Lecture 6: Liver

Question 1

What happens to the cholesterol when it reaches the liver?

Answer 1

Secreted unchanged into the bile
OR
Oxidized to cholic acid or chenodeoxycholic acid

Question 2

What are the primary bile acids and how are they made?

Answer 2

Cholic acid or chenodeoxycholic acid react with glycine or taurine to produce 4 primary bile acids.

Glycocholic acid (pKa ~ 4)
Taurocholic acid (pKa ~ 2)
Glycochenodeoxycholic acid (pKa ~ 4)
Taurochenodeoxycholic acid (pKa ~ 2)

Question 3

What method isused to measure bile acids?

Answer 3

Gas Liquid Chromatography (main)

Others:
HPLC
Enzymatic Assay
RIA
ELISA
MS

Question 4

*** What is the physiological significance of bile acids?

Answer 4

1. Cholesterol elimination
2. Solubilization of cholesterol
3. Emulsification of fats to facilitate the action of pancreatic lipas
4. Facilitate the absorption of fat soluble vitamins

Question 5

What is the clinical significance of bile acids?

Answer 5

1. Used to aid in diagnosis of:
   - Intrahepatic cholestasis of pregnancy
   - Genetic defects of bile acid metabolism

Question 6

When are bile acids elevated?

Answer 6

1. Gall stones
2. Primary biliary cirrhosis
3. Sclerosing cholangitis (disease of the bile ducts)
4. Neoplasms

Question 7

What are the different markers of hepatic injury?

Answer 7

1. AST
2. ALT
3. ALP
4. GGT

Question 8

What is the function of AST and what is its coenzyme?

Answer 8

AST catalyzes the transfer of an amino group from aspartic acid to 2-oxoglutaric acid

Coenzyme is pyridoxal-5’-phosphate (P-5’-P)

Question 9

Why can AST be detected in the plasma/serum?

Answer 9

Even though it is an intracellular enzyme, continuous cell breakdown allows a small amount of AST to be present in the plasma/serum

Question 10

What are the major tissues that express AST?

Answer 10

Heart
Liver
Skeletal Muscle
Kidney

Question 11

How is AST measured?

Answer 11

Coupled enzymatic reaction.
Activity is proportional to deltaA/deltat at 340 nm which monitors the disappearance of NADH.

Uses L-Aspartate to oxoloacetate.
Oxoloacetate to L-Malate with help of MDH. At the same time, NADH + H -> NAD

Question 12

What are 3 preanalytical considerations when measuring AST?

Answer 12

1. Increases with increasing BMI
2. Up to 3x increase with vigorous exercise
3. Significant increase with hemolysis

Question 13

What is the reference interval for AST?

Answer 13

At 30 C:
1. 8-20 U/L in normal serum
2. 10-30 U/L when P-5’-P is added

Question 14

What is the function of ALT? Coenzyme?

Answer 14

Catalyzes transfer of amino group from alanine to 2-oxoglutarate
P-5’-P

Question 15

What is the difference between AST and ALT?

Answer 15

ALT exclusively cytoplasmic unlike AST.

Question 16

What are the major tissues expressing ALT?

Answer 16

Liver
Kidney
Skeletal muscle

Question 17

How is ALT measured?

Answer 17

Coupled enzymatic reaction.
Uses LDH instead of MDH.

Question 18

What are the pre-analytical considerations when measuring ALT? (3)

Answer 18

1. Increases with increasing BMI
2. Lower in those who exercise regularly
3. Hemolysis leads to moderate increase in activity

Question 19

Reference interval for ALT?

Answer 19

At 37 C:
1. 10-40U/L when P-5’-P is added

Question 20

What is the function of ALP?

Answer 20

Metabolite transfer across cell membranes

Question 21

Where is ALP found (in decreasing order)?

Answer 21

Placenta
Ileal mucosa
Kidney
Bone
Liver

Question 22

What is the difference between bone, liver and kidney ALP differ?

Answer 22

Carbohydrate content

Question 23

Why does cholestasis increase ALP levels?

Answer 23

Causes synthesis of ALP by hepatocytes and bile salts stimulate the release of ALP from biliary tract cell membranes

Question 24

How is ALP measured?

Answer 24

By means of its action on p-nitrophenyl phosphate at pH 10.
Reaction turns the colourless compound into a yellow quinoid whose absorption is measured at +/- 405 nm.

Question 25

What are the pre-analytical considerations when measuring ALP? (3)

Answer 25

1. Increases with increasing BMI 2. Increases 2-3 fold in the 3rd trimester 3. Complexing agents such as EDTA, citrate, oxalate, coordinate the cofactors Zn and Mg leading to falsely low values

Question 26

Describe the electrophoretic techniques in the analysis of ALP isoenzymes.

Answer 26

1. Polyacrylamide/starch gel used 2. Performed at alkaline pH 3. Liver migrates rapidly towards anode, followed by more diffuse bone, diffuse intestinal and kidney when present 4. Placental runs with liver and bone 5. HMW form of ALP is present in tissue extracts and serum which migrates faster to the anone than the main liver fraction on non-seiving gels such as cellulose acetate - Referred to as "fast liver" fraction - Does not migrate at all in seiving gels

Question 27

How do we improve liver and bone ALP electrophoretic separation?

Answer 27

1. Expose to wheat germ lectin: retards bALP more than liver 2. Expose to neuraminidase removes sialic acids residues and bALP is retarded more than liver

Question 28

What are other methods to analyse ALP isoenzymes other than electrophoretic techniques?

Answer 28

1. Heat inactivation - LALP is more stable than bALP in the presence of heat - If <20% of activity remains, then sample is mostly bone 2. Urea inhibition - Bone susceptibility > LALP > iALP/pALP 3. Immunoassays - Cross reactivity is a big problem

Question 29

What is the function of GGT?

Answer 29

Transfers gamma glutamyl from peptides to acceptor molecule

Question 30

Where is the typical reaction for GGT?

Answer 30

Glutathione + aa -> gamma-glutamyl-aa + cysteinylglycine

Question 31

Where is GGT found?

Answer 31

PT Liver Pancreas Intestine

Question 32

How to measure GGT?

Answer 32

Assay uses artifical substrate which produces the chromogen p-nitroanilide whose absorbance can be monitored at 410 nm

Question 33

Pre-analytical considerations for GGT? (3)

Answer 33

1. Increased by many drugs 2. Increased by smoking 3. Direct relationship with ethanol intake

Question 34

How to determine bilirubin?

Answer 34

1. Reaction of bilirubin with a diazonium salt of sulfanilic acid 2. Chromogen azobilirubin is blue in color and is measured at 560 nm/600 nm 3. Reaction will only detect fractions of bilirubin which are water soluble

Question 35

What are the 4 bilirubin fractions?

Answer 35

1. alpha - uncojugated to glucuronic acid 2. beta - monoconjugated to glucuronic acid 3. gamma - diconjugated 4. delta - covalent albumin bilirubin conjugate

Question 36

What is the difference between direct and indirect acting bilirubin?

Answer 36

Direct: 1. Water soluble forms of bilirubin - beta, gamma, delta 2. React with diazo compound directly 3. Delta fraction reacts slowly; not detected by standard direct assays in hospital Indirect: 1. Alpha fraction (unconjugated bilirubin) 2. Not water soluble, and bound (not covalently) to albumin

Question 37

Why unconjugated bilirubin cannot react with diazotized sulfanilic acid? What can we do about it?

Answer 37

Not water soluble Use accelerators (cmpds that solubilize the unconjugated bilirubin and cause it to react) - Traditional: ethanol - Now: Caffeine benzoate

Question 38

Pre analytical considerations of bilirubin?

Answer 38

1. Hemolysis - false increase 2. Hemolytic anemia increases unconjugated portion 3. 48 hr fast will increase by up to 2x

Question 39

How to measure bilirubin in neonates? How does it work?

Answer 39

Direct spectrophotometry Absorbance measured at 454 nm and then absorbance at 540 nm is subtracted to correct for HbO2 In adults cannot use this method because there are too many pigments that interfere at 454 nm, such as carotene.