Bilirubin Metabolism - Abali 2/22/16

Question 1

bile metabolism: related disease

bilirubin metabolism: related disease

Answer 1

bile: cholestasis
bilirubin: bilirubinemia/jaundince/icterus

Question 2

bilirubinemia

Answer 2

jauncice, icterus

increased bilirubin in circulation

• yellowing of eyes, hands
  
  • if no eyes…might be hyperbetacarotenemia (high levels of beta-carotene : precursor of vit A)

Question 3

key functions of heme in the body

Answer 3

• hemoglobin: oxygen transport in blood
• myoglobin: oxygen storage in muscle
• cytochrome c: e transport chain
• cytochrome P450s: toxin breakdown
• catalase: H2O2 breakdown
• Trp pyrrolase: oxidation of Trp

Question 4

types of hemolysis

• proportions, location

Answer 4

extravascular (90%): macrophage phagocytosis in liver/spleen

and

intravascular (10%): in vasculature

Question 5

extravascular hemolysis

Answer 5

• 90% of hemolysis
• RBCs phagocytized by macrophages in liver and spleen
• characterized by spherocytes (round cells - not discs)

why?

• senescent RBCs
• RBCs with membrane abnormalities (bound Ig)
• RBCs with physical abnormalities that prevent exit from spleen (sickle cell, thallasemia, PK deficiency)
  *

Question 6

intravascular hemolysis

Answer 6

• 100% of hemolysis
• RBC lysis within circulation
• characterized by fragmented cells called schistocytes

why?

• trauma
• complement fixation
• toxic damage to RBC
• Favism (G6PD deficiency)

Question 7

extravascular erythrocyte destruction

Answer 7

• accounts for daily 1% normal RBC turnover
• reticuloendothelial system is responsible
  
  • monitors RBCs
  • macrophages of liver, spleen, and bone marrow engulf RBCs before they lyse

Question 8

salvage of intravascular hemolysis products

Answer 8

hemolysys releases RBC contents including Hb and heme

• Hb binds to haptoglobin
  
  • Hb-Hp binds to receptor on macrophage, gets endocytosed
• heme binds to hemopexin
  
  • ​heme-hemopexin binds to receptor on macrophage, gets endocytosed

in macrophage, Hb and heme undergo lysosomal degredation releasing…

• CO
• Fe+2 (recycled)
• heme → biliverdin → bilirubin

Question 9

symptom: low/no haptoglobin in circulation

Answer 9

potentially hemolysis taking place somewhere → all haptoglobin is bound to hemoglobin that needs to be degraded, so none is free in circulation

Question 10

heme degradation

Answer 10

in the macrophages of the reticuloendothelial system (liver, spleen, bone marrow)

same enzymes whether intra/extravascular hemolysis

heme [purple] → biliverdin [green]

• via heme oxygenase
• uses oxygen; gives off Fe+3, CO

biliverdin [green] → bilirubin [red-orange]

• via biliverdin reductase

Question 11

bilirubin transport and modification in liver

Answer 11

released from site of production (macrophages of RES) into circulation

• it’s hydrophobic! need something for it to be bound to in circ: albumin

in liver,

• GST-B (glutathione S transferase B, aka ligandin) traps bilirubin in liver [“indirect”/unconjugated bilirubin]
• conjugation occurs via UDP-GT (UDP gucuronyl transferase aka UGT1A1)
  
  • attaches 2 glucoronic acid to bilirubin [“direct” bilirubin-monoglucuronide, “direct” bilirubin-diglucuronide]
• conjugated bilirubin moves into gallbladder → can be secreted into sm intestine

Question 12

bilirubin in intestinal tract, fates

Answer 12

in colon

• bacterial enzymes deconjugate bilirubin-diglucuronide back to bilirubin
• bacterial enzymes take bilirubin → urobilinogen
  
  • 10% urobilinogen reabs and sent back to liver via hepatic portal circ → central circ to kidney → oxidized to urobilin (yellow, gives urine its color), excreted in urine
  • 90% unabsorbed, further modified to stercobilin, urobilin (gives red-brown color to feces), excreted in feces

Question 13

urobilinogen: antibiotics and urine concentration

Answer 13

oral admin of broad spectrum antibiotics drastically drops production of urobilinogen

high urine concentration of urobilinogen associated with hemolytic disorders/hepatocellular disease

Question 14

direct vs. indirect bilirubin

Answer 14

direct = conjugated

indirect = unconjugated

named based on test, which makes use of van den Bergh rxn in which bilirubin is coupled with diazonium salt to make colored complex

• direct bilirubin: fraction which complexes with diazonium salt in absence of an accelerator
  
  • approximates conjugated bilirubin (normal : 0-0.3 mg/dL)
• total bilirubin: bilirubin which complexes with diazonium salt in presence of an alcohol accelerator
• indirect bilirubin: diff between total and direct bilirubin
  
  • ​approximates unconjugated bilirubin (normal : 0.3-1.9 mg/dL)

Question 15

why is direct bilirubin supposed to be near-absent in circulation?

Answer 15

macrophages that bring bilirubin to liver bring it in circulation as indirect bilirubin

conjugation to direct bilirubin occurs in the liver

Question 16

heme oxygenase

Answer 16

enzyme found in macrophages of RES (liver, spleen, bone marrow)

converts heme to biliverdin

• produces Fe+3 and CO (only rxn that produces CO)
• uses NADPH

Question 17

biliverdin

Answer 17

heme to biliverdin + CO, catalyzed by heme oxygenase in RES macrophages

• biliverdin and CO are cytoprotective
  
  • ​biliverdin = antioxidant
  • CO = signaling mol, vasodilator

Question 18

biliverdin reductase

Answer 18

catalyzes biliverdin to bilirubin in RES macrophages

• uses NADPH
• bilirubin produced is released from macrophage into circ bound to albumin

Question 19

reactions catalyzed by UDP-GT

consequence of reduced UDP-GT activity

Answer 19

UDP-GT conjugates bilirubin with glucoronic acid in liver

1. bilirubin + UDP-glucoronic acid → BMG
2. BMG + UDP-glucoronic acid → BDG (conjugated bilirubin)

under normal conds, the second reaction is rate limiting step

• if there’s a reduction in UDP-GT activity (ex. in genetic conds), you’ll see buildup of BMG

Question 20

bilirubin transport

• into hepatocytes
• into bile canaliculi
• efflux from hepatocytes
• reuptake by hepatocytes from circ

Answer 20

influx

• unconj bilirubin into hepatocytes: OATP1B1
  
  • acted on by GST-B to prevent auto-efflux
• reabs conj bilirubin, into hepatocyltes: OATP1B1, OATP1B3

efflux

• into bile canaliculi: MRP2
• sinusoidal efflux: MRP3 [uses ATP]

OATP = organic anion transporting polypeptide

Question 21

jaundice overview

Answer 21

French jaune = yellow

= icterus = hyperbilirubinemia

• consequence of malfunction in heme degradation
• not a disease!!! indicator that something else might be wrong
  
  • ​can be a serious problem in newborns

Question 22

jaundice

• clinical features
• biochemical features

Answer 22

clinical features

• yellowing of skin and eyes
  
  • due to affinity of elastin for bilirubin
  • reason why you DO NOT see yellowing of eyes in hyperbetacarotenemia! (sclera x carotene)

biochemical features

• accumulation of bilirubin in hydrophobic compartments of periph tissues
  
  • usually in cytoplasmic membranes
• bilirubin is harmless in older children and adults, not in newborns
  
  • ​has neurotoxic props in newborns, leads to KERNICTERUS

Question 23

causes of jaundice

Answer 23

prehepatic

• hemolysis : autoimmune or abnormal Hb : rare

intrahepatic

• infection
• chemicals/drugs
• genetic errors : bilirubin metab [4 syndromes]
• genetic errors : specific proteins (Wilson’s disease/Cu toxicity)
• autoimmune : chronic active hepatisis
• neonatal : physiologic : common

posthepatic

• intrahepatic bile ducts
• posthepatic bile ducts

Question 24

physiological jaundice

Answer 24

newborns achieve adult levels of UDP-GT in approx 1 month

• premature babies have less to begin with, so it takes them longer to catch up
  
  • buildup of unconjugated/indirect bilirubin in the catchup period is normal for all babies, but if a baby starts with a lower stock of UDP-GT, the increased buildup can reach dangerous levels

Question 25

kernicterus

Answer 25

condition in newborns in which bilirubin accumulates in parts of the brain (kern = nucleus), causes neurotoxicity **yellow-staining of deep nuclei of brain** _symptoms_ * early * extreme jaundice * absent startle reflex * poor feeding/sucking * extreme lethargy * midstage * high pitched cry * arched back, neck hyperextended * bulging fontanel * seizures * late state (full neurological syndrome) * high freq hearing loss * intellectual disability * muscle rigidity * speech difficulty * seizures * movement disorder

Question 26

treatment for neonatal jaundice

Answer 26

bilirubin builds up in part bc it's so insoluble: making it more soluble would allow it to be excreted in urine **phototherapy** (blue light oxidizes bilirubin, makes it into more soluble products: _yellow photoisomers_ and _colorless oxidation pdts)_ * photoisomers → excreted in bile (main pathway) * oxidation products → excreted in urine (minor pathway)

Question 27

intrahepatic causes of jaundice due to genetic errors in bilirubin metabolism

Answer 27

1. Rotor's syndrome * absence of OATP1B1, OATP1B3 (influx of reabs conj bilirubin) 2. Gilbert's syndrome [1 in 20] * defect in UDP-GT : normal protein, but less of it 3. Crigler-Najjar syndrome * defect in UDP-GT : either _no protein_ or _less protein_ 4. Dubin-Johnson syndrome * absence of MRP2 (efflux of conj bilirubin from hepatocyte into bile canaliculus)

Question 28

Criggler-Najjar syndrome Type 1

Answer 28

* complete absence of UDP-GT gene * no enzyme activity * **jaundice:** severe, causes kernicterus

Question 29

Criggler-Najjar syndrome Type 2

Answer 29

* mutation in UDP-GT gene * some enzyme activity * **jaundice:** benign

Question 30

Dubin-Johnson syndrome

Answer 30

* mutation in MRP2 * diffuse deposition of course, dark-brown granular pigment in hepatocytes * some enzyme activity * **jaundice:** only symptom \*appears during adolescence/early childhood \*more common in Iranians, Morrocan Jews of Israel

Question 31

Rotor syndrome

Answer 31

* biallelic inactivation mutation of OATP1B1, OATP1B3 * no enzyme activity * **jaundice:** intermittent; mild conjugated/unconjugated jaundice \*appears shortly after birth/in childhood

Question 32

Gilbert syndrome

Answer 32

* mutation in promoter of UDP-GT gene → reduced transcription * some enzyme activity * **jaundice:** mild, with physiologic stress \*appears during/after adolescence \*common (2-10% of pop)

Question 33

types of jaundice overview

Answer 33

* **prehepatic:** hemolytic * **intrahepatic** * neonatal * hepatocellular * **posthepatic** * **​**obstructive

Question 34

hemolytic jaundice

Answer 34

prehepatic * massive hemolysis : heme released in quantities that overwhelm liver's capacity to degrade it * see **increased indirect and increased direct bilirubin** * buildup in blood and periph tissues

Question 35

neonatal jaundice

Answer 35

* esp in premature infants * low levels of hepatic UDP-GT at birth * low levels of GST-B * **increased levels of indirect bilirubin**

Question 36

obstructive jaundice

Answer 36

* obstruction of bile duct, prevents passage of conjugated bilirubin into intestines * feces have characteristic pale clay color due to absence of stercobilin

Question 37

hepatocellular jaundice

Answer 37

more complicated * damage to liver cells (via hepatitis, liver disease, alcohol) leading to decrease in normal fx * cant handle even normal loads * both liver uptake and conjugation can be affected * **increased unconjugated bilirubin**

Question 38

liver function tests: AST, ALT, alkaline phosphatase

Answer 38

AST/ALT will usually work in tandem BUT * ALT \> AST in viral hep * AST \> ALT in alcoholic hep alk phos highest levels in biliary obstruction, infiltrative liver disease

Question 39

**do bilirubin wrapup questions**