Lab 6 Flashcards by Helene Berg

Total protein concentration in blood (TP) is dependent on?

intake, synthesis, transformation, catabolism, and hydration status (dehydration, hyperhydration)
measurments can be done by chromatography, electrophoresis and refractometry

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TP concentration of plasma:

60-80 g/l

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Biuret test:

TP is commonly measured photometrically
reagent contains: KNaSCN, CuSO4, KI and NaOH
schematic chemical reaction: CO-NH + Cu2+ + alkaline = purple complex
Wave length: 546 nm

Method:

Buiret reagent (ml): Sample 3.0ml, blank 3,0 ml
Serum: Sample 0.05 ml, no blank
physiol. NaCl: No sample, blank 0.05ml
wait fo 30 min
use calibration curve or the formula if you have standard (60 g/l)
Esample / Estandard x 60 = total protein g/l

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Ultrasensitive total protein analysis

Na-molibdate, and pirogallol-red reagnet forms a complex molecule by binding proteins
the complex can be measured on 600nm wave length
sensitivity is 0.2 g/l - 4 g/l
standards are: 0.25, 0.5, 1.2 g/l

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Refractometry:

light is refracted when reaching the border of media with different specific gravity.
the light reflraction characteristics of a solution is dependent on its specific gravity.
the total protein content in blood plasma or serum is the factor having biggest influence on its specific gravity (being present in 2-3x higher concentration than other substances e.g ions)
in the refractometer the specific gravity of one media is given (glass), so the changes in light reraction depend on the quality of plasma/serum.
specific gravity is also dependent on temperature.
after calibration (destilled water), 1 droplet of plasma/serum is placed on the glass, the cover is closed and looking in the visor the result is read:

–> the horizontal line intercepting the scale of serum/plasma total protein

the procedure should be performed in room temp.
the method is quick and easy, but less precise than spectrophotometry.
can be used in range of 25-95 g/l, may give biased result in haemolysis or lipaemia.

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Protein fractions:

major fractions are albumin, globulin and fibrinogen.
Fibrinogen is in the smallest quantity (1/20, 1/25 of TP), so globulin concentration is generally calculated by the difference of the TP and albumin concentration.

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Plasma TP in Dog, cat, horse, cattle, swine and sheep

Dog: 67-70 g/l
Cat: 70-75 g/l
Horse: 68-70 g/l
Cattle: 75-85 g/l
Swine: 65-77 g/l
Sheep: 58-60 g/l

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Albumin concentration measurment by Spectrophotometry

Bromocresol green is used as reagent.
This reagent binds to albumin on pH 4.2, and forms a blue-green complex which is measurable on 578 nm wave length

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Albumin concentration measurment by Serum Electrophoresis:

used in combination with TP measrument for determining albumin concentration
the cost is higher compared to spectrophotometry, but it is used when Protein Fraction analysis is the basic aim.
this method provides albumin as a % of the Total Protein content of the sample, so it is necessary to know the TP concentration

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Changes of albumin concentration: Decrease and Increase

Decrease:

decreased intake of proteins, decreased absorption (maldigestion, malabsorption)
decreased synthesis - liver failure, acute inflammation (its a negative acute phase protein)
Increased utilisation: pregnancy, work, exercise, production (milk, egg etc), and chronic diseases
Increased loss: via kidneys (protein loosing nephropathy - PLN), Gastro intestinal tract (protein loosing enteropathy - PLE), skin (burn), whole blood loss, sequestration into body cavities - NOT the decrease of colloid pressure (cardiac disease, lymphangectasia, portal hypertension, other vascular disorders, peritonitis e.g perforation in intestines, gall bladder, translocation of bacteria)
Other (relative decrease): hyperhydration (may be iatrogen)

Increase:

dehydration

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Globulin concentration measurment:

Method 1:

is calculated roughly by the difference of TP and albumin concentration of serum

Method 2:

Serum electrophoresis. Same way as Albumins

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Albumin/Globulin ratio

The decrease of Albumin/Globulin ratio is most frequently caused by the increase of Globulin concentration, e.g: inflammatory processes or processes related to neoplasia.
This inflammatory reaction can be evaluated using the RBC sedimentation test and the Glutaric-Aldehyde test.
The other cause for the decrease of Alb/Glob ratio is the decrease of Albumin concentration (see own flash card for this)

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Explain the theory of Electrophoresis:

Serum protein electrophoresis is a simple, affordable method of separating blood proteins based on electrical charge, size and shape.
In the blood serum there are two major categories of proteins: albumin and globulin. Albumin has the largest concentration and globulines are in a smaller amount, but they are the basis for electrophoresis.
There are 5 categories of globulins: alpha-1, alpha-2, beta-1, beta-2 and gamma.
The patient’s serum is placed on a special medium (e.g., cellulose, agar) and then an electric charge is applied for a period of time when the proteins will migrate from the negative pole to the positive one.
Albumin migrates and reaches the positive pole and gamma globulins will remain at the negative pole, resulting in the fractions specific to each protein.
With the help of the staining step, the fractions are highlighted and can be interpreted by means of readers that measure optical density. The results are expressed for each type of protein in part and expressed as: normal, low, increased.

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Explain the two forms of Protein Electrophoresis:

Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Isoelectric focusing (IEF).

In SDS-PAGE the proteins samples are first deatured by heating in SDS.
This results in the coating of the polypeptides with negatively charged SDS molecules, leaving each protein highly negatively charged.
The denatured proteins are then applied to one end of a slab of polyacrylamide. Electrodes are then attached to opposite ends of the slab with the cathode (-) at the end where the denatured protein was applied and the anode (+) at the other end.
apllication of voltage across the gel forces the negatively charged proteins to migrate towards the anode.
in the highly viscous polyacrylamide, larger denatured proteins will exerience greater frictional drag than smaller dentured proteins - and move through the gel matric at a much slower rate.
after a predetermined period of time, the electrodes are removed and the gel slap is stained with dye to show the locations of proteins in the gel.
the smaller proteins are found closer to the anode and the larger ones closer to the cathode.
an inverse log-linear relationship of protein molecular weight to the distance traveled is observed.

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Detection of the results after Electrophoresis:

After staining the separated proteins fractions, the densitomer can detect the results.
Serum contains: 60% of albumin, 40% of globulin
Plasma contains: 50% of albumin, 30% of globulin and 20% of fibrinogen.
by their electrophoretic character we distribute plasma protein fractions to Albumin, alpha1, alpha2, beta1, beta2, gamma1, gamma2
alpha-globulins: are acute phase proteins
Beta-globulins: are immuniglobulins (IgA, IgM), and some other proteins i.e LDLD.
Gamma-globulins: are Immunoglobulins (IgG).
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.

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Increase of globulin concentration:

Polyclonal gammopathy
Monoclonal gammopathy

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Polyclonal gammopathy:

Beta and gamma globulins derived from different clones. Generally occurs during inflammatory processes or some immune mediated diseases.
this is seen as a borad-based peak in the beta and/or gamma region.
some common causes: chronic inflammatory diseases (infectious, immunde mediated), liver disease, FIP (alpha-2 globulins elevated), occult heartworm disease and Ehrlichiosis.
Beta-gamma bridning corrus in disorders with increased IgA and IgM, such as lymphoma, heartwork disease and chronic active hepatatis.

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Monoclonal gammopathy:

One protein fraction derived from one clone. Occurs during immune mediated or neoplastic conditions.
Seen as a sharp spike i the beta or gamma region.
the peak can be compared to the albumin peak, same narrowing.
both neoplastic and non-neoplastic disorders can produce a monoclonal gammopathy.

Neoplasia causing Monoclonal Gammopathy:

Multiple myeloma is the most common cause (producing IgG or IgA monoclonal).

Multiple Myeloma is a disorder of plasma cells that have undergone antigenic stimulation in peripheral lymphnodes. It is a cancer of plasma cells, a type of white blood cells which normally produce antibodies.
The plasma cells can also form a mass in the bone marrow or soft tissue. - the bone marrow produces appropriate growth factors that support growth of Myeloma cells.
Therefore Myeloma is characteristic as a bone marrow disorder, with osteolytic bone lesions. In 50% of Canine cases, and Berre-Jones proteinuria.

Other associated with Monoclonal Gammopathy:

Lymphoma (IgM or IgG), and chronic lymphocytic leukemia (IgG)
Extramedullary Plasmacytomas: Solid tumours found in the skin of dogs. Can be found in the GI tract and liver of cats and dogs.
Macroglobulinemia: increase of IgM
Waldenstrom´s Macroglobulinemia: is a neoplasm of B-cells (lymphoma) that has a different presentation from multiple myeloma. Pasients usually have Splenomegaly and/or Heptomegaly and lack osteolytic lesions.

Non-neoplastic disorders causing Monoclonal Gammopathy:

(rare)

Usually IgG Monoclonal gammopathies:

occult heartworm disease, FIFV (rarely), Ehrlichia canis, lymphoplasmacytic enteritis, lymphoplasmacytic dermatitis and amyloidosis.
this causes should be ruled out before a diagnosis of multiple myeloma is made in a patien with an IgG monoclonal gammopathy.

Causes of Hypoglobinaemia:

Decreased intake of Globulins:
- in neonates before drinking colostrum, absorption disorders of neonates
Decreased synthesis of Globulins:
- Acquired or inherited immunodeficiency
- liver function impairment
Increased loss:
- PLE, PLN,
- via skin (burns, inflammations),
- bleeding

Fibrinogen Concentration Method 1:

If both plasma and serum TP concentration is measured, the difference of plasma and serum TP-concentration gives Fibrinogen concentration

Fibrinogen Concentration, Method 2:

Based on the heat labile character of Fibrinogen.
One part of plasma is used for TP measurement (e.g by Biuret test), other part is heated to 56-58 * C and the plasma is centrifuged, then TP concentration is measured.
In this method TP analysis can be easily determined by using refractometry. Capillary tubes can be used.

Fibrinogen concentration, Method 3:

the test used for Thrombin Time (TT), can be used for establishing fibrinogen concentration, as in this test the values are primarily dependent on the fibrinogen concentration.
The reagent containes bovine thrombin and Ca2+.
the clot formation can be detected by using standards of different fibrinogen concentrations.

Causes of fibrinogen concentration changes:

Increase:

Acute inflammation (especially ruminants), dehydration

Decrease:

liver function impairment, advanced protein deficiency, DIC, sequestration after bleeding to body cavity, chronic bleeding, blood loss, inherited afibrionogenaemia (St. Bernard dog)

Glucose measurment by using a glucometer:

It measures the electric conductance in the fresh droplet of blood.
the electric concuctance changes not only with changing glucose concentration, but is also influenced by the ratio of cellular elements (mostly RBCs).
the reason for this is that cells also conduct electricity but have a bigger resistance compared to plasma.
the handheld glucometers are calibrated for the physiological cell counts, thus if the patient has anaemia the glucose concentration is lower.
if the patient has polycythaemia the measured glucose is higher.
In this condition the glucose concentration can be measured using the enzymatic method.

Glucose measurment by using the Enzymatic method:

Plasma glucose is quickly catabolised by the enzymes of the RBCs. Therefore we have to determine blood glucose concentration quickly after sampling, or we have to ensure to avoid in vitro catabolism of the glucose samples:

Store the sample cooled until measured blood glucose
separate plasma from blood quicly (plasma can be stored for longer by keeping it at 2-4*C.)
coaglulate RBC by 3% Trichloric acetic acid (100ul blood into 900ul 3% trichloric-acetic-acid solution). The dilution must be considered in the calculation.
take blood samples in tube containing NaF (NaF inhibits enolase in RBCs by the reaction between Mg and F).

Glucose measurment in GOD/POD reaction:

Chemical reaction:

Glu + H2O —-glucose oxydase ——– = gluconic acid + H2O2

H2O2 ——– peroxydase ——– = O + H2O

O oxidises a stain (m cresol and aminophenasone) which has a reduced form of no colour.
The oxidation changes the colour of the stain. The depth of colour depends on the glucose concentration.
This test is used for spectrofotometrical and for refractometrical (urine teststrips) analysis.

Causes of changes in Increased glucose concentration:

Transient increase:
- laboratory erris (haemolysis, lipaemia, icterus)
- stress (cats, may even have > 15mmol/l
- food intake (dogs and humans)
- Xylazin effect
- Cranial trauma or inflammation (Rabies, Aujeszky disease)
- after/during administration of glucose containing fluid therapy
Constant hyperclycaemia:
- Diabetes Mellitus
- Hyperadrenocorticism and Glucocorticosteroid therapy
- Progesterone effect (atrogen or endogenous -> insulin resistance)
- enterotoxaemia (sheep)

Causes of changes in Decreased glucose concentration:

laboratory error (incorrect storage/transport of the sample)
decreased energy status (ketosis of ruminants, growing pigs; baby pig diseaes, puppies small breed. Starvation, strenuous exercise; hunting dogs, racehorses etc.
insulin overdose (sometimes beta-receptor blockers in heart insuffieciency)
insulinoma
anabolic steroid effect
liver failure, terminal stage
acute liver failure (fast depletion of liver flycogen after a very short hyperglycaemic phase)
hypoadrenocorticsm
septicaemia
hyperthyroidism
paraneoplastic syndrome

Intravenous glucose tolerance test:

is performed when we suspect the onset of latent diabetes mellitus or insulinoma.
If we take two starving blood samples (with exclusion of stressors) and the glucose concentration is above 11 mmol/l, we suspect Diabetes Mellitus.
the test should not be performed if blood glucose concentration is very high, > 20 mmol/l.

The test can be performed after the animal has starved for 24 hours:

blood sampling (0 min), then 1g/kg for dog, and 5mg/kg for cat, 40% glucose solution is infused IV for 30-45 seconds.
Blood sampling at 5, 15, 30, 45 and 60 min. after the infusion.
Blood glucose should be normalised at 30-60 min sampling point.

Oral glucose tolerance test:

is performed when we suspect chronic bowel disease, exocrine pancreatic insufficiency, OR it can be insted of IV glucose tolerance test.

Test can be performed after starvation for 24 hours:

Blood sampling (0.min), then 26/kg 12,5% glucose solution is given orally.
blood sampling at 15, 30, 60, 90, 120 min after administration.
blood glucose should be increased twice as normal value at 30 mon, and should be normalised at 120 min.

Evaluation of constant Hyperglycaemia:

The glucose molecules in the blood can attach to various proteins, like alpha-amylase, haemoglobin.
Macroamylase = when glucose is bound to alpha-amylase the size of the molecule is bigger.

–> due to its big size it cannot be excreted by the kidneys, so it circulates in the blood for a long time.

Ketoamines = Glucose molecules bound to more than one protein.

–> Fructosamine. Its concentration is not influenced by short-term hypergycaemia, eg stress in cats or food intake in dog, but increases if Hyperglycaemia is long-term. Its concentration represents the glucose average concentration in the 2-3 week period before sampling.

over prolonged periods of time: Glycated haemoglobin (haemoglobin A1c, HbA1c, A1C or Hb1c) is a form of haemoglobin that is measured primarily to identify the average plasma glucose concentration.

it is formed in a non-enzymatic glycation pathway by haemoglobins exposure to plasma glucose.
normal levels of glucose produce a normal amount of glycated haemoglobin.
as the average amount of plasma glucose increases, the fraction of glycated haemoglobin increases in a predictable way.
this serves as a marker for average blood glucose levels over the previous 2-3 month prior to measurment.

Appearance of Keton bodies:

is due to energy deficiency in liver cells
it can be caused by decreased intake of carbohydrates or deceased insulin production (diabetes ketoacidosis)
concentration of acetone, and acetoacetic acid can be estaimated by the Ross reaction (B-hydrocy butirate is not determined by this method)

Ross reagent (found on test strips too):

1g of nitroprussid - Na
100g of (NH4)2SO4
50g of Na2CO3

Ross reagent contaminated by ketone bodies changes its colour from white (grey) to purple.

the depth of the colour depends on the keton concentration.

Several samples can be used for determination of ketone bodies:

plasma, urine, milk.
If milk containes 1mmol, plasma containes 3-5mmol, urine containe 10mmol.
it means that keton bodies are found concentrated in urine samples.
Drip some drops of sample onto the Ross-reagent, wait one minute and see the results.

Urea concentration of milk and plasma:

Energy status of dairy cows can be estimated by the urea concentration analysis from milk and plasma.
if rumen has energy deficiency due to decreased carbohydrate intake, NH3-level increases in the rumen, this results in increased urea production by the liver, so urea concentration increases in milk from 2-5mmol/l to 8-10 mmol/l, and in plasna from 8-10mmol/l to 15-17 mmol/l.

Resence of lipaemia:

to differentiate chylomicrons from other lipids in blood plasma, freeze the plasma on -18*C and wait 12-24 hours. Then warm it up slowly and centrifuge.
the protein part of the chylomicrons coagulates.
if the layer under the fat (ontop of the plasma) is clear after the centrifugation, it means that the lipaemia has been caused by the food intake. In this case it can be used for the measurment
if the plasma is not clear after the centrifugation, it means that there is an increased lipid metabolism from the far storages.

Causes of hyperlipidaemia:

hyperlipidaemia of ponies
increased fat content in diet
diabetes mellitus (decreased FFA influx to cells)
hypothyroidism
hyperadrenocorticism or glycosteroid therapy
nephrotic syndrome
septicaemia (energy deficiency)
pancreatitis (lipase activation)
iodopathic - familiar hyperlipidaemia in miniature schnauzers, beagles.

Causes of decreased lipid content:

starvation (long term)
liver failure (e.g PSS)
malabsorption, maldigestion (eg. EPI)

Lipid absorption test:

used to determine whether there is existing malabsorption, maldigestion or when there is chronic bowel disease.

when there is fast lipid intake in normal conditions, plasma triglycerol (TG) concentration rises to twice the normal value (normal for digs: 1mmol/l)

Test can be performed after starvation for 24 hours:

blood sampling (0.min), then 3ml/kg corn oil given orally.
blood sampling at 1st, 2nd, 3rd, 4th, 5th hours after administration.
blood should be lipaemic, and TG concentration must show minimal 2 fold rise from the normal value.

If there is no such change, we have to repeat the test by giving predigested corn oil.

We mix corn oil with: pancreatic enzyme extract (2-3 teaspoons) to 200ml corn oil and incubate at 37*C.
give the same dose and check lipaemia every hour.
if the result is increased TG conc. and lipaemia = we suspect that the original problem is exocrine pancreatic insufficiency (EPI).
if the result is no change in TG conc. and no lipaemia = we suspect intestinal absorption defect.

Total cholesterol and cholesterol-ester:

cholesterol measurments is used for detection of increased fat mobilisation: in this case total cholesterol value increases (hypothyroidism, hyperadrenocorticism, nephrotic syndrome, DM etc.) and cholesterol catabolism decreases.
the cholesterol-ester is 40% of the total cholesterol value.
decreased esterification of cholesterol as a result of impaired liver-function (and increased apolipoprotein production), causes decreased total cholesterol concentration.
average value for cholecterol conc. = 2-6mmol/l.

Causes of Hypocholesterolaemia:

malnutrition
liver failure (decreased synthesis)
neoplastic disease
hyperthyreosis (increased usage)
decreased apolipoprotein synthesis

Causes of hypercholesterolaemia:

increased dietary fat content
hypothyroidism
hyperadrenocorticism
diabetes mellitus
nephrotic syndrome (concurrent low TP)
cholestatic disease (increased leakage from liver du to bile duct ibstruction)
idiopathic - primary dyslipidosis

Free fatty acids:

FFA or NEFA conc. measurment could be usefull to detect increased or decreased lipid mobilisations.

non specific tissue lipase is the major enzyme breaking down lipids from Triglycerols in tissue due to energy deficiency.
in ruminatns a severe energy deficiency can cause increased blood FFA concentration as a result fo the energy need.
from fat storages FFA is mobilised in order to cover energy (carbohydrate) deficiency.
FFA can compensate energy deficiency until liver is able to produce enough Ocal-Acetic-Acid for the Beta-oxidation.
as a result of starvation and as a consequence of glycogen deficiency of the liver, or lipid mobilisation syndrome and hepatic lipidosis; Total lipid (TL) concentration will decrease beacuse the liver can not produce enough Apolipoporteins for transporting lipids, However FFA concentration is increased, because it is transported by albumin.

Average normal values of lipids:

FFA: 0,1-0,3 mmol/l
TL: 5-7mmol/l
TG: 0,6-1,2 mmol/l (sheep: 1,5-4mmol/l)
cholesterol: 2-6 mmol/l

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