Heme Synthesis and Degradation Flashcards
Hemoprotein functions
Oxygen transport (Hb, Mb)
Drug metabolism, steroid hormone synthesis, and metabolism of unsaturated FAs (eicosanoids)
P450 CYPs
Oxidative phosphorylation (cytochromes):
Succinate dehydrogenase (cytochrome b)
cytochromes b and c1
cytochrome c oxidase
Decomposition of hydrogen peroxide:Peroxidases and Catalase
Heme Synthesis
Location
A majority of heme is produced in a bone marrow (~85%) to form hemoglobin.
High steady-state level of heme synthesis depends on iron availability.
Heme is also produced in hepatocytes and o_ther cells that use cytochromes_ (~15%).
A biosynthesis is turned over rapidly to respond to the changing metabolic requirements.
Heme Synthesis
General Pathway
expression patterns of the enzymes
Synthesized from: Succinyl-CoA and Glycine
2nd, 3rd, and 4th enzymes have genes with dual promotor (erythroid specificity)
tissue-specific expression
The single genes for ALAD, PBGD and URO3S utilize tissue-specific promoters and alternative splicing to generate either identical proteins (ALAD, URO3S) or tissue-specific proteins (PBGD).
5 Processes of heme synthesis
- Formation of the pyrrole.
- Assembly of the tetrapyrrole (porphyrin).
- Modification of the porphyrin side chains.
- Oxidation of protoporphyrinogen IX to protoporphyrin IX .
- Insertion of iron to form a heme.
First rxn of heme synthesis
enzyme
Cofactor
Inhinition
1st reaction is the RLS
g-aminolevulinic acid synthase
pyridoxal phosphate
ALS is inhibited by heme
Location of the reactions
Heme synthesis
First and the last 3: Mitochondria
The remaining rxns: cytoplasm
Photosensitivity
accumulation of intermediates.(starting at hydroxymethylbilane)
ð-Aminolevulenic Acid synthesis
Starting compounds
enzyme (cofactor) & assc deficiency
Condensation of Succinyl-CoA and Glycine. ALA synthase (tissue spc. genes),requires pyridoxal phosphate (B6). Deficiency of vitamin B6 reduces heme synthesis and can cause sideroblastic anemia.
Genes for ALA synthase in humans
Housekeeping ALAS-1 is expressed throughout the body:
chromosome 3.
Its expression changes in response to changed demands for the heme.
Erythroid-specific ALAS-2 is expressed _in RBC precursor_s:
chromosome X.
Expression is induced only during active heme synthesis
ALAS-2 synthesis regulated by iron availability to prevent accumulation of porphyrin intermediates.
Regulation of ALA synthase activity
ALA synthase activity is regulated by the heme:
Heme acts as a negative allosteric regulator
High level of free heme → ALAS heme complex (too big to enter Mt) ► reducing heme synthesis.
Infusion of hematin can abort attacks of porphyria in patients with an inborn error of heme synthesis.
Drugs that are metabolized by Cyt P450 monooxygenase system enhance hepatic ALA synthase activity.
Regulation of ALAS-2 trascription
Transcription of ALAS-2 is controlled by Fe2+-binding element.
Prevents excessive formation of porphyrin intermediates in the absence or low levels of iron.
Hepatic ALA synthase & Cyt P450
Drugs that are metabolized by Cyt P450 monooxygenase system enhance hepatic ALA synthase activity.
An increased requirement for Cyt P450 monooxygenase system need an increased demands for heme and stimulates its synthesis.
Sideroblastic anemia
Sideroblasts are abnormal nucleated erythroblasts.
Mutations in ALAS2 gene is the most common congenital cause
Although it is X-linked disorder, almost half of patient are women because of skewed X-inactivation.
Treated with pyridoxine (cofactor of ALA synthase).
There are no diseases associated with ALAS1 gene mutations.
Most likely due to the importance of the heme for cellular survival.
Effects of fasting or low carb diet on PGC1-a
Glucagon and fasting induce PGC-1α synthesis because of the increased demand in cytochromes required for the production of ATP in the liver necessary for gluconeogenesis.
PGC-1α induces the synthesis of enzymes involved in
- FA oxidation
- Gluconeogenesis
- Heme synthesis (ALAS-1) because of the increased demands in cytochromes required for the production of ATP in the liver.
Insulin and glucose inhibit PGC-1α synthesis because of the decreased demand in cytochromes required for the production of ATP in the liver.
PGC-1a
PGC-1α, a coactivator of nuclear receptors and transcription factors, regulates mitochondrial biogenesis and oxidative metabolism.
It induces the synthesis of the genes invlolved in:
- FA ox
- Gluconeogenesis
- Heme synthesis (ALAS-1)
Synthesis of pyrrole ring
Pyrrole ring is formed by the condensation of two molecules of d-ALA. Catalyzed by d-ALA dehydratase (ALAD)
disorders associated with deficiency of ALAD and elevated levels of ð-ALA
There are three disorders associated with deficiency of ALAD and elevated levels of ð-ALA :
ALAD porphyria is due to mutations in ALAD gene.
Hepatorenal tyrosinemia can lead to the accumulation of succinylacetone, which is a potent competitive inhibitor of ALAD.
Lead poisoning leads to anemia and accumulation of d-ALA.
Lead inhibits ALAD activity by displacing zinc atoms from the enzyme.
The effects of elevated levels of ð-ALA on CNS
Elevated levels of d-ALA can have a neurotoxic affect on both central and peripheral nerve systems.
Memory loss and confusion
The final step of heme synthesis
Final step of heme synthesis is the introduction of Fe2+ into protoporphyrin IX.
Occurs spontaneously, but very slow.
The reaction is enhanced by ferrochelatase.
The enzyme is inhibited by lead (lead poisoning).
Deactivating mutations of ferrochelatase can lead to erythropoetic protoporphyria.
deactivating mutations of ferrochelatase
Deactivating mutations of ferrochelatase can lead to erythropoetic protoporphyria.
ferrochelatase enhances the introduction of Fe<strong>2</strong>+ into protoporphyrin IX.
A potent competetive inhibitor of ALAD
succinylacetone
Hepatorenal tyrosinemia can lead to the accumulation of succinylacetone, which is a potent competitive inhibitor of ALAD.
Lead poisoning and anemia
Lead inhibits ALAD activity (displaces Zn)
Lead poisoning leads to anemia and accumulation of ð-ALA.
Elevated levels of ð-ALA are neurotoxic: memory loss and confusuion
Synthesis of HMB
Hydroxymethylbilane (HMB) is produced by condensation of four porphobilinogen molecules.
Catalyzed by PBG-deaminase also known as HMB-synthase.
This is the first intermediate of heme synthesis pathway an accumulation of which will lead to light sensitivity.
The reaction catalyzed by PBG-deaminase
Hydroxymethylbilane (HMB) is produced by condensation of four porphobilinogen molecules.
PBG-deaminase also known as HMB-synthase.
This is the first intermediate of heme synthesis pathway an accumulation of which will lead to light sensitivity.
the first intermediate of heme synthesis pathway an accumulation of which will lead to light sensitivity
hydroxymethylbilane (HMB)
Iron functions in human
Iron content in human body:
Total 3-4 g (2,5 g in hemoglobin)
~2 g in ferritin complexes that are present in all cells
The main stores are: liver, bone marrow, and spleen.
Functions:
Transport of oxygen
Detoxification through cytochrome P450
Cofactor in iron-containing proteins
Heme iron:
Hemoglobin, myoglobin, cytochrom-c oxidase, and catalase.
Non-heme iron:
Fe-S complexes (xanthine oxidase), DNA synthesis (ribonucleotide reductase)
Hereditary hemochromatosis
Defects in proteins regulating iron absorption may lead to iron overload.
Iron content is regulated at the level of absorption.
Foods that facilitate iron absorption
Foods containing ascorbic acid like citrus fruits, broccoli & other dark green vegetables can facilitate iron absorpthion because ascorbic acid reduces iron from ferric to ferrous forms, which is absorbed in deodenum.
Inhibitors of iron absorption and Iron deficiency
High intake of inhibitors of iron absorption such as polyphenol compound found in certain foods:
Cereals like sorghum & oats.
Vegetables such as spinach and spices.
Beverages like tea, coffee, cocoa and wine.
Porphyrias
Most are resulted from the inherited defects in heme synthesis.
Enzyme deficiency can lead to accumulation of intermediate and increased excretion of porphyrins.
Most porphyrias are autosomal dominant disorders, excepts
x-linked dominant protoporphyria
X-linked dominant protoporphyria
X-linked dominant protoporphyria: caused by the gain of function mutation in ALAS2 gene.
A frameshift deletion affects last 19-20 C-terminus of ALAS enzyme, which significantly increases its activity.
Accumulation: free an Zn-protoporphyrin IX
causes: photosensitivity and liver damage.
ALA dyhydratase porphyria
recessive, caused by the deficiency of ALA dehydratase in RBC.
Sx: abd. pian NV
Congenital erythropoetic porphyria (gunther disease)
recessive, caused by deficiency of uroporphyrinogen III synthase in RBC
Affected enzyme: UROS
Accumulated products: Uroporphyrinogen I and Corpoporphyrinogene I
Symp: Photosensitiviy, abd. pain and NV
Classification of Porphyrias
Based on causes and location of the deficienct enzymes
erythropoietic or hepatic
Based on whether the enzyme deficiency is in bone marrow or in the liver.
Hepatic porphyrias can be acute and chronic.
Erythropoietic porphyrias are usually chronic.
Increased activity of ALA synthase
Treatment: IV injection of hemin (trade name Panhematin).
Tx of porphyrias caused by increased activity of ALA synthase
Treatment: IV injection of hemin
Acute Intermittent Porphyria
Most common form of acute (AD)
Causes by deficiency of porphobilinogen deaminase (PBGD).
Results in elevated levels of PBG and d-ALA in urine.
PBG in urine is oxidized to colored bright red colored porphobilin.
Used in diagnosis of acute intermittent porphyria during attack.
Does not have light sensitivity symptoms.
Congenital Erythropoietic Porphyria
Mode if inheritance
affected enzyme
accumulated product
symptoms
treatment
AR
Causes by deficiency of uroporphobilinogen III synthase (UROS).
Results in elevated levels of uroporphyrinogen I and corpoporphyrinogene I in urine.
Symptoms
Patients have severe cutaneous sensitivity is present in most.
Porphyrins also accumulate in the bone and teeth, resulting in erythrodontia.
Hemolytic anemia is another common symptom.
Treatment:
limit or completely eliminate sun exposure.
Bone marrow transplantation.
Porphyria Cutanea Tarda (PCT)
affected enzyme
accumulated product
inheritance pattern
symptoms
treatment
Caused by a deficiency of uroporphyrinogen decarboxylase (UROD).
Autosomal dominant.
Patients with PCT often develop photosensitivity in forms of blisters and erosions.
Cutaneous sensitivity is caused by accumulation of porphyrins under skin, which become phototoxic upon activation by light.
Treatment: avoidance of alcohol consumption, iron supplements, excess exposure to sunlight.
Sources of bilirubin
~85% produced from the breakdown of old RBCs.
~15% produced from other heme-containing enzymes, cytochromes, myoglobin, and immature RBCs.
Life span of RBCs in blood stream is 60-120 days.
Senescent RBCs are phagocytized and/or lysed.
Normally, RBCs are degraded extravascularly in the reticulo-endothelial system, typically the spleen.
Lysis can also occur intravascularly (in blood stream).
The first 3 reactions of Hb degradation
The steps in the subsequent metabolism of the hemoglobin are:
- Globulin fraction is removed from hemoglobin, leaving heme.
- Heme oxygenase: Heme——->biliverdin
- Biliverdin reductase: Biliverdin——> bilirubin
Transport of unconjucated bilirubbin
The unconjugated (water insoluble) bilirubin is transported from the RE system to the liver in complex with albumin.
Hb degradation at the level of hepatocytes
At the level of the hepatocyte, metabolism occurs in three stages:
- Transfer of bilirubin-albumin complex inside of hepatocyte and release of bilirubin.
- Transfer to intracellular binding proteins called ligandins, which transport bilirubin to the ER.
- Conjugation of bilirubin with glucuronic acid via glucuronyltransferase.
The resulting bilirubin diglucuronide is more polar and water soluble and therefore can now be secreted into the bile.
Heme degradation
In normal adults a daily load of bilirubin is 250-300 mg.
Normal plasma bilirubin concentrations are less then 1 mg/dL.
Bilirubin is hydrophobic – transported by albumin to the liver for further metabolism prior to its excretion.
Two glucuronate residues are transferred to bilirubin forming “conjugated bilirubin”.
More soluble conjugated bilirubin is transported into the bile for excretion.
N. bilirubin metabolism
Uptake of bilirubin by the liver
Conjugation of bilirubin with glucoronic acid
“Conjugated” bilirubin is secreted into the bile.
It is converted to urobilinogen (colorless) by bacteria in the gut, which is absorbed and excreted in urine as its oxidized form, urobilin (yellow).
Urobilinogen is oxidized to stercobilin (brown colored) and excreted in feces.
Heme degradation and Jaundice
Jaundice is the imbalance between production and excretion of bilirubin results in its increased plasma concentrations of (> 3 mg/dL).
It is characterized by the yellowish tingles of skin and eye.
Causes of Jaundice
Causes of neonatal jaundice
An excessive hemolysis due to trauma or infection.
A failure of liver to transport, store or conjugate bilirubin.
Deficiencies of enzymes involved in bilirubin metabolism.
Cirrhosis or hepatitis.
Neonatal jaundice is due to a combination of two factors:
Excessive intravascular hemolysis.
Underdeveloped liver.
Prehepatic (hemolytic) jaundice
Prehepatic jaundice can result from excessive hemolysis (beyond the livers ability to conjugate it).
Excessive RBC lysis can be a result of:
Autoimmune disease
Hemolytic disease of the newborn (Rh- or ABO- incompatibility)
Structurally abnormal RBCs (Sickle cell disease)
High plasma concentrations of unconjugated bilirubin (normal concentration ~1 mg/dL) is indicator of prehepatic (hemolytic) jaundice.
Interhepatic Jaundice
Intrahepatic jaundice can be caused by impaired uptake, conjugation, or secretion of bilirubin.
It reflects a generalized liver (hepatocyte) dysfunction.
In this case, hyperbilirubinemia is usually accompanied by other abnormalities in liver function.
Enlarged liver
Elevated plasma liver enzymes
Can be caused by viruses, toxins, or genetic errors.
Posthepatic Jaundice
Caused by an obstruction of the biliary tree.
Leads to Increased level of plasma conjugated bilirubin and other biliary metabolites ( bile acids).
Characterized by
Pale colored stools (absence of fecal stercobilin).
Dark urine is caused by an increased levels of conjugated bilirubin in urine.
Elevated plasma ALP levels
In a complete obstruction, urobilin is absent from the urine.
Can be caused by gallstones or tumors.
differential dx of Jaundice
Hyperbilirubinemias
Hyperbilirubinemias are inherited disorders in bilirubin metabolism associated with high plasma levels of unconjugated or conjugated bilirubin
Unconjugated hyperbilirubinemias
Unconjugated hyperbilirubinemias are usually more severe because of low solubility of unconjugated bilirubin.
They can be caused by:
Insufficient conjugation due to enzyme deficiency.
Excessive hemolysis due to trauma, infection or autoimmune disease.
conjugated hyperbilirubinemias
Conjugated hyperbilirubinemia are less severe because conjugated bilirubin is more soluble than unconjugated.
They can be caused by:
Liver disease - cirrhosis
Infection – hepatitis
Inherited disorders of bilirubin transport to the bile.
Gilber Syndrome
Gilbert Syndrome is a relatively mild genetic disorder.
It is characterized by periods of elevated levels of bilirubin in the blood (hyperbilirubinemia) and nonhemolytic jaundice.
It is caused by the l_ow activity of glucuronyltransferase_ (25-30% of normal).
Episodes are usually caused by dehydration, fasting or illness.
Usually is recognized in adolescence.
~30% of people with Gilbert syndrome have no signs or symptoms.
Gilbert Syndrome can show two different inheritance patterns:
Mutations in promoter region result in autosomal recessive inheritance.
Missense mutation in one of UGT1A1 gene results in autosomal dominant inheritance.
It is estimated that Gilbert Syndrome affects 3-7% of Americans.
Gilbert Syndrome Inheritance pattern
Gilbert Syndrome can show two different inheritance patterns:
Mutations in promoter region result in autosomal recessive inheritance.
Missense mutation in one of UGT1A1 gene results in autosomal dominant inheritance.
Cringer-Najjar syndrome
More severe than Gilbert syndrome and has even lower activity of glucuronyltransferase (0-10% of normal).
AR
It is caused by mutation in both UGT1A1 genes.
Extremely rare, with high incidence is in Amish and Mennonites.
The disease is characterized by complete absence or marked reduction in bilirubin conjugation.
A severe unconjugated hyperbilirubinemia that usually presents at birth.
Affected individuals are at a high risk for kernicterus, a damage to the brain centers of infants caused by increased levels of bilirubin.
It is fatal when the enzyme is completely absent.
Dubin-Johnson Syndrome
Impaired biliary secretion of conjugated bilirubin.
Presence of conjugated bilirubin in the blood.
Dubin-Johnson Syndrome results from mutations in the ABCC2 gene coding for bile canalicular multispecific organic anion transporter.
This transporter is involved in the excretion of many non-bile organic anions by an ATP-requiring process.
Lysosomal accumulation of pigment causes the liver to turn black.
It is inherited in an autosomal recessive pattern.
Rotor Syndrome
Impaired biliary secretion of conjugated bilirubin.
Presence of conjugated bilirubin in the blood.
Rotor Syndrome is symptomatically similar to DJS except that in Rotor Syndrome, the liver cells are not pigmented.
It caused by deficiency of anion transporter in hepatocytes cause by mutation in SLCO1B1 and SLCO1B3 genes that provide instructions for making organic anion transporting polypeptide.
Bilirubin uptake by the liver is less efficient that leads to its accumulation in the blood and jaundice.
It is inherited in an autosomal recessive pattern.
AAT
The alpha 1-antitrypsin (AAT)
It is a natural inhibitor of variety of proteases.
Manifold increased levels during acute inflammation.
Is the most important inhibitor of leukocyte’s elastase.
Unchecked elastase activity can lead to extensive destruction of the connective tissues in such organs as lungs and liver.The inherited disorders of AAT secretion can cause live disease (cirrhosis):
AAT accumulates in ER of hepatocytes.
Plasma [AAT] is 10-15 % of normal.
Characterized by elevated plasma levels of conjugated and unconjugated bilirubin.
Conditions can be exacerbated by the unhealthy diet and alcohol.
Neonatal Jaundice
Neonatal jaundice is very common, particularly in premature infants.
Transient (resolves in the first 10 days)
60% of newborns develop “neonatal jaundice” caused by:
Lysis of large number of RBCs after birth.
Immaturity of the enzymes involved in bilirubin conjugation (UDP-glucuronyltransferase).
High levels of unconjugated bilirubin are toxic to the newborn because it:
Can cross the not fully developed blood-brain barrier.
Can damage the brain in infants by increased levels of bilirubin.
Phototherapy in neonatal jaundice
If bilirubin levels are too high, then phototherapy to convert it to a water soluble, non-toxic forms
Phototherapy works by using a blue light (420-470 nm) that converts bilirubin into two other compounds, called lumirubin and isolumirubin
Lumirubin and isolumirubin are isomers of bilirubin that can be removed from the body without conversion into conjugated bilirubin
Neonatal Jaundice
Treatment
If necessary, blood transfusion is used to remove excess bilirubin.
Phenobarbital is often administered to Mom prior to an induced labor of a premature infant
Drug crosses the placenta and induces the synthesis of
UDP glucuronyl transferase.
Jaundice within the first 24 hrs of life or which takes longer then 10 days to resolve is usually pathological and needs to be further investigated.
Determination of Bilirubin Concentration
Van der Bergh reaction
Conjugated bilirubin reacts rapidly with reagent – direct reaction.
Direct bilirubin
Normal value = 0.0 - 0.4 mg/dl
Total bilirubin is measured by addition of alcohol
Unconjugated bilirubin is calculated
Indirect bilirubin
Normal value = 0.1 - 1.0 mg/dl
Summary
Free heme is toxic – synthesis should be tightly regulated (d-ALA catalyzes rate-limiting step).
Genetic defects in enzymes of heme synthesis or lead poisoning can cause porphyrias and anemias.
Porphyrias can be hepatic, erythroid or mixed.
Heme disposal should be tightly regulated because of toxic nature.
Accumulation of bilirubin – jaundice.
Can be caused by extensive hemolysis, genetic disorders of bilirubin metabolism or liver disease (cirrhosis).
Untreated neonatal jaundice can lead to kernicterus.
Untreated neonatal jaundice can lead to
kernicterus
