Lab 9. Flashcards
Blood bilirubin measurment:
Diaso method is the “van den Bergh” or “Grof-Jendrassik” reaction. - Reagents are: sulphanilic acid and NaNO2 (sodium-nitrite)
- Sample: serum (Acidic reagent causes the coagulation of fibrinogen in the plasma and the coagulated particles can disturb the spectrophotometric measuring method, therefore serum samples are recommended.)
Spectrophotometric measurement, wave length: 530 nm
Extinction x 100 = Br in umol/l
- Indirect Br = Total Br - Direct Br
- Direct Br: 0.2-3 umol/l
- Normal values: Total Br: 8 umol/l, horse: < 40 µmol/l
- after starvation it can be physiologically increased
Causes of increased BrI level in the serum
Excess production of BrI due to increased RBC destruction:
- acute haemolysis
- absorption of haemoglobin folowing massive internal haemorrhage, or after big haematoma formation (= “resorption icterus”)
- transfusion of stored blood, which contanes many dying or dead RBCs
Decreased uptkae of BrI from the blood by liver cells:
- impared hepatic function
- (acute haemolysis)
Decreased rate of conjugation of BrI by the liver cells:
- impared hepatic function
Causes of increased BrII level in the serum
A few days after severe acute intravascular haemolysis (increased production)
Decreased excretion of BrII by liver cells:
- impared hepatic function
Obstruction of bile canaliculi within the liver, often due to inflammation causing swelling of cells, or connective tissue proliferation (fibrosis):
- impared hepatic function
Obstruction of bile canaliculi due to blockage or compression of the bile ducts:
- rupture of the biliary vessels or duct or gall bladder (this leads to a very severe type of peritonitis, so called bile-peritonitis)
Test for examination of biirubin derivates in urine
Gmelin test:
- C.c. HNO3 has to be carefully layered under urine in a test tube, and the width of the differently coloured layers at the meeting of the two fluid phases has to be evaluated. Differently coloured layers from the highest layer:
condensed material on the surface of the glass tube - acidic urea yellow - urine itself
white (opaque) – protein: physiological in horse. Only present in others in case of kidney probl.
purple - indicane (indol-sulphate): physiological in horse. only in other species if there is a problem with the intestines
green – biliverdin: Physiological in dogs, increase in icterus
brown - urobilinogen (UBG): physiological
HNO3 (nitric acid)
Ehrlich test:
- UBG can be determined by using a semi-quantitative method with Ehrlich reagent. Put some drops of Ehrlich reagent (2 g p-dimethyl-amino-benzaldehyde in 100 ml 20% HCl) into 2 ml urine sample:
Normal:
colour by looking the tube from above - a mild reddish discolouration can be seen
colour by looking the tube from the side - no colour change (from the original colour of urine)
Abnormal:
colour by looking the tube from above - intensive red colour can be seen
colour by looking the tube from the side - mild or intense red
colour change (from the original colour of urine)
Examination of bilirubin derivates in faeces
Increased Br II excretion leads to increased UBG and thus increased stercobilin formation. Increased stercobilin amount leads to an intense orange colour of the faeces.
- Hyperchromic, pleichrom, hypercholic - intense colour, due to i.e. haemolysis
Hypochromic, hypocholic - decreased colour (light brown), due to liver failure (as less Br II is excreted by the bile)
- Hypochromic, acholic – very light colour (grey), due to bile duct obstruction
Summary of the results of bilirubin metabolism measurments:
(table)
Tests for examination of the hepatic excretory function
- Brom-sulphalein (sulphobromtalein) (BSP) retetion test
- Measurment of BSP half life
- Indocyane green (ICG) retention test
Brom-sulphalein- (sulphobromtalein-) (BSP) retention test
- In order to examine excretory function of liver we can perform the BSP-test. BSP-test is based upon the administration of brom-sulphalein (sulphobromtalein) dye intravenously and examination of the clearance of it from the plasma by hepatocytes.
- BSP has a purple colour in alkaline pH. If liver function is normal, BSP is not retained in the plasma and it is split to brom and thioether by the hepatocytes. Then brom is conjugated with glucuronic acid and excreted in the bile. The test actually gives information about UDP-glucuronyl transferase activity of liver cells.
- In case of carnivores we administer 0.063 mol (5 %) 0.1 ml/kg BW (= 5 mg/kg BW) BSP. Normally liver eliminates 95% of BSP from blood within 30-45 minutes. In dogs and sheep BSP is eliminated within 30, in cattle and horse within 40-45 minutes.
- The pitfall of this test is that in case of cats it is not liver specific, as some of the BSP can be excreted through the kidney, too. Another problem is that BSP can be dangerous as it can cause unexpected anaphylactic reactions.
Sampling procedure:
1) Blood sampling for basal value (blank)
2) Administration of BSP iv (5 mg/kg body weight in 5% solution)
3) Blood sampling after 3 minutes for 100% value 9
4) Blood sampling after 27 minutes (30 minutes after BSP administration) for the %-age in question
Measurment of BSP half life
In case of large animals we perform the test similarly, but we administer 20-30 ml of 5% BSP solution iv and do not calculate the amount for body weight. The reason is that BSP is expensive drug, and the test would cost too much.
In this test we take blood samples before and after the administration of BSP every 5 minutes until the 45th minutes.
After performing the laboratory analysis and getting percentages a curve can be formed. It is recommended to perform the test in some healthy animals and get the standard curve. Then we can repeat it in the questioned sick animal, that has a suspected liver failure.
Indocyane green (ICG) retetion test
Similarly to BSP ICG can be used, as it is more liver specific, especially good for cats.
Moreover it is less dangerous (generally does not cause anaphylactic reactions) but more expensive.
Procedure: 1 mg/kg bw - dog, 1.5 mg/kg BW - cat in 0.5% solution, blood sampling before and after 3 and 30 minutes of administration of ICG.
Normal is 20-25% retention in dog, and 15% in cat.
Causes of increased BSP retention
Primary liver failure:
liver cirrhosis
liver tumour
hepatic lipidosis
“lipid mobilisation syndrome”
Decreased hepatic perfusion:
right sided heart failure
portosystemic shunt
arteriole-venous fistulas in liver
blockage in portal vessels
Other causes:
decreased UDP-glucuronyl transferase activity in liver cells (congenital disorder)
Determination of Bile acids:
different types:
- HPLC. measures every value, can use serum and plasma
- RIA
- or total bile acids (TBA): only serum
spectrophotometric method.
(Measurement can not be accurately performed if in the blood sample heparin is used as an anticoagulant, or the sample is haemolysed or lipaemic)
Sampling methods in checking bile acids:
There are two ways of sampling:
1) after 12 hours starvation,
2) after eating, postprandial value - postprandial value can be 10 times (dog), 5-6 times (cat) more than the value measured after starvation.
Na-EDTA or -citrate can be used as an anticoagulant, because heparin disturbs the measurement.
Normal value (fasting): <20 µmol/l (carnivores) 20-30 µmol/l (other animals).
Causes of increased bile acid level in the blood
these are specific parameters, not sensitive! because 60-70% of the liver needs to not function
liver injury, hepatic cell damage - increased outflow of bile acids from the damaged hepatocytes to the blood,
bile duct obstruction or bile endothelial cell damage - decreased secretion of bile acids to the bile, increased outflow to the plasma instead,
decrease in liver function, therefore decreased uptake of the absorbed bile acids (note: increased urobilinogen level !)
biliary stasis (cholangiohepatitis cirrhosis, hepatic or pancreatic neoplasm, pancreatitis)
portosystemic shunt (absorbed bile acids bypass liver tissue)
Causes of decreased bile acid level in the blood
decreased absorption from the intestines - intestinal wall damage or surgical removal of the ileum or lymphangiectasia
severe liver cirrhosis (or other cause of severe liver cell damage) - decreased synthesis of bile acids
