Detoxicol

SDLS 2008
Medicine for the intoxicated
File Created on 8/19/2004 10:08 PM Transcriber(s): Glenn and d gang Editors: JC Tayco, Mark Lomboy, Lulu Velazco, Pre Ausan 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 rings e.g Fe Porphyrins heme Mg Porphyrins chlorophyll Hemoproteins Proteins that contain heme Examples: Protein Function Hemoglobin Transport of O2 in blood Myoglobon Storage of O2 in muscle Cytochrome c Involvement in electron transport chain Cytochrome P450 Hydroxylation of xenobiotics Catalase Degradation of hydrogen peroxide Tryptophan Oxidation of pyrrolase 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 Krebs cycle o Glycine o Pyridoxal phosphate -coenzyme, for activation of glycine o ALA synthase- rate controlling (limiting), key regulatory enzyme o Product: delta-Aminolevulinic acid Porphobilinogen (PBG) o Precursor of pyrole Topic: Hemoglobin Lecturer: Dr. Ma. Esperanze E. Uy No. of pages: 5 o 4 molecules of PBG for the formation of a cyclic tetrapyrole (porphyrin) derived from breakdown of Aminolevulinic acid by ALA dehydratase in the CYTOSOL Hydroxymethlybilane

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Formed by Uroporphyrinogen I synthase, also removes NH3 Uroporphyrinogen I

Formed by hydroxymethylbilane Uroporphyrinogen III

Formed by uroporphyrinogen III synthase (most exclusive) Coporphophyrinogen III o Formed by uroporphyrinogen decarboxylase, acting on the acetate group Protoporphyrinogen IX

Formed by coporphyrinogen oxidase, back in the MITOCHONDRIA Protoporphyrin IX o Formed by protoporphyrinogen oxidase Heme molecule

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

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 synthase o Uroporophyrinogen decarboxylase o Ferrochelatase Sign and symptoms of the porphyrias:

*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 ALA 1. ALAS1 hepatic form rate limiing reaction of heme synthesis in the liver (positive and negative feedback mechanism) affected by administration of drugs 2. ALAS2 form by drug undergo feedback mechanism erythroid not induced does not

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 Ferrochelatase o 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 Heme Hemoglobin Heme + Iron + Globin Reuse Amino Acid (Iron Pool) Biliverdin Biliverdin Reductase Bilirubin (unconjugated) Attaches to serum albumin New Proteins

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

Glucoronic Acid LIVER

Bilirubin (Conjugated)

Urine Secretion into Bile Urobilinogens Urine Stool

organic soln 3.Van Den Bergh Reaction 4. Site 5. Bind 6. Conjugation 7. Brain tissue affinity 8. Presence w/ jaundice in urine 9. associated w/ jaundice (hemolytic)

Indirect RES Albumin Unconjugat ed High Affinity Negative Positive

Direct Liver Cholesterol Conjugated Low Affinity Positive Nrgative

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 thats 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!?

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 UDPglucoronyltransferase (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 (-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?

Remember: Unconjugated Bilirubin before passing and in the liver also known as: o Indirect Bilirubin o B1 o Prehepatic Bilirubin o Free Bilirubin Conjugated Bilirubin after passing the liver also known as: o Direct Bilirubin o B2 o Posthepatic Bilirubin Point of Comparison B1 1. Solubility in H2O Insoluble 2. Alcohol & other Non polar B2 Soluble Polar

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 compound A. 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 Kernicterus o 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 disease o Deficit in conjugation o Treatment:

E.g chloroform, acetaminophen

B. Increase of Conjugated Bilirubin (B2) Obstruction of Biliary Tree o Due to blockage of common bile duct o Often due to gallstone of CA of the head of the pancreas o Cholesthiasis Dubin Johnson syndrome Due to excretion deficit Defect in the hepatocyte mebrane o Mutation in the gene encoding MRP-2 o Bilirubin contains abnormal black pigment Rotor syndrome o Thought of due to abnormality in hepatic storage

C. Increase of Both Type Hepatitis o Drug induced o Alcoholic o Viral o 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 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

Phenobarbital induces the Cytocrome P450 enzyme system thus increasing the rate of conjugation Phototheraphy - hepatic excretion of Bilirubin. Bilirubin is photosensitive, easily breaks down.

Gilberts Syndrome o Bilirubin transport deficit o Transport of bilirubin into the hepatocyte Toxic hyperbilirubinemia o Due to toxin induced liver function


Hb

Heme group is w/o O2 Contains BPG in the chain of

Binds 1 proton for every 2 O2 molecules released (BOHR effect) b. obin (Relax state) affinity conformation Oxyhemogl R state High

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 mmHg o 98% saturation occurs In the musles o Able to release O2 at a lower tension (20 mmHg) o 33% saturation in working muscle o delivers 65% of O2

Heme group is associated with O2 Doesnt 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 conc = 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 compensate 2. 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 and chains Blood CO2 is present in 3 major form 1. Bicarbonate 78% 2. Carbamino Hb 13% 3. Dissolved CO2 9% Factors affect O2 binding 1. Temperature Increase temperature such as in fever weakens Hbs 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 (22) normal adult Hb

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

HbA2(A2D2) minor adult Hb HbF (A2G2) fetal Hb HbS (A2S2) Sickle cell

o Hb becomes stronger acid and

releases H+ when it becomes oxygenated Clinical correlation: Cyanosis o Purple magenta o Increase deoxyhemoglobin o Caused by disease of cardiac or pulmonary system o Inadequate oxygenation of blood HbA1c o Information for the management of diabetes mellitus o Reflects mean blood glucose concentration for 6-8 weeks o Based on the half life or the RBCs

Mutant Hemoglobin Termed as hemoglobinopathies Due to mutations in the genes that encode the and subunits of Hb Methemoglobin (Hi) o Also termed as hemoglobin o Ferrous iron is oxidized in the ferric state, resulting inability of Hi to combine with O2 o Polypeptide chains are not altered o Upto 1.5% is present in normal person o Increase to 10% conc of this will result to cyanosis Hemoglobin M o Hereditary Methemoglobinemia o Alteration of either or globin chain o Causing asymptomatic cyanosis Hemoglobin S o Due to alteration of chain o Glutamic acid in the 6th position of chain is replaced by valine o Generates hydrophobic sticky patch o Decrease O2 supply in the RBC

*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)

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 pH o provided by immidazole side chains (38 histidine per tetramer) o histidine amino acid involve in gas exchange o 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 mechanism