2. Water 91%
Proteins 7%
Other solutes
Plasma 55%
Buffy coat %
Erythrocytes 45 %
Albumin:55-60%
Globulin: 36%
Fibrinogen: 4%
Trace proteins
PLASMA PROTEINS
3. PLASMA Vs SERUM
Total proteins estimated from serum will
be 5% less than the total proteins
estimated from the plasma
SERUM = Plasma – Fibrinogen & Clotting Factors
4. PLASMA PROTEINS
• Method of Estimation of total plasma proteins
– Biuret method
– Lowry method
– Bradford method
– Kjeldhal method
NORMAL VALUE
Dog: 5.4-7.1 g/dL
Cat: 5.4-7.8 g/dL
Cattle: 6.7-7.4 g/dL
5. Separation of Plasma Proteins
• Salting out: using sodium or ammonium sulfate
– 3 major groups of proteins – Albumin, Globulin and
Fibrinogen will be precipitated at different concentrations of
the salt
• Electrophoresis
• Immunoelectrophoresis
• Ultracentrifugation
• SDS-PAGE
• Proteomics
8. Classification of Plasma Proteins
• Electrophoresis
5 Fractions of plasma proteins
– Albumin
– α1 Globulin
– α2 Globulin
– β Globulin
– γ Globulin
9. Plasma proteins
300 types of proteins
Synthesized in the liver
except for gamma globulin
10. Albumin
• Most abundant plasma protein
• 55 % of plasma proteins
• Molecular weight 65 – 69 kDa
• Iso-electric pH 4.7
• Has a strong net negative charge
• Synthesis: Liver (15 g /day)
– estimation of serum Albumin forms a part of LFT
• Half-life: 20 days
• Also present in CSF and interstitial fluids
11. Functions of Albumin
Maintenance of Colloid Osmotic Pressure
• Colloid osmotic pressure: osmotic pressure exerted by proteins in
blood plasma that pulls water into the capillaries
– Because large plasma proteins cannot easily cross through the capillary walls
– 60-80% of the total plasma osmotic pressure
• Plays an important role in blood volume and body fluid distribution
12. Maintenance of Colloid Osmotic Pressure by Albumin
A decrease in plasma albumin level results in a fall in COP,
causing enhanced fluid retention in tissue spaces (edema)
13. Functions of Albumin
• Transport
– Metal ions: calcium and copper
– Free fatty acids: albumin binds to free fatty acids released by
adipose tissue and facilitates their transfer to other tissue
– Bilirubin: helps to protect against the toxic side effects of
unconjugated bilirubin
– Bile acid
– Hormones: thyroid and steroid hormones
14. Functions of Albumin
Buffering function
–Maintenance of acid-base balance
–Albumin has the maximum buffering capacity among the
plasma proteins
–Buffering capacity is less than the bicarbonate buffering
system
15. Functions of Albumin
Nutritive function
• Source of amino acids for tissue protein synthesis,
during malnutrition
• Transport form of essential amino acids from the liver
to extrahepatic cells
16. Albumin: Clinical Aspects
Normal Range of Albumin
Dog: 2.6-3.5 g/dL
Cat: 2.8-3.9 g/dL
Cattle: 3-3.5 g/dL
Method of Estimation: Bromocresol Green
17. HYPOALBUMINEMIA
HYPOALBUMINEMIA: decrease in the level of Albumin in blood
Mild 2.0 - 2.6 g/dL Few clinical signs
Moderate 1.5 – 2.0 g/dL Few clinical signs
Severe <1.5 g/dL Oedema, Ascites, Pleural effusion
19. Hypoalbuminemia: Consequences
– Oedema/Ascites
– Hypocalcaemia
• total calcium level will be low, but no tetany because the ionized
calcium levels will be normal
– Binding with drugs
• Sulfonamides can cause the release of unconjugated bilirubin from
albumin by competitive binding. If given to infants, sulfonamides
may lead to kernicterus
– Alteration in the level of protein-bound substance
Albumin is therapeutically useful for the treatment of burns and
hemorrhage
23. α1- Globulins
1. α1- Fetoprotein (AFP)
– Molecular Weight: 65000 Da
– AFP - measured in amniotic fluid for diagnosis of open
neural tube defects and Down’s syndrome (prenatal defects)
– In adults: a tumor marker for hepatoma
– Increase: Hepatoma, Pregnancy
– Decrease: Liver & chronic pulmonary disease
24. 2. α1- Antitrypsin (ATT)
– Molecular weight: 45,000 Da
– Major constituent of α1- globulin fraction of plasma protein
– serine protease inhibitor: combines with trypsin, elastase and other
protease and inhibits them
– Genetic deficiency leads to emphysema
– Increase: Acute phase response
– Decrease: Liver & chronic pulmonary disease
α1- Globulins
25. α1- Globulins
3. α1-Acid
Glycoprotein
43,000 Da Immunomodulator,
binds ligands &
drugs
Increase: Acute phase
response
Decrease: Liver disease,
nephrotic syndrome,
malnutrition
4. α1-
Antithrombin III
65,000 Da Thrombin inhibitor Increase: possible acute
phase response in cats
Decrease:coagulopathies,
PLN, PLE sepsis
5. α1-Lipoprotein
(HDL)
200,000
Da
Lipid transport Decrease: Acute Phase
response
26. 1. α2-Macroglobulin
– Molecular weight: 62,000 Da
– a major constituent of α2 fraction
– Binds insulin
– A panprotease inhibitor: combines with and inhibits many
proteases
– Increase: Nephrotic syndrome, chronic active liver disease
α2- Globulins
27. 2. Ceruloplasmin
– Molecular weight: 151,000 Da
– a blue-coloured, copper-containing α2 fraction
– Carries 90% of plasma copper tightly so that copper is not readily
exchangeable (Albumin carries the other 10%)
– Low level of ceruloplasmin is associated with Wilson’s disease
(hepatocellular degeneration)
– Increase: acute phase response
α2- Globulins
28. 3. Haptoglobulin
– Molecular weight: 100,000 Da
– binds with the free hemoglobin (extra-corpuscular Hb) in a tight
noncovalent complex Hp-Hb during haemolysis.
• Hp-Hb (155 kDa) cannot pass through the glomeruli of the kidney while
free Hb (65kDa) can and Hp prevents the loss of free Hb into the urine
Increase: acute phase response, glucocorticoids in dogs
Decrease: intravascular haemolysis
α2- Globulins
30. β-Globulins
1. Transferrin (Tf)
– Molecular weight: 76,000 Da
– Transports iron in plasma as ferric ions (Fe3+)
– Protects the body against the toxic effects of free iron
Increase: Fe deficiency, acute liver disease, nephrotic syndrome
Decrease: Iron storage disease, acute phase response,
inflammatory response
31. 2. β2-lipoprotein
(LDL)
2,750,0
00 Da
Lipid transport Increase: nephrotic
syndrome, hypothyroidism,
hepatocanalicular disease
3. Ferritin 465,00
0 Da
Iron transport Increase: Iron storage
disease, acute phase response
Decrease: Fe deficiency
4. Hemopexin 80,000
Da
Heme transport Decrease: haemolytic
anaemia, chronic active liver
disease
5. C-reactive
protein (CRP)
100,00
0 Da
Active complement, useful
to differentiate bacterial
from viral infections,
coronary artery diseases
Increase: acute phase
response
β-Globulins
32. • Immunoglobulins
• Antibodies
• Produced by plasma cells or lymphocytes
• Glycoproteins
• Light and Heavy chains
• Based on the type of H chain- IgG, IgA, IgM, IgE, IgD
• Bence-Jones proteinuria (Light chains) - multiple myeloma
γ-Globulins
33. Fibrinogen
• A protein of high molecular weight
• Can be precipitated easily
• Synthesised in the liver
• Converted to fibrin and helps in blood clotting
34. Interpretation of Serum Protein Profiles
Physiological Influences
1. Age, Development, Breed
2. Hormonal and Sexual influences
3. Pregnancy and Lactation
4. Nutritional influences
5. Stress and Fluid loss
35. Changes in Serum Electrophoretic Pattern
Multiple Myeloma
M-spike
36. Changes in Serum Electrophoretic Pattern
Protein synthesizing capacity of liver is compromised and concentrations of albumin
and proteins in the alpha and beta bands are decreased.
An additional common finding is beta-gamma bridging due to increased IgA
Liver Cirrhosis
37. Nephrotic syndrome
• Urinary protein loss
• α2 macroglobulin is sufficiently large
so that it is not filtered and increased
synthesis (from the general hepatic
protein synthesis) causes its
accumulation
Changes in Serum Electrophoretic Pattern
39. Dysproteinemias
• Refers to any abnormality in the protein content of blood
• The concentration of a protein in blood depends on the ratio
between the rate of its synthesis and breakdown or excretion
• If one or more fractions of globulins becomes more prominent,
the ratio albumin-globulin - A/G coefficient changes
• Normal A/G ratio: 1.5 - 2
40. Dysproteinemias: General causes
• Congenital (mutation or loss of a gene) or acquired
• Loss of certain protein fractions:
• Kidneys - proteinuria, damage to glomeruli
• Skin - serious burns, extensive infections
• Intestine - exsudative enteropathy
• Disorders of protein synthesis:
• Liver - loss of function in cirhosis, hepatitis
• Insufficient dietary protein intake
• Haemoconcentration by loss of water
41. Classification of Dysproteinemias
I . Normal A: G
1. Hyperproteinemia: dehydration
2. Hypoproteinemia
-Overhydration
-Acute blood loss
-External plasma loss: extravasation from burns,
abrasions, exudative lesions, external parasites, GI diseases
- Internal plasma loss: vasculitis
42. II. Decreased A: G
1. Decreased Albumin
– Selective loss of Albumin: Glomerulonephritis, nephrosis,
nephrotic syndrome, GI diseases
– Decreased synthesis of Albumin: Chronic liver disease,
malnutrition, chronic inflammatory disease
Classification of Dysproteinemias
43. 2. Increased Globulin
a. Increased α-Globulin
– Increased α1 & α2 globulins- acute inflammatory response
– α2 Macroglobulin- acute hepatitis and nephritis, nephrotic syndrome
– Haptoglobin –glucocorticoids in dogs
b. Increased β-globulin
– Active liver disease, suppurative dermatopathies, nephrotic
syndrome
Classification of Dysproteinemias
45. III. Increased A:G
1. Increased albumin: does not occur except in dehydration
2. Decreased globulins
a. Fetal serum
b. Pre-colostral neonate
c. Combined immunodeficiency of Arabian foals
d. Aglobulinemia
Classification of Dysproteinemias
47. ACUTE PHASE PROTEINS (APP)
• Proteins that change their serum concentration by >25% in response
to inflammation, infection, trauma or some other conditions like
neoplasms
• ACUTE PHASE REACTANTS
• Used as quantitative markers for disease diagnosis, prognosis and
monitoring responses to therapy, general health screening, etc
• ‘Molecular Thermometer’; Markers of inflammation
• Synthesis:
– Liver
– Non-hepatic synthesis: lungs, intestine and mammary gland
49. ACUTE PHASE RESPONSE
• A nonspecific response to the stimulus of tissue following
trauma, infection, inflammation
• Part of the innate immune system
• Play a role in mediating such systemic effects as fever,
leukocytosis, increased cortisol, decreased thyroxine, decreased
serum iron, and many others
• Defense of animal to pathological damage and restoration of
homeostasis
50. TYPES OF ACUTE PHASE PROTEINS
Positive APP
APP
Negative APP
Decrease in response to
inflammation
Increase in concentration
during acute phase response
MODERATE APP
-Increase 5 to 10 fold
-Peak 2 – 3days
MINOR APP
-Increase 50 – 100 %
-Decrease gradually
MAJOR APP
-Increase 100 to 1000 fold
-Peak 24-48 hrs
51. TYPES OF ACUTE PHASE PROTEINS
Positive APP
• C-reactive protein (CRP)
• Serum Amyloid A (SAA)
• Haptoglobin (Hp)
• Ceruloplasmin
• α2-Macroglobin
• α1-Acid Glycoprotein
• Fibrinogen
• Complement (C3,C4)
Negative APP
• Albumin
• Transferrin
• Transthyretin
• Retinol Binding
Protein
• Adiponectin
52. Species Major APP Moderate APP
Cat Serum Amyloid A (SAA)
Acid Glycoprotein (AGP),
Haptoglobin (Hp)
Dog
C-Reactive Protein (CRP)
Serum Amyloid A (SAA)
Acid Glycoprotein (AGP),
Haptoglobin (Hp),
Ceruloplasmin (Cp)
Horse Serum Amyloid A (SAA) Haptoglobin (Hp)
Cow
Haptoglobin (Hp)
Serum Amyloid A (SAA)
Acid Glycoprotein (AGP
Pig
CRP,
Pig Major APP (Pig-MAP)
Haptoglobin (Hp),
Ceruloplasmin (Cp)
Mouse Serum Amyloid A (SAA)
Haptoglobin (Hp),
Acid Glycoprotein (AGP)
Rat α2-Macroglobulin
Haptoglobin (Hp)
Acid Glycoprotein (AGP)
53. Acute Phase Proteins
Protein Main function
Alpha-1-acid
glycoprotein
Antiinflammatory and immunomodulatory agent: has
antineutrophil and anticomplement activity and increases
macrophage secretion of IL-1 receptor antagonist.
Binds to lipophilic and acidic drugs.
C-reactive
protein
On bacteria, it promotes the binding of complement,
facilitating phagocytosis.
Induction of cytokines, Inhibition of chemotaxis and
modulation of neutrophil function
Dogs, Pigs: Major APP; Cattle, Cat: Minor APP
54. Protein Main function
Haptoglobin
Binds free hemoglobin (limiting Hb iron availability for
bacterial growth)
Hb has peroxidase activity- binding with Hp prevents
oxidative damage to tissues
Ruminants: major APP; Others: moderate
Serum amyloid
A (SAA)
Associated with HDL-Reverse cholesterol transport,
inhibition of phagocyte oxidative burst, platelet activation
and other immunomodulatory (via the inflammasome)
Inhibition of myeloperoxidase release and lymphocyte
proliferation
Horses: Major increase following surgery, aseptic
inflammation, arthritis, pneumonia, Colic
Cattle: Marker of inflammation
55. Acute Phase Proteins
Protein Main function
Pig-Major Acute Phase
Proteins (P-MAP)
Pig: increases during infection, inflammation,
transportation
Ceruloplasmin
Copper transport (for wound healing, collagen
formation and maturation),Antioxidant
Reduces the number of neutrophils attaching to
endothelium
