Eicosanoid synthesis

Describes the different pathways involved in the synthesis of different eicosanoids like prostaglandins, leukotrienes, lipoxins etc along with different enzymes involved.

1. Eicosanoid synthesis Dr. Radhakrihna G Pillai Department of Life Sciences University of Calicut, India 673 635

2. Eicosanoids The term " eicosanoids " is used as a collective name for molecules derived from 20-carbon fatty acids Fatty acids of the n-6 family deriving from linoleic acid are the main source of eicosanoids Arachidonic acid (20:4n-6) being the major precursor Eicosanoids are synthesized in vivo through several routes some compounds being formed by more than one mechanism In the plant kingdom, several potent derivatives from linolenic acid ( octadecanoid -derived compounds) are found and have hormone-like functions ( phytohormones )

3. Lipid number Lipid numbers. 18:3. ... Lipid numbers take the form C:D, where; C is the  number of carbon atoms in the fatty acid and D is the number of double bonds in the fatty acid 18:4 ω-3 or 18:4 n−3 indicates; an 18-carbon chain with 4 double bonds and with the first double bond in the third position from the CH 3   end Fig: source https://upload.wikimedia.org/wikipedia/commons/e/e4/Fatty_acid_numbering.png

4. Eicosanoid synthesis Eicosanoids consists of prostaglandins, thromboxanes, leukotrienes and lipoxins Prostaglandins and thromboxanes are identified as prostanoids The number of C-C double bonds are used as subscripts with the name of the prostanoids Majority of biologically active prostaglandins and thromboxanes are referred as series 2 molecules – presence of 2 C-C double bonds Predominant Leukotrienes are series 4 molecules -4 C-C double bonds Important series 1 prostaglandins and thromboxanes are also present Prostaglandins are originally shown to be synthesised in the prostate gland Thromboxanes from platelets ( thrombocyte ) Leucotrenes from leucocytes Lipoxins are synthesised through lipoxygenase interactions

5. Names of Prostaglandins Prostaglandins are originally shown to be synthesised in the prostate gland Thromboxanes from platelets and Leukotrienes from leukocytes Their names are derived from the place of synthesis https://www.google.co.in/search?q=platelets&rlz=1C1VFKB_enIN599IN599&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjS45uNr53QAhUIvo8KHVv1AJUQ_AUICCgB&biw=1280&bih=621#imgrc=O0EbkI9EC2Sl8M%3A Source of Fig https ://www.google.co.in/search?q=leukocytes&rlz=1C1VFKB_enIN599IN599&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjP2a_Mr53QAhXFo48KHeeZAeYQ_AUICCgB&biw=1280&bih=621#imgrc=NswWAwoMbeBGIM%3A

6. Eicosanoid synthesis Two main pathways involved in the biosynthesis of eicosanoids PG and TX are synthesised by the cyclic pathway The leukotrenes by the linear pathway Cyclic pathway initiated by Prostaglanding G/H synthase This enzyme has 2 activities – COX and peroxidase Two forms of COX- COX1 an dCOX2 COX-1 expressed constitutively – gastric mucosa, kidney, platelets and vascular endothelial cells COX-2 is inducible-in macrophages and monocytes express in response to inflammation COX-1 and COX-2 catalyse – arachidonic acid to PGG2 and PGH2

7. Cyclic pathway Numerous stimuli- epinephrine, thrombin and bradykinin activates phospholipase A2-(PLA2) PLA2 hydrolyses arachidonic acid from cell membrane phospholipids Bradykinin receptor coupled with G-protein activation Increased intracellular calcium ions Protein kinase C activated PKC phosphorylation and Ca2+ ions activate ER membrnane - associated cPLA2 isoform

8. Synthesis of prostaglandins Hydrolysis of arachidonic acid form phosphatidylinositol bisphosphate (PIP2) Arachidonic acid is converted to PGH 2 –action of COX-1 and COX-2 Prostaglandin is synthesised from PGH 2 Action of different PGE enzymes catalyse the synthesis of different PGs Thromboxanes are synthesised from PGH2 by thromboxane synthase

9. Linear Synthetic pathways Linear pathway initiated through arachidonate lipoxygenase (LOXs) 3 forms of LOXs Arachidonate 5-lipoxygenase (5-LOX), 12-LOX and 15-LOX 5-LOX help to produce leukotrenes – synthesised by leukocytes, mast cells, lung, spleen, brain and heart 12-LOX and 15-LOX involved in the synthesis of lipoxins

10. Synthesis of Leukotrienes Numerous stimuli- epinephrine, thrombin, bradykinin activates phospholipase A2-(PLA2) PLA2 hydrolyses arachidonic acid from cell membrane phospholipids Bradykinin receptor coupled with G-protein activation Increased intracellular calcium ions Protein kinase C activated PKC phosphorylation and Ca2+ ions activate ER membrnane - associated cPLA2 isoform

11. Leukotrienes Activated cPLA2 isoforms Hydrolyse arachidonic acid from phosphatidylinositol biphosphate (PIP2) Enzyme 5-LOX + 5-LOX activating protein catalyse the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) 5-hydroperoxyeicosatetraenoic acid (5-HPETE) is spontaneously reduced to 5-hydroxyeicosatetraenoic acid (5-HETE) Then to LTA4 LTA4 unstable – converted to LTC4

12. Synthesis of Lipoxins Synthesised through the concerted actions of 15-LOX on arachidonic acid in epithelial cells, 5-LOX in leukocytes and 12-LOX in platelets Three pathways Classic pathway is 5-LOX activity in leukocyte followed by 12-LOX in platelets 15-LOX in epithelial cells followed by 5-Lox in leukocytes Action of Aspirin on COX-2 in epithelial or endothelial cells –produce 15-epi-lipoxins (aspiring triggered lipoxins ATLS)

13. Porphyrins Large heterocyclic organic ring structures Composed of 4 modified pyrrole subunits connected by methine (=CH-) bridges Heme an example of naturally occurring porphyrin Heme in biological system consits of Fe2 + ions complexed with 4N of porphyrin molecules Three structurally distinct hemes in human; heme a, heme b and heme c heme is critical for biological functions of several enzymes – eg cytochromes of oxidative phosphorylation Cytochrome P450 family (CYP)

14. Heme synthesis First reaction in mitochondria Condensation of one succinyl-coA by pyridoxal phosphate requiring enzyme (vitamin B6) – δ aminolevulinic acid synthase (ALAS) forming δ aminolevulinic acid (5 aminoleuvinic acid) This is the rate limiting reaction in heme synthesis ALA is transported to cytosol ALA dehydratase ( porphobilinogen synthetase ) dimerises 2molcules of ALA Forms Porphobilinogen Head-to-tail condensation of 4 molecules of porphobilinogen -form linear tetrapyrrole intermediate – hydroxymethylbilane

15. Heme synthesis Enzyme for Head-to-tail condensation of 4 molecules of porphobilinogen is porphobilinogen deaminase (PBG deaminase ) Hydroxymethylbilane has two fates One due to enzymatic action Other due to non-enzymatic action Hydroxymethylbilane is enzymatically converted to uroporphyrinogen III Mediated by the enzyme uroporphyrinogen III synthase uroporphyrinogen III is decarboxylated by uroporphyrinogen decarboxylase Forms coproporphyrinogens Coproporphyrinogen III is most important in heme synthesis Coproporphyrinogen III transported to the interior of the mitochondria

16. Formation of conjugated ring 2 propionate residues Coproporphyrinogen III are decarboxylated Protoporphyrinogen IX formed Catalysed by coproporphyrinogen -III oxidase

17. Insertion of Fe 2+ Protoporphyrinogen IX converted to protoporphyrin IX – protophyrinogen IX oxidase Oxidation reaction requires molecular oxygen R ing system -Loss of 6protons and 6 electrons – produce a completely conjugated Responsible for the red colour of heme Final reaction in heme synthesis takes place in mitochondria Insertion of Fe 2+ into the ring system Enzyme involved is ferrochelatase

18. Respiratory burst in phagocytes Respiratory burst (oxidative burst) -rapid release of reactive oxygen species (superoxide radical and hydrogen peroxide) from different types of cells Release of these chemicals from immune cells, e.g., neutrophils and monocytes, as they come into contact with different bacteria or fungi. They are also released from the ovum of higher animals after the ovum has been fertilized. Respiratory burst plays an important role in the immune system Crucial reaction that occurs in phagocytes to degrade internalized particles and bacteria NADPH oxidase , an enzyme family in the vasculature (in particular, in vascular disease), produces superoxide, which spontaneously recombines with other molecules to produce reactive free radicals

19. Phagocytic killing Oxygen dependent killing Oxygen independent killing Oxygen dependent phagocytic killing Activated phagocytes produce a number of reactive oxygen intermediates and Nitrogen intermediates When exposed to certain stimuli- phagocytes (Neutrophils, oesinophils , and macrophages) increase oxygen uptake- upto 50 fold Oxygen burst

20. Superoxide radical Elimination of invading micro-organisms by neutrophils , monocytes , and macrophages depends heavily on the generation of reactive oxygen species during the phagocytosis -associated respiratory burst NADPD oxidase also called respiratory burst oxidase Present in phagocyte membrane toxic oxidants are released to the inside and outside of the cell Catalyse reduction of O 2 - by adding electron 2O2 + NADPH 2O 2 - + NADP + + H + 2O 2 - + H + H 2 O 2 - + O 2 the oxidants superoxide anion (O2), hydrogen peroxide (H2O2), hypochlorous acid, and hydroxyl radical, created in this process- carry the potential to damage the phagocytes themselves as well as other cells at sites of inflammation

21. Oxygen-dependent myeloperoxidase -independent intracellular killing During phagocytosis , glucose is metabolized via the pentose monophosphate shunt, with formation of NADPH Cytochrome B from the granulocyte-specific granule combines with and activates plasma membrane  NADPH oxidase The activated  NADPH oxidase  then employs oxygen to oxidize the formed NADPH with resultant production of superoxide anion A portion of the superoxide anion is converted to H2O2 plus singlet oxygen by superoxide dismutase Superoxide anion can react with H2O2, resulting in the formation of hydroxyl radical plus more singlet oxygen Together these reactions produce the toxic oxygen compounds; superoxide anion (O2-), H2O2 singlet oxygen ( 1 O2) and hydroxyl radical (OH•).

22. Oxygen-dependent myeloperoxidase -dependent intracellular killing Fusion of azurophilic  granules with the phagosome causes release of myeloperoxidase into the phagolysosome azurophilic  granules -A large, coarse, blue-purple membrane-bound organelle in  progranulocytes ,  mylocytes and  neutrophils , which acts as a  reservoir  for digestive and  hydrolytic enzymes before delivery to a  phagosome Myeloperoxidase utilizes H2O2 and halide ions (usually Cl - ) to produce highly toxic hypochlorite Some hypochlorite spontaneously breaks down to yield singlet oxygen Together these reactions produce toxic hypochlorite ( OCl -) and singlet oxygen ( 1 O2)

23. Detoxification reactions Neutrophils  and macrophages are able to protect themselves by detoxifying the toxic oxygen intermediates that they generate Granulocyte self-protection is achieved in reactions employing the dismutation of superoxide anion to hydrogen peroxide by  superoxide dismutase  and The conversion of hydrogen peroxide to water by  catalase