DetoxicolSDLS 2008

Medicine for the intoxicated

File Created on 8/19/2004 10:08 PM Topic: HemoglobinTranscriber(s): Glenn and d gang Lecturer: Dr. Ma. Esperanze E. UyEditors: JC Tayco, Mark Lomboy, Lulu Velazco, Pre Ausan No. of pages: 5

Porphyrins metabolic intermediates in the biosynthetic pathway that has

HEME as its principal product. cyclic compounds formed by linkage of 4 pyrole rings and four

methenyl bridges.

Metalloporphyrins complexes of porphyrin with metal ions bound to the Nitrogen

atom of the pyrole ringse.g Fe Porphyrins – heme Mg Porphyrins – chlorophyll

Hemoproteins Proteins that contain heme Examples:

Protein FunctionHemoglobin Transport of O2 in bloodMyoglobon Storage of O2 in muscleCytochrome c Involvement in electron

transport chainCytochrome P450 Hydroxylation of

xenobioticsCatalase Degradation of hydrogen

peroxideTryptophan pyrrolase Oxidation of tryptophan

Isomeric forms of porphyrins Clinical significant porphyrins: uroporphyrins, coproporphyrins,

protoporphyrins Four isomeric forms can exist for each porphyrins Naturally occurring porphyrins are of either type I or type III

isomer type(more abundant) Type III isomer have been shown to play a functional role in

the biosynthesis of heme Heme and protoporphyrin IX are both type III porphyrin

Heme synthesis – takes place first in the mitochondria

Starting materials:o Succinyl CoA-from Kreb’s cycleo Glycine o Pyridoxal phosphate -coenzyme, for activation of

glycineo ALA synthase- rate controlling (limiting), key

regulatory enzymeo Product: delta-Aminolevulinic acid

Porphobilinogen (PBG)o Precursor of pyrole o 4 molecules of PBG for the formation of a cyclic

tetrapyrole (porphyrin)o derived from breakdown of Aminolevulinic acid by

ALA dehydratase in the CYTOSOLo Hydroxymethlybilane

Formed by Uroporphyrinogen I synthase, also removes NH3

o Uroporphyrinogen I Formed by hydroxymethylbilane

o Uroporphyrinogen III Formed by uroporphyrinogen III synthase (most

exclusive)

Coporphophyrinogen IIIo Formed by uroporphyrinogen decarboxylase, acting on the acetate group

Protoporphyrinogen IXo Formed by coporphyrinogen oxidase, back in the

MITOCHONDRIA

Protoporphyrin IXo Formed by protoporphyrinogen oxidase

Heme moleculeo Formed by ferrochelatase and Iron (ferrous state)

* Mature Erythrocytes(RBC) does not undergo heme synthesis due to absence of mitochondria*Lifespan of RBC- 120 days

*Question:1. what is the enzyme that is rate controlling (limiting), key regulatory enzyme?2. what is the amino acid present in the formation of heme?

Forms of ALA1. ALAS1

hepatic form rate limiing reaction of heme synthesis in the liver (positive and

negative feedback mechanism) affected by administration of drugs

2. ALAS2 erythroid form

not induced by drug does not undergo feedback mechanism

Spectrophotometry a device used to measure or transmitted light energy visible wavelength: 350-700nm in porphyrias, Coproporphyrins and Uroporphyrins are

excreted in urine or feces, hence identified in this method

Fluoresce of Porphyrins Soret Band – sharp absorption of porphyrin band near 400nm

(red Fluoresence) Distinguishing feature or all porphyrins: the double bonds

present the joins the pyrrole rings Porphyrinogens are COLORLESS

Clinical correlation:Porphyrias

Disorders due to abnormalities in the pathway of biosynthesis of heme

Can be genetic or acquired Most prevalent porphyrias involves the enzymes:

o Uroporphyrinogen I synthaseo Uroporophyrinogen decarboxylaseo Ferrochelatase

Sign and symptoms of the porphyrias:

Anemia Decrease RBC count and decreased hemoglobin Due to lead poisoning Lead can affect heme metabolism by combining with SH

groups in enzyme such as:o Ferrochelataseo ALA dehydratase

Affects porphyrin metabolism

*Question:1. what enzyme in the heme synthesis that liberates NH3?2. what are the 3 enzymes that are prevalent that causes porphyrias?3. what is the wavelength of visible light?

Catabolism of HemeHemoglobin Heme + Iron + Globin

Reuse Amino Acid (Iron Pool)

Biliverdin New Proteins

Biliverdin Reductase

Bilirubin (unconjugated)Attaches to serum albumin

Bilirubin (Conjugated)

Urine

Secretion into Bile

Urobilinogens

Urine Stool

Globin – degraded to amino acids Heme – formation of bilirubin Iron – return to iron pool for reuse Porphyrins – degraded by the Reticuloendothelial (RES) cells

in the liver, bone marrow, and Mainly in the Spleen

Under physiologic condition: 1-2 x 108 erythrocytes are destroyed per hour approximately 6 grams of hemoglobin are turned over

Catabolism of Heme carried out by microsomal fractions of cells by a complex

enzyme – heme oxygenase, in the mitochondria Biliverdin is produced by further reduction of heme, NADPH

acts as coenzyme

Nice to know:*In birds and amphibia, Biliverdin reductase is absent that’s why their poopoo is colored green

Biliverdin reductase acts on the methenyl bridge between pyrrole III and pyrrole IV to a methylene group of biliverdin, hence producing the YELLOW pigment Bilirubin

It is estimated that 1gm of bilirubin yields 35mg of bilirubin Dialy bilirubin formation: about 250 – 350 mg Example: in hematoma, the chemical conversion of heme to

bilirubin by the reticuloendothelial cells can be observed in vivo as the purple color of the heme is slowly converted to yellow pigment of bilirubin…. Gets!?

Remember:Unconjugated Bilirubin

before passing and in the liver also known as:

o Indirect Bilirubino B1o Prehepatic Bilirubino Free Bilirubin

Conjugated Bilirubin after passing the liver also known as:

o Direct Bilirubino B2o Posthepatic Bilirubin

Point of ComparisonB1 B2

1. Solubility in H2O Insoluble Soluble2. Alcohol & other organic sol’n

Non polar Polar

3.Van Den Bergh Reaction Indirect Direct4. Site RES Liver5. Bind Albumin Cholesterol6. Conjugation Unconjugated Conjugated7. Brain tissue affinity High Affinity Low Affinity8. Presence w/ jaundice in urine

Negative Positive

9. associated w/ jaundice (hemolytic)

Positive Nrgative

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Glucoronic AcidLIVER

Transport of Bilirubin Plasma Albumin for transport Transported into the liver (parenchymal cells)

Bilirubin in the Liver Fascilitated tranport system, removal of Bilirubin from the

albumin transport, where no energy is used Cytosolic proteins in the hepatocyes: Ligandin and Protein Y

o They help keep bilirubin in solubilized form prior to conjugation

o Also help prevent efflux of bilirubin back in the bloodstream

Conjugation of bilirubin occurs, making it water soluble, polar form, achieved by addition of glucoronic acid to it

Conjugation of bilirubin is catalyzed by UDP-glucoronyltransferase (UDP-Uridine diphosphate) in the smooth endoplasmic reticulum

After conjugation, bilirubin is readily secreted into bile and small intestine

Bilirubin secreted into Bile Occurs by active transport mechanism Protein involve:

o Multidrug resistance-like protein 2 (MRP-2), also called Multispecific organic anion transporter (MOAT)

o Located in the plasma membrane of the bile canalicular membrane

Bilirubin diglucoronide – form of bilirubin excreted here

Bilirubin in the Small Intestine Bilirubin reaches the terminal ileum and large intestine Glucoronides are removed by bacterial enzymes (β-

glucoronidases) Urobilinogen is formed. Colorless pigment Urobilinogen can be reabsorb back to the liver to constitute

enterohepatic urobilinogen cycle Oxidation of urobilinogens causes darkening of feces

*Questions:1. what enzyme is not present in birds/amphibians that make their poopoo green?2. conjugation occurs in what organelle in the liver?3. can B1 go back to the bloodstream? How about B2?

Terms:1. Hyperbilirubinemia

increase serum bilirubin exceeds 1mg/dl

2. Jaundice Yellow discoloration of the skin

3. Icteric The degree of icteresia or yellowishness of the

serum or plasma in cases of jaundice

Hyperbilirubinemia Due to production of more bilirubin than the normal liver

excrete May result from failure of a damaged liver to excrete bilirubin in

normal amounts Obstruction of the excretory ducts of the liver

Van Den Bergh Reaction Quantitative analysis of bilirubin Uses diazo reagent, bilirubin produces reddish purple azo

compoundA. Direct

Reaction of conjugated bilirubin Without the use of methanol

B. Indirect Reaction of unconjugated bilirubin With the use of methanol

Clinical correlation

A. Increase of Unconjugated bilirubin (B1) Hemolytic Anemia

o Increase RBC destruction hence production of large amounts of B1

Neonatal “Physiologic Jaundice” or Kernicteruso Results from accelerated hemolysis the time of birth

and an immature hepatic system for the uptake, conjugation and excretion of B1

o Capable of penetrating Blood Brain Barrier (BBB)o Can result to mental retardation

Crigler-Najjar Syndrome (type I & II)o Congenital diseaseo Deficit in conjugationo Treatment:

Phenobarbital – induces the Cytocrome P450 enzyme system thus increasing the rate of conjugation

Phototheraphy - hepatic excretion of Bilirubin. Bilirubin is photosensitive, easily breaks down.

Gilbert’s Syndromeo Bilirubin transport deficito Transport of bilirubin into the hepatocyte

Toxic hyperbilirubinemiao Due to toxin induced liver functiono E.g chloroform, acetaminophen

B. Increase of Conjugated Bilirubin (B2) Obstruction of Biliary Tree

o Due to blockage of common bile ducto Often due to gallstone of CA of the head of the

pancreaso Cholesthiasis

Dubin Johnson syndromeo Due to excretion deficito Defect in the hepatocyte mebraneo Mutation in the gene encoding MRP-2o Bilirubin contains abnormal black pigment

Rotor syndromeo Thought of due to abnormality in hepatic storage

C. Increase of Both Type Hepatitis

o Drug inducedo Alcoholico Viralo Autoimmune

*Questions:1. what is the term used when the serum is yellowish?2. what is bilirubin is increased if patient is suffering from cholesthiasis?3. what is kernisterus?4. Illustrate the biosynthetic pathway of heme synthesis!!!!! NOW NA!!!! +5 sa unang matapos! GUD LUCK!

HEMOGLOBIN AND MYOGLOBIN

Hemoglobin (Hb) Main component of RBC Conjugated protein Transports O2 and CO2

In full saturation, 1gm of Hb holds 1.34ml of O2

In adult, approx. 600gm of Hb is capable of carrying 800 ml of O2

* O2 is not soluble in plasma, therefore it needs Hb for its delivery

Structure of Hb Contains 2 pairs of polypeptide chains (“globin”) 4 pyrrole rings (prosthetic heme groups

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each heme group is capable of carrying 1 mole of O2, hence each Hb molecule can transport 4 moles of O2

each pyrrole ring contains 1 atom of Ferrous Iron

Clinical correlation: Carbon monoxide (CO) is toxic to hemoglobin Cyanide is toxic to Cytochrome oxidase

Unusual property of a physiological O2 carrier In the lungs

o Binds O2 at tension of 100 mmHgo 98% saturation occurs

In the musleso Able to release O2 at a lower tension (20 mmHg)o 33% saturation in working muscleo delivers 65% of O2

The O2 binding curve for Hb is sigmoidal

Allosteric Proeperty Due to its quarternary structure.

1. Cooperative binding Phenomenon wherein permits Hb to maximize both the

quantity of O2 loaded at the PO2 of the lungs and the quantity of O2 released at the PO2 of the peripheral tissue

2. P50 Partial pressure of O2 that half saturates a given Hb It expresses the affinity of Hb to O2

Tension is around 27 mmHg

3. Oxygenation of Hb Accompanied by conformational change

a. Deoxyhemoglobin T state (Taut state) Low affinity conformation Heme group is w/o O2

Contains BPG in the β chain of Hb Binds 1 proton for every 2 O2 molecules released

(BOHR effect)

b. Oxyhemoglobin R state (Relax state) High affinity conformation Heme group is associated with O2

Doesn’t accommodate BPG

What is BPG? 2,3 Biphosphoglycerate causes inhibitory effect on Hb

binding with O2. Therefore, it will allow Hb to readily give up O2 ↑

BPG con’c = ↑ O2 delivery Tissue hypoxia – state of tissue wherein there is an insufficient

supply of O2

↑ BPG during tissue hypoxia

Clinical correlation: in Lung CA

1. BPG will not compensate2. Very low Partial pressure of O2

4. After release of O2 Hb transports CO2 and protons (H+) Carbamates formed with the amino terminal nitrogens Carbamates change the charge of amino acid: from positve to

negative; hence favoring salt bond formation between the α and β chains

Blood CO2 is present in 3 major form1. Bicarbonate – 78%2. Carbamino Hb – 13%3. Dissolved CO2 – 9%

Factors affect O2 binding1. Temperature

Increase temperature such as in fever weakens Hb’s O2 affinity

Shifts dissociation curve to the right Increase pressure to Hb to readily release O2 due to

increase metabolic function of the tissues

2. Level of BPG Increase BPG – increase O2 release

3. Low pH (acidic pH) O2 is released Increase O2 demand

*increase metabolic rate will result to increase production of CO2

*muscular exercise will result to tissue hypoxia and formation of lactic acid

Tetrameric structures of common Hb: HbA (α2β2) – normal adult Hb HbA2(A2D2) – minor adult Hb HbF (A2G2) – fetal Hb HbS (A2S2) – Sickle cell

Mutant Hemoglobin Termed as hemoglobinopathies Due to mutations in the genes that encode the α and β subunits of

Hb Methemoglobin (Hi)

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o Also termed as hemoglobino Ferrous iron is oxidized in the ferric state, resulting

inability of Hi to combine with O2

o Polypeptide chains are not alteredo Upto 1.5% is present in normal persono Increase to 10% con’c of this will result to cyanosis

Hemoglobin Mo Hereditary Methemoglobinemia o Alteration of either α or β globin chaino Causing asymptomatic cyanosis

Hemoglobin So Due to alteration of β chaino Glutamic acid in the 6th position of β chain is replaced

by valineo Generates hydrophobic “sticky patch”o Decrease O2 supply in the RBC

Processes that regulate H+ from CO2 transport Buffering effect

o Halimbawa:Pag ang mag syuta, nag aaway, ang taong nagpapabati sa kanila, tawag dun booper (c/o Dra Uy) Gets?!

o pk values are close to intraerythrocyte pHo provided by immidazole side chains (38 histidine per

tetramer)o histidine – amino acid involve in gas exchangeo example:

Hb – most important non bicarbonate buffer in the blood. - controls excess H+ during CO2 transport

o Buffering:By Hb – 50%Isohydric mechanism –40%

o Isohydric mechanismo Hb becomes stronger acid and releases H+

when it becomes oxygenatedClinical correlation:

Cyanosiso Purple magentao Increase deoxyhemoglobino Caused by disease of cardiac or pulmonary systemo Inadequate oxygenation of blood

HbA1co Information for the management of diabetes mellituso Reflects mean blood glucose concentration for 6-8

weekso Based on the half life or the RBC’s

*Questions:1. what is BPG? What are its effect?2. is Methemoglobin present in normal person?3. did u understand this transcription?

Greetings!!!!Welcome to all the new members of SDLS 2008!!!

Announcement!!!To all new members, please select a group leader, and give it to Erika, our secretary on Friday…

Good luck sa evals on Monday…

Sa mga hindi pa nagbabayad hanggang sa paglabas ng tranx na ito… magbayad ka na… (u know hu u r)

To all your comments… keep it to yourself…. –glenn

To all group leaders, ask nyo members nyo kung gusto nila ng shirt, socks, jackets for our batch ng SDLS, survey nyo group nyo,

and again bigay nyo kay Erika yung result sa Friday…. (this was suggested)

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