1. Metabolism of tetrapyrrols Pavla Balínová

2. Tetrapyrrols circular compounds binding a metal ion (most frequently Fe 2+ and Fe 3+ ) consist of 4 pyrrol rings interconnected via methine bridges Examples: heme (Fe 2+ ) chlorophyll (Mg 2+ ) vitamin B 12 (Co 2+ ) Figure was assumed from http://en.wikipedia.org/wiki/Porphyrin

3. Where can we find a heme?? Hemoproteins Hemoglobin (Hb) Myoglobin (Mb) Cytochrome c Catalases (decomposition of H 2 O 2 to H 2 O and O 2 ) Figure was assumed from http://en.wikipedia.org/wiki/Heme

4. Heme structure pyrrol methine bridge Figure was assumed from a book T. M. Devlin et al.: Textbook of Biochemistry With Clinical Correlations, 4th ed., Wiley ‑ Liss, Inc., New York, 1997.

5. Biosynthesis of heme Organ location: bone marrow 85% and liver Subcellular location: mitochondria and cytosol Substrates: succinyl-CoA and glycine Important intermediates: δ-aminolevulinic acid (ALA), porphobilinogen, uroporphyrinogen III, protoporphyrin IX Key regulatory enzyme: ALA synthase

6. Heme biosynthesis Figure was assumed from http://www.porphyrin.net/mediporph/_netbiochem/synthesis/_synthmain.html

7. Delta-aminolevulinic acid (ALA) synthesis of heme starts in mitochondria succinyl-CoA and glycine (Gly) undergo condensation → δ-aminolevulinic acid (ALA) reaction is catalyzed by enzyme ALA synthase - OOC-CH 2 -CH 2 -CO-S-CoA + NH 3 + -CH 2 -COO - CO 2 - OOC-CH 2 -CH 2 -CO-CH 2 -NH 3 +

8. Porphobilinogen (PBG) ALA leaves the mitochondria → cytoplasm 2 molecules of ALA condense to form porphobilinogen reaction is catalyzed by enzyme porphobilinogen synthase Figure was assumed from http://en.wikipedia.org/wiki/Porphobilinogen pyrrol ring

9. Uroporphyrinogen III Figure was assumed from book T. M. Devlin et al.: Textbook of Biochemistry With Clinical Correlations, 4th ed., Wiley ‑ Liss, Inc., New York, 1997.

10. Uroporphyrinogen → coproporphyrinogen III enzyme hydroxymethylbilane synthase catalyzes the linkage of 4 PBG molecules and cleavage of 4 NH 4 + to yield uroporphyrinogen III 4 acetate residues are decarboxylated into methyl groups → coproporphyrinogen III returns to the mitochondria again

11. Protoporphyrinogen IX Figure was assumed from book T. M. Devlin et al.: Textbook of Biochemistry With Clinical Correlations, 4th ed., Wiley ‑ Liss, Inc., New York, 1997.

12. Protoporphyrinogen IX → protoporphyrin IX oxidation of protoporphyrinogen IX produces the conjugated π-electron system of protoporphyrin IX Figure was assumed from book T. M. Devlin et al.: Textbook of Biochemistry With Clinical Correlations, 4th ed., Wiley ‑ Liss, Inc., New York, 1997.

13. Heme Fe 2+ is incorporated into protoporhyrin IX reaction is catalyzed by enzyme ferrochelatase Figure was assumed from http://www.porphyrin.net/mediporph/_netbiochem/synthesis/http://www.porphyrin.net/mediporph/_netbiochem/synthesis/ ferrochelatase.html

14. Regulation of heme biosynthesis ALA synthase is a key regulatory enzyme it is an allosteric enzyme that is inhibited by heme = feedback inhibition requires pyridoxal phosphate certain drugs and steroid hormones can increase heme synthesis Porphobilinogen synthase is inhibited by lead ions Pb 2+ in case of lead poisoning. Ferrochelatase can be also inhibited by Pb 2+. Its activity is influenced by availability of Fe 2+ and ascorbic acid.

15. Porphyrias are hereditary or acquired disturbances of heme synthesis in all cases there is an identifiable abnormality of the enzymes which synthesize heme this leads to accumulation of intermediates of the pathway and a deficiency of heme → excretion of heme precursors in feces or urine, giving them a dark red color ● accumulation of porphyrinogens in the skin can lead to photosensitivity the neurological symptoms

16. Heme degradation around 100 – 200 million aged erythrocytes per hour are broken down in the human organism Organ location: RES (reticuloendothelial cells) in the spleen, liver and bone marrow Hb is degraded to: ● globin → AAs → metabolism ● heme → bilirubin ● Fe 2+ → transport with transferrin and used in the next heme biosynthesis Not only Hb but other hemoproteins also contain heme groups which are degraded by the same pathway.

17. Conversion of heme to bilirubin Figure was assumed from http://web.indstate.edu/thcme/mwking/heme-porphyrin.html

18. Bilirubin Bilirubin (Bil) is released from RES into the blood. BUT! Bil is only poorly soluble in plasma, and therefore during transport it is bound to albumin. ↓ LIVER In the hepatocytes, Bil is conjugated by 2 molecules of glucuronic acid → bilirubin diglucuronide (soluble in water, „conjugated Bil“). Conjugation is catalyzed by UDP-glucuronosyltransferase.

19. Bilirubin diglucuronide Figure was assumed from http://web.indstate.edu/thcme/mwking/heme-porphyrin.html

20. Bile pigments bilirubin diglucuronide ↓ BILE ↓ INTESTINE Bil is reduced to urobilinogen and stercobilinogen by bacteria → oxidation to urobilin and stercobilin Bile pigments are mostly excreted in feces, but a small proportion is resorbed (enterohepatic circulation). Small amount of urobilinogen is excreted with urine.

21. Determination of bilirubin in serum Blood tests Bil reacts directly when dyes are added to the blood sample → conjugated bilirubin = direct free Bil does not react to the reagents until alcohol (methanol) or caffeine is added to the solution. Therefore, the measurement of this type of bilirubin is indirect → unconjugated bilirubin = indirect Total bilirubin measures both unconjugated and conjugated Bil (normal value up to 20 µmol/L).

22. Hyperbilirubinemias Hyperbilirubinemia = an elevated bilirubin level (> 10 mg/L) → Bil can diffuses from the blood into peripheral tissues and gives it a yellow color (jaundice = icterus) Jaundice can have various causes: Increased erythrocyte degradation – hemolytic jaundice Impaired conjugation of bilirubin in the liver – hepatocellular jaundice Disturbance of bile drainage (gallstones) – obstructive jaundice In the urine, only conjugated bilirubin can be present.

23. Icterus Icterus is the yellow coloration of skin and mucus membranes of jaundice (hyperbilirubinemia with various ethiology) Hemolytic icterus: elevated level of unconjugated Bil in blood Neonatal jaundice usually appears after a few days after birth (elevated hemolysis, decreased activity of UDP-glucuronosyltransferase → ↑ unconjugated Bil) In severe cases, unconjugated Bil can cross the blood- brain barrier and lead to brain damage (kernicterus).