Blood Chemistry

Liver and GIT

Portal vein carries blood from the GIT to the liver

Hepatic artery provides oxygenated blood
Bile duct transports bile to the gallbladder
Function
Metabolism
Complete digestion and absorption of dietary nutrients
Detoxification
Storage
Stores glycogen, which can be broken down into glucose
Production
Proteins, bile, urea, cholesterol, glycogen-glucose
Regulation
Homeostasis
Signs of Liver Diseases:
Jaundice or icterus (accumulation of bilirubin)
Ascites (accumulation of fluid in the abdomen)
Examples of Liver Disease
Congenital absence of enzymes that convert ammonia to urea
Congenital portosystemic shunt (defects in the portal vein)
Viral parasitic, protozoal, and bacterial infections
Hepatic lipidosis
Steroid hepatopathy
Hepatoxins (heavy metals, anti-inflammatory drugs, certain antibiotics and anesthetics, anticonvulsant medications, and certain anti-parasite
drugs and dips)
Chronic active hepatitis
Cirrhosis (scar tissue replaces healthy liver cells)
Cholangohepatitis (inflammation of the bile-carrying structures and the surrounding liver tissue)
Both benign and malignant (hemangiosarcoma, lymphosarcoma, and metastatic tumors)
Diagnostic Tests
Initial Tests: complete blood count, biochemistry profile, and urinalysis
Bile acids: blood tests that assess the function of the liver and the amount of the liver that is diseased
Blood ammonia, blood steroid, and amino acid levels
Serology tests for certain viruses, protozoa, and fungal diseases
Abdominal radiography (x-ray): may show changes in liver size and shape
Abdominal ultrasonography
Clotting profile (evidence of chronic and severe liver disease)
Advanced imaging tests: MRI, CT scan, dye contrast studies
Liver biopsy: under the guidance of an ultrasound, through laparoscopy, or by surgical opening of the abdomen

Total Protein (g/dL)

Sum concentration of all serum proteins (albumin, globulin)

Can be seen through protein electrophoresis
Separates serum proteins based on their mobility into fractions of albumin, alpha-, beta-, and gamma-globulins
Determine their concentrations
Plasma 55% (Proteins 7% = Albumin (58%) + Globulins (38%) + Fibronogen 4%) + Formed elements 45% (RBCs, WBCs, Platelets)
Measurement: refractometry, biuret method, and turbidometric
Refractometry
Plasma protein in EDTA plasma
Variation in other serum components "solids (sodium, chloride, phosphate, glucose, etc.)," colloids such as heterostarch and gelatin
Lipemia and severe hemolysis
Colorimetric assay or Biuret method: Blue (no protein) to violet (has protein that reacts with divalent copper in an alkaline medium -> Cu
protein complex)
All proteins; not accurate for the range of 1-10 g/dL
Not sensitive enough for low concentrations found in some body fluids, such as CSF, urine, and many body cavity effusions
 TP is usually higher than that provided from the chemical analyzer due to the contribution of total solids to the refractive index, and the
contribution of fibronogen to total protein content in plasma compared to serum
Turbidometric methods
Quantitation of protein in CSF, urine, and other low-protein fluids
Urinary dipsticks can be used to estimate protein in CSF samples (not as accurate as turbidometric techniques)
Precipitation methods include trichloroacetic acid and sulfosalicylic acid, or on precipitation of protein by benzethonium chloride (sensitive
to as little as 6 mg/dL of protein)
Dye-binding methods uses dyes, such as Coomassie blue and pyrogallol red-molybdate
Sample Considerations
Sample Type: Serum and plasma should be separated from the clot within 4 horus of collection.
Plasma: has anticoagulant and clotting factors (fibrinogen)
Serum: no anticoagulant and clotting factors
Anticoagulants: Ethylenediamine tetraacetic acid (EDTA) or heparin
Stability (in humans): 3 days at 2 to 8 C or 6 months at -15 to -25 C
Interferences
Lipemia and Hemolysis: BUN levels will increase with severe lipemia and severe hemolysis (hemoglobin will react as a protein in the assay)
Icterus: Severe icterus may decrease concentrations
Drugs: 4% succinylated gelatin will falsely increase results
Test Interpretation
Specimen required ultracentrifugation due to severe lipemia
The results are not interpreted in isolation
Factors Affecting Total Protein Level
Age, diet, pregnancy, disease, and dehydration
Hypoproteinemia
Chronic malnutrition
Malabsorption (not properly absorbed)
Protein loss due to kidney or liver disease
Liver disease: affects production of serum proteins
Kidney disease: affects filtration of serum proteins
Specific decrease in the albumin fraction: liver disease, chronic malnutrition, or dental disease
Hyperproteinemia
Dehydration, chronic infection, shock, or a metabolic imbalance
Chronic infection: increased proteins from microbes
Shock: decrease in blood flow
Technical issues, such as assay method and sample handling
Albumin
First fraction to appear by electrophoresis; predominant serum protein
Produced in the liver
Highly soluble in water
Half-life: 15-19 days
Maintain colloidal osmotic pressure within the intravascular compartment
Part of ECF, CSF, urine, amniotic fluid, and interstitial fluid
Increase 300 times in nephrotic syndrome
Binds and transport: Free fatty acids, amino acid, metallic ions, phospholipids, drugs, hormones, bilirubin
Globulin
Alpha: increase during acute infection; demonstrate the most rapid electrophoretic mobility
Alpha 1: Alpha-1 antitrypsin, serum amyloid A, ceruplasin, corticosteroid-binding globulin (transcortin), retinol-binding proteins
Alpha 2: Haptoglobin, ceruloplasmin, protein C, angiotensinogen (blood pressure)
Beta: binding proteins in the plasma, part of the clotting process
Transferrin (transports ion), C3 and C4 (complementary proteins during inflammation), Beta-2 microglobulin
Gamma: primarily antibodies and increase in response to infection
CRP, immunoglobulins (IgM, IgG, IgA), RF, CRP
Albumin and Globulin Ratio
The normal ratio in most species is 1:1
Can be interpreted only in light of the total protein concentration
High TP with normal A:G ratio - dehydration
High TP with low A:G ratio - hypovolemia
Same protein with a low A:G ratio - hyperglobulinemia
Low ratio: decrease albumin or increased globulins due to myeloma or autoimmune disease
 High ratio: may be caused by low immunoglobulin levels as occurs in some leukemias

Alanine Aminotransferase (ALT) or Serum Glutamate Pyruvate Trransaminase (GPT)

Catalyzes the transfer of alpha amino group of alanine to alpha-ketoglutaric acid, resulting in the formation of pyruvic and glutamic acid
Located in the cytoplasm with small amounts in mitochondria
Found in the liver, skeletal and cardiac muscle, kidneys, and erythrocytes
Fairly liver specific in dogs, cats, rabbits, rats, and primates
Requires pyridoxal phosphate (active form of B6) for maximum performance
Serum half life: 59 hours in dogs, 2.8 to 4.4 hours in cats
Following acute hepatic injury, serum enzyme activity peaks at about 48 hours and begins to decrease
Increases in the enzyme occur due to cell damage (increased membrane permeability, blebbing, or necrosis)
Some increases are possible in severe muscle diseases
Colorimetric method
Units of measurement: U/L
The amount of enzyme that catalyzes the conversion of 1 μmol of substrate per minute under specified conditions
Sample considerations
Sample type: Serum and plasma should be separated from the clot within 4 hours of collection
Anticoagulant: Heparin or EDTA
Stability: 3 days at 15 to 25 C, 7 days at 2 to 8 C, more than 7 days at -80 C
Interferences
Lipemia/turbidity, icterus: no significant inteferences
Hemolysis:
In cats and pigs, intravascular on in vitro hemolysis may increase due to the ALT release from eythrocytes
In pigs, hemolysis of > 100 units increased ALT by a median of 8 U/L
Drugs
Anticonvulsants (primidone,phenobarbitone, dilantin) can increase ALT activity up to 4x normal
Corticosteroids increase ALT to approximately 2-3x normal
Activity is higher in dogs with steroid hepatopathy (where actual hepatocellular injury occurs
Any drugs that can cause hepatoxicity (tetracycline in cats, caparsolate in dogs, acetaminophen) can result in increase ALT activity
Some drugs may decrease ALT (and AST) activity, by impairing activation of vitamin B6 to P5P (e.g. cephalosporin, cyclosporin,
isoniazide)

Urea Cycle

Occurs in the liver

Carbonyl phosphate synthesis
BUN levels
Overhydration leads to electrolyte imbalance or less salt
Steps
Carbamoyl Phosphate Synthetase I Initiates Urea Biosynthesis
Carbamoyl Phosphate + Ornithine -> Citrulline
Citrulline + Aspartate -> Argininosuccinate
Cleavage of Argininosuccinate -> Arginine + Fumarate
Cleavage of Arginine -> Urea -> Ornithine
HCO3- + 1 NH3 + Asp + 3 ATP + H2O -> Urea + Fumarate + 2 ADP + AMP + 2 Pi + 1 PPi

SDS Page and Western Blot: Application in Animal Disease Diagnosis

Terminologies

Western blot (protein immunoblot): an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract
Southern blot: a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples
Northern blot: a technique used in molecular biology research to study gene expression by detection of RNA
Southwestern blotting: a lab technique which involves identifying and characterizing DNA-binding proteins
Based along the lines of Southern blotting (which was created by Edwin Southern) and first described by B. Bowen and colleages in 1980

Western Blot

Allows investigators to determine the MW of a protein and to measure relative amounts of the protein present in different samples
Proteins are separated by gel electrophoresis, usually SDS-PAGE
The proteins are transferred to a sheet of special blotting paper called nitrocellulose
The proteins retain the same pattern of separation they had on the gel
The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose
 An antibody is then added to the solution, which is able to bind to its specific protein
The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it, which cannot be seen at this time
The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that
can be seen and photographed
Steps
SDS Polyacrylamide Gel Electrophoresis: separate protein sample by electrophoresis
Electro-transfer: transfer proteins to membrane
Blocking: block non-specific sites to increase sensitivity and to prevent non-specific signal
Several measures should be followed to decrease the non-specific reactions to a minimum
Blocking step is the incubation of the membrane with solution containing BSA or fat-free milk or casein for a sufficient time with shaking
Primary and Secondary Detection Reagents: incubate the membrane with antibody
For direct transfer, choices are
Monoclonal antibodies
Fluorescent probes and labeling kits
Enzyme labeling kits
Primary Antibody labeling
The immobilized proteins on the surface of the membrane can be detected using a specific labeled antibody
Labeling of the antibody can be performed using a radioactive or non-radioactive method
Primary Antibody probing
The blot is first incubated with a primary antibody followed by the addition of a labeled secondary antibody that has species specificity
for the primary one
For example, probing of the membrane using mouse primary antibody and anti-mouse secondary antibody
Enzyme Substrates: add the detection reagent
Film: expose the membrane to X-ray film
Can detech one protein in a mixture of any number of proteins while giving you information about the size of the protein
The method, however, is dependent on the use of a high-quality antibody directed against a desired protein
The antibody is used as a probe to detect the protein of interest
Proteins are separated using SDS-PAGE, which separates proteins by size
Nitrucellulose membrane is placed on the gel
The actual blotting process may be active (electroblotting) or passive (capillary)
Electroblotted is used for faster and more efficient transfer of protein from gel to membrane
Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette, which is placed between platinum electrodes
An electric current is passed through the gel, causing the proteins to electrophorese out of the gel and onto the nitrocellulose membrane

SDS-PAGE (Polyacrylamide Gel Electrophoresis)

A technique widely used in biochemistry, forensics, genetics, and molecular biology:

To separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight)
To separate proteins accordin to their size, and no other physical feature
SDS (Sodium dodecyl sulfate): a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it
Before SDS: Protein incubated with the denaturing detergent SDS, showing negative and positive charges due to the charged R-groups in the
protein
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges, which overwhelms any positive charges the
protein had due to positively charged R-groups
The resulting protein had been denatured by SDS (reduced to its primary structure-amino acid sequence) and as a result has been linearized
SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges, thus overwhelming positive charges in the
protein
The detergent binds to hydrophobic regions in a cosntant ratio of about 1.4 g of SDS per gram of protein
Therefore, if a cell is incubated with SDS, the membrane will be dissolved, all the proteins will be solubalized by the detergent and all the
proteins will be covered with many negative charges
PAGE (Polyacrylamide gel electrophoresis): If the proteins are denature and put into an electric field (only), they will all move towards the positive
pole at the same rate with no separation by size
However, if the proteins are put into an environment that will allow different size proteins to move at different rates
The environment is polyacrylamide
Small molecules move through the polyacrylamide faster than big molecules, and big molecules stay near the well
End result of SDS-PAGE has two important features
All proteins contain only primary structure
All proteins have a large negative charge, which means they will all migrate towards the positive pole when place in an electric field
The actual bonds are equal in size, but the proteins within each band are of different sizes
After electrophoresis
Fix the proteins in the gel and stain them
Electrophoretic transfer to a membrane and then probe with antibodies (Western blotting)