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Lab D > Lab 4 - Examinations on metabolic disorders > Flashcards

Lab 4 - Examinations on metabolic disorders Flashcards

1
Q

What are the different metabolic parameters

A

PROTEIN METABOLISM
CARBOHYDRATE METABOLISM
LIPID METABOLISM

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2
Q

CHANGES OF METABOLIC PARAMETERS
PROTEIN METABOLISM
What is TP

A

Total protein concentration in blood (TP)
- of the plasma (serum)

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3
Q

CHANGES OF METABOLIC PARAMETERS
PROTEIN METABOLISM
What is TP dependent on

A

The intake, synthesis, transformation, catabolism, and hydration status (dehydration, hyperhydration).

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4
Q

CHANGES OF METABOLIC PARAMETERS
PROTEIN METABOLISM
How can TP measurements be performed?

A

Measurements can be performed by chromatography, electrophoresis, and refractometry

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5
Q

What is the Biuret method used for

A

There are various TP measurements, i.e. Biuret method involves CuSO4 containing
reagent or we can use refractometry.

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6
Q

What is the best detection range of the Biuret method which is generally used, or
the refractometry

A

20-100 g/l

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7
Q

What is included in the range of the Biuret method?

A

What is The average TP concentration of plasma

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8
Q

What is The average TP concentration of plasma

A

(60-80 g/l)

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9
Q

Smaller TP concentrations of the urine, cerebrospinal fluid, body cavity fluids or tissue homogenates can be determined correctly by using these
methods. (Biuret + TP average conc of plasma)
True or false

A

false

Smaller TP concentrations of the urine, cerebrospinal fluid, body cavity fluids or tissue homogenates cannot be determined correctly by using these
methods. (Biuret + TP average conc of plasma)

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10
Q

How to determine Smaller TP concentrations

A

They can be determined by the 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.

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11
Q

Lowry method

A

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.

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12
Q

In which method is the Folin-phenol reagent is used,

A

Lowry method
Folin-phenol reagent is used,

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13
Q

Biuret test
TP is commonly measured how?

A

Photometrically.
The reagent contains: KNaSCN, CuSO4, KI
and NaOH.

Schematic chemical reaction: CO-NH+ Cu2++ alkaline = purple coloured complex
Wave length: 546 nm

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14
Q

Characterize the methods of Total protein concentration in blood (TP)

A

The total protein content of the plasma (serum) is dependent on the intake, synthesis,
transformation, catabolism, and hydration status (dehydration, hyperhydration).

Measurements can be performed by chromatography, electrophoresis and refractometry.

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15
Q

What are the different test of TP

A

Biuret method involves CuSO4 containing reagent
Ultrasensitive total protein analysis

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16
Q

What is characteristic about Refractometry

A

Light is refracted when reaching the border of media with different specific gravity.

The light refraction characteristics of a solution are dependent on its specific gravity. The total protein content in blood plasma or serum is the factor having the biggest influence on its specific gravity. The
changes in light refraction depend on the quality of plasma/serum. Specific gravity is
also dependent on temperature.

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 at room temperature.
  • The method is quick and easy, but less precise than
    spectrophotometry.
  • Can be used in the range of 25-95 g/l, may give biased results in hemolysis or lipaemia.
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17
Q

important:
TP concentration of blood plasma depends not only on the protein metabolism but also on the water balance.

During dehydration, TP decreases
True or false

A

False
During dehydration TP increases

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18
Q

During hyperhydration, TP decreases.
True or false

A

True
During hyperhydration, TP decreases.

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19
Q

For the Biuret test, what is the calculation to determine the TP?

A

Use calibration curve or the formula if you have standard (60 g/l)

E sample / E standard x 60 = total protein g/l

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20
Q

What can not be detected with buiret or refractometry

A

Smaller TP concentrations of the urine, cerebrospinal fluid, body cavity fluids, or tissue homogenates cannot be determined correctly by using these
methods

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21
Q

What is the characteristics of Ultrasensitive total protein analysis?

A

Ultrasensitive total protein analysis
Na-molybdate and pyrogallol-red reagent form a complex molecule by binding proteins.
- The complex can be measured on 600 nm wave length.
- Sensitivity is 0.2 g/l - 4g/l.
- Standards are: 0.25, 0.5, 1. 2 g/l

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22
Q

Charachteristics of Biuret test

A

Measured photometrically.

Reagent: KNaSCN, CuSO4, KI and NaOH.

Schematic chemical reaction:
CO-NH+ Cu2++ alkaline = purple coloured complex

Wave length: 546 nm

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23
Q

How long should you wait for the buirett test?

A

Wait for 30 minutes!

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24
Q

What are the Protein fractions

A

Major fractions are

  • albumin,
  • globulin and
  • fibrinogen. The latter 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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25
Q

Globulin concentration is generally calculated by the difference of the TP and albumin concentration.
True or false

A

True
Generally

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26
Q

Plasma TP g/l different species

A

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

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27
Q

Plasma TP in diff species
Serum albumin

A

Serum albumin
Dog 25-34 - 48-64%
Cat 25-45 - 43-63%
Horse 27-40 - 40-60%
Cattle: 23-40 - 35-55%
Swine: 27-39 - 40-62%
Sheep: 24-30 -50-60%

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28
Q

Plasma TP in diff species
Serum globulin

A

Serum globulin
Dog 30-48 11-21%
Cat 30-48 8-24%
Horse 40-62 15-20%
Cattle 30-55 12-17%
Swine 30-65 12-25%
Sheep 30-58 11-16%

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29
Q

Plasma TP in diff species
Albumin/Globulin

A

Albumin/Globulin
Dog 1,083
Cat 1,083
Horse 0,61
Cattle 0,81
Swine 0,54
Sheep 0,724

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30
Q

Plasma TP in diff species
Fibrinogen g/l

A

Fibrinogen g/l

Dog 1-4
Cat 1-4
Horse 2-4
Cattle 2-5
Swine 2-4
Sheep 2-4

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31
Q

Characteristics of Albumin method 1

A

Method 1
Albumin concentration can be measured by spectrophotometry.
Reagent: Bromocresol green.
- This reagent binds to albumin on pH: 4.2 and forms a blue-green complex which is measurable on
578 nm wavelength.

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32
Q

Characteristics of Albumin method 2

A

Serum electrophoresis in combination with TP measurement is a reliable method 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, for this reason, it is necessary to know TP concentration.

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33
Q

Changes in albumin concentration:

Decrease

A

Decrease:
 Decreased intake of proteins, decreased absorption (maldigestion, malabsorption)
 Decreased synthesis - liver failure, acute inflammation (it is a negative acute-phase protein)

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34
Q

Changes in albumin concentration:
Increase

A

 Increased utilization – physiologic conditions cause mild changes: pregnancy, work, exercise, production (milk, egg, etc.), and chronic diseases
(chronic inflammation, neoplasm)

 Increased loss –
- via the kidneys (protein-losing nephropathy - PLN),
- gastrointestinal tract (protein-losing enteropathy- PLE),
- skin (burn),
- whole blood loss,
- sequestration into body cavities – NOT the decrease of colloid pressure (cardiac disease, lymphangiectasia, portal hypertension, other vascular disorders,
peritonitis e.g. perforation in intestines, gall bladder, translocation of bacteria)

 Other (relative decrease):
hyperhydration (may be iatrogenic)

Increase:
 dehydration

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35
Q

Characteristics of Globulins and causes
Method 1

A

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

Alb/Glob 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 (see practical Evaluation of inflammatory processes).

The other cause for the decrease of alb/glob ratio is the decrease of albumin
concentration

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36
Q

Characteristics of Globulins and causes
Method 2
*LONG

A

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.

Electrophoresis
Electrophoresis is 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.

Serum protein electrophoresis (SPEP) is a laboratory test that examines specific proteins in the blood called globulins. Electrophoresis is a laboratory technique where the blood serum (the fluid portion of the blood after the blood has clot) 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
    1. albumin,
    2. alpha-1-globulins,
    3. alpha-2-globulins,
    4. beta globulins, and
    5. gamma globulin.

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.

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37
Q

In Serum protein electrophoresis (SPEP) components are devided into five classifications by:
- size and electrical charge, those being serum
What are they

A
  1. albumin,
  2. alpha-1-globulins,
  3. alpha-2-globulins,
  4. beta globulins, and
  5. gamma globulin.
38
Q

How are the different components distinguishable in Serum protein electrophoresis (SPEP)

A

Small and highly charged molecules will move a greater
distance than will large and lower charged molecules in the same period of time.

The efficiency of electrophoretic separation of ions is thus determined by the relative charge
densities (charge per unit volume) of the ions in the mixture.

In-gel electrophoresis the
medium is typically polyacrylamide or agarose, a vicious media that is required to minimize diffusion of the constituent ions.

39
Q

What are the two most commonly used forms of protein electrophoresis?

A
  1. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
  2. Isoelectric focusing (IEF)
40
Q

What are the characteristics of SDS-PAGE

A
  • In SDS-PAGE, 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.
  • 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.
  • 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.
  • After staining separated protein fractions the densitometer can detect the results. The square below the curve depends on the concentration of the protein fraction.

The serum contains 60% of albumin, 40 % of globulin
Plasma contains 50% of albumin, 30% of globulin, 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 imunoglobulins (IgA, IgM), and some other proteins i.e. LDL,
  • gamma-globulins are immunglobulins, too (IgG).
41
Q

What kind of proteins are alpha globulins

A
  • alpha globulins are acute-phase proteins,
42
Q

What kind of proteins are beta globulins

A
  • beta-globulins are immunoglobulins (IgA, IgM), and some other proteins i.e. LDL,
43
Q

What kind of proteins are gamma globulins

A
  • gamma-globulins are immunglobulins, too (IgG).
44
Q
  • 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.
    Important:
A

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.

45
Q

The causes of the Increase in globulin concentration
1. Polyclonal gammopathy

A
  1. Polyclonal gammopathy
    This is seen as a broad-based peak in the beta and/or gamma region. 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

46
Q

The causes of the Increase in globulin concentration
Monoclonal gammopathy

A

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.

Both neoplastic and non-neoplastic disorders can produce a monoclonal gammopathy

47
Q

Serum protein electrophoresis with monoclonal gammopathy
1) Neoplasia:

A

1) Neoplasia: Multiple myeloma is the most common cause (producing an IgG or IgA monoclonal).

Other neoplastic disorders associated with 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).

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:

  1. splenomegaly and/or
  2. hepatomegaly and
  3. lack osteolytic lesions.

In contrast, 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.

48
Q

Serum protein electrophoresis with monoclonal gammopathy
2) Non-neoplastic disorders (rare):

A

2) Non-neoplastic disorders (rare): Monoclonal gammopathies (usually IgG) have been reported with:
1. occult heartworm disease,
2. FIPV (rarely),
3. Ehrlichia canis,
4. lymphoplasmacytic enteritis,
5. lymphoplasmacytic dermatitis and
6. amyloidosis.

These causes should be ruled out before a diagnosis of multiple myeloma is made in a patient with an IgG monoclonal gammopathy.

49
Q

Serum electophoresis examinations, according to some examples

a?

A

(a) physiological protein separation,

50
Q

Serum electophoresis examinations, according to some examples

b

A

(b) hypoalbuminaemia,

51
Q

c

A

(c) polyclonal gammopathy (FIP),

52
Q

Serum electophoresis examinations, according to some examples

d

A

(d) monoclonal gammopathy (plasmacell myeloma)

53
Q

Causes of Hypoglobulinaemia 

A

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 the skin (burns, inflammation), bleeding

54
Q

Fibrinogen concentration Method 1

A

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.

55
Q

Fibrinogen concentration

Method 2

A

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),

another 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.

56
Q

Fibrinogen concentration

Method 3

A

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.

57
Q

Causes of fibrinogen concentration changes

A

Increase

  • acute inflammation (especially ruminants),
  • dehydration

Decrease

  • liver function impairment,
  • advanced protein deficiency,
  • DIC,
  • sequestration after bleeding to the body cavity,
  • chronic bleeding,
  • blood loss
  • inherited afibrinogenemia (St. Bernard dog)
58
Q

CARBOHYDRATE METABOLISM

What are the parameters

A
  1. Glucose (The evaluation of constant hyperglycaemia)
  2. Ketons
  3. Urea concentration of milk and plasma
59
Q

CARBOHYDRATE METABOLISM

Why Glucose?

A

Glucose of the basis of the energy household, which can be detected in plasma. Hyper- or hypoglycaemias are detectable from the blood sample.

60
Q

What are the characteristics of methods for measuring Glucose

A

Currently, the 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.

The electric conductance changes not only with changing glucose concentration but is greatly 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

  • anemia the glucose concentration measured is lower (compared to the gold standard GOD/POD spectrophotometry), if the patient has
  • Polycythemia the measured glucose is higher. In such a condition the glucose concentration can be reliably measured using the below enzymatic method.
61
Q

Why using the Plasma glucose measurement, and what to remmmember

A

Plasma glucose is quickly catalysed 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.

62
Q

How to avoid in vitro glucose catabolism:

A

 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)

63
Q

Biochemical reaction for glucose measurement is the

A

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 color.

The oxidation changes the color of the stain. The depth of color depends on the glucose concentration.

This test is used for spectrofotometrical and for refractometrical (urine teststrips) analysis.

64
Q

When is the GOD/POD reaction test used

A

This test is used for spectrofotometrical and for refractometrical (urine teststrips) analysis.

65
Q

Causes of changes in glucose concentration:

Increased glucose concentration:

A

 Transient increase:

o laboratory errors (haemolysis, lipaemia, icterus)

o stress (cats! may even have >15 mmol/l)

o food intake (dogs and humans!)

o xylazine effect

o cranial trauma or inflammation (Rabies, Aujeszky disease)

o after/during the administration of glucose-containing fluid therapy

 Constant hyperglycaemia

o diabetes mellitus - DM (see internal med for types of DM)

o hyperadrenocorticism and glucocorticosteroid therapy! o Progesterone effect (iatrogenic or endogenous – insulin resistance!)

o enterotoxaemia (sheep)

66
Q

Causes of changes in glucose concentration:

Decreased glucose concentration:

A

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

 septicemia

hyperthyroidism

paraneoplastic syndrome

67
Q

Intravenous glucose tolerance test

when do we perform it

A

An 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 samples (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 e.g. >20 mmol/l.

68
Q

When can you perform an intravenous glucose tolerance test, and how?

A

The 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 normalized at 30-60 min sampling point.

69
Q

How offen do you sample the blood after infusion of glucose for the IV glucose tolerance test?

When will the glucose be normalized

A

Blood sampling at 5, 15, 30, 45, 60 min. after the infusion. Blood glucose should be normalized at 30-60 min sampling point.

70
Q

When do you perform the Oral glucose tolerance test (glucose absorption test)

A

Oral glucose tolerance test (glucose absorption test) Glucose absorption test is performed when we suspect chronic bowel disease, exocrine pancreatic insufficiency, or it can be used instead of iv glucose tolerance test.

71
Q

How do you perform the oral glucose test?

When do you take blood sample?

When will the values normalize?

A

The 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 normalized at 120 min after po administration.

72
Q

Characteristics for The evaluation of constant hyperglycaemia

A

The glucose molecules in the blood attach to some degree to various proteins e.g. alpha-amylase and hemoglobin.

When glucose is bound to a-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 hemoglobin (hemoglobin A1c, HbA1c, A1C, or Hb1c) is a form of hemoglobin 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 hemoglobin’s exposure to plasma glucose. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin 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.

73
Q

Characteristics when glucose binds to alpha-amylase

A

When glucose is bound to a-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 hyperglycemia 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.

74
Q

Characteristics when glucose binds to glycated hemoglobin

A

Similarly, glycated hemoglobin (hemoglobin A1c, HbA1c, A1C, or Hb1c) is a form of hemoglobin 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 hemoglobin’s exposure to plasma glucose. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin 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.

75
Q

What could be the cause of a shortterm hyperglycemia

A

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 hyperglycemia

e.g. stress in cats or food intake in dogs, but increases if hyperglycaemia is long-term.

76
Q

Characteristics of KETONS

appearance due to?

From which consentrations are measured?

It is estimated by ?

A

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).

Concentration of acetone, and acetoacetic acid can be estimated by the Ross reaction,

For the detection of keton bodies use Ross-reagent. This reagent is found on test strips (i.e. Ketostix, Bili-labstix), too.

77
Q

Characteristics of Ross reagent test for ketone bodies

Contain

Contaminated by

Colour

From where can we make the sample for the Ross reagent

A

Ross-reagent contains:

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

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.

Several samples can be used for the determination of ketone 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.

78
Q

Urea concentration of milk and plasma can estimate what in which species?

From what kind of sample?

Which levels will increase where and why
?

Resulting in?

A

Energy status of cattle (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 2-5 mmol/l to 8-10 mmol/l, and in plasma from 8-10 mmol/l to 15-17 mmol/l.

79
Q

LIPID METABOLISM

Characteristics of the Presence of lipaemia

A

To differentiate chylomicrons (postprandial) 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.

If the layer under the fat (which is located on the top of the plasma) is clear (transparent) after the centrifugation = 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 mobilization from the fat stores.

80
Q

Causes of hyperlipidemia

A

 hyperlipidemia 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

81
Q

Causes of decreased lipid content:

A

starvation (long term)

liver failure (e.g. PSS)

malabsorptio, maldigestio (e.g EPI)

82
Q

Characteristics of Lipid absorption test

To determine what

TG level rise

When can we perform the test

What is given how

When do you collect the blood sample

How mutch rise of TG

If TG is incr + Lipaemia =

No change in TG and Lipaemia not seen =

A

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.

When there is fast lipid intake in normal conditions, plasma triglycerol (TG) concentration rises to twice as normal value (normal for dogs: 1 mmol/l). The 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 hour.

If the result is increased TG concentration and lipaemia, = exocrine pancreatic insufficiency (EPI).

If the result is no change in TG concentration and lipaemia is not seen, = intestinal absorption defect.

83
Q

The characteristics of Total cholesterol and cholesterol-ester

Detection of what

What is important about the Cholesterol-ester

Decreased esterification of cholesterol as a result of

Average normal value for cholesterol concentration:

A

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.

Important: The cholesterol-ester is 40% of the total cholesterol value.

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

84
Q

Causes of hypocholesterolaemia:

A

 malnutrition

 liver failure (decreased synthesis)

 neoplastic disease

 hyperthyreosis (increased usage)

 decreased apolipoprotein synthesis

85
Q

Causes of hypercholesterolaemia:

A

 increased dietary fat content

hypothyroidism

 hyperadrenocorticism

diabetes mellitus

nephrotic syndrome (concurrent low TP)

cholestatic diseases (increased leakage from the liver due to bile duct obstruction)

idiopathic - primary dyslipidosis

86
Q

Characteristics of Free fatty acids

To detect?

Major enzyme breaking down lipid?

In ruminants?

Fat stores, function

How long can FFA compensate for energy deficiency?

TL increase or decrease

FFA increase or decrease

FFA is transported by?

A

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.

In ruminants, a severe energy deficiency can cause increased blood FFA concentration as a result of the energy need.

From fat stores, FFA is mobilized in order to cover energy (carbohydrate) deficiency.

FFA can compensate for energy deficiency until the liver is able to produce enough OAA (oxal-acetic-acid) for beta-oxidation.

As a result of starvation (energy deficiency) and as a consequence of glycogen deficiency of the liver, or lipid mobilization syndrome and hepatic lipidosis, total lipid

(TL) concentration decrease because the liver can not produce enough apolipoproteins for transporting lipids, however, FFA concentration is increased, because it is transported by albumin.

87
Q

Average normal values:

free fatty acid, FFA:

total lipid, TL:

triacyglycerol, TG:

cholesterol, Chol:

A

FFA = 0,1-0,3 mmol/l

TL= 5-7 mmol/l

TG= 0,6-1,2 mmol/l (sheep: 1,5-4 mmol/l)

Cholesterol= 2-6 mmol/l

88
Q

lipid fractions: apolipoproteins

Triacyl-glycerol

A

Triacyl-glycerols - VLDL

89
Q

lipid fractions : apolipoproteins

Phospholipids

A

phospholipids - HDL

90
Q

lipid fractions : apolipoproteins

Cholesterol

A

cholesterol - LDL/HDL

91
Q

lipid fractions : apolipoproteins

Cholesterol-ester

A

Cholesterol-ester - (HDL)

92
Q

lipid fractions : apolipoproteins

free fatty acid

A

free fatty acid - albumin

Lab D (11 decks)

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