HEME DEGRADATION AND JAUNDICE

xiaoli

Molecular Biochemistry II

RBCs last 120 days, degraded by reticuloendothelial (RE) system

most heme from RBCs (85%) - rest fro

m turnover of cytochromes, p450s, immatu

re erythrocytes

Under physiologic conditions in the human adult, 1~2×108 erythrocytes are destroyed per hour. Thus, in 1 day, a 70kg human turns over approximately 6g of hemoglobin.

When hemoglobin is destroyed in th

e body, globin is degraded to its cons

tituent amino acids, which are reused,

and the iron of heme enters the iron p

ool, also for reuse.

globin

Heme

Amino acids

iron + Bile pigments

iron Bile pigments

Bile pigments

Any of several coloured compounds der

ived from porphyrin that are found in bile;

principally bilirubin and biliverdin.

Bilirubin MetabolismBilirubin formation

Transport of bilirubin in plasma

Hepatic bilirubin transport Hepatic uptake

Conjugation

Biliary excretion

Excrect through intestine system

microsomalcytosol

Bilirubin formation

The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells of the liver, spleen, and bone marrow.

Heme oxygenase

Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. This produces biliverdin, iron, and carbon monoxide.

The first step

Heme oxygenase

There are three known isoforms of heme oxygenase. Heme oxygenase 1 (HO-1) is an inducible isoform in resp

onse to stress such as oxidative stress, hypoxia, heavy metals,

cytokines, etc. Its activity is induced by its substrate heme an

d by various nonheme substances.

Heme oxygenase 2 (HO-2) is a constitutive isoform which

is expressed under homeostatic conditions. Both HO-1 and H

O-2 are ubiquitously expressed and catalytically active.

A third heme oxygenase (HO-3) is not catalytically active,

but is thought to work in oxygen sensing.

In mammalian cells Heme oxygenase (HO1) has two basic functions: 1. it recycles iron supplies within the cell to maintain homeostasis. 2. biliverdin and biliruben (its reduced form), are powerful antioxidants believed to aid in the prevention of oxidative cell damage.

CH2

CH2

HOOC

NH

CHONH

CH2 NH

CHNH

O

M M M MV VCH2

CH2

COOH M： -CH2

V： -CH=CH2

Bilirubin is lipophilic and therefore insoluble in aqueous solution.the solubility in water is less.

The building of intermolecular hydrogen bonds by the NH and COOH groups is spatially hided.

Albumin + free Bilirubn Bilirubin ~ Albumin Complex

Bilirubin on release from macrophages circulates as unconjugated bilirubin in plasma tightly bound to albumin.

Transport of Bilirubin in Plasma

Why bound to albumin?

Significance:

★Increase the solubility of whole molecule

★ Prevent unconjugated bilirubin freely come into other tissue, cause damage.

unconjugated bilirubin

Transport of Bilirubin in Plasma

can be replaced byOther organic anions

PH UB

MolarRatio

BilirubinH affinity binding sites

2:1

Bilirubin

Plasma proteinAlbumin

L affinity binding sites>2:1

Albumin has two binding sites for bilirubin---a high affinity site and a low affinity site.

Albumin per 1L of plasma can bind about 342 ～ 427.5μmol bilirubin, but 1.7 ～ 17.1μ bilirubin per 1L .

blood

[bilirubin-albumin]

Other antibiotics [albumin]↑

[bilirubin]

membrance cytosol

[bilirubin-lipids]

[bilirubin]Certain drugs as sulfonamides and salicylates compete with bilirubin for albumin binding and displace bilirubin to enter into the brain in neonates and increase the risk of kernicterus (a type of brain damage that can result from high levels of bilirubin in a baby’s blood. It can cause athetoid cerebral palsy and hearing loss) .

Hepatic phase

On coming in contact with the hepatocyte surface, unconjugated bilirubin is preferentially metabolized which involved 3 steps:

Hepatic uptake

Conjuation

Secretion in bile

Hepatic uptake of BilirubinBilirubin ~ Albumin Complex

Albumin

Bilirubin(lipid soluble)

carrier proteinBilirubin

transfer

ER

ligation (Y protein, Z protein)

(be) taken up by membrane of the liver

(be) bound to

Conjugation of bilirubin

Conjugation occur in endoplasmic reticulum UDP-glucuronosyl transferase, Product: mono or diglucuronides.

The process of conjugation can be induced by drugs phenobarbital.

Bilirubin +UDP-gluconic acidBilirubin monoglucuronides + UDP

UDPGT

UDPGT

Bilirubin diglucuronide + UDP

UDP-gluconic acid

CH2

CH2

HOOC

NH

CHONH

CH

2NH

CHNH

O

M M M MV VCH2

CH2

COOH

H H H

CH2

CH2

CO

NCHO

NCH2 N

H

CHN

O

M M M MV VCH

2

CH2

COO O

OH

HO

COOH

OH

OOH

OHHOOC

OHO bilirubin diglucuronides

conjugated bilirubin

★Increase the solubility of whole molecule

★ delete the toxicity of bilirubin.

Unconjugated bilirubin:

Bilirubin that are not conjugated with gluconic acid , also called hemobilirubin, indirected bilirubin.

conjugated bilirubin:

Bilirubin that are conjugated with gluconic acid, also called hepatic bilirubin, directed bilirubin.

Major differences between unconjugated and conjugated bilirubinFEATURE Unconjugated biliru

binCONJUGATED BILIRUBIN

Normal serum level More Less (less than 0.25mg/dl)

Water solubility Absent Present

Affinity to lipids (alcohol solubilty)

Present Absent

Serum albumin binding High Low

Van den Bergh reaction Indirect (Total minus direct)

Direct

Reanal excretion Absent Present

Affinity to brain tissue Present (kernicterus) Absent

Excretion of bilirubin into bile

Bilirubin diglucuronide is actively transported against a concentration gradient into bile duct.

This energy-dependent, rate –limiting step is susceptible to impairment in liver disease. Uncojugated bilirubin is normally not excreated.

Formation of urobilins in the intestine Bilirubin diglucuronide is hydrolysed and reduced by

bacteria in the gut to yield urobilinogen, a colorless compound.

Most of the urobilinogens of the feces are oxidized by intestinal bacteria to stercobilin, which gives stools their characteristic brown color.

Some urobilinogen is reabsorbed from the gut into the portal blood and transported to the kidney, where it is converted to the yellow urobilin and excreted, giving urine its characteristic color. (bilinogen enterohepatic circulation)

Bilirubin diglucuronide urobilinogen

urobilin

stercobilinbilin

Portal vein

10 ～ 20%

reabsorbed

bilinogen

Sterobilin excrection

80% ～ 90%

bilinogen

prototype

Conjugated bilirubin

bilinogen enterohepatic circulation

0.5 ～ 4mg/day

blood

urobilin

BLOOD CELLS

LIVER

Bilirubin diglucuronide(water-soluble)

2 UDP-glucuronic acid

via bile duct to intestines

Stercobilin excreted in feces

Urobilinogen formed by bacteria

KIDNEY

Urobilin excreted in urine

CO

Biliverdin IX

Heme oxygenase

O2

Bilirubin (water-insoluble)

NADP+

NADPH

Biliverdinreductase

HemeGlobin

Hemoglobin

reabsorbed into blood

Bilirubin (water-insoluble)via blood

to the liver

INTESTINE

Catabolism of hemoglobin

unconjugated

Summary of bilirubin metabolism Senescent red cells are major source of hemeproteins

Breakdown of heme to bilirubin occur in macrophage of reticuloendithelial system ( tissue macrophages, spleen and liver).

Unconjugated bilirubin is transported through blood ( complex to albumin) to liver.

Bilirubin is taken into liver and conjugate with glucuronic acid.

Bile is secreted into intestine where glucuronic acid is removed and the resulting bilirubin is converted to urobilinogen.

A portion of urobilinogen is reabsorbed into blood, where it is converted to the yellow urobilin and excreted by kidneys.

Urobilinogen is oxidized by intestinal bacteria to the brown stercobilin.

＊ normal range of bilirubin:

1 ～ 16mol/l (0.1 ～ 1mg/dl)

4/5 are unconjugated bilirubin, others are conjugated bilirubin.

<1mg/dl normal1-2mg/dl occult>2mg/ dl jaundice Hyperbilirubinemia

Occult: the concentration of blood bilirubin are increased , but have no clinic sympotom, normally 1-2mg/dl.

Jaundice : ( also called icterus) refers to the yellow color of the sk

in and scleare caused by deposition of bilirubin, secondry to inc

reased bilirubin levels in the blood.

Although not a disease itself, jaundice is usually a symptom

of an underlying disorder.

Hyperbilirubinemia: the concentration of blood bilirubin are more than 1mg/dl.

Based on pathophysiology, jaundice may result fro

m one or more of the following mechanism:1. Increased bilirubin production ( excessive red cell destruct

ion)

2. Decreased hepatic uptake ( ligandin, drug, prolonged star

vation, and sepsis)

Decreased hepatic conjugation (enzyme,drugs, cirrhossis)

3. Decreeased excretion of bilirubin into bile ( gallstone, tum

our)

Simple Classification of jaundice

Accordingly, a simple classification of jaundice is to divided into 3 predominant type:

① Pre-hepatic (hemolytic jaundice)

② Hepatic jaundice

③ Post – hepatic cholestatic (obstructive jaundice)

Hemolytic jaundice

massive lysis of red blood cells (for example, in patients with sickel cell anemia or malaria) may produce bilirubin faster than the liver can conjuagte it. More bilirubin is excreted into the bile, the amount of the urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased. Unconjugated bilirubin is elevated in blood.

Causes of hemolytic jaundice Malaria Side effects of certain drugs :antibiotic and anti-tuberculosis medicines, levo

dopa, Certain drugs in combination with a hereditary enzyme deficiency known as

glucose-6-phosphate dehydrogenase (G6PD) Poisons Snake and spider venom, certain bacterial toxins, copper, and som

e organic industrial chemicals directly attack the membranes of red blood cells

Artificial heart valves Hereditary RBC disorders sickle cell disease Enlargement of the spleen Diseases of the small blood vessels Immune reactions to RBCs cancer Transfusions Kidney failure and other serious diseases Erythroblastosis fetalis

Hepatocellular jaundice Damage to liver cells( for example in patient with cir

rhosis or hepatitis) causes a decrease in both bilirubin uptake and production of conjuagted bilirubin.

Unconjugated bilirubin occur in the blood and increased urobilinogen in the urine.

The urine is dark in color and stool are pale, clay color.

Plasma level of AST and ALT are elevated and the patient experience nausea and anorexia.

Obstructive jaundice

In this instance jaundice is results from obstruction of the bile duct.

For example, the presence of a hepatic tumor or bile stone may block the bile ducts, preventing passage of bilirubin into the intestine, patients with obstructive jaundice experience GI pain, nausea and produce stools that are a pale, clay color.

65 year-old lady presenting with obstructive jaundice

Biliary stones

Sample Indices Normal Obstructive Jaundice

Hemolytic Jaundice

Hepatic Jaundice

Serum Total Bil ＜ 1mg/dl ＞ 1mg/dl ＞ 1mg/dl ＞ 1mg/dl

Direct Bil 0 ～ 0.8mg/dl

↑↑ ↑

Indirect Bil ＜ 1mg/dl ↑↑

Urine Color normal deep deeper deep

Bilirubin — ＋＋ — ＋＋

Urobilinogen a 　 little ↓ ↑ uncertain

Urobilin a 　 little ↓ ↑ uncertain

Stool Color normal Argilous (complete obstr

uction)

deeper lighter or normal

Diagnoses of Jaundice