Lecture set 2 Flashcards

Question

how are enzymes measured

Answer 1

-Product formation (end point) -substrate (how much used, formation or depletion based on enzyme activity) -continuous monitoring , instrument takes multiple readings, change in absorbance over time, can use NAD or NADP

Answer 2

international unit-ne (1) micromole of substance (or substrate) per minute, U/L or IU/L *same thing -amount of enzymes = activity NOT concentration

Answer 3

-convert AA and alpha keto acids by transfer of AA group AST transfer amino group between aspartate (substrate) and alpha-ketoglutarate > Product is oxaloacetate ALT transfer amino group between alanine (substrate) and alpha-ketoglutarate > Product is Pyruvate both part of Kerb cycle physiological reactions -both enzymes are substrate-and stereo specific in that they only act on L forms of AAs

Answer 4

AST- heart, liver, muscle, kidney, pancreas and RBC (affected by hemolysis false increase) ALT - most liver specific, kidney, heart, muscle, serum

Answer 5

increased in -hepatocellular disorders like viral hep, cirrhosis -cardiac muscle involvement ; rises in 6-8 hours of heart attack -skeletal muscles - muscular dystrophies and inflammatory conditions 10-30 U/L

Answer 6

-hepatic disorders -viral diseases like hepatitis ALT will be higher than AST except when its alcohol induced -ALT is elevated longer than AST due to longer half life 0-55u/l

Answer 7

-through coupled rxn where the analyzer continuously measures change in A at 340 when NADH is oxidized to NAD- a change in absorbance is proportional to enzyme activity first rxn- aspartate + alpha-ketoglutarate (in the reagent) and PT sample with AST = oxalacetate then in 2nd rxn oxalacetate gets used and analyzer measures for NAD at 340 NADH absorbs light and while at the same wavelength NAD does not Optimal pH: 7.3 - 7.8 Sources of error: Hemolysis: falsely ↑ AST results

Answer 8

coupled rxn -first reaction produces the product PA -looking for a decrease proportional to enzyme activity Optimal pH: 7.3 - 7.8 Sources of error: Unaffected by hemolysis

Answer 9

transfer of a γ-glutamyl group from glutathione γ-glutamyl peptides to amino acids, H2O & other small peptides found in kidney, brain , prostate, pancreas and liver (non specific of liver testing)

Answer 10

-most sensitive of all liver enzymes -increased in hepatobiliary (liver) diseases (this is bc location of GGT is in canaliculi – epithelial lining - of hepatic cells, easy access) -highest increases seen in biliary tract obstruction - Cholestasis s-slowing of bile due to obstruction -increased in chronic alcoholism- early marker of abuse even if there is no liver damage, levels can go back to normal after person stops drinking

Answer 11

-non specific for liver disease because it is increased in diabetes, MI, pancreatitis , in PT that get liver enzyme inducing drugs warfarin barbs -can differentiate source of increased ALP because ALP is increased both liver disease & musculo-skeletal disease therefore is GGT is not increased itll rule out liver disease because in liver disease they are both increased

Answer 12

ggt in patient , gamma glutam - reagent -measure Abs of the dye P-nitro-aniline is a coloured dye and absorbs light at 405-420 -so increase in A is proportional to GGT activity in pt -no affect in hemolysis -Males: 2-30 U/L -Females: 1-24 U/L

Answer 13

acts as catalyst between Lactate and pyruvate -Works by transferring hydrogen using the co-enzyme NAD forward pH 8.3- 8.9 -L -lactic acid oxidized to pyruvic with NAD as electron acceptor LA oxidized and NAD reduced reverse pH 7.1-7.4, 3 times faster than forward pyruvic acid reduced to L-LA with NADH as electron donor

Answer 14

heart*, liver*, skeletal muscle*, kidney*, RBC*☆, lung, smooth muscle, brain -5 isoenyzmes -tetramer w/ 4 polypep chains H for heart and M for muscle LDH-1 is fastest, LDH-5 is slowest (electrophoretic mobility – how fast it migrates to anode )

Answer 15

non specific for liver disease because its found in too many tissues - not recommend in liver profile -increased in liver and biliary disease highest in liver carcinoma -high in cardiac disease = MI -high in skeletal disease, renal diseases -hematologic: hemolytic anemias, Pernicious anemia

Answer 16

- hemolysis is UNACCEPTABLE -unstable in serum - asap -store at PT loss of activity at 4 degrees -LD concentration in RBC is high so hemolysis increases it -concentration of LD in RBC is 100-150x what is found in serum

Answer 17

Forward - l-LA + NAD = PA +NADH (increase ABs) and reverse (decrease ABs) substrate must be in excess -continously monitored reaction -measure increase or decrease at 340 because at 340 NAD does not absorb -NADH formation or depletion proportional to amount of enzyme (LD activity) in the specimen

Answer 18

Hemolysis: falsely elevated improper anticoagulants: falsely decreased freezing specimen: falsely decreased reference varies based on forward or reverse reactions

Answer 19

electrophoresis not done usually because its expensive

Answer 20

-located on the sinusoids and bile canaliculi membranes of liver -Bone, ALP is in the osteoblasts so it’s involved in bone formation -Catalyzes the hydrolysis of various phosphomonoesters Tissue Source Found in intestine, liver, bone, spleen, placenta and kidney

Answer 21

-non specific for liver disease -increased in hepatobiliary disorder -highest in obstructive (extra hepatic conditions like gallstones -sensitive to impaired bile flow; cholestasis increases synthesis of ALP -Moderate increase in hepatrocellular diseases (e.g. hepatitis, cirrhosis) ↑ bone disorders (e.g. Paget’s disease) ↑ pregnancy

Answer 22

p-Nitrophenylphosphate (colorless) is hydrolyzed with ALP and h2o at pH10.2 to p-nitrophenol (yellow) -increase in ABs at 405 proportional to ALP in pt -RI depends on age/sex, higher in children due to increased bone growth Sources of error: Hemolysis may cause slight ↑ Run ASAP – activity in serum increases at RT

Answer 23

abnormal accumulation of bilirubin in blood -bile will accumulate in the skin, eyes mucous membranes and causes yellow discolouration - Jaundice and icterus can be used interchangeably -icterus is more for the yellow color of serum and plasma

Answer 24

1. How much is present? (conc’n ?) we measure Total Bilirubin = unconjugated + conjugated 2. What type of bilirubin is present? unconjugated: found in serum bound ot ALB and insoluble in water conjugated: majority normally excreted into bile 3 types of jaundice: pre-hepatic, hepatic, and post-hepatic

Answer 25

RBC destroyed conversion of Heme to Biliverdin (intermediate product), then conversion of Biliverdin to Bilirubin (unconjugated) -Bilirubin will combine with albumin in plasma (this is unconjugated) -In the liver. Unconjugated is converted into conjugated via UDP glucorinde transferase -Conjugated is carried through either gall bladder for storage or its in the small intestine -in the small intestine the conjugated gets converted into URObili and then urobilin secreted into stool -a little bit of the URO gets reabsorbed to liver, to kidney to be filtered and excreted in urine

Answer 26

hemolytic anemias - RBC destruction (ex. Thalassemia, hereditary spherocytosis, hereditary elliptocytosis, autoimmune hemolytic anemia, sickle cell anemia) transfusion reaction (i.e. incompatible blood leads to increased RBC destruction) Burns, Poisoning (trauma may lead to increased RBC destruction)

Answer 27

-rate of hgb breakdown exceeded and there is accumulation of unconjugated bilirubin in serum = a pre-hepatic disorder -conjugated bilirubin stays with RI -hepatocyte function is normal only capacity for handling bilirubin is overloaded The liver can conjugated can conjugate up to 2X its normal load. When the liver is working at max, then there will be more un conjugated bili going to the small intestine and kidneys so we will see more urobilinogen in urine and urobilin in stool

Answer 28

urine & stool darker in colour due to increased excretion of urobilinogen & urobilin -mild jaundice -bilirubin overproduction causes gallstone formation (composed of bilirubin) -if there is an increase in RBC then there will be an increase in unconjugated bili causing jaundice

Answer 29

plasma / Serum total bilirubin ↑ = unconjug + conjug unconjugated bilirubin ↑ conjugated bilirubin → Urine urobilinogen ↑ urine dipstick testing (qualitative testing) – due to increase in load -conjugated bilirubin (water soluble): negative feces urobilin ↑ not routinely performed – due to increase in load Liver Enzymes: Since liver is operating normally AST, ALT, ALP, GGT: within reference intervals

Answer 30

-Crigler-Najjar syndrome Gilbert’s syndrome (difference is where the mutation takes place) -mutation in gene that produces UDP which affects conversion to conjug -therefore bili cannot be conjug leading to increase of unconjuga

Answer 31

-Dubin-Johnson syndrome -Defect in conjugated bilirubin excretion. Bilirubin conjug is normal but removal from liver and excretion is affected -Increase in conjug

Answer 32

-affect depends on level of necrosis or impairment of hepatocytes -in early phase bili is still getting conjug but not as effectively. Damaged liver cells will prevent reabsorption of urobilinogen, so we expect increase in urine urobilinogen excretion -in later phases there is more unconjugated because the liver cant do its job so there is alot of both - mixed

Answer 33

Plasma/Serum: total bilirubin ↑ generally unconjugated bilirubin ↑ generally conjugated bilirubin ↑ Urine: urobilinogen ↑ (later as liver is not functioning, ↓ or absent bc less conjugation occurring thus less conversion into urobilinogen) conjugated bilirubin: positive (if increased in serum) Feces: urobilin ↓ (or absent): depends on degree of obstruction (bc less conjugation occurring thus less conversion into urobilinogen and then into urobilin) Liver Enzymes: AST, ALT, GGT: ↑ ALP ↑ or →

Answer 34

cholestasis (slowing or stopping of bile flow) -excretory pathway of bile (intra-hepatic & extra-hepatic ducts) physically obsrtucted preventing flow carcinoma of pancreas gallstones - common bile duct obstructed liver tumor- liver CA POST hepatic disorder Liver is still functioning, still getting conjugated bilirubin but it’s not being properly excreted. if bile flow blocked, it accumulates in canaliculi causing rupture & increased permeability of the walls of the small bile passages -bile mostly conj -bilirubin diglucuronide leaks into sinusoids then circulation -high conj in plasma/serum -very high unconju bili (bound to alb) because of liver backup

Answer 35

Plasma / Serum: total bili ↑ unconjugated bilirubin ↑ conjugated bilirubin ↑ ↑ Urine: urobilinogen ↓ or absent conjugated bilirubin: positive Feces: urobilin ↓ or absent (stools paler) – bc bile doesn’t enter small intestine Liver Enzymes: ALP, GGT: ↑ ↑ (ALT, AST variable) Increased production of ALP & GGT in bile canaliculi due to blockages in bile flow (cholestasis)

Answer 36

-third fraction of bilirubin that contributes to “Total Bilirubin” if present -conjugated bound to ALB -the molecule is too large so it cant be filtered by the glomerulus and is not detected in urine so instead you see it in plasma -you only see if there is SIGNIFICANT hepatic obstruction aka in babies w/o bile ducts