Heme Synthesis and Degradation COPY

Question 1

Hemoprotein functions

Answer 1

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

Question 2

Heme Synthesis

Location

Answer 2

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.

Question 3

Heme Synthesis

General Pathway

expression patterns of the enzymes

Answer 3

Synthesized from: Succinyl-CoA and Glycine

2^nd, 3^rd, and 4^th 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).

Question 4

5 Processes of heme synthesis

Answer 4

1. Formation of the pyrrole.
2. Assembly of the tetrapyrrole (porphyrin).
3. Modification of the porphyrin side chains.
4. Oxidation of protoporphyrinogen IX to protoporphyrin IX .
5. Insertion of iron to form a heme.

Question 5

First rxn of heme synthesis

enzyme

Cofactor

Inhinition

Answer 5

1st reaction is the RLS

g-aminolevulinic acid synthase

pyridoxal phosphate

ALS is inhibited by heme

Question 6

Location of the reactions

Heme synthesis

Answer 6

First and the last 3: Mitochondria

The remaining rxns: cytoplasm

Question 7

Photosensitivity

Answer 7

accumulation of intermediates.(starting at hydroxymethylbilane)

Question 8

ð-Aminolevulenic Acid synthesis

Starting compounds

enzyme (cofactor) & assc deficiency

Answer 8

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.

Question 9

Genes for ALA synthase in humans

Answer 9

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.

Question 10

Regulation of ALA synthase activity

Answer 10

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.

Question 11

Regulation of ALAS-2 trascription

Answer 11

Transcription of ALAS-2 is controlled by Fe²⁺-binding element.

Prevents excessive formation of porphyrin intermediates in the absence or low levels of iron.

Question 12

Hepatic ALA synthase & Cyt P450

Answer 12

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.

Question 13

Sideroblastic anemia

Answer 13

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.

Question 14

Effects of fasting or low carb diet on PGC1-a

Answer 14

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.

Question 15

PGC-1a

Answer 15

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:

1. FA ox
2. Gluconeogenesis
3. Heme synthesis (ALAS-1)

Question 16

Synthesis of pyrrole ring

Answer 16

Pyrrole ring is formed by the condensation of two molecules of d-ALA. Catalyzed by d-ALA dehydratase (ALAD)

Question 17

disorders associated with deficiency of ALAD and elevated levels of ð-ALA

Answer 17

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.

Question 18

The effects of elevated levels of ð-ALA on CNS

Answer 18

Elevated levels of d-ALA can have a neurotoxic affect on both central and peripheral nerve systems.

Memory loss and confusion

Question 19

The final step of heme synthesis

Answer 19

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.

Question 20

deactivating mutations of ferrochelatase

Answer 20

Deactivating mutations of ferrochelatase can lead to erythropoetic protoporphyria.

ferrochelatase enhances the introduction of Fe^{<strong>2</strong>+} into protoporphyrin IX.

Question 21

A potent competetive inhibitor of ALAD

Answer 21

succinylacetone

Hepatorenal tyrosinemia can lead to the accumulation of succinylacetone, which is a potent competitive inhibitor of ALAD.

Question 22

Lead poisoning and anemia

Answer 22

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

Question 23

Synthesis of HMB

Answer 23

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.

Question 24

The reaction catalyzed by PBG-deaminase

Answer 24

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.

Question 25

the **first intermediate** of heme synthesis pathway an accumulation of which will lead to **light sensitivity**

Answer 25

hydroxymethylbilane (HMB)

Question 26

Iron functions in human

Answer 26

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)

Question 27

Hereditary hemochromatosis

Answer 27

Defects in proteins regulating iron absorption may lead to iron overload. Iron content is regulated at the _level of absorption_.

Question 28

Foods that facilitate iron absorption

Answer 28

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.

Question 29

Inhibitors of iron absorption and Iron deficiency

Answer 29

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.

Question 30

Porphyrias

Answer 30

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

Question 31

X-linked dominant protoporphyria

Answer 31

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.

Question 32

ALA dyhydratase porphyria

Answer 32

recessive, caused by the deficiency of ALA dehydratase in RBC. Sx: abd. pian NV

Question 33

Congenital erythropoetic porphyria (gunther disease)

Answer 33

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

Question 34

Classification of Porphyrias Based on causes and location of the deficienct enzymes

Answer 34

**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).

Question 35

Tx of porphyrias caused by increased activity of ALA synthase

Answer 35

Treatment: IV injection of hemin

Question 36

Acute Intermittent Porphyria

Answer 36

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.

Question 37

Congenital Erythropoietic Porphyria ## Footnote Mode if inheritance affected enzyme accumulated product symptoms treatment

Answer 37

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.

Question 38

Porphyria Cutanea Tarda (PCT) ## Footnote affected enzyme accumulated product inheritance pattern symptoms treatment

Answer 38

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.

Question 39

Sources of bilirubin

Answer 39

~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).

Question 40

The first 3 reactions of Hb degradation

Answer 40

The steps in the subsequent metabolism of the hemoglobin are: 1. Globulin fraction is removed from hemoglobin, leaving heme. 2. Heme oxygenase: Heme-------\>biliverdin 3. Biliverdin reductase: Biliverdin------\> bilirubin

Question 41

Transport of unconjucated bilirubbin

Answer 41

The unconjugated (water insoluble) bilirubin is transported from the RE system to the liver in complex with albumin.

Question 42

Hb degradation at the level of hepatocytes

Answer 42

At the level of the hepatocyte, metabolism occurs in three stages: 1. Transfer of bilirubin-albumin complex inside of hepatocyte and release of bilirubin. 2. Transfer to intracellular binding proteins called ligandins, which transport bilirubin to the ER. 3. 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.

Question 43

Heme degradation

Answer 43

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**.

Question 44

N. bilirubin metabolism

Answer 44

**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.

Question 45

Heme degradation and Jaundice

Answer 45

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.

Question 46

Causes of Jaundice Causes of neonatal jaundice

Answer 46

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.

Question 47

Prehepatic (hemolytic) jaundice

Answer 47

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.

Question 48

Interhepatic Jaundice

Answer 48

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.

Question 49

Posthepatic Jaundice

Answer 49

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.

Question 50

differential dx of Jaundice

Answer 50

Question 51

Hyperbilirubinemias

Answer 51

Hyperbilirubinemias are inherited disorders in bilirubin metabolism associated with high plasma levels of unconjugated or conjugated bilirubin

Question 52

Unconjugated hyperbilirubinemias

Answer 52

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.

Question 53

conjugated hyperbilirubinemias

Answer 53

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.

Question 54

Gilber Syndrome

Answer 54

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.

Question 55

Gilbert Syndrome Inheritance pattern

Answer 55

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.**

Question 56

Cringer-Najjar syndrome

Answer 56

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.

Question 57

Dubin-Johnson Syndrome

Answer 57

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.

Question 58

Rotor Syndrome

Answer 58

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 transporte**r 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.

Question 59

AAT

Answer 59

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.

Question 60

Neonatal Jaundice

Answer 60

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.

Question 61

Phototherapy in neonatal jaundice

Answer 61

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

Question 62

Neonatal Jaundice Treatment

Answer 62

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.

Question 63

Determination of Bilirubin Concentration

Answer 63

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

Question 64

Summary

Answer 64

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.

Question 65

Untreated neonatal jaundice can lead to

Answer 65

kernicterus