Lecture 3: Plasma proteins and dysproteinemia

What is the primary difference between serum and plasma?

Plasma contains all proteins and clotting factors, whereas serum is plasma minus the clotting factors (mainly fibrinogen and clotting proteins).

What is the composition of plasma proteins?

Plasma proteins are comprised of hundreds of different proteins with diverse structures and functions.

What are the primary sites of plasma protein synthesis?

Liver → produces most plasma proteins.

Lymphoid organs → produce immunoglobulins.

What are the major functions of plasma proteins?

-Transport of nutrients, small hormones, waste, and drugs.

-Maintenance of colloid osmotic pressure.

- Acid-base buffering.

- Regulatory roles (e.g., cell production, inflammation).

- Immune defense (immunoglobulins).

- Hemostasis (clotting factors).

How do total plasma protein concentrations differ between neonates and adults?

Neonates: 4-6 g/dL.

Adults: 6-8 g/dL.

Why do neonates have lower total plasma protein compared to adults?

Neonates lack immunoglobulins until ingestion and absorption of colostrum; total plasma protein rises as immunoglobulin concentrations increase with antigen exposure.

How does exposure to antigens influence plasma protein concentrations in young animals?

Immunoglobulins continue to increase as the immune system encounters a wide variety of antigens, raising total plasma protein closer to adult levels.

What are the three main categories of plasma protein measurement methods?

Physical (refractometry), biochemical (spectrophotometry), and fractionation (electrophoresis).

How does refractometry measure plasma proteins?

By determining the refractive index of plasma, which correlates with the amount of dissolved solids. The refractometer subtracts the constant contribution of nonprotein solids (~1.5 g/dL), displaying the remaining protein concentration.

Why is refractometry commonly used in veterinary medicine?

It is quick, inexpensive, routinely available, and provides a fast screening of total plasma protein. It can also measure urine specific gravity.

What is the principle of light refraction in refractometry?

Light travels at different velocities in different media and bends at the interface of two materials ("refraction"). The degree of bending (refractive index) correlates with the concentration of dissolved solids.

What are the limitations of refractometry for measuring plasma protein?

Nonprotein solutes (e.g., electrolytes, glucose, urea) can artifactually increase readings, especially in hyperglycemia or azotemia.

How are plasma proteins measured biochemically?

Via spectrophotometric assays that measure total protein and albumin separately in serum (or plasma). Total globulin concentration is calculated as:

[Totalprotein]-[Albumin]=[Globulins]

Why is albumin measured separately in spectrophotometry?

Albumin has distinct chemical properties that allow specific quantification, while globulins are too diverse to be measured directly and must be calculated.

What is electrophoresis used for in plasma protein measurement?

To fractionate proteins into α-, β-, and γ-globulin bands for qualitative and semi-quantitative analysis.

When is serum protein electrophoresis (SPE) clinically indicated?

When unexplained hyperglobulinemia is present.

When immunoglobulin deficiency is suspected.

Why are abnormalities in serum protein electrophoresis often nonspecific?

Because many diseases can cause similar alterations in protein fractions, requiring correlation with clinical findings and other diagnostics.

What information can be obtained from examining plasma in a microhematocrit tube?

PCV (Hct) for red cell mass.

Buffy coat for leukocyte and platelet estimation.

Plasma appearance: icterus (yellow), lipemia (turbid), hemolysis (red).

Plasma protein concentration (via refractometer).

Fibrinogen concentration (with heat-precipitation test in large animals).

What are the general structural features of albumin?

Albumin is a single, homogeneous protein with a small molecular weight (~66 kDa) and minimal carbohydrate content.

Why is albumin the most important plasma protein for maintaining colloid osmotic pressure?

Its high concentration, small molecular weight, and lack of glycosylation allow it to exert the greatest oncotic pressure among plasma proteins.

What clinical consequence results from hypoalbuminemia?

Edema or ascites, due to reduced plasma colloid osmotic pressure that allows fluid to shift into interstitial or body cavities.

Why does hypoglobulinemia not typically cause edema?

Globulins are larger molecules and contribute less to colloid osmotic pressure compared to albumin; their loss or reduction has minimal impact on fluid balance.

What is the relationship between albumin and calcium in plasma?

Albumin binds most of the plasma calcium (especially total Ca²⁺); hypoalbuminemia leads to a decrease in measured total calcium, though ionized (biologically active) calcium may remain normal.

What substances are transported by albumin?

- Cations (especially Ca²⁺)

- Metabolites

- Certain hormones

- Poorly soluble drugs

- Toxic substances

What does it mean that albumin is a negative acute-phase protein?

Albumin concentrations decrease during inflammation as hepatic protein synthesis shifts toward positive acute-phase proteins (e.g., fibrinogen, haptoglobin).

What defines an acute-phase protein (APP)?

Proteins whose serum concentrations change by >25% in response to inflammatory cytokines (IL-1, TNF-α, IL-6).

How are acute-phase proteins categorized?

Positive APPs: increase in serum concentration during inflammation.

Negative APPs: decrease in serum concentration during inflammation.

Why are positive APPs clinically useful?

They are sensitive indicators of inflammation, often increasing before an inflammatory leukogram is evident.

In which species is APP measurement particularly helpful and why?

Cattle and manatees, because they often lack prominent leukogram changes in response to inflammation.

What is the major acute-phase protein in all domestic mammals?

Serum Amyloid A (SAA).

What are the functions of Serum Amyloid A (SAA)? (supplemental)

Transports cholesterol to the liver for bile excretion, recruits immune cells to inflammation, and induces enzymes that degrade extracellular matrix.

What is the major acute-phase protein in dogs and humans?

C-reactive protein (CRP).

What are the functions of C-reactive protein (CRP)? (supplemental)

Binds lysophosphatidylcholine on dead/dying cells and some bacteria; activates complement.

What is the major acute-phase protein in ruminants?

Haptoglobin.

What are the functions of haptoglobin?

Binds free hemoglobin irreversibly, prevents Hb loss in urine, protects against bacterial infections, has antioxidant activity.

What are the functions of fibrinogen?

Promotes platelet aggregation, serves as coagulation factor I (precursor of fibrin), and provides scaffolding for inflammatory cells and fibroblasts.

How is fibrinogen classified as an APP?

A moderate positive APP, especially important in horses, cattle, and goats.

What is the role of ceruloplasmin?

Copper transport, ferroxidase activity (mobilizes iron from stores), and plasma antioxidant function.

What is transferrin and how does it act as an APP?

An iron-binding metalloprotein that transports iron and correlates with TIBC. It is a negative APP, decreasing slightly during inflammation.

What is ferritin's clinical significance as an APP?

Normally reflects body iron stores, but plasma ferritin also increases as a positive APP during inflammation, limiting its specificity for iron status.

What is the role of hormone-binding proteins in plasma?

They bind small MW hormones (e.g., corticosteroids, thyroxine, sex hormones) to prevent rapid renal clearance.

Which plasma proteins serve as protease inhibitors during inflammation?

Antithrombin, α1-protease inhibitor (α1-antitrypsin), and α2-macroglobulin.

What is the role of antithrombin?

Inhibits thrombin and other coagulation factors; requires GAGs (e.g., heparin) for optimal activity.

What is the role of α1-protease inhibitor (α1-antitrypsin)?

Inhibits proteases released during inflammation and some coagulation factors.

What is the role of α2-macroglobulin?

Inhibits proteases released during inflammation, pancreatic proteolytic enzymes, and some coagulation factors.

Which proteins are considered negative acute-phase proteins?

Albumin and transferrin.

Why does albumin decrease during inflammation?

Hepatic protein synthesis shifts toward positive APPs, conserving amino acids; albumin reduction is more noticeable in chronic inflammation.

Why does transferrin decrease slightly during inflammation?

To help reduce iron availability for microbes, thereby limiting bacterial growth.

What are the two broad causes of hyperproteinemia?

Dehydration (relative increase) and hyperglobulinemia (absolute increase).

Why does dehydration cause hyperproteinemia?

Only plasma water is lost, while proteins and erythrocytes remain, resulting in relative hyperproteinemia and erythrocytosis.

What are the main categories of causes of hypoproteinemia?

- Dilution (overhydration)

- Decreased productionIncreased loss

- Sequestration in body cavities

What is the term for simultaneous decreases in albumin and globulins?

Panhypoproteinemia.

How is the albumin-to-globulin (A:G) ratio used in hypoproteinemia evaluation?

- Normal A:G ratio → panhypoproteinemia (both decreased equally).

- Low A:G ratio → hypoalbuminemia with preserved/increased globulins.

- High A:G ratio → hypoglobulinemia with preserved albumin.

What effect does external hemorrhage have on plasma proteins and hematocrit?

All blood components are lost equally, but fluid is replaced faster than proteins/cells → hypoproteinemia + anemia.

What are the major causes of hypoalbuminemia?

1. Excessive fluid therapy (dilution; globulins also decreased).

2. Decreased synthesis (chronic liver failure, inflammation, or secondary to hyperglobulinemia).

3. Loss from the body (protein-losing nephropathy, enteropathy, hemorrhage, exudative dermatopathy).

4. Sequestration (protein-rich effusions in body cavities, e.g., peritonitis).

How does chronic liver failure cause hypoalbuminemia?

The liver is the primary site of albumin synthesis; chronic hepatic insufficiency reduces albumin production.

Why is albumin decreased in inflammation?

Albumin is a negative acute-phase protein; hepatic production decreases as resources are redirected toward positive APPs.

How does protein-losing nephropathy (PLN) typically affect albumin and globulins?

Albumin is preferentially lost due to its smaller size, while globulins may remain normal.

How does protein-losing enteropathy (PLE) usually affect plasma proteins?

Both albumin and globulins are lost → often panhypoproteinemia.

How does hemorrhage affect albumin and globulins?

Both decrease due to whole blood loss.

How can severe exudative dermatopathies affect albumin and globulins?

Both may be lost, but globulins can be normal or increased if inflammation drives immunoglobulin production.

What are the two main causes of hyperalbuminemia?

Dehydration (with proportional globulin increase, normal A:G ratio) and assay artifact.

Why is true hyperalbuminemia (beyond dehydration) extremely rare?

Albumin synthesis is tightly regulated, and the body does not overproduce albumin even in disease states.

What are the main causes of hypoglobulinemia?

- Increased loss (albumin also low): hemorrhage, protein-losing enteropathy.

- Failure of passive transfer of immunoglobulins via colostrum.

- Overhydration (albumin also low).

- Decreased production: rare humoral immunodeficiency.

What is the most common cause of hypoglobulinemia in neonatal animals?

Failure of passive transfer of immunoglobulins from colostrum.

What are the main causes of hyperglobulinemia?

- Hemoconcentration (dehydration).

- Increased immunoglobulin production (immune-mediated response or neoplastic lymphoid cells, usually plasma cells).

- Increased acute-phase proteins (haptoglobin, fibrinogen in plasma).

Why do most acute-phase proteins contribute minimally to hyperglobulinemia?

Their serum concentrations are generally too low to significantly alter the total globulin concentration.

What is the role of serum protein electrophoresis (SPE) in hyperglobulinemia evaluation?

It differentiates polyclonal from monoclonal increases in immunoglobulins.

What does a polyclonal hyperglobulinemia look like on SPE?

A blunt, broad-based peak in the gamma region.

What causes polyclonal hyperglobulinemia?

Immunoglobulin production from many different lymphoid clones, usually due to antigenic stimulation (infections, immune-mediated disease, immune response to neoplasia).

What does a monoclonal hyperglobulinemia look like on SPE?

A narrow, sharp peak in the gamma region.

What causes monoclonal hyperglobulinemia?

Immunoglobulin (or Bence Jones protein) production from a single lymphoid clone, usually due to neoplasia (multiple myeloma, lymphoma, leukemia).

Can monoclonal hyperglobulinemia occur in non-neoplastic conditions?

Rarely, yes (occasionally chronic infections or immune stimulation), and rarely biclonal.

How can you distinguish polyclonal vs monoclonal gammopathies clinically?

Polyclonal: broad peak, multiple lymphoid clones, suggests chronic inflammation or immune response.

Monoclonal: sharp peak, single clone, strongly suggests plasma cell or B-cell neoplasia.

What are three main causes of erroneously increased plasma protein concentration on lab results?

Hemolysis (free Hb interferes, but Hb is not a plasma protein), lipemia (interference with light transmission), and markedly increased nonprotein solids (e.g., glucose in diabetes, urea nitrogen in kidney disease).

How do you interpret a normal HCT with low TPP?

Suggests protein loss (GI loss, proteinuria) or decreased production (liver disease).

How do you interpret a normal HCT with normal TPP?

Normal findings.

How do you interpret a normal HCT with high TPP?

Increased globulin synthesis or dehydration masking anemia.

How do you interpret a high HCT with low TPP?

Protein loss combined with relative or absolute erythrocytosis.

How do you interpret a high HCT with normal TPP?

Splenic contraction, absolute erythrocytosis, or dehydration masking hypoproteinemia.

How do you interpret a high HCT with high TPP?

Dehydration.

How do you interpret a low HCT with low TPP?

Substantial recent or ongoing blood loss, or overhydration.

How do you interpret a low HCT with normal TPP?

Anemia due to increased RBC destruction, decreased RBC production, or chronic hemorrhage.

How do you interpret a low HCT with high TPP?

Anemia of inflammatory disease, multiple myeloma, or other lymphoproliferative disease.

How does inflammation alter plasma proteins?

- Increased loss: albumin (due to vascular permeability, catabolism).

- Increased synthesis: positive acute-phase proteins, immunoglobulins.

- Decreased synthesis: negative acute-phase proteins (albumin, transferrin).

Where are lipoproteins synthesized?

Gastrointestinal tract and liver.

What is the primary function of lipoproteins?

Transport of water-insoluble lipids in blood.

What is the composition of lipoproteins?

High MW complexes of lipids (triglycerides, cholesterol, phospholipids) and apolipoproteins. Structure is pseudomicellar: hydrophilic proteins + phospholipids outside, hydrophobic triglycerides + cholesterol esters inside.

How are lipoproteins classified?

By density (ultracentrifugation) and migration on electrophoresis.

What are chylomicrons and where are they formed?

Very large, low-density lipoproteins formed in the duodenal/jejunal mucosal cells after dietary fat digestion.

How do chylomicrons enter circulation?

Secreted into lacteals → lymphatic vessels → thoracic duct → bloodstream.

What happens to chylomicron triglycerides in circulation?

Hydrolyzed by lipoprotein lipase in muscle/adipose capillaries; fatty acids + glycerol are metabolized.

What causes postprandial lipemia in carnivores/omnivores?

Chylomicronemia after eating a fat-containing meal → white, cloudy plasma. Not seen in herbivores.

Why do chylomicrons form a "cream layer" in stored plasma?

They are less dense than water, so they rise to the top after refrigeration overnight.

Why are chylomicrons not normally seen in fasting samples?

They have a short half-life in circulation and are rapidly cleared postprandially.

Where are very low density lipoproteins (VLDLs) primarily synthesized?

In the liver (with some intestinal formation).

What is the primary function of VLDLs?

Transport the bulk of endogenous triglycerides.

What happens to VLDLs after triglyceride hydrolysis?

Residual VLDLs are metabolized into LDLs.

How does plasma appear with excess VLDLs when refrigerated overnight?

Cloudy/milky plasma, but no cream layer (unlike chylomicrons).

From what lipoprotein are LDLs derived?

VLDLs.