Lab D 6 Metabolic parameters; proteins Flashcards
Total protein concentration in blood, dependent on?
Total content in plasma (serum) is dependent on the intake, synthesis, transformation , catabolism and hydration status (dehydration, hyperdration)
Measurements of the total protein concentration of blood?
Chromatography, electrophoresis and refractometry
Normal range of total protein in blood
60-80 g/L
Where can we find smaller total protein content than the normal range?
Urine, cerebrospinal fluids, body cavity fluids or tissue homogenates
What method to see total protein content of fluids with lower than normal range values?
Lowry method, with Folin-phenol reagent is used
or
Ultrasensitive total protein method, where proteins are bound directly to stain molecules
Sensitivity of different methods measuring total protein content, whats the range of Biuret method or refractometry?
20-100 g/L
Biuret test
- Total protein measurement
- Measured photometrically
- Reagent contains: KNaSCN, CuSO4, KI and NaOH
- Chemical reaction: CO-NH + CU2+ + alkaline = purple complex
- Wavelength: 546 nm
Ultrasensitive total protein analysis
- Total protein in lower concentration
- Na-molibdate and pirgallol-red reagent forms a complex molecule by binding proteins
- Complex measured on 600nm wavelenght
- Sensitivity is 0.2-4 g/L
- Standards are 0.25, 0.5, 1.2 g/L
Refractometry
- Total protein (TP) analysis
- Light is refracted when reaching border of media with diff in specific gravity
- TP in blood plasma or serum is the biggest influencing factor on specific gravity (2-3 x higher conc than for eks ions)
- In refractometer the specific gravity of one media is given (glass), so the change in light refraction depend in quality of plasma/serum, also dependent on temperature
Refractometry, method
- Calibration, using distilled water
- 1 droplet plasma/serum on glass, close cover
- Looking in visor: the horizontal line on the scale tells us about the total protein content of blood sample
- Performed in room temp
Refractometry, false results?
- Less precise than spectrophotometry, but quicker
- Can be used in range of 25-95 g/L
- May give biased results in haemolysis or lipaemia
TP concentration of blood plasma depends on:
- Protein metabolism
- Water balance
TP concentration of blood plasma under dehydration
Total protein increases
TP concentration of blood plasma under hyperhydration
Total protein decreases
Protein fractions
- Major fractions are albumin, globulin and fibrinogen
- Fibrinogen in smallest amount
- Globulin is calculated by the difference of the TP and albumin concentration (Fibrinogen being 1/20 or 1/25 of TP)
Albumin measurement
- Method 1
- Albumin conc. can be measured by spectrophotometry
- Bromocresol green as reagent
- Reagent binds to albumin on pH 4.2 and forms blue-green complex -> measurable on wavelength 578nm
Albumin measurement
- Method 2
- Serum electrophoresis in combination with TP measurement
- More expensice than spectrophotometry, but used when protein fraction analysis is the basic aim
- Necessary to know TP because albumin is given in % of the total protein content of the sample
Changes in albumin concentration: decrease
- Decreased intake of proteins, decreased absorption (maldigestion, malabsorption)
- Decreased synthesis - liver failure, acute inflammation (it is a neg acute phase protein)
- Increased utilization - physiological conditions cause mild changes: pregnancy, work, exercise, production (milk, egg) and chronic diseases (chronic inflammation, neoplasm)
- Increased loss: via kidneys (protein loosing nephropathy; PLN), GI tract (protein loosing enteropathy; PLE), skin (burns), whole blood loss, sequestration into body cavities - NOT decrease of colloid pressure (cardiac disease, lymphangiectasia, portal hypertension, other vascular disorders, peritonitis ex. perforation in intestines, gall bladder, translocation of bacteria)
- Other relative decrease: hyperhydration (may be iatrogen, ex. excessive infusion)
Changes in albumin concentration: increase
Dehydration
Globulin measurement:
- Method 1
- Calculated roughly by the diff of TP and albumin conc. of serum
Globulin measurement:
- Method 2
- Serum electrophoresis is used if protein fraction analysis is the basic aim
- This method provides percentage values, so objective concentration (g/L) has to be calculated knowing the TP concentration
Albumin/Globulin ratio
- Decrease og A/G ratio is most frequently caused by increase of globulin conc. in ex. inflammatory processes or processes related to neoplasia (abnormal growth).
- This inflammatory reaction can be evaluated using RBC sedimentation test and glutaric-aldehyde test
Albumin/Globulin ratio, reasons for decreased ratio
- Decrease og A/G ratio is most frequently caused by increase of globulin conc. in ex. inflammatory processes or processes related to neoplasia (abnormal growth)
- Can also be decreased due to decrease of albumin, (the very long list of reasons)
Electrophoresis (globulins), basics
- Based on fact; proteins have amphoteric character (acidic amino acids - Asp and Glu go to the + pole, alkaline amino acids go to the -pole)
- Serum is placed on paper treated with agarose gel and exposed to an el.current to separate the serum protein components into five classifications by size and electrical charge
SPEP test analyses?
- Serum protein electrophoresis is a lab test that examines specific proteins in blood -> globulins
- El.phoresis is a process where ions move through a medium in response to an applied electric field
- The rate of motion of the charged particles in the applied el.field is proportional to their charge and inversely proportional to their size and the viscosity of the medium.
Electrophoresis method is separating the serum proteins i to five classifications
- serum albumin
- alpha-1-globulins
- alpha-2-globulins
- beta globulins
- gamma globulins
Electrophoresis result,
- Small and highly charged molecules will move a greater distance, than the large and lower charged molecules
Normal media in gel electrophoresis?
Polyacrylamide or agarose gel
The two msot commonly used forms of protein electrophoresis?
- SDS-PAGE (sodium docecyl sulfate-polyacrylamide gel el.phoresis)
- IEF (isoelectric focusing)
SDS-PAGE (sodium docecyl sulfate-polyacrylamide gel el.phoresis)
- Protein samples first denatured by heating in sds -> coating -> highly neg. charged proteins
- Then applied to one end of a polyacrylamide slab, with electrodes on each side
- Voltage -> negatively charged proteins migrate towards the anode
- After time, gel slab is stained to show locations of proteins. Smaller proteins are closer to anode
- An inverse log-linear relationship of protein molecular weight to the distance travelled is observed
After staining SDS-PAGE (sodium docecyl sulfate-polyacrylamide gel el.phoresis), what is used to see results
- Separated protein fractions can be detected by the densitometer, depending on the conc. of the protein fraction
- By their electrophoretic character we distribute plasma protein fractions to albumin
Serum contains what amounts of glubulin and albumin
60% albumin
40% globulin
Plasma contains what amounts of glubulin, albumin and fibrinogen
50% albumin
30% globulin
20% fibrinogen
By their electrophoretic character we distribute plasma protein fractions to
Albumin, alpha-1, alpha-2, beta-1, beta-2, gamma-1, gamma-2
Alpha globulins are
Acute phase proteins
Beta globulins are
immunoglobulins (IgA, IgM) and some other proteins ex. LDL
Gamma globulins are
Immunoglobulins (IgG)
Proteins, especially immunoglobulins are derived from
Special lymphoid cells (plasma cells)
Proteins, especially immunoglobulins are derived from special lymphoid cells (plasma cells), one cell group of the same origin is a clone, produces the same proteins
- Polyclonal gammopathy
- Monoclonal gammopathy
Polyclonal gammopathy, short
- Beta and gamma globulins derived from different clones
- Generally occurs during inflammatory process or some immune mediated diseases
- Seen as broad-based peak in beta and/or gamma region
Monoclonal gammopathy, short
- One protein fraction derived from one clone
- Occurs during immune mediated or neoplastic conditions
- Seen as large spike in the beta or gamma region
- Spike can be compared to the albumin peak, a monoclonal gammopathy has a peak as narrow as that of albumin
Polyclonal gammopathy, some common causes and beta-gamma bridging causes
- Various chronic inflammatory disease (infectious or immune mediated)
- Liver disease
- FIP (alpha-2 globulins are often concurrently elevated, see adjacent ELP tracing)
- Occult heartworm disease
- Erlichiosis
- Beta-gamma brigding occurs in disorders with increased Ig AND IGM, such as lymphoma, heartworm disease and chronic active hepatitis
Monoclonal gammopathy, common causes
- Both neoplastic and non-neoplastic disorders can produce monoclonal gammopathy
Serum protein electrophoresis with monoclonal gammopathy, 1) Neoplasia
- Multiple myeloma is the most common cause, producing an IgG or IgA monoclonal.
- Lymphoma (IgM or IgG) and chronic lymphocytic leukemia (usually IgG)
- Extramedullary plasmacytomas
- Macroglobulinaemia (increase in IgM)
- Multiple myeloma
Extramedullary plasmacytomas
- Solid tumours composed of plasma cells, usually found in skin of dogs or in GI tract of cats and dogs
- Can be associated with a monoclonal gammopathy, or even a biclonal gammopathy if there are multiple tumours
Macroglobulinaemia and Waldenstroms macroglobinaemia
- An increase in IgM
- Waldenstroms: is a neoplasm of B-cells (lymphoma) that has different presentation from multiple myeloma.
- Patients usually have splenomegaly and/or hepatomegaly and lack osteolytic lesions
Multiple myeloma
- In contrast to macroglobulinaemia, multiple myeloma is a disorder of plasma cells that have undergone antigenic stimulation in peripheral lymph nodes and then home in the bone marrow (the marrow produces appropriate growth factors that support growth of myeloma cells)
- Myeloma is therefore characterized as a bone marrow disorder, with osteolytic bone lesions (in 50% of canine cases) and Bence-Jones proteinuria.
- Extramedullary infiltrate of plasma cells are uncommon, but can occur in terminal phases of the disease
Serum protein electrophoresis with monoclonal gammopathy, 2) non-neoplastic disorders
- Rare
- Usually IgG
- Occult heartworm diseae
- FIPV (rarely)
- Ehrlichia canis
- Lymphoplasmacytic enteritis
- Lymphoplasmacytic dermatitis
- Amyloidosis
- These causes should be ruled out before a diagnosis of multiple myeloma is made in a patient with an IgG monoclonal gammopathy
Hypoglobulinaemia
- Decreased intake of globulins: in neonates before drinking colostrum, absorption disorders of neonates
- Decreased synthesis of globulins: acquired of inherited immunodeficiency, liver function impairment
- Increased loss: PLE, PLN, via skin (burns, inflammation), bleeding
Fibrinogen concentration, Method 1)
If both plasma (plasma separated from anticoagulated blood) and serum (serum separated from clotted blood) TP conc. is measured, the difference of plasma and serum TP conc. gives fibrinogen conc.
Fibrinogen concentration, Method 2)
- Test is based on heat labile character of fibrinogen
- One part of plasma is used for TP measurement (ex. Biuret test), other part is heated to 55-58 C and centrifuged, then TP conc. is measured
- Difference between the two measurements is equal to the fibrinogen conc.
- In this method TP analysis can be determined by refractometry, capillary tubes can be used
Fibrinogen concentration, Method 3)
- The test for thrombin time can be used for establishing fibrinogen conc., because the values are primarily dependent on the fibrinogen conc.
- Reagent contains bovine thrombin and Ca2+.
- Clot formation can be determined by using standards of different fibrinogen concentrations
Cause of fibrinogen concentration changes, Increase
Acute inflammation (especially ruminants), dehydration
Cause of fibrinogen concentration changes, Decrease
Liver function impairment, advanced protein deficiency, DIC, sequestration after bleeding to body cavity, chronic bleeding, blood loss, inherited afibrinogenaemia (st. Bernard dogs)
