biochem 5.1 porphyrins and bile trans

Novemeber 7 2015

Biochemistry Dr.Pacifico Eric E. Calderon

Biochemistry of Porphyrins and Bile Pigments

Baquiran, Garcia, Imperial 1 1 of 8

TRANS OUTLINE

I. Heme Synthesis II. Heme Catabolism III. Fate of Bilirubin IV. Clinical Notes

I. Heme Synthesis Biomedical Importance: Heme: Synthesized from Porphyrins & Iron Porphyrins Cyclic compounds formed by linkages

of 4 pyrrole rings through methyne bridges. Heme: Produces Bile Pigments & Iron when degraded

Biosynthesis of Heme: Occurs in the mitochondria & cytoplasm of RBC

precursors. Begins with the condensation of Glycine & Succinyl

CoA. Glycine is activated by Pyridoxal Phosphate. 1st Product of Glycine & Succinyl CoA is a-Amino-B-

ketoadipic acid this product is rapidly decarboxylated to form a-Aminolevulainate (ALA).

Decarboxylation process of a-Amino-B-ketoapidic acid is catalyzed by: Aminolevulinate Synthase (ALAS)

ALA Dehydratase + ALA = Porphobilinogen ALA Dehydratase is aka: Porphobilinogen Synthase. ALA Dehydratase is a Zinc containing enzyme

sensitive to inhibition by Lead.

Porphobilinogen Deaminase (aka: hydroxymethylilane synthase or Urophorphyrinogen 1 Synthase) + porphobilinogen = Hydroxyl methylbilane or Urophorphyrinogen 1

Urophorphyrinogen 1 is converted to Uriphorphyrinogen III by Urophorphyrinogen III Synthase

Urophorphyrinogen III converted to Coporphyrinogen III by acetate decarboxylation. This acetate decarboxylation action will convert

acetate to Methyl substituents. Urophorphyrinogen III conversion to

Coporphyrinogen III is catalyzed by Urophorphyrinogen Decarboxylase.

Corpophyrinogen III enters the Mitochondria where it is converted to Protophorphyrinogen III. Coprophyrinogen Oxidase catalyzes the production

of Porphopyrinogen. (This enzyme only acts on Coprophyrinogen III).

Protophorphyrinogen III converted to Protophyrin III. An oxidation process catalyzed by

Protophophyrinogen Oxidase Enzyme.

FINAL STEP OF HEME SYNTHESIS: Ferrous Iron is incorporated into the Protophyrin. This reaction is catalyzed by: Ferrochelatase (aka:

Heme Synthase)- a mitochondrial enzyme.

** ALA Synthase is the key regulatory enzyme in hepatix biosynthesis of heme.

Heme is a ubiquitous molecule that is Involved in many essential biological processes:

oxygen transport respiration photosynthesis, detoxification drugs (cytochrome ezymes) signal transduction. (enzymes that use heme proteins

Most of these reactions are carried out by redox

reactions of heme iron.

The heme biosynthetic and catabolic pathways generate pro- and antioxidant compounds, and consequently, influence cellular sensitivity to oxidants.

Why regulate Heme?

Free heme is a potent pro-oxidant, leading to the formation of reactive oxygen species that can damage a variety of biological molecules

Heme precursors (-aminolevulinic acid, porphyrins) generate reactive oxygen species (ROS) from autoxidation and photochemical reactions, respectively

Heme can associate with phospholipid membranes, altering bilayer structure and thus, causing cell disruption For this reason, the cells strictly regulate heme homeostasis

To regulate Heme, you need to break it.

Heme Catabolism As the heme is not recycled, most cells containing heme proteins have the microsomal mixed function oxygenase, heme oxygenase, which enzymatically degrades heme to biliverdin, carbon monoxide, and iron

The catabolism of heme from all heme proteins is carried out in the microsomal fraction of cells by heme oxygenase

Heme Catabolism Heme oxygenase- is operational in various tissues such as spleen, liver , kidney and macrophages.

Plays a role in heme catabolism

II. HEME CATABOLISM

8 of 8 Biochemistry: Baquiran, Garcia, Imperial

Degrades heme to :

biliverdin carbon monoxide iron

Fig. 1. Heme Catabolism Products

Heme Catabolism- the only endogenous/ natural biological reaction in the body that produces Carbon monoxide (CO)

The conversion of of one mole of heme Fe3+ to bilivedin, CO, and Fe3+ consumes three molecules of O2 plus seven e- provided by NADH and NADPH cytochrome P450 reductase.

Fe3- Heme + 3 O2 + 7 e- bliverdin + CO+ Fe3- Why breakdown heme. then it has to be processed again?

Heme breakdown can alter physiological and biochemical reactions of the cells.

Because each of the products of the heme breakdown has its own importance. That why, we regulated its breakdown as well as its synthesis.

Products of Heme Catabolism

Carbon monoxide (CO)

Because CO is the product of incomplete combustion of hydrocarbons.

If so, how do release Carbon monoxide?

It is released through he breath

Clinical and phyiological significance of CO: CO in the breath can be a marker for heme breakdown.

Has high affinity for heme Fe2-

The CO that produced does not severley inhibit

heme oxygenase

Iron/ Ferritin (Fe)

The iron of the heme that reaches heme oxygenase has been oxidized to its ferric form (hemin)

Biliverdin

Has biliverdin reductase that reduces the central methylene bridge of biliverdin to a methyl group, producing the yellow pigment Bilirubin. Biliverdin + NADPH +H+ Bilirubin + NADP+

Effects of hemooxygenase activity In mammals, CO, a gaseous messenger, has anti- inflammatory and anti-apoptotic effects Biliverdin and its reduced product bilirubin may function as important antioxidants

Heme oxygenase Humans harbor two distinct heme oxygenase genes identified as HMOX1 and HMOX2 The heme oxygenase enzyme encoded by the:

HMOX1 gene- is the rate-limiting enzyme of heme catabolism Both HMOX1 and HMOX2 genes are constitutively expressed, however, the activity of

the HMOX1 encoded enzyme is inducible by heme, heavy metals, and conditions of stress such as hypoxia

Meaning both HMOX1 and HMOX2 genes are expressed continuously.

HMOX1 encoded enzyme is inducible expression only occurs when need arises.

Factors for Heme Degration:

Heme Heavy metals Hypoxia

Over production of these factors can cause heme degradation.

Heme catabolism The red cell with the largest pool of heme protein, hemoglobin, contains no heme oxygenase Enzymatic degradation of the red cell heme occurs only after the senescent red cells are removed by the reticuloendothelial system. RBC is devoid of heme oxygenase because:

It carries its own oxidation

Heme Catabolism


Gene is not expressed continuously because RBC doesnt have genes and it is devoid of nucleus. RBC Degradation:

Globin- degraded to Amino Acids Iron- enters the iron pool and will be reused Iron-free porphrin- degraded into the

reticuloendothelial cells of liver, spleen and bone marrow.

Heme - breaks down into: Bile Iron CO

Fig. 2. Degradition of RBC (Heme+globin)

Heme catabolism The largest repository of heme in the human body is in RBCs which have a life span of about 120 days There is a turnover of about 6 g/day of hemoglobin, which presents 2 problems:

The porphyrin ring is hydrophobic and must be solubilized to be excreted

Iron must be conserved for new heme synthesis

Heme is not recycled Fe is recycled, for new heme synthesis to occur

So, Heme and Fe should part ways. L

Sources of Heme metabolites Roughly 80% of heme destined for degradation and excretion comes from senescent erythrocytes 20% comes from premature erythrocytes in the bone marrow which are destroyed prior to release into the circulation and a minor component is derived from other cell types

Sources: Senescent RBC Premature RBC Bone Marrow

Heme breakdown into Bilirubin Within hepatic and splenic macrophages, heme is first converted to bilirubin in a twostep enzymatic process which employs biliverdin as an intermediate (enzyme: biliverdin reductase)

Fig.4. Oxidative metabolism of heme by HO and biliverdin reductase, giving rise to CO, iron, biliverdin, and bilirubin Biliverdin is insoluble in water so for it to be excreted, it has to be converted to bilirubin through biliverdin reductase .

This results to the oxidation and opening of heme ring.

Macrophage will excrete resulted bilirubin into the plasma.

Heme breakdown into bilirubin

These steps result in oxidation and opening of the heme ring Macrophages then excrete the resultant bilirubin into the plasma as unconjugated bilirubin

Bilirubin redcutase There are two biliverdin reductase genes in humans identified as BLVRA and BLVRB The enzyme encoded by the BLVRA gene is pricipally responsible for the catabolism of biliverdin The enzyme encoded by the BLVRB gene catalyzes the reduction of not only biliverdin but also a variety of flavins, such as riboflavin, FAD or FMN, and methemoglobin aka NADPHdependent flavin reductase


Fig. 3. Bilirubin reductase genes

Bilirubin is significantly less extensively conjugated than biliverdin causing a change in the color of the molecule from blue-green (biliverdin) to yellow-red (bilirubin) Peripherally arising bilirubin is transported to the liver in association with albumin, where the remaining catabolic reactions take

Bilirubin is sparingly water souble (between being soluble and insoluble) but when it is bounded by albumin, it will readily transported to the liver.

Albumin both have a high affinity site and low affinity site for bilirubin.

Since most of the bilirubin is loosely attached to albumin, it will detached and diffuse into the tissues.

Liver- where HEPATIC CATABOLISM OF BILIRUBIN takes place. 3 Stages of Bilirubin Metabolism: - Uptake by the liver - Conjugation with glucorinic acid, - Secretion in the bile

1. Uptake of bilirubin by liver parenchymal cells

Bilirubin is removed in the albumin, then it will be taken up to at the sinusoidal surface of the hepatocytes by Facilitated Transport System (malaking molecule ang bilirubin), once inside it will bind to cytosol proteins(glutathione S-transferase or know as ligandin) to prevent reentering the blood stream.

2. Conjugation of bilirubin with glucoronate in the endoplasmic reticulum Bilirubin is non polar, to make it more polar it will be converted to glucuronic acid by conjugation. Within the hepatocyte, the enzyme UGT1A1 (UDP-glucuronyltransferase) covalently attaches one or two molecules of glucuronic acid to bilirubin, either bilirubin mono- or di-glucuronide is generated to form acylglucoronate

Fig. 4. Conjugation of Bilirubin

Fig. 5. UGT1A1 in hepatocytes

Glucuronyltransferase - Enzyme for conjugation, uses UDP-glucuronic

acid as the glucuronosyl donor and referred to as bilirubin-UGT.

Unconjugated bilirubin - Not yet process in the liver Conjugated bilirubin - direct reacting bilirubin

Fate of bilirubin

BLVRA BLVRB NADPH dependent flavin

redcutase

biliverdin reductase genes

Catabolism of:

-Biliverdin

Catabolism of :

-Bilive-Biliverdin rdin -Flavins

Eg. Riboflavin FAD FMN

-MethHG


- already processed in the liver - glucuronic acid-attached species of bilirubin - increased water solubility of the tetrapyrrole

facilitates its excretion with the remainder of the bile as the bile pigment

Glucuronide - Increases polarity and solubility

Glucuronidation - Addition of glucuronide - Most important of conjugation reaction - Reaction does not proceed spontaneously - Requires the activated form of glucuronic acid

(glucuronic acid uridine diphosphate)

Glucuronidation and Grey Baby Syndrome When Chloramphenicol is given without regard for

infants diminished capacities for hepatic detoxification and renal elimination hypothermia, vomiting, acidosis, cyanosis, grey discoloration occur

- Very versatile in baby - Immaturity of the liver

Immature glucuronosyl transferase activity - Toxic to the cells - Immaturity of the enzyme (not yet fully

expressed) 3. Secretion of conjugated bilirubin into the bile

- Occurs by active transport - Protein involved is MRP-2 (Multidrug

resistance like protein 2) or MOIT (Multispecific organic Ion Transporter) located in the plasma membrane of the bile canalicular membrane.

As the conjugated bilirubin reaches the terminal ileum and large intestine, the glucoronides are removed by -glucuronidases (bacterial enzymes). The pigment is then reduced to Urobilinogen (a group of colorless tetrapyrroles), urobilinogens is reabsorbed in the terminal ileum and large intestine through the liver via enterohepatic urobilinogen cycle.

When the senescent RBC goes into the liver or spleen, the Hemoglobin will be degraded into Heme and Globin.

Globin, since its a protein, will be broken down into Amino acids recycled, for another synthesis of Erythropoeisis.

Heme, will be broken down into Carbon Monoxide, Fe and Biliverdin by the enzyme Heme Oxygenase

Biliverdin, will be converted into Bilirubin by the enzyme Bilirubin reductase

When bilirubin becomes conjugated, using

glucorinic acid, from being sparingly water soluble (between being soluble and insoluble) it will now become water-soluble and secreted in the bile.


RBC will circulate the body for 120 days, when these RBC reached their life span, Old RBC it will be phagocytosed by macrophages in spleen and bonemarrow .

RBC now will release to its Hemoglobin molecule Hemoglobin degraged into globin and heme Globin is a protein and it will breakdown into AMINO ACIDS and be reuse for

Erythropoiesis HEME will be broken down to unconjugated bilirubin and Iron Iron will re-enter the circulation and reused for Erythropoiesis like the Globin Unconjugated bilirubin is not recycled and thus needs to be excreted because it is toxic. It

has a yellowish orange color For the Unconjugated bilirubin to be Lipid soluble when it is in the blood, it requires

protein for it to be carried around. This protein is called Albumin Albumin will carry the unconjugated bilirubin into the liver for further metabolism Liver has own macrophage Kupffer cells which break donwn other old/senescent RBC

Unconjugated Bilirubin is lipid soluble LIVER is the site for conjugation (lipid soluble to water soluble) Unconjugated Bilirubin will form into conjugated by glucoronic acid

Conjugated Bilirubin will be water soluble for it to be excreted by bile contains which

contains Conjugated Bilirubin and Bile salt Conjugated Bilirubin and Bile salt will be excreted into small intestine Conjugated Bilirubin will go to the small intestine through the common bile duct Conjugated Bilirubin travels in large intestine Ileum where Conjugated Bilirubin is converted by intestinal bacteria to become

Urobilinogen by removing glucorinic acid Urobilinogen lipid soluble 10-15 percent bound to albumin and reabsorbed Stercobilin brown colored pigment Conjugated Bilirubin excreted

Uptake by the Liver

Conjugation with Glucorinic acid

Secretion of Bilirubin into the Bile Bile

Fig. 5. General View of Bilirubin Metabolism

NOTE: In general view or process only. Para lang madaling intindihin J


Fig.6.Heme Degradation

Two types of Clinical problems associated with heme metabolism:

1. Porphyrias - Disorders that arise from defects in the

enzymes of heme biosynthesis - Both inherited and acquired - Classified as either erythroid or hepatic,

depending upon the principal site expression of the enzyme defect

- Cause elevation in serum and urine

Type Deficient enzyme Gene Gene locus

ALA-Dehydratase

ALA-Dehydratase ALAD 9q34

Acute Intermittent Porphyria

Hydroxymethylbilane synthase (Porphobilinogen deaminase

HMBS 11q23

Congenital Erythropoietic Porphyria (CEP)

Uroporphyrinogen III synthase

UROS 10q25-26

Porphyria Cutanea Tarda (PCT), familial form

Uroporphyrinogen decarboxylase

UROD 1p34

Hepatoerythropoietic Porphyria (HEP)

Uroporphyrinogen decarboxylase

UROD 3q12

Hereditary Coproporphia (HCP)

Coproporhyrinogen oxidase

CPOX 1q22

Variegata Porphyria (VP)

Protoporphyrinogen oxidase

PPOX 18q21

Erythropietic (EPP)

Ferrochelatase FECH Xp11.21

X-linked Protoporphyria (XLP)

-Aminolevulinate synthase 2

ALAS2

2. Bilirubinemias

- Inherited disorders of bilirubin metabolism

Hyperbilirubunemia and Jaundice - Occurs when bilirubin in the blood exceeds

1mg/dL (17.1 mol)

- Due to excess bilirubin production or liver failure

- Obstruction of the excretory ducts of the liver

- When bilirubin reaches 2-2.5 mg/dL in blood, it diffuses into tissues causing Jaundice

Types of Hyperbilirubinemia 1. Retention hyperbilirubinemia due to over

production of bilirubin (unconjugated bilirubin) 2. Regurgitation hyperbilirubinemia due to reflux

into the bloodstream secondary to biliary obstruction (conjugated bilirubin)

Kernicterus

- Because of its hydrophobicity, only unconjugated bilirubin can cross the blood-brain barrier into the CNS

- Encephalopathy due to hyperbilirubinemia (kernicterus) can occur in only with unconjugated bilirubin, as found in retention hyperbilirubinemia

Jaundice - Conjugated bilirubin can appear in urine

Choluric jaundice - Occurs only in regurgitation hyperbilirubinemia

Acholuric jaundice - Occurs only in the presence of an excess of

unconjugated bilirubin

Unconjugated Hyperbilirubinemia

Neonatal Physiological Jaundice - Unconjugated hyperbilirubinemia of neonatal

physiological jaundice results from accelerated hemolysis and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin.

- Decreased bilirubin-glucosyltransferase activity and UDP-glucuronate synthesis

- Plasma concentration of unconjugated bilirubin exceeds that which can be tightly bound by albumin (20-25 mg/dL) can penetrate the blood brain barrier.

Crigler-Najjar Syndrome Type I - Congenital nonhemolytic jaundice - Autosomal recessive - Due to mutations in the gene encoding

bilirubin-UGT activity in hepatic tissues - Often fatal within the first 15 months

Crigler-Najjr Syndrome Type II - Like type I but more benign

Gilbert Syndrome - Also has mutations in genes encoding bilirubin-

UGT but retains 30% of enzyme activity Toxic Hyperbilirubinemia

- Caused by chloroform, arsphenamines, carbon tetrachloride, acetaminophen, hepatitis virus, cirrhosis, or Amanita mushroom poisoning.

- Can result from toxin-induced liver dysfunction - Hepatic parenchymal cell damage which

impairs bilirubin conjugation. Hemolytic Anemia

Clinical Notes


Conjugated Hyperbilirubinemia

Biliary obstruction - Regurgitation into hepatic veins and lymphatics - Conjugated bile in blood urine

Dubin-Johnson Syndrome - Benign autosomal recessive - Mutation in the gene encoding MRP-2 for thee

secretion of conjugated bilirubin into bile Rotor Syndrome

- Chronic conjugated hyperbilirubinemia and normal liver

https://www.rarediseasesnetwork.org/porphyrias/patients/learnmore/index.htm Harpers Illustrated Biochemistry 30th Edition Dr. Pacifico Eric E. Calderons Lecture

References

biochem 5.1 porphyrins and bile trans

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