Lab 4 - Examinations on metabolic disorders Flashcards

Question

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

Answer 1

True Generally

Answer 2

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

Answer 3

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%

Answer 4

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%

Answer 5

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

Answer 6

Fibrinogen g/l Dog 1-4 Cat 1-4 Horse 2-4 Cattle 2-5 Swine 2-4 Sheep 2-4

Answer 7

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.

Answer 8

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.

Answer 9

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

Answer 10

 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

Answer 11

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

Answer 12

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.

Answer 13

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

Answer 14

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.

Answer 15

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

Answer 16

- 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).

Answer 17

- alpha globulins are acute-phase proteins,

Answer 18

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

Answer 19

- gamma-globulins are immunglobulins, too (IgG).

Answer 20

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.

Answer 21

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

Answer 22

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

Answer 23

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.

Answer 24

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.

Answer 25

(a) physiological protein separation,

Answer 26

(b) hypoalbuminaemia,

Answer 27

(c) polyclonal gammopathy (FIP),

Answer 28

(d) monoclonal gammopathy (plasmacell myeloma)

Answer 29

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

Answer 30

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.

Answer 31

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.

Answer 32

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.

Answer 33

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)

Answer 34

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

Answer 35

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

Answer 36

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.

Answer 37

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.

Answer 38

 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)

Answer 39

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.

Answer 40

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

Answer 41

** 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)**

Answer 42

**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**

Answer 43

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**.

Answer 44

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.

Answer 45

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

Answer 46

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.**

Answer 47

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.

Answer 48

The glucose molecules in the blood attach to some degree to various proteins e.g. **alpha-amylas**e 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 e**xcreted 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 i**ncreases 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.**

Answer 49

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.

Answer 50

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.**

Answer 51

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 dog**s, but increases if hyperglycaemia is long-term.

Answer 52

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.

Answer 53

Ross-reagent contains: ## Footnote 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.

Answer 54

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.

Answer 55

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.**

Answer 56

 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**

Answer 57

 **starvation** (long term)  **liver failure** (e.g. PSS)  **malabsorptio**, **maldigestio** (e.g EPI)

Answer 58

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, = i**ntestinal absorption defect.**

Answer 59

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 c**holesterol 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**

Answer 60

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

Answer 61

 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

Answer 62

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.**

Answer 63

**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

Answer 64

Triacyl-glycerols - VLDL

Answer 65

phospholipids - HDL

Answer 66

cholesterol - LDL/HDL

Answer 67

Cholesterol-ester - (HDL)

Answer 68

free fatty acid - albumin

Lab 4 - Examinations on metabolic disorders Flashcards

(92 cards)