6. Changes of metabolic parameteres Flashcards
1
Q
The total protein content of the plasma (serum) is dependent on:
A
- the intake
- synthesis
- transformation
- catabolism
- hydration status (dehydration, hyperhydration).
2
Q
Which measurments are used to find the TP?
A
Measurements can be performed by:
-chromatography
- electrophoresis
- refractometry
3
Q
What is TP?
A
The total protein concentration in the blood.
4
Q
What are the detection range of the refractometry?
A
20-100 g/l
5
Q
What is the average TP conc of plasma?
A
60-80 g/l
6
Q
where are the smaller TP concentrations?
A
Smaller TP concentrations:
- urine
- cerebrospinal fluid
- body cavity fluids
- tissue homogenates.
7
Q
How do we measure the smaller concentrations?
A
- Lowry method in which the Folin-phenol reagent is used
- or by the so called Ultrasensitive TP method in which proteins are bound directly to stain molecules.
8
Q
Biuret test reagent
A
Reagent contains: KNaSCN, CuSO4, KI and NaOH.
9
Q
Ultrasensitive total protein analysis:
What happens?
How is it measured?
What is the sensitivty and standards?
A
Na-molibdate, and pirogallol-red reagent forms a complex molecule by binding proteins.
- Measured on 600 nm wave length.
- Sensitivity is 0.2 g/l - 4g/l.
- Standards are: 0.25, 0.5, 1. 2 g/l.
10
Q
Teory behind Refractometry:
A
- Light is refracted when reaching the border of media with different specific gravity, which light refraction is dependent on.
- The total protein content in blood plasma or serum influences its specific gravity.
- In the refractometer the specific gravity of one media is given (glass), so the changes in light refraction depend on the quality of plasma/serum.
- Specific gravity is also dependent on temperature.
11
Q
How do we do refractometry?
A
After calibration (distilled 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 temperature.
12
Q
What is good about refractometry?
A
The method is quick and easy, but less precise than spectrophotometry.
Can be used in the range of 25-95 g/l, may give uncorrect result in haemolysis or lipaemia.
13
Q
TP concentration of blood plasma depends not only on the protein metabolism but ….
A
also on the water balance. During dehydration TP increases, or hyperhydration TP decreases.
14
Q
What are some major fractions of proteins?
A
albumin, globulin and fibrinogen.
15
Q
How do we measure albumin? (method 1)
A
Spectrophotometry! Bromocresol green is used as a reagent. It binds to albumin on pH 4.2 and forms a blue-green complex. Measured on 578 nm wave length.
16
Q
How do we measure albumin? (method 2)
A
Serum electrophoresis used in combination with TP is a reliable method. The cost is higher, but it is used when protein fraction analysis is the basic aim. This method provides albumin as %, thats why we need to knoe the TP concentration.
17
Q
Decreased albumin conc:
A
- Decreased intake of proteins
- decreased absorption (maldigestion, malabsorption)
- Decreased synthesis
- liver failure, acute inflammation (it is a negative acute phase protein)
- Increased utilisation
- physiologic conditions cause mild changes: pregnancy, work, exercise, production (milk, egg etc.) and chronic diseases (chronic inflammation, neoplasm)
-Increased loss :
via the kidneys (protein loosing nephropathy - PLN), gastrointestinal tract (protein loosing enteropathy- PLE),
skin (burn),
whole blood loss,
sequestration into body cavities
-Other (relative decrease): hyperhydration (may be iatrogen)
18
Q
Increased albumin conc:
A
dehydration
19
Q
How do we calculate globulins? (method 1)
A
Globulin concentration is calculated roughly by the difference of TP and albumin concentration of serum.
20
Q
How do we calculate globulins? (method 2)
A
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.
21
Q
Explain the Alb/Glob ratio
A
The decrease of albumin/globulin ration 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.
22
Q
Theory of Electrophoresis:
A
Electrophoresis based on the fact that the proteins have amphoteric character (acidic amino acids - Asp, Glu go to the + pole (charge) alkaline amino acids go to the - pole.) Electrophoresis is a laboratory technique where the blood serum is placed on special paper treated with agarose gel and exposed to an electric current to separate the serum protein components into five classifications by size and electrical charge, those being serum albumin, alpha-1-globulins, alpha-2-globulins, beta globulins, and gamma globulins.
23
Q
What is SPEP?
A
Serum protein electrophoresis (SPEP) is a laboratory test that examines specific proteins in the blood called globulins.
24
Q
What is blood serum?
A
blood serum is the fluid portion of the blood after the blood has clot.
25
Explain the separation of ions by electrophoresis:
Electrophoresis is the process whereby ions move through a medium in response to an applied electric field. Separation of ions by electrophoresis exploits the fact that the rate of motion of charged particles in any particular applied electric field is directly proportional to their charge and inversely proportional to their size and the viscosity of the medium.
26
Which molecules will move in a greater distance in electrophoresis?
Small and highly charged molecules will move a greater distance than will large and lower charged molecules in the same period of time.
27
The efficiency of electrophoretic separation of ions is determined by....
.. by the relative charge densities (charge per unit volume) of the ions in the mixture.
28
Which gel is used in gel electrophoresis?
In gel electrophoresis the medium is typically polyacrylamide or agarose, a viscous media that is required to minimize diffusion of the constituent ions.
29
What are the most used electrophoresis methods?
- sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
- isoelectric focusing (IEF).
30
When is the proteins denatured? What happens to them?
Protein samples are first denatured by heating in SDS. Denaturation in SDS results in the coating of the polypeptides with negatively charged SDS molecules, rendering 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 (+) to the other end.
31
What happens if you apply voltage across the gel ?
Application of voltage across the gel forces the negatively charged proteins to migrate towards the anode. In the highly viscous polyacrylamide, larger denatured proteins will experience greater frictional drag than smaller denatured proteins and move through the gel matrix at a much slower rate.
32
When do you stain during electrophoresis?
After a predetermined period of time, the electrodes are removed and the gel slab is stained with dye to show the locations of proteins in the gel. The smaller proteins are found closer to the anode than the larger ones. An inverse log-linear relationship of protein molecular weight to the distance traveled is typically observed.
33
What happens after staining in electrophoresis?
After staining separated protein fractions the densitometer can detect the results. The square below the curve depends on the concentration of the protein fraction. Serum contains 60% of albumin, 40 % of globulin Plasma contains 50% of albumin, 30% of globulin, 20% of fibrinogen
34
Which types of globulins do we have?
a1, a2, b1, b2, gamma1, gamma2. alfa globulins are acute phase proteins, beta globulins are imunoglobulins (IgA, IgM), and some other proteins i.e. LDL, gamma globulins are immunglobulins, too (IgG).
35
Proteins, especially immunglobulins are derived from:
special lymphoid cells (plasma cells). One cell group of the same origin is a clone, produces the same proteins.
36
What are Polyclonal gammopathy and monoclonal gammapathy?
Polyclonal gammopathy (beta and gamma globulins derived from different clones) generally occurs during inflammatory processes or some immune mediated diseases and monoclonal gammopathy occurs during (one protein fraction derived from one clone) immune mediated or neoplastic conditions.
37
Common causes of Polyclonal gammopathy:
Some common causes include various chronic inflammatory diseases (infectious, immune-mediated), liver disease, FIP (alpha-2 globulins are often concurrently elevated - see adjacent ELP tracing), occult heartworm disease, and Ehrlichiosis. Beta-gamma bridging occurs in disorders with increased IgA and IgM such as lymphoma, heartworm disease and chronic active hepatitis. Serum protein electrophoresis with polyclonal gammopathy
38
How does Monclonal gammopathy look on an diagram?
This is seen as a sharp spike in the beta or gamma region. The peak can be compared to the albumin peak - a monoclonal gammopathy has a peak as narrow as that of albumin.
39
What can cause monoclonal gammapathy?
Both neoplastic and non-neoplastic disorders can produce a monoclonal gammopathy.
40
How does polyclonal gammapathy look on a diagram?
This is seen as a broad-based peak in the beta and/or gamma region
41
Explain neoplasia:
Neoplasia: Multiple myeloma is the most common cause (producing an IgG or IgA monoclonal). Other neoplastic disorders associated with a monoclonal gammopathy include lymphoma (IgM or IgG) and chronic lymphocytic leukemia (usually IgG). Extramedullary plasmacytomas are solid tumors composed of plasma cells that are usually found in the skin of dogs. They have also been reported in the gastrointestinal tract and liver of cats and dogs. They can be associated with a monoclonal gammopathy, or even a biclonal gammopathy (if there are multiple tumors).
42
What is macroglobulinemia ?
An increase in IgM is called macroglobulinemia. Waldenstrom's macroglobulinemia is a neoplasm of B-cells (lymphoma) that has a different presentation from multiple myeloma. Patients usually have splenomegaly and/or hepatomegaly and lack osteolytic lesions.
43
What is myeloma?
Multiple myeloma is a disorder of plasma cells that have undergone antigenic stimulation in peripheral lymph nodes and then home in on the bone marrow (the marrow produces appropriate growth factors that support growth of myeloma cells). Therefore, myeloma is characterized as a bone marrow disorder, with osteolytic bone lesions (in 50% of canine cases) and Bence-Jones proteinuria. Extramedulllary infiltrates of plasma cells are uncommon but can occur in terminal phases of the disease.
44
Explain Non-neoplastic disorders (rare):
Monoclonal gammopathies (usually IgG) have been reported with occult heartworm disease, FIPV (rarely), Ehrlichia canis, lymphoplasmacytic enteritis, lymphoplasmacytic dermatitis and amyloidosis. These causes should be ruled out before a diagnosis of multiple myeloma is made in a patient with an IgG monoclonal gammopathy.
45
Reasons for 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
46
how do we find fibrinogen concentration? (method 1)
If both plasma (plasma separated from anticoagulated blood) and serum (serum separated from clotted blood) TP concentration is measured, the difference of plasma and serum TP-concentration gives fibrinogen concentration.
47
how do we find fibrinogen concentration? (method 2)
This test is 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 oC and the plasma is centrifuged then TP concentration is measured. The difference between the two measurements is the fibrinogen concentration. In this method TP analysis can be easily determined by using refractometry. Capillary tubes can be used.
48
how do we find 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 contains bovine thrombin and Ca2+. The clot formation can be determined by using standards of different fibrinogen concentrations.
49
Causes of fibrinogen concentration increases:
Increase acute inflammation (especially ruminants), dehydration
50
Causes of fibrinogen concentration decreases:
Decrease liver function impairment, advanced protein deficiency, DIC, sequestration after bleeding to body cavity, chronic bleeding, blood loss, inherited afibrionogenaemia (St. Bernard dog)
51
How do we measure glucose in plasma?
Most frequent method of glucose measurement is using a handheld glucometer. The older types may use the below described GOD/POD enzymatic method, however the currently in used glucometers measure the electric conductance in the fresh droplet of blood.
52
The electric conductance changes not only with changing glucose concentration, but also:...
it is greatly influenced by the ratio of cellular elements (mostly RBCs). The reason of this is that cells also conduct electricity but have a bigger resistance compared to plasma.
53
what happens to the glucose conc if the patients is anaemic?
measures with the handheld glucometer is low
54
what happens to the glucose conc if the patients is polycytheamic?
higher measures.
55
Why is it important to determine blood glucose conc quickly after sampling?
Plasma glucose is quickly catabolysed by the enzymes of the red blood cells (RBC). Therefore we have to determine blood glucose concentration quickly after sampling, or we have to ensure to avoid in vitro catabolism of the glucose in blood samples.
56
What should we avoid in vitro catabolism of glucose?
Store the sample cooled until measured blood glucose
- Separate plasma from blood quickly (plasma can be stored for longer by keeping it at 2-4 oC)
- Coagulate RBC by i.e. 3% trichloric acetic acid (100 µl blood into 900 µl 3% trichloric-acetic-acid solution (dilution must be considered in calculation)
- Take blood samples in tubes containing NaF (NaF inhibits enolase in RBC-s by the reaction between Mg and F)
57
Increased glucose concentration: (transient increase)
Transient increase:
- laboratory errors (haemolysis, lipaemia, icterus)
- stress (cats! may even have \>15 mmol/l)
- food intake (dogs and humans!)
- xylazin effect
- cranial trauma or inflammation (Rabies, Aujeszky disease)
- after/during administration of glucose containing fluid therapy
58
Increased glucose concentration: (constant hyperglycaemia)
Constant hyperglycaemia
- diabetes mellitus - DM (see internal med for types of DM)
- hyperadrenocorticism and glucocorticosteroid therapy!
- Progesterone effect (iatrogen or endogenous – insulin resistance!)
- enterotoxaemia (sheep)
59
Decreased glucose concentration:
- laboratory error (incorrect storage/transport of sample)
- decreased energy status (ketosis of ruminants, growing pigs /baby pig diseases/, puppies small breed!, starvation, strenuous exercise /hunting dogs, racehorses etc./
- insulin overdose (sometimes β-receptor blockers in heart insufficiency)
- insulinoma
- anabolic steroid effect
- liver failure, terminal stage
- acute liver failure (fast depletion of liver glycogen after a very short hyperglycaemic phase)
- hypoadrenocorticism septicaemia
- hyperthyroidism
- paraneoplastic syndrome
60
Why to perform a Intravenous glucose tolerance test:
Intravenous glucose tolerance test is performed, when we suspect the onset of latent diabetes mellitus or insulinoma. Suspicion of DM appears if in two consecutive starving blood sample (with the exclusion of stressors) glucose concentration was above 11 mmol/l. The test should not be performed if blood glucose concentration is very high
61
How do we perform an intravenous glucose tolerance test?
Test can be performed when the animal starved for 24 hours: Blood sampling (0 min.), then 1g/kg bw for dog, and 5mg/kg bw for cat 40% glucose solution is infused intravenously for 30-45 seconds. Blood sampling at 5, 15, 30, 45, 60 min. after the infusion. Blood glucose should be normalised at 30-60 min sampling point.
62
Why is the oral glucose tolerance test performed?
Glucose absorption test is performed when we suspect chronic bowel disease,
63
How is the oral glucose tolerance test performed?
Test can be performed when the animal starved for 24 hours: Blood sampling (0 min.), then 2g/kg bw 12,50% 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 min. and should be normalised at 120 min after po administration.
64
The evaluation of constant hyperglycaemia:
- The glucose molecules in the blood attach to some degree to various proteins e.g. amylase, haemoglobin.
- When glucose is bound to amylase the size of the complex molecule is bigger which is called macroamylase.
- Due to the bigger size it cannot be excreted via the kidneys, so is circulates in the blood for a longer time.
The glucose molecules can be bound more than one protein, thus ketoamines are formed.
- One of the known ketoamines is fructosamine. Its concentration is not influenced by short term hyperglycaemia e.g. stress in cats or food intake in dogs, but increases if hyperglycaemia is long-term.
- Its concentration represents the glucose average concentration in the 2-3 weeks period before sampling.
- Similarly glycated haemoglobin (haemoglobin A1c, HbA1c, A1C, or Hb1c) is a form of haemoglobin that is measured primarily to **identify the average plasma glucose concentration over prolonged periods of time.**
- It is formed in a non-enzymatic glycation pathway by haemoglobin's 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 months prior to the measurement.
65
The appearance of keton bodies is due to..
energy deficiency in liver cells. It can be caused by decreased intake of carbohydrates or decreased insulin production (diabetes ketoacidosis see, at acid base analysis, too).
66
When do we use the Ross-reagens?
To detect ketone bodies: actone and acetoacetic acid. not betahydroxy butyrate.
67
What does Ross-reagent contain?
1 g of nitroprussid - Na, 100 g of (NH4)2SO4, 50 g Na2CO3
68
How does the ross reagens change?
Ross-reagent contaminated by keton bodies (acetone, acetoacetic-acid) changes its colour from white (grey) to purple. The depth of the colour depends on the keton concentration.
69
Several samples can be used for determination of keton bodies:
plasma, urine, milk If milk contains 1 mmol, plasma contains 3-5 mmol, urine contains 10 mmol. It means that keton bodies are found concentrated in urine samples. Drip some drops of milk, plasma or urine samples onto the Ross-reagent, wait one minute and see the results.
70
Urea concentration of milk and plasma
Energy status of cattle (dairy cows) can be estimated by the urea concentration analysis form 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 2-5 mmol/l to 8-10 mmol/l, and in plasma from 8-10 mmol/l to 15-17 mmol/l.
71
how to differentiate chylomicrons (post prandial) from other lipids in blood plasma?
To differentiate chylomicrons (post prandial) from other lipids in blood plasma freeze the plasma on -18 oC wait 12-24 hours then warm it slowly again and centrifuge. The protein part of the chylomicrons coagulates.
72
What do the result of the chylomicron differentiation mean?
If the layer under the fat (which is located on the top of the plasma) is clear (transparent) after the centrifugation, it means that the lipaemia had been caused by the food intake, in this case the plasma is ready for the measurement. If the plasma is not clear after the centrifugation it means that there is an increased lipid mobilisation from the fat stores.
73
Causes of hyperlipidaemia:
- hyperlipidaemia of ponies
- increased fat content in diet
- diabetes mellitus (decreased free fatty acid /FFA/ influx into the cells)
- hypothyroidism
- hyperadrenocorticism or glucocorticosteroid therapy
- nephrotic syndrome
- sepitcaemia (energy deficiency)
- pancreatitis (lipase activation)
- idiopathic – familiar hyperlipidaemia in miniature schnauzers, beagles
74
Causes of decreased lipid content:
starvation (long term) liver failure (e.g. PSS) malabsorptio, maldigestio (e.g EPI)
75
Why do we perform the lipid absorption test?
In order to determine whether there is existing malabsorption, maldigestion (especially in exocrine pancreatic insufficiency) or when there is chronic bowel disease we perform this test.
76
How do we perform the lipid absorption test?
Test can be performed when the animal starved for 24 hours: Blood sampling (0 min.), then 3ml/kg bw corn oil given peroraly. Blood sampling at 1 st, 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 by pancreatic enzyme extract (2-3 tea spoonful) to 200 ml corn oil and incubate it on 37 oC give the same dose and check lipaemia every hours. If the result is increased TG concentration and lipaemia, we can suspect that the original problem is exocrine pancreatic insufficiency (EPI). If the result is no change in TG concentration and lipaemia is not seen, we can suspect intestinal absorption defect.
77
Why do we use cholesterol measurments ?
In the veterinary practice we use cholesterol measurements for the detection of increased fat mobilisation - in this case the total cholesterol value increases (hypothyroidism, hyperadrenocorticism, nephrotic syndrome, diabetes mellitus etc.) and cholesterol catabolism decreases.
78
What is the decreased esterfication of cholesterol a result of?
Decreased esterification of cholesterol as a result of impaired liver-function (and decreased apolipoprotein production) causes decreased total cholesterol concentration. Average normal value for cholesterol concentration: 2-6 mmol/l
79
Causes of hypocholesterolaemia:
malnutrition liver failure (decreased synthesis) neoplastic disease hyperthyreosis (increased usage) decreased apolipoprotein synthesis
80
Causes of hypercholesterolaemia:
- increased dietary fat content
- hypothyroidism
- hyperadrenocorticism
- diabetes mellitus
- nephrotic syndrome (concurrent low TP)
- cholestatic diseases (increased leakage from liver due to bile duct obstruction)
- idiopathic - primary dyslipidosis
81
Why can FFA or NEFA (non-esterised-fatty-acid) concentration measurement be useful ?
FFA or NEFA (non-esterised-fatty-acid) concentration measurement could be useful to detect increased or decreased lipid mobilisation. Non specific tissue lipase is the major enzyme breaking down lipids from triglycerols in tissues due to energy deficiency.
82
What is NEFA?
non-esterised-fatty-acid
83
In ruminants a severe energy deficiency can cause :
increased blood FFA concentration as a result of the energy need.
84
Where and why is FFA mobilised from?
From fat stores FFA is mobilised in order to cover energy (carbohydrate) deficiency. FFA can compensate energy deficiency until liver is able to produce enough OAA (oxal-acetic-acid) for the beta oxidation
85
What can be a result from starvation ?
As a result of starvation (energy deficiency) and as a consequence glycogen deficiency of the liver, or lipid mobilisation syndrome and hepatic lipidosis, total lipid (TL) concentration decreases because liver can not produce enough apolipoproteins for transporting lipids, however FFA concentration is increased, because it is transported by albumin.
86
Normal values: 1.FFA 2.total lipid TL 3.TG 4.Chol
Average normal values: free fatty acid, FFA: 0,1-0,3 mmol/l total lipid, TL: 5-7 mmol/l triacyglycerol, TG: 0,6-1,2 mmol/l (sheep: 1,5-4 mmol/l) cholesterol, Chol: 2-6 mmol/l
87
Give the lipid fractions and the apolipoproteins
triacil-glycerols - VLDL phospholipids - HDL cholesterol - LDL/HDL cholesterol-ester - (HDL) free fatty acid - albumin
88
Explain the glutaric aldehyde test
- Examines the increase of fibrinogen and globulin conc in plasma. (in adult cattle)
- Fibrinogen is an acute phase protein, increase if there is an acute inflammation.
- Glutaric aldehyde solution causes a rapid coagulation of fibrinogen and labile globulins, so blood mixed with this solution coagulants in seconds if there is an acute inflam. (even blood with anticoagulants)
- We use heparinised blood and mix the sample with the same amount of 1,25% glutaric aldehyde solution.
- Check for coagulation every 30 seconds.
- Test is done to diagnose reticuloperitonitis, severe mastitis or endometeritis of cattle.
