Lecture 6: Liver Flashcards
What happens to the cholesterol when it reaches the liver?
Secreted unchanged into the bile
OR
Oxidized to cholic acid or chenodeoxycholic acid
What are the primary bile acids and how are they made?
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)
What method isused to measure bile acids?
Gas Liquid Chromatography (main)
Others:
HPLC
Enzymatic Assay
RIA
ELISA
MS
*** What is the physiological significance of bile acids?
- Cholesterol elimination
- Solubilization of cholesterol
- Emulsification of fats to facilitate the action of pancreatic lipas
- Facilitate the absorption of fat soluble vitamins
What is the clinical significance of bile acids?
- Used to aid in diagnosis of:
- Intrahepatic cholestasis of pregnancy
- Genetic defects of bile acid metabolism
When are bile acids elevated?
- Gall stones
- Primary biliary cirrhosis
- Sclerosing cholangitis (disease of the bile ducts)
- Neoplasms
What are the different markers of hepatic injury?
- AST
- ALT
- ALP
- GGT
What is the function of AST and what is its coenzyme?
AST catalyzes the transfer of an amino group from aspartic acid to 2-oxoglutaric acid
Coenzyme is pyridoxal-5’-phosphate (P-5’-P)
Why can AST be detected in the plasma/serum?
Even though it is an intracellular enzyme, continuous cell breakdown allows a small amount of AST to be present in the plasma/serum
What are the major tissues that express AST?
Heart
Liver
Skeletal Muscle
Kidney
How is AST measured?
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
What are 3 preanalytical considerations when measuring AST?
- Increases with increasing BMI
- Up to 3x increase with vigorous exercise
- Significant increase with hemolysis
What is the reference interval for AST?
At 30 C:
1. 8-20 U/L in normal serum
2. 10-30 U/L when P-5’-P is added
What is the function of ALT? Coenzyme?
Catalyzes transfer of amino group from alanine to 2-oxoglutarate
P-5’-P
What is the difference between AST and ALT?
ALT exclusively cytoplasmic unlike AST.
What are the major tissues expressing ALT?
Liver
Kidney
Skeletal muscle
How is ALT measured?
Coupled enzymatic reaction.
Uses LDH instead of MDH.
What are the pre-analytical considerations when measuring ALT? (3)
- Increases with increasing BMI
- Lower in those who exercise regularly
- Hemolysis leads to moderate increase in activity
Reference interval for ALT?
At 37 C:
1. 10-40U/L when P-5’-P is added
What is the function of ALP?
Metabolite transfer across cell membranes
Where is ALP found (in decreasing order)?
Placenta
Ileal mucosa
Kidney
Bone
Liver
What is the difference between bone, liver and kidney ALP differ?
Carbohydrate content
Why does cholestasis increase ALP levels?
Causes synthesis of ALP by hepatocytes and bile salts stimulate the release of ALP from biliary tract cell membranes
How is ALP measured?
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.
What are the pre-analytical considerations when measuring ALP? (3)
- Increases with increasing BMI
- Increases 2-3 fold in the 3rd trimester
- Complexing agents such as EDTA, citrate, oxalate, coordinate the cofactors Zn and Mg leading to falsely low values
Describe the electrophoretic techniques in the analysis of ALP isoenzymes.
- Polyacrylamide/starch gel used
- Performed at alkaline pH
- Liver migrates rapidly towards anode, followed by more diffuse bone, diffuse intestinal and kidney when present
- Placental runs with liver and bone
- 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
How do we improve liver and bone ALP electrophoretic separation?
- Expose to wheat germ lectin: retards bALP more than liver
- Expose to neuraminidase removes sialic acids residues and bALP is retarded more than liver
What are other methods to analyse ALP isoenzymes other than electrophoretic techniques?
- Heat inactivation
- LALP is more stable than bALP in the presence of heat
- If <20% of activity remains, then sample is mostly bone
- Urea inhibition
- Bone susceptibility > LALP > iALP/pALP
- Immunoassays
- Cross reactivity is a big problem
What is the function of GGT?
Transfers gamma glutamyl from peptides to acceptor molecule
Where is the typical reaction for GGT?
Glutathione + aa -> gamma-glutamyl-aa + cysteinylglycine
Where is GGT found?
PT
Liver
Pancreas
Intestine
How to measure GGT?
Assay uses artifical substrate which produces the chromogen p-nitroanilide whose absorbance can be monitored at 410 nm
Pre-analytical considerations for GGT? (3)
- Increased by many drugs
- Increased by smoking
- Direct relationship with ethanol intake
How to determine bilirubin?
- Reaction of bilirubin with a diazonium salt of sulfanilic acid
- Chromogen azobilirubin is blue in color and is measured at 560 nm/600 nm
- Reaction will only detect fractions of bilirubin which are water soluble
What are the 4 bilirubin fractions?
- alpha - uncojugated to glucuronic acid
- beta - monoconjugated to glucuronic acid
- gamma - diconjugated
- delta - covalent albumin bilirubin conjugate
What is the difference between direct and indirect acting bilirubin?
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
Why unconjugated bilirubin cannot react with diazotized sulfanilic acid? What can we do about it?
Not water soluble
Use accelerators (cmpds that solubilize the unconjugated bilirubin and cause it to react)
- Traditional: ethanol
- Now: Caffeine benzoate
Pre analytical considerations of bilirubin?
- Hemolysis - false increase
- Hemolytic anemia increases unconjugated portion
- 48 hr fast will increase by up to 2x
How to measure bilirubin in neonates? How does it work?
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.
