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 CH3 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
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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 PGH2 –action
of COX-1 and COX-2
• Prostaglandin is synthesised from PGH2
• Action of different PGE enzymes catalyse the
synthesis of different PGs
• Thromboxanes are synthesised from PGH2 by
thromboxane synthase

10. 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

11. 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

12. 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

14. 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)

16. 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)

17. Heme synthesis
• First reaction in mitochondria
• Condensation of one succinyl-coA by pyridoxal phosphate
– requiring enzyme (vitamin B6) – δaminolevulinic 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

18. 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

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

20. Insertion of Fe2+
• Protoporphyrinogen IX converted to protoporphyrin
IX –protophyrinogen IX oxidase
• Oxidation reaction requires molecular oxygen
• Ring 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 Fe2+ into the ring
system
Enzyme involved is ferrochelatase

21. 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

22. 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

23. 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 O2
- by adding electron
• 2O2 + NADPH 2O2
- + NADP+ + H+
• 2O2
- + H+ H2O2
- + O2
• 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

24. 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 (1O2) and
– hydroxyl radical (OH•).

25. 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 (1O2)

26. 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