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Chapter 7 The Plasma Protein. Tu Jiancheng. contents. 1. Concept of Plasma Protein 2. Total Protein: Acute-phase Reactants Individual Proteins and Disease States 3. Methods of Protein Analysis. 1. Concept of Plasma Protein. - PowerPoint PPT Presentation
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Chapter 7 The Plasma Protein
Tu Jiancheng
contents
1. Concept of Plasma Protein
2. Total Protein: Acute-phase Reactants Individual Proteins and Disease States
3. Methods of Protein Analysis
1. Concept of Plasma Protein
The Plasma Proteins Are A Diverse Group of Molecules ThatPerform A Variety Of Functions
* Need to Be Distinguished from Proteins That Occur Only Incidentally in The Blood.
* Often Classified Based on Their Electrophoretic Mobility
* Most of Them are Synthesized in the liver
Densitometric scan of a normal serum protein electrophoresis pattern showing the relative position of the albumin,α1,α2,β and γregions
Selected Majora and Minor Plasma Proteins (TABLE 7-2 )
Albumin zone Albumin Prealbumin
α1 Zone
α1 –Antitrypsin
High-density lipoprotein (α-lipoproteins) α1-Antichymotrypsin
Orosomucoid α-Fetoprotein
α2 Zone
α2 –Macroglobulin
Haptoglobin Ceruloplasmin Gc-Globulin
βZone Low-density lipoprotein (β-lipoproteins) Transferrin C3 β2-Microglobulin
Hemopexin Fibrinogen (may be inγZone)
γZone Immunoglobulins C-Reactive protein Fibrinogen Lysozyme
a In boldface
Plasma protein abnormalities arise from one or more of the following:
* Congenital abnormalities affecting a specific protein
* Acquired abnormaltities affecting a specific protein
* Alterations affecting multiple proteins reflecting variations in the physiologic state
* Alterations affecting multiple proteins secondary to disease
2. Total Protein
Factors Which Affect Protein Concentration: * Fluid Balance * Changes in Synthesis or Catabolism
* Protein losses.
* Prolonged Tourniquet Application during Venipuncture Increase the Total Protein by as much as 5g/L.
* Recumbent Subjects 10% lower than in Ambulatory Ones * Normal Diurnal Variation of 4 g/L
* Seasonal Fluctuation: Peak in Nov. and Lowest in June. * Exercise increases the serum protein concentration by as much as 10%, but this effect is transient.
Preanalytic Errors Which May Alter the Serum Protein value
* The total serum protein includes both albumin (60%) and the globulins (40%)
* The total protein concentration may be altered by changes in fluid balance
* Changes in the amounts of plasma proteins may result from alterations in synthesis or catabolism or from protein losses
Hypoproteinemia
* decreased protein synthesis
* malnutrition
* chronic liver disease
Nephritic syndrome
* the serum albumin level may fall to less than 5 g/L
ACUTE-PHASE REACTANTS(APRs)
A Group of Proteins Whose Plasma Concentration Changes In Response To A Variety of Inflammatory Sates Including: * Infection * Surgery * Trauma* Myocardial Infarction * Malignancy * Any Condition Associated with Tissue Necrosis
Cytokines
* Major Stimulator: Interleukin-6 (IL-6) is the
* Other Cytokins Which Upreugulate APRs: Interleukin-1β(IL-1β), Tumor Necrosis Factor-α(TNF-α) Interferon Gamma (IFN-γ) Transforming growth factorβ(TGF-β)
Regulation of APRs
Positive Negative
α1-Antitrypsin Albumin
α1-Antichymotrypsin Prealbumin
Haptoglobin Retinol binding protein
Ceruloplasmin Transferrin (rises in late acute phase)
Fibrinogen
C3
C-Reactive protein
Hemopexin
Serum amyloid A protein
Major Acute Phase Reactants (TABLE 7-3 )
* Measurement of APRs, especially C-reactive protein (CRP), may be useful to detect and follow patients with acute inflammatory disorders.
The changes in plasma proteins seen
during the acute-phase response
potentially serve a variety of functions
* Positive APRs: AAT,α1-Antichymotrypsin.
* Protect against reactive oxygen species: haptoglobin, hemopexin
Individual Proteins and Disease States
PrealbuminAlbuminα1-Antitrypsinα2-Macroglobulin CeruloplasminHaptoglobinTransferrinβ-Lipoproteinβ2-MicroglobulinC-Reactive Protein
* Prealbumin is a tryptophan-rich tetrameric non-glycosylated protein named for its anodal migration relative to albumin on protein electrophoresis.
Prealbumin(1)
[MW]: 55 kD [Synthesized]: in Liver[Synonyms]: Transthyretin and Thyroid-Binding Prealbumin. [Function]: Carrier Protein for Thyroid Hormone and for vitamin A (with Retinal Binding Protein) [Half-Lives]: 1.9 days. [Reference Range]: 195 - 358 mg/L
Prealbumin(2)
* Prealbumin levels reflect hepatic synthesis and can serve as an index of liver function
* Prealbumin is a compact molecule that can cross the blood-brain barrier and may also be synthesized by cells of the choroids plexus.
* Prealbumin can be measured by a variety of immunoassays, including radial immunodiffusion and nephelometry.
Albumin(1)
The most abundant protein in plasma, comprising approximately 60% of the total protein concentration.
[MW] 66.3kD, Most Abundant protein in Plasma, [Synthesized] almost exclusively by the liver,[Half-life] 19 days.[Functions]1. 80% of the plasma COP. 2. Amino Acid Source to a Variety of Cells 3. Major Transport Protein 4. Lipid Metabolism [Reference Range] 31 to 43g/L.
Albumin (2)
[measure]
Electrophorectic
Immunochemical
dye-binding techniques
bromocresol purple
Bromocresol green
Albumin(3)
α1-Antitrypsin (AAT)(1)
One of the major plasma proteins, comprising nearly 90% of the α1 globulin region on SPE.
Deficiency of AAT has been associated with pulmonary emphysema and hepatic cirrhosis.
[MW]: 52kD, Major Plasma Proteins, [Function]: a Serine Proteinase Inhibitors(Serpins )[Synthesizied]: in Liver. Seen in Plasma, Tears, Lymph, Bile, Semen, and Amniotic [Acquired decreases]:•Protein-losing syndromes, •malnutrition, •severe liver damage, •respiratory diseases: neonatal respiratory distress syndrome [Congenital Deficiency]:•Pulmonary Emphysema and Hepatic Cirrhosis[Reference Range]: 900~1500mg/L
α1-Antitrypsin (AAT)(2)
* Genes encoding the AAT protein comprise an autosomal allelic system containing at least 75 codominant genes inherited on a single locus called Pi for proteinase inhibitor.
α1-Antitrypsin (AAT)(3)
• The PiZ allele is the most common variant associated with AAT deficiency.
PiZZ : 10% to 15% (normal)
PiMZ: 60% (normal)
PiSS and PiMS: 63% and 83%(normal)
α1-Antitrypsin (AAT)(4)
* Consists of four identical subunits and is one of the largest serum proteins
* Can inhibit Several endopeptidases
* Can bind numerous growth factors, hormones, and cytokines
α2-Macroglobulin(AMG)(1)
[MW] 720 kD, Largest Serum Protein [Synthesized] in Liver [Half Life] 5 Day in Serum
[Function] 1. Defense Against Proteolytic Enzymes (Irreversibly Binds Proteinases ) 2. Transport Protein? (Binds to Growth Factors, Hormones, and Cytokines)
[Reference Range]125 to 215 mg/dL
α2-Macroglobulin(AMG)(2)
Ceruloplasmin(CER)(1)
* A glycoprotein synthesized by the liver as a single polypeptide chain to which six to eight copper atoms are attached.
* The pure protein is blue because of its high copper content. Increased levels may impart a green tinge to plasma samples.
[MW] 132kD, a Glycoprotein, Synthesized by the Liver, not Normally Visible on Routine Gels[Clinical Significants]: Diagnosis of Wilson’s Disease (Hepatolenticular Degenetation)
[Reference Range]: 27 to 50 mg/dL Highest in young children and somewhat Lower in both Infants and Adults. Less than 10 mg/dL are Suggestive of Wilson’s Disease,
Ceruloplasmin(CER)(2)
Haptoglobin(1)
* A glycoprotein synthesized by the liver that migrates as an α2 globulin.
* In addition to its well-known function in the binding of free hemoglobin, this APR might also play an important role in the control of local inflammation.
Glycoprotein [Synthesized] in the liver [Migrates] as α2 globulin [Function] Binding of Free Hemoglobin, Control of Local Inflammation.[Clinical Significatns] Decreased Associated with Intravascular Hemolysis, (Hemolytic Anemias, Hemoglobin-opathies, Hemolytic Transfusion Reactions, Extensive Burns, Malaria, Disseminated Intravascular Coagulation, and Exercise-Induced Hemolysis). [Reference range] 16 to 199 mg/dL.
Haptoglobin(2)
Transferrin
[MW]: 80kD, a Glycoprotein[Synthesized] by Liver(most), reticuloendothelial system, the gonads, and the submaxillary gland [Function] Transport of Iron from Intestine (or Hemoglobin catabolism) to Red Cell[Reference Range] 191 to 365 mg/dL (1.91 to 3.65g/L).
β-Lipoprotein(1)
* Lipoproteins constitute a family of molecules composed of lipids and proteins whose function is to transport cholesterol, triglycerides, and phospholipids in the blood.
β-Lipoprotein(2)
[Components] Chylomicrons(CM), [Very Low Density Lipoproteins (VLDL) [Low-Density Lipoproteins (LDL) [High-Density Lipoproteins (HDL).
β2-Microglobulin(1)
* A small protein (MW 11,818) comprising the common light chain of the class I major histocompatibility complex antigen on the surfaces of all nucleated cells.
[MW] ~11,818 Dalton, Noncovalently Linked to the human lymphocyte antigen (HLA) heavy chain. [Distribution] Plasma, Urine, and CSF[Function] Necessary for Insertion of the HLA into Cell Membrane, and Stabilize the HLA heavy chain. It Regulation of Some Lymphocyte Functions.[The normal Reference Range] Serum 2.8mg/L Urine 0.2mg/L
β2-Microglobulin(2)
* CRP was first identified in 1930 as a substance present in the sera of patients with pneumococcal pneumonia that could bind to C-polysaccharide isolated from Streptococcus pneumoniae, producing a flocculation reaction.
C-Reactive Protein(CRP)(1)
[MW] 118,000 Dal [Physiology] Elevated in a Variety of Acute Inflammatory siseases. [Half Life] 19 hours. [Synthesized] by the liver and released into the plasma. Peripheral(Small Amount)[Function] Recognition of Microbial Organisms An immunomodulator in Host Defense. Recognition of Necrotic Tissues. [Reference Range] Less than 0.8mg/dL..
C-Reactive Protein(CRP)(2)
MTEHODS OF PROTEIN ANALYSIS
* Measurement of Total Protein
* Serum Protein Electrophoresis
* Other methods
Measurement of Total Protein
☆Kjeldahl procedure
☆ biuret method
☆ Lowry method
☆ dye-binding procedures
☆ turbidimetric method
☆ nephelometric method
Serum Protein Electrophoresis
* Principles of Electrophoresis
* Agarose Gel Electrophoresis
* Capillary Elecrophoresis
Principles of Electrophoresis
* support media * buffer ions * pH * the size and shape of the molecule * the strength of the electric field * the temperature * the effects of convection and diffusion * the ionic and pore properties of the * electrophoresis medium
Typical serum protein Electrophoresis Reference Pattern (TABLE 7-4)
ZonePercentage of Total Protein (mean)
Albumin 45.8-68.2 (57.0)
α1 2.1-6.1 (4.1)
α2 8.8-18.0 (13.4)
β 7.8-13.0 (10.4)
γ 7.8-22.6 (15.2)
Representative patterns of serum protein seen on serum protein electrophoresis
Capillary Elecrophoresis
* Electrophoretic separation of analytes is carried out in fused-silica capillaries with an inner diameter of 50 μm (range, 20 to 200 μm), an outer diameter of 375 μm, and an effective length of approximately 50 cm (range, 7 to 100 cm).
* Ion Exchange Chromatogrphy
* Affinity Chromatogrphy
* Isoelectric Focusing
Other Methods