Jaundice

JAUNDICE GROUP 8 Sheila Santos, Louissainne Sison Rie Schnell Jayvee Sumera Jessah Tamayo

LEARNING OBJECTIVES 1. Heme and bilirubin metabolism 2. Jaundice and the different types and causes of Jaundice (including genetic disorders) 3. Occurrence of jaundice in newborn, hemolytic anemia, polycythemia. Blood extravasation, abnormal coagulation, ingestion of breast milk, obstruction of the biliary tract 4. Catabolism of heme 5. The different phases of bilirubin metabolism 6. The role of proteins (albumin, ligandins, etc.) in bilirubin metabolism 7. The difference between unconjugated and conjugated metabolism

OVERVIEW OF THE TOPIC The biochemistry of the topics porphyrins and of the bile pigments are closely related to one another. Heme is synthesized from porphyrins and the iron, and the products of degradation of heme are the bile pigments and iron. Jaundice is a consequence of an elevated level of plasma bilirubin, due to either to overproduction of bilirubin or to failure of its excretion. Jaundice occurs in numerous diseases ranging from hemolytic anemias to viral hepatitis and to cancer of the pancreas (Rodwell et al., 2015).

HEME ● prosthetic group of hemoglobin and cytochromes, that is consists of an iron atom contained in the center of a large heterocyclic organic ring called porphyrin ● Porphyrins ● clinically significant; associated with a number of genetic diseases with deficiencies of the enzymes used in its biosynthesis and catabolism

HEME

BIOSYNTHESIS OF HEME ● Sites of heme biosynthesis ○ Occurs in most mammalian cells, except in mature erythrocytes (85% in erythroid precursors in BM) ○ Hepatocytes ● Initial reaction and the last three steps in the synthesis occur in mitochondria, whereas the intermediate steps occur in the cytosol (cytosolic and mitochondrial event)

BIOSYNTHESIS OF HEME

Succinyl- CoA + Glycine

δ-aminolevulinate

Porphobilinogen

Heme

Hydroxymethylbilane

Uroporphyrinogen III

Protoporphyrin III Protoporphyrinogen IX

Mitochondria

Coproporphyrinogen III

Cytosol

Biosynthesis  of  Heme Succinyl-CoA + Glycine CoA-SH CO2

δ-­aminolevulinate synthase

Δ-Aminolevulinate 2 H2O

δ-­aminolevulinate dehydratase

Porphobilinogen 4 NH3

Hydroxymethylbilane synthase

Hydroxymethylbilane H2O

Uroporphyrinogen III  s ynthase

Uroporphyrinogen III Uroporphyrinogen decarboxylase

4 CO2

Coproporphyrinogen III Coproporphyrinogen oxidase

2 CO2 2 H2O

Protoporphyrinogen III 3 H2O2

Protoporphyrinogen oxidase

Protoporphyrin III 2 H+

Ferrochelatase

Heme

Succinyl-CoA + glycine → δ-aminolevulinate + CoA-SH + CO2 ● Catalyzed by the mitochondrial δaminolevulinate synthase (ALA synthase) ● Pyridoxal phosphate-dependent reaction - Humans express two isozymes of ALA synthase: ALAS1 and ALAS2 2 δ-aminolevulinate → porphobilinogen + 2 H20 ● Catalyzed by cytosolic dehydratase

4 Porphobilinogen + H20 → hydroxymethylbilane + 4NH3 ● Catalyzed by cytosolic hydroxymethylbilane synthase ● Involves head-to-tail condensation of four molecules of porphobilinogen to form the linear tetrapyrole hydroxymethylbilane Hydroxymethylbilane → Uroporphyrinogen III + H20 ● Cyclization of hydroxymethylbilane ● Catalyzed by uroporphyrinogen III synthase

Uroporphyrinogen III → coproporphyrinogen III + 4 CO2 ● Side chains modified by the enzyme Uroporphyrinogen decarboxylase Side chains of uroporphyrinogen are Propionate and Acetate

Coproporphyrinogen III + O2 + 2 H+ → Protoporphyrinogen III + 2 CO2 + 2 H20 ● Coproporphyrinogen III enters mitochondria to protoporphyrinogen III ● Catalyzed by coproporphyrinogen oxidase

Protoporphyrinogen III + 3 O2 → protoporphyrin III + 3 H2O2 ● Oxidation to protoporphyrin III catalyzed by protoporphyrinogen oxidase Protoporphyrin III + Fe3+ → Heme + 2 H+ ● Final step ● Incorporation of ferrous iron and is

catalyzed by ferrochelatase

● 200 billion erythrocytes per day are destroyed in human adults; approximately 6 g of hemoglobin ● They are removed from the circulation by the reticuloendothelial cells of the liver, spleen and bone marrow. ● The red blood cells are hemolyzed and hemoglobin comes outglobin and heme molecules ● Globin is hydrolyzed into a free amino acid while heme gives out iron and bilirubin

COMPONENTS OF HEMOGLOBIN globin amino acids recycled iron recovered and reused heme bilirubin

HEMOGLOBIN It is the iron-containing chemical in red blood cells that carries oxygen, is released from the destroyed red blood cells after the iron it contains is removed. The chemical that remains in the blood after the iron is removed becomes bilirubin

HEME CATABOLISM PRODUCES… BILIRUBIN ●Bilirubin comes from red blood cells ●One of the liver's functions is to remove toxic chemicals or waste products from the blood, and bilirubin is a waste product. The liver removes bilirubin from the blood ○ Conjugated bilirubin vs. unconjugated bilirubin

STAGES OF HEME CATABOLISM ●Formation of bilirubin ●Uptake of bilirubin by the liver ●Conjugation of bilirubin with glucuronate ●Secretion of bilirubin to bile ●Urobilin formation in intestines ●Urobilinogen excretion in stool and urine

STAGES OF HEME CATABOLISM A. FORMATION OF BILIRUBIN ● The catabolism of heme from all heme proteins is carried out in the microsomal fraction of cells by heme oxygenase ● Heme serves as both as a substrate and as cofactor for the reaction ● Iron oxidized to its ferric form (hemin)

STAGES OF HEME CATABOLISM Fe3+-Heme + 3 O2 + 7 electrons → Biliverdin + CO + Fe - Bilverdin: green pigment, is reduced forming the red-orange bilirubin - Heme reductase Biliverdin + NADPH + H+ → Bilirubin + NADP+ - Catalyzed by Biliverdin reductase

STAGES OF HEME CATABOLISM Hemoglobin

HEME

Macrophage Heme oxygenase

NADPH + O2 NADP+ + Fe3+ + CO

BILIVERDIN NADPH NADP+

Biliverdin reductase

BILIRUBIN

globin

amino acids

STAGES OF HEME CATABOLISM B. UPTAKE OF BILIRUBIN BY THE LIVER ● Bilirubin is transported to the liver by noncovalently binding to albumin. Then the bilirubin dissociates from the carrier albumin molecule and enters a hepatocyte via facilitated diffusion. It binds to intracellular proteins, particularly the protein ligandin (Ferrier et al., 2014).

STAGES OF HEME CATABOLISM C. CONJUGATION OF BILIRUBIN WITH GLUCORONATE ● Bilirubin is conjugated with one or two molecules of glucuronic acid forming bilirubin monoglucuronide and bilirubin diglucuronide ● Catalyzed by bilirubin endoplasmic reticulum

specific UDP-glucosyl

transferase

of

the

Bilirubin + UDP-glucuronate → bilirubin monoglucuronide + UDP Bilirubin monoglucuronide + UDP-glucuronate → bilirubin diglucuronide + UDP

STAGES OF HEME CATABOLISM D. SECRETION OF BILIRUBIN TO BILE ● Bilirubin diglucuronide is actively transported against concentration gradient into the bile canaliculi and then into the bile ○ rate -limiting process of hepatic bilirubin metabolism ○ susceptible to impairment in liver disease ○ MOAT: multi-specific organic anion transporter

STAGES OF HEME CATABOLISM E. UROBILIN FORMATION IN INTESTINES ● Intestinal bacteria act on bilirubin diglucoronide leading to: ○ Removal of glucoronides ○ Reduction to colorless compounds called urobilinogen ● A small fraction of urobilinogen is reabsorbed, goes back to the liver and re-excreted in the bile

STAGES OF HEME CATABOLISM F. UROBILINOGEN EXCRETION IN STOOL AND URINE ● Stercobilin gives the stool its brown color ● Urobilin with urochrome gives the urine its yellow color

From the stages of heme catabolism, the different phases of bilirubin metabolism can be summarized to:

Blood

Uptake of Bilirubin + Albumin

Hepatocyte

Conjugation of Bilirubin to Bilirubin Diglucuronide

Bile duct

Secretion of Bilirubin diglucuronide

ROLES OF PROTEIN IN BILIRUBIN METABOLISM • Albumin and ligands: carriers for transport • Albumin: carrier of free bilirubin to liver – hepatocytes (via portal system) o Trans form - insoluble in water (bound to protein) • Y and Z proteins and Ligandins o Bilirubin binds with Y and Z and then to ligandin (transported to SER for further metabolism and bilirubin acts as substrate for enzyme Glucirinyl Transferase)

UNCONJUGATED BILIRUBIN • Tetrapyrrole, lipid soluble and toxic o Conversion o Binds to albumin for transport o Unconjugated bilirubin is bound to hydrophilic acceptors in liver

CONJUGATED BILIRUBIN • Uridine diphosphate (UDP) glucuronyl transferase TO glucuronides or conjugated bilirubin • Polar • Water soluble excreted easier in bile by ATP dependent transporter

UNCONJUGATED BILIRUBIN

CONJUGATED BILIRUBIN

Present normally in the plasma

Present normally in the bile

Attached non-covalently to albumin

Conjugated to glucuronic acid

Has high molecular weight and cannot be filtered through the kidney

Has smaller molecular weight and cannot be filtered through the kidney

Non-polar, insoluble in plasma and can cross the blood-brain barrier in neonates causing brain damage

Polar, insoluble in plasma and cannot cross the blood brain barrier

JAUNDICE • Icterus • Sign rather than a disease, causes yellow discoloration of the skin and the sclera • The yellow discoloration of the skin and sclera is caused by the elevated level of bilirubin in the blood, hyperbilirubinemia • Exceeding 1.5mg/dL (Normal value: 0.5mg/dL)

CATEGORY Pre-hepatic/Hemolytic Jaundice

CAUSES Bilirubin lysed from RBCs

Hepatic/Hepatocellular Liver defect Jaundice Posthepatic/Obstructive Jaundice

Obstruction of bile duct

PRE-HEPATIC/HEMOLYTIC JAUNDICE • Liver capacity to conjugate: 3,000 mg/day • Normal bilirubin production: 300mg/day • Massive lysis or RBC produces bilirubin faster than conjugated = inc. unconjugated bilirubin levels = jaundice • Urobilinogen entering enterohepatic circulation is increased = urinary urobilinogen is increased • E.g. Hemolytic anemia due to malaria

PRE-HEPATIC/HEMOLYTIC JAUNDICE • Urine: absent bilirubin • Serum: Increased unconjugated bilirubin • Kernicterus

HEPATIC/HEPATOCELLULAR JAUNDICE • Damage to liver cells • Dec. conjugation = inc. unconjugated bilirubin • Conjugated bilirubin not efficiently secreted to bile, rather it “leaks“ into blood • Urobilinogen increased in urine • Urine is dark; stools are pale clay color • Hepatic damage decrease enterohepatic circulation = enter blood & filtered into urine

HEPATIC/HEPATOCELLULAR JAUNDICE • E.g. Acute hepatitis, hepatotoxicity and alcoholic liver disease o Liver metabolism decreased = increased bilirubin in bloo • Less common causes o Gilbet‘s syndrome, Crigler-Najjar syndrome, metastatic carcinoma and Niemann-Pick disease

POST-HEPATIC JAUNDICE/OBSTRUCTIVE JAUNDICE • Obstruction of bile duct o Gallstones – most common o Pancreatic CA – head of pancreas o Liver flukes – parasites in CBD

• GI pain, nausea, pale clay-colored stools, dark urine, pruritus (severe itching), elevated serum cholesterol • Regurgitates conjugated liver into blood (hyperbilirubinemia) and excreted in urine

POST-HEPATIC JAUNDICE/OBSTRUCTIVE JAUNDICE • Others:

o Bile duct, biliary atresia, ductal carcinoma, pancreatitis, pancreatic pseudocysts o Rare: Mirizzi’s syndrome

• Combination of liver tests to diagnose or differentiate classifications of jaundice

Choluric vs Acholuric Jaundice 1. Choluric Jaundice: there is presence of bile derivatives and occurs in regurgitation hyperbilirubinemia; obstructive type 2. Acholuric Jaundice: absence of bile in the urine and occurs in retention hyperbilirubinemia; hemolytic type

RECALL: UNCONJUGATED vs CONJUGATED BILIRUBIN Unconjugated Bilirubin

Conjugated Bilirubin

Free bilirubin

Water soluble

Fat-soluble waste product

moves through the bile duct to the intestines as bile salts

Attaches to albumin to travel the liver where it reacts with an enzyme glucuronyl transferase to become water soluble, conjugated bilirubin

Converted to urobilinogens which are excreted from the body

UNCONJUGATED BILIRUBIN UNCONJUGATED HYPERBILIRUBINEMIA ● Condition that results from a high level of unconjugated form of bilirubin in the blood ● Caused by several factors: ○ Increased production of bilirubin ○ Decreased in the conjugation which is a result of an absence of decrease in glucuronyl transferase

CONJUGATED BILIRUBIN CONDITIONS INVOLVING CONJUGATED HYPERBILIRUBINEMIA Conjugated hyperbilirubinemia: high level of conjugated bilirubin and is usually caused by inefficiency in the excretion process A. Hereditary transferase conjugation deficiency: ● Gilbert Syndrome (Milk Transferase Deficiency) ○ hepatic UDP-glucuronosyl transferase activity is consistently decreased ○ affected individuals may have no symptoms but may have mild icterus and jaundice

● Crigler-Najjar Syndrome Type 1 (Absence of Transferase) ○ Hepatic bilirubin UDP-glucuronosyl transferase activity is undetectable ○ No conjugated bilirubin is formed and the bile is colorless ○ Unconjugated bilirubin will accumulate ○ Fatal ● Crigler-Najjar Syndrome Type II (Moderate Transferase Deficiency) ○ reduction of hepatic bilirubin UGT activity is incomplete

IMPAIRED HEPARIC EXCRETION (INTRAHEPATIC DEFECTS) A. Familial/ Hereditary Disorders ● Dubin-Johnson Syndrome ○ Interferes with the body’s ability to move bilirubin from the liver ○ When bilirubin is not properly processed, it builds up in the bloodstream and causes jaundice

IMPAIRED HEPARIC EXCRETION (INTRAHEPATIC DEFECTS) ● Rotor Syndrome ○ belongs to a family of disorders that result as a consequence of defects in the metabolism/ excretion of bilirubin ○ exact molecular defect(s) resulting in Rotor Syndrome is not known ● Recurrent ( Benign) intrahepatic cholestasis ● Cholesterol jaundice of pregnancy

IMPAIRED HEPARIC EXCRETION (INTRAHEPATIC DEFECTS) B. Acquired Disorders ● Hepatocellular Disease ●

Drug-induced cholestasis

● Alcoholic liver disease ● Sepsis ● Post-operative state ● Parenteral nutrition ● Bilateral cirrhosis

IMPAIRED HEPARIC EXCRETION (EXTRAHEPATIC DEFECTS) A. Intraductal Obstruction

B. Compression of Biliary ducts

● Gallstones

● Malignancy

● Biliary malformation

● Inflammation (e.g.pancreatitis

● Infection ● Malignancy ● Hemobilia( trauma, tumor) ● Sclerosing cholangitis

OCCURRENCE OF JAUNDICE Physiologic Jaundice of the Newborns

●

○

Immature Liver leading to insufficient Bilirubin conjugation

○

Normal to a certain degree

○

Kernicterus

Causes ○

Increase bilirubin load on the hepatocytes

○

Defective uptake

○

Defective bilirubin conjugation

○

Decrease rate of bilirubin excretion

OCCURRENCE OF JAUNDICE HEMOLYTIC ANEMIA • Increase rate of erythrocytes destruction whether intravascularly or extravascularly. • RBC Destruction is greater than RBC Production leading to Anemia • Accumulation of Bilirubin from increase RBC destruction leads to Jaundice Causes • Immune regulated; antibodies towards RBCs • Hyperactivity of the Spleen (Hypersplenism) • Trauma to erythrocytes

OCCURRENCE OF JAUNDICE POLYCYTHEMIA VERA ● Pancytosis; uncontrolled Production of Cells ● Increase rate of destruction within the Spleen, decrease life span of RBCs <120 days ● Bilirubin is produced from increased hemolysis ● Symptoms are seen more in individuals with pre existing hepatic disorders

OCCURRENCE OF JAUNDICE BLOOD EXTRAVASATION ● Escape of blood from blood vessels towards the adjacent tissues ● Seen in burn patients and hypersensitive reactions a. Cephalohematoma breakdown in neonates lead to the production of bilirubin, and without a fully mature liver jaundice occurrence b. Ingestion of blood by neonates lead to production of bilirubin as well c. Jaundice is significantly seen in individuals with preexisting hepatic disorders (cirrhosis, hepatitis, etc.)

OCCURRENCE OF JAUNDICE BREAST MILK JAUNDICE ● Commonly seen in newborns Causes a. Presence of unusual progesterone metobolites inhibiting glucoronyl transferase b. Increase non-esterified fatty acids inhibiting UDPGA c. Minimum ammount of normal florand increae activity of beta glucorinidase

OCCURRENCE OF JAUNDICE BREAST MILK JAUNDICE e. Defect in gene coding f. Mutation in SLCO1B1; solute carrier g. Inflammatory cytokines h. High levels of Epidermal growth factor

OCCURRENCE OF JAUNDICE Breastfeeding Jaundice ● Physiologic jaundice of newborns which is manifested in the first life due to insufficient production or intake of breast milk

OCCURRENCE OF JAUNDICE Defective Conjugation of Bilirubin a. Crigler-Najar Syndrome - conjugation deficit; Increased unconjugated bilirubin (B1) b. Dubin- Johnson and Rotor Syndrome - bilirubin excretion deficit; Increased Total Bilirubin c. Gilbert’s Syndrome - bilirubin transport deficit; Increased unconjugated bilirubin (B1) d. Lucey- Driscoll Syndrome - familial form of hyperbilirubinemia due to circulating inhibitors; increase unconjugated bilirubin (B1)

OCCURRENCE OF JAUNDICE Biliary Obstruction ● Blockage within the bile duct restricting the transport of bile. ● Increase conjugated bilirubin

OCCURRENCE OF JAUNDICE Intrahepatic Jaundice ● Damage towards the hepatocytes compromising metabolism process Causes 1. Hepatocellular damage 2. Drug Induced (Hepatotoxic) 3. Cirrhosis 4. Alcoholic liver Disease

OCCURRENCE OF JAUNDICE Extrahepatic ( Posthepatic) ● Obstructing the excretion of bile ● Increase of conjugated bilirubin (B2) Causes a. Gallstones - most common b. Tumors/Malignancy c. Inflammation

SIGNS & SYMPTOMS PHYSICAL EXAMINATION: - Color of skin and sclera of the eyes : yellow discoloration of the sclerae and skin

SIGNS & SYMPTOMS PHYSICAL EXAMINATION: - abdominal examination: enlarged liver, enlarged spleen, ascites and palpable gallbladder - look for evidence of chronic liver disease: reddening of the palms, easy bruising, muscle wasting, testicular atrophy, enlargement of the breasts in male

DIAGNOSIS Blood tests ● Bilirubin, direct <0.3 mg/dL ● Bilirubin, total 0.2-1.3 mg/dL

DIAGNOSIS Urine Analysis and microscopy Radiologic investigations: ❏Upper abdominal ultrasound scan ❏CT scan of abdomen ❏Magnetic resonance imaging (MRI) ❏ERCP( Endoscopic retrograde cholangiopancreatography) ❏Liver biopsy

HOW IS JAUNDICE TREATED? ● The treatment of jaundice requires a diagnosis of the specific cause, e.g., removal of a gallstone blocking the bile duct. ● If the liver is found to be seriously damaged, a transplant may be an option.

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