleischer rings– Low ceruloplasmin– High liver Cu– High urinary Cu– Low 64Cu incorporation into ceruloplasmin

Serum Cu – low or low normalCeruloplasmin may be low normal in presence of acute

phaseGenerally KF rings + low ceruloplasmin = Wilsons

Wilsons disease

• Treatment– Chelation of Cu – penicillamine, trientine– Reduce Cu absorption eg oral Zn

ReferencesClinical Chemistry in Diagnosis and Treatment. Zilva, Pannall, Mayne.

Ch.16

The value of acute phase protein measurements in clinical practice. Thompson D et al. Ann Clin Biochem 1992;29:123

Proteins used in nutritional assessment. Spiekerman AM. Clin Lab Med 1993; 13:353

Use of a highly purified α1-antitrypsin standard to establish ranges for the common normal and deficient α1-antitrypsin phenotypesBrantly ML et al. Chest 1991; 100:703

The Cytokines: physiology and pathophysiological aspects. Evans SW, Whicher JT. Adv Clin Chem 1993; 30: 1-88

Serum albumin: touchstone or totem?Margarson MP, Soni N. Anaesthesia 1998; 53: 789

Referencesα1-antitrypsin deficiency. Stoller JK. Lancet 2005; 365: 9478

Molecular pathology of α1-antitrypsin deficiency and its significance tp clinical medicine. Kalsheker NA. Q J Med 1994; 87: 653

α1-antitrypsin deficiency: a model for conformational disease. Carroll RW, Lomas DA. N Engl J Med. 2002; 346: 45

Diagnosis and treatment of Wilson’s disease. Schilsky ML. Pediatr Transplantation 2002; 6: 15

Wilson’s disease: acute and presymptomatic laboratory diagnosis and monitoring. Gaffney D et al. J Clin Pathol 2000; 53: 807