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Lab D > Lab 10. Liver enzymes > Flashcards

Lab 10. Liver enzymes Flashcards

1
Q

Examination of protein synthesising ability of the Liver

  • parameters to be measured:
A
  • TP, albumin and fibrinogen concentration by spectrophotometric, refractometric methods
    • fibrinogen by thrombin time
  • Edema can be expected if albumin conc. is <20 g/l
  • already Edema formation if albumin conc. is <11 g/l
  • Alpha and beta globulins can be measured by electrophoresis
  • Coagulation factors by the APTT and PT
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2
Q

Determination of ammonia concentration in the blood is required in:

A
  1. in carnivores to diagnose severe decrease of liver function (i.e chirrosis), portosystemic shunt (where blood bypasses liver and NH3 is not detoxified)
  2. In ruminants as a consequence of ruminal alkalosis, or decomposition of ruminal fluid, severe systemic alkalosis can develop due to hyperammoniaemia
  3. in case of horse and rabbit, pathologic breakdown of ingest in the colon or cacum of in case of liver failure
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3
Q

Metabolism of Ammonia

A
  • Ammonia is produced in the intestines by the bacteria
  • it is absorbed to the portal vein and then liver detoxifies it by the ornitin cycle and by the action of GLDH utilised alpha-keto-glutaric acid, or glutamic acid.
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4
Q

How to take ammina concentration measurments:

A
  • we generally take amminia conc. measurments as a liver function test.
  • before taking blood samples animal should starve for 24 hours.
  • we take blood to EDTA or citrate tubes
  • samples should be taken by avoiding air contamination (“astrup-samples”), because NH3 may evaporate from the sample to the air, or it can be dissolved in the sample from the air.
  • determination should be performed right after sampling.
  • blood samples can be stored in ice baths for max. 20 minutes, or plasma samples can be stored deep frozen (-20*C) for 2 days
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5
Q

Normal values for ammonia concentration:

A

Dog: 26,4-70,5 umol/l

Cat: 17,6-58,7 umol/l

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6
Q

Ammonia measurment (standard) method:

A
  • ammonia wil alpha-ketoglutaric acid and NADH + H+ and GLDH enzyme produces glutamic acid and NAD+
  • the reduction of absorbancy can be measured spectophotometrically
  • this standard method is generally complicated by many preanalytical problems, therefore it is not widely used.
  • another, light-refraction based method is used more frequently (ammonia-checker).
  • plasma samples are needed for the standard method, while whole blood samples can be used with the (portable) ammonia-checker.
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7
Q

Ammonia tolerance test:

A
  • Performed when basal NH3-concentration values do not show alteration and the suspect of portosystemic shunt is strong
  • after 24 hours starvation and pretreatment with neomycin (in order to reduce bacterial NH3 production in the intestines), we take a basal blood sample, and give NH4Cl in 5%-water solution (100 mg/kg per os, max 3g), then blood samples are taken 30 or 45 minutes after treatment.
  • if liver function is normal, ammonia concentration is <120 umol/l in dogs, and <175 umol/l in cats
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8
Q

Causes of increased NH3 concentration in the blood, or positive ammonia tolerance test results:

A
  • impared liver function - decreased production of urea in case of peracute liver failure, liver chirrosis or neoplasia, portosystemic shunt, hepatic lipidosis, lipid mobilisation disease
  • Ruminal alkalosis or ammonia toxicosis - due to absolute or relative protein overload (with the feed), or the intake of rotten feed.
  • Intestinal overgrowth of ammonia producing bacteria - in ruminants, horses, rabbits, swine or carnivores.
  • congenital enzymopathies - decreased activity of i.e OCT.
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9
Q

Location and function of AST

A

AST = aspartate-aminotransferase

Location:

  • mitochondria of liver cells, muscles (heart too), red blood cells (false increase)
  • in herbivores liver specific enzyme

Function:

  • converts aloha-keto-glutaric acid to L-glutamic acid, and L-aspartate to oxalitc acetic acid

Basis of the measurment method:

  • AST enzyme with L-aspartate and alpha-ketoglutarate produces oxalic-acetate and L-glutamate.
  • Oxalix-acetate with NADH+ H+ and malate-dehydrogenase (this is the reagent) produces malate and NAD+.
  • This NADH + H+ –> NAD+ change cause absorbancy reduction. The speed of it is in correlation with AST activity
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10
Q

Causes of increased activity of AST:

A

- from muscle cells: intensive exersice, training, muscle necrosis, muscular inflammation (myostitis), muscle injury (IM injection), myocarditis (CK, LDH also increased), neoplasm of the muscles (rhabdomyosarcoma AST/ALT > 1). In case of muscle cell damage, generally LDH and CK enzymes are also elevated.

- from the liver cells: ethanol consumption (human beings, ethanol is especially harmful for the mitochondria of the liver cells, AST/ALT > 1), hepatopathy (in herbivores), severe parenchymal damage (in carnivores, eg. lipotic degeneration, hepatitis, or toxic liver damage).

- From RBCs: haemolysis

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11
Q

Causes of decreased AST activity:

A
  • metronidasol (as antimicrobial drug, which can cause liver function problems)
  • Vitamin B6 deficiency

(practically no diagnostic importance of this)

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12
Q

ALT location and function:

A

ALT = Alanine-amonitransferase

Location:

  • liver cells (in carnivores liver specific), red blood cells (false increase), in the cytoplasm (half life of the enzyme is short), in herbivores not liver specific (small amounts are found in heart-and striated muscles, kidney cells)

Function:

  • converts alpha-keto-glutaric acid to L-glutamic acid, and L-alanine to pyruvic acid.

Determination:

  • produced pyruvic acid is converted to lactic acid by lactate dehydrogenase (LDH) that is the reagent.
  • this process causes NADH + H+ –> NAD transformation that causes discoloration of a chromophor 8and absorbancy reduction)
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13
Q

Causes of increased activity of ALT:

A
  • liver cell damage (especially in carnivores)
  • chronic active hepatitis (CAH), cholangiohepatitis (CH)
  • virus hepatitis (humans) (AST/ALT < 1)
  • hepatic lipidosis
  • chirrosis, bile duct obstruction
  • septicaemia, neoplasm
  • drugs: barbiturates, glucocorticoids, salicylates, tetracyclines as a result of cell damage
  • copper storage disroder: Dobberman pincher, West hihland white and Bedlingtone terrier
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14
Q

GLDH Location and Function:

A

GLDH = Glutamate-dehydrogenase

Location:

  • liver specific, small amount is found in nerves and muscles in mitochondria only
  • liver specific in ruminants, horses and dogs

Function:

  • binds NH3 to form glutamic acid

Determination:

  • GLDH converts alpha-keto-glutaric acid to L-glutamic acid while this process causes NADH + H+ –> NAD transformation that causes discoloration of a chromophor.
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15
Q

Causes of increaed activity of GLDH

A
  • severe liver cell necrosis that leads to mitochondrial membrane damage
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16
Q
  1. Liver cell (parenchymal) enzymes:
  2. Bile duct obstruction enzymes:
  3. Other liver specific enzymes:
A
  1. AST, ALP, GLDH
  2. ALKP, GGT
  3. OCT, Ar, SDH
17
Q

ALKP location:

A

ALKP = AP-alkaline-phosphatase

  • potentially appears in every cell membrane, produced by different organs (placenta, bones, liver - hepatocytes, biliary epithelial cells, intestines, kidney tubular epithelial cells) - conversion in bile endothelial cells.
  • Only hepatic and bone ALKP appears in the blood
  • in cats not liver specific (half life is short, excretion via kidney)
  • tubular cell damage causes increased activity of ALKP in urine
18
Q

ALKP Isoenzymes:

A

there are different isoenzymes - distinction between isoenzymes of liver and bone origin

  • Liver originated ALKP is heat-stabile,
  • Bone originated ALKP is heat-labile.
  • heating the plasma to 65*C for 5 min inactivates bone ALKP
  • one of the heat stabile isoenzymes is Steroid induced Alkaline phosphatase (SIAP) which is produced in the licer
  • isoenzymes can be differentiated by many methods: electrophoresis, specific blocking (L-fenilalanine, ELISA, chromatographic)
19
Q

Function of ALKP

A

However it exists in every cell membrane it is not active enzyme as its pH optimum is 10 I, which is too high in vivo.

  • they are phosphotransferases and not phsophatases
  • generally phosphate-esters are translocated to one alcohol or phenol hydroxyl groups to another by ALKP

Determination:

  • 4-nitrophenil phosphate and H2O is converted to 4-nitrophenol and phosphate ion (bound to buffer solution used in the reagent)
  • produced 4-nitrophenol is yellow and can be detected spectrophotometrically
20
Q

Causes of increased ALKP activity:

A
  • Bone originated: young dogs (> 8 months), new born animals, pregnant animals (from bones and placenta), bone tumours, osteomyelitis, bone fractures, healing of fractures (callus formation)
  • paraneoplastic processes: lymphoid, lung and hepatic tumors
  • liver originated: cholestasis (bilirubin has a direct, ALKP-activity increasing effect on the biliary enpithelial cells), bile acids (increasing the synthesis of ALKP, and help in liberating it from membranes), acute hepatic necrosis, liver chirrosis, intra- or extrahepatic biliary obstruction, chlangiohepatitis, hepatic lipidosis, barbiturates (increasing synthesis of ALKP), salicilates (liver cell and biliary epithelial cell damage)
  • in connection with increased SIAP synthesis: hyperadrenocorticism, iatrogenous or endogenous (cushings), chronic stress
21
Q

Other liver specific enzymes:

A
  • SDH: liver specific in swine, cattle and horse
  • OCT: in dogs
  • AR: dogs and horses
22
Q

What to measure?

A
  • Dog: ALT, (AST, GLDH), ALKP, GGT
  • Cat: ALT, (AST, GLDH), GGT, (ALKP in acute processes)
  • Ruminants: AST, GLDH, (GGT)
  • Horse: AST, GGT, (Arg)
  • Swine: AST, GGT, ALKP, OCT, SDH
23
Q

Examination of change in lipid metabolism due to impaired liver function:

A
  1. Decreased total cholesterol concentration:
    - cause: decreased esterification ability, decreased apolipoportein synthesis
  2. Increased free faty acid concetration: in case of increased energy demand (lipaemia)
    - cause: decreased FFA utilisation especially in case of increased energy demand
  3. Lipid accumulation in the liver:
    - cause: decreased ability to perform beta-oxidation (decreased lipid breakdown), decreased synthesis of lipid-transporting apolipoprotein molecules
24
Q

Other diagnostic approaches

A

Liver biopsy or aspiration cytology

  • routine cytology or histology and laboratory evaluation of the lipid, protein, glycogen etc. content of the liver

Lab D (11 decks)

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