1
Lipid metabolism II
Phospholipids and glycolipids
Eicosanoids
Cholesterol and bile acids
Biochemistry I
Lecture 9 2008 (J.S.)
2
Schematic structure of complex lipids
The "head“ group
GlycolipidsSphingophospholipids
The "head“ group
Plasmalogens
The "head“ group
Glycerophospholipids
Sphingolipids:
3
In spite of the difference in the structures of glycerophosphoplipids and
sphingophospholipids, the over-all shape of the both types of
phospholipid molecules is very similar:
Glycerophospholipid Sphingophospholipid
Simplified icon of
a phospholipid molecule
Polar head
Two hydrophobic
chains
4
The major glycerophospholipids
The "head“ group
The simplest glycerophospholipid is phosphatidic acid
(phosphatidate, sn-1,2-diacylglycerol 3-phosphate).
Only very small amounts of phosphatidate
are present in membranes. However, the
molecule is a key intermediate in the biosynthesis
of the other glycerophospholipids.
Glycerophospholipids
5
6
Biosynthesis of glycerophospholipids
The synthesis is localized on the membranes of endoplasmic reticulum.
The competent enzymes are integral membrane proteins, the active sites
are accesible on the cytoplasmic side of ER.
The new molecules formed in the outer layer of ER membranes are
transferred into the inner layer by the action of flipases,
transported into other membranes in the form of membrane vesicles,
released by means of phospholipid-transfer proteins into the cytoplasm.
The initial steps in the synthesis are similar to those
of the triacylglycerol synthesis:
7
GLYCEROPHOSPHOLIPIDS
TriacylglycerolsPhosphatidate
CH2–O–CO–R
CH2–O–PO 3
2–
CH–O–CO–R
CH2–O–CO–R
CH2–OH
CH–O–CO–R
CH2–O–CO–R
CH2–O–CO–R
CH–O–CO–R
R-CO-S-CoA CoA-SH
Pi
hydrolase
H2O
1,2-Diacylglycerol
Addition of the
head group
There are two mechanisms of addition of the head group.
In both cases, the reaction is driven by CTP (cytidine triphosphate):
1 – diacylglycerol can accept CDP-activated choline or ethanolamine
(synthesis of phosphatidyl choline, phosphatidyl ethanolamine, resp. phosphatidyl serine).
2 – phosphatidate is activated to CDP-diacylglycerol that can accept the
head group (synthesis of phosphatidyl inositol or cardiolipin),
8
Diacylglycerol accepts CDP-activated choline or ethanolamine.
Activation of choline in two steps:
1 Synthesis of phosphatidyl choline, phosphatidyl
ethanolamine, and phosphatidyl serine
Choline + ATP → choline phosphate + ADP
Choline phosphate + CTP → CDP-choline + PPi
CDP-choline plays a part formally similar to that of UDP-glucose in the
synthesis of glycogen.
CH3
CH3
OH OH
O
N
NO
NH2
O–
CH2
O–
O O
CH3–N–CH2–CH2–O–P–O–P–O–
+
O–
O
+
CH3–N–CH2–CH2–O–P–O–
CH3
CH3
9
Cytidine diphosphate (CDP) is used as a carrier, from which
choline phosphate is transferred, the acceptor being a 1,2-diacylglycerol.
1,2-diacylglycerol + CDP-choline CMP + phosphatidyl choline (PC)
The biosynthesis of phosphatidyl ethanolamine (PE) is similar.
O–
CH2
O
P–O–
R–CO–O–CH2
R–CO–O–CH
CH2–O
–CH2–N–CH3
CH3
CH3
+
O–
O
P–O–CH2–CH2–NH2
R–CO–O–CH2
R–CO–O–CH
CH2–O
N-Methylation of PE (in the
liver, the donor of methyl group is
S-adenosylmethionine) to give
PC is not as important in higher
animals as incorporation of
choline de novo.
10
Phosphatidyl ethanolamine + serine →
phosphatidyl serine + ethanolamine
Phosphatidyl serine (PS) is not, in animals, formed directly in this
way, but as exchange of serine for the ethanolamine of PE:
Phosphatidyl serine can be also decarboxylated to form PE.
CH2–CH–NH2
O–
O
P–O–
R–CO–O–CH2
R–CO–O–CH
CH2–O
COOH
O–
O
P–O–CH2–CH2–NH2
R–CO–O–CH2
R–CO–O–CH
CH2–O
Serine
Ethanolamine
CO2
11
Phosphatidic acid is activated in a reaction with CTP to CDP-diacylglycerol:
CDP
2 Synthesis of phosphatidyl inositol and cardiolipin
Phosphatidic acid + CTP → CDP-diacylglycerol + PPi
OH OH
O
N
NO
NH2
O–
CH2
O–
O O
P–O–P–O–
R–CO–O–CH2
R–CO–O–CH
CH2–O
12
CDP-diacylglycerol + inositol CMP + phosphatidyl inositol (PI)
CDP-Diacylglycerol then reacts
with free inositol to give phosphatidyl inositol (PI), or
with glycerol phosphate to form phosphatidyl glycerol /PG), resp.
Phosphatidyl inositol:
Further phosphorylations of PI generate
phosphatidyl inositol bisphosphate (PIP2) which is an intermediate of the
phosphatidyl inositol cycle generating important intracellular messengers
IP3 and diacylglycerol.
13
Cardiolipin
(1,3–bisphosphatidyl glycerol)
Cardiolipin (constituent of the inner mitochondrial membrane)
CDP-diacylglycerol
CMP
Phosphatidyl glycerol
R–CO–O–CH2
CH2–O
R–CO–O–CH
O
OHO–
P–O–CH2–CH–CH2OH
O–
O
CH2–O–CO–R
CH2–O
CH–O–CO–R
CH2–O–CO–R
CH2
CH–O–CO–R
O–
O
OHO–
P–O–CH2–CH–CH2–O–P
Glycerol
CDP-diacylglycerol + glycerol 3-phosphate
phosphatidyl glycerol 3-phosphate + CMP
Pi
14
Glycerophospholipids
are
– essential structural components of all biological membranes,
– essential components of all types of lipoproteins in extracellular fluids,
– supply polyunsaturated fatty acids for the synthesis of eicosanoids,
– act in anchoring of some proteins to membranes,
– serve as a component of lung surfactant
– phosphatidyl inositols are precursors of second messengers (PIP2, DG),
etc.
15
Anchoring of proteins to membrane
The linkage between the COOH-terminus
of a protein and phosphatidylinositol fixed
in the membrane lipidic dilayer exist in
several ectoenzymes (alkaline phosphatase,
acetylcholinesterase, some antigens).
16
Lung surfactant
The major component of lung surfactant is dipalmitoylphosphatidylcholine.
It contributes to a reduction in the surface tension within the alveoli (air spaces) of
the lung, preventing their collapse in expiration. Less pressure is needed to reinflate
lung alveoli when surfactant is present.
The respiratory distress syndrome (RDS) of premature infants is caused, at least
in part, by a deficiency in the synthesis of lung surfactant.
17
Phosphatidyl inositol phosphates
(PIP, PIP2, PIP3) are minor components of plasma membranes, and their
turnover is stimulated by certain hormones.
A specific phospholipase C, under hormonal control, hydrolyses phosphatidyl
4,5-bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5-trisphosphate
(IP3), both of which have second messenger functions.
Second messengers
are intracellular compounds the concentration of which raises as a consequence of binding
of the hormone or the neurotransmitter to the membrane receptor. The hormone-receptor
complex controls the synthesis (or release) of the second messenger and this control is
mediated by a third type of protein, called G-protein.
IP3
Inositol 1,4,5-trisphosphate
R–CO–O–CH
R–CO–O–CH2
CH2-O-PO
O-
O-
R–CO–O–CH
R–CO–O–CH2
CH2-OH
O-PO3
2-
2-
O3PO
OH
OH OH
2-
O3P-O
O-PO3
2-
2-
O3PO
OH
OH OH
PIP2
PI 3,4-bisphosphate
DG
18
Phosphatidyl inositol cascade
Phospholipase CReceptor
Gαq
βγ
Specific ligand
PIP2 DG
Activation of
proteinkinase C
Phosphorylation of
intracellular proteins
Increase of Ca2+
concentration in cytosol
Endoplasmic
reticulum Ca2+
IP3-receptor
Ca2+
-ion channel
IP3
19
Plasmalogens
are modified glycerophospholipids – called alkoxylipids or
ether glycerophospholipids.
Plasmalogens represent about 20 % of glycerophospholipids.
Choline plasmalogen is found in myocard, in the liver (~1 %), and
ethanolamine plasmalogen in myelin (~ 23 %).
O–
O
P––
CH2–O–CH=CH–R
R–CO–O–CH
CH2–O choline (in myocard)
ethanolamine (in myelin)
serine
Alkenyl
Ether bond
20
Synthesis of plasmalogens
(ether glycerophospholipids, alkoxylipids)
Dihydroxyacetone phosphate
Acyl-dihydroxyacetone phosphate
Alkyldihydroxyacetone phosphate
1-Alkylglycerol 3-phosphate
1-Alkyl-2-acylglycerol phosphate
1-Alkyl-2-acylglycerol
1-Alkyl-2-acylglycerophosphoethanolamine
1-Alkenyl-2-acylglycerophosphoethanolamine
Ethanolamine plasmalogen
Acyl-CoA
Acyl-CoA
Fatty alcohol
Fatty acid
O2 + NADH
2H2O
H2O
Pi
NADPH+H+
NADP+
CDP-ethanolamine
CMP
Exchange of the acyl
for an alcohol and the
desaturation of it
21
PAF (platelet activating factor)
is an unusual alkoxylipid in which the alkenyl group of plasmalogens was
reduced to saturated alkyl and the fatty acyl at position 2 was exchanged for
acetyl.
PAF induces aggregation of blood platelets and vasodilation and exhibits
further biological effects, e.g. it is a major mediator in inflammation, allergic
reaction and anaphylactic shock.
Acyl reduced to
alkyl
Acetyl in place of
the fatty acyl
O–
O
P–O–CH2–CH2–N–CH3
CH2–O–CH2–CH2–R
CH3–CO–O–CH
CH2–O
CH3
CH3
+
22
Catabolism of glycerophospholipids
Enzymes catalysing hydrolysis of glycerophosholipids are called
phospholipases. Phospholipases are present in cell membranes or in
lysosomes. Different types (A1, A2, C, D) hydrolyse the substrates at
specific ester bonds:
O–
O
P–O–X (head group)
R–CO–O–CH2
R–CO–O–CH
CH2–O
C
A1
A2
D (only in brain and plants)
23
Phospholipase A1 (PL A1) exhibits preference for phosphatidyl
ethanolamines.
Phospholipase A2 obviously prefers phosphatidyl cholines and is of
special importance because it liberates arachidonic acid as a precursor
of eicosanoids.
Either PL A1 or A2 set free only one acyl residue and leaves
a lysophospholipid which is not further attacked by either enzyme.
The remaining acyl group is removed by the action of
lysophospholipase-transacylase (formerly called phospholipase B).
The enzyme removes the remaining acyl group from the
lysophosholipid, and transfers it either to water (hydrolysis), or to a
second lysophospholipid (transacylation).
Phospholipase C is stimulated by some hormonal signals and some
neurotransmitters. It hydrolyses PIP2 to IP3 and DG – the crucial step in
phosphatidyl inositol cascade.
24
Sphingolipids – schematic structure
A glycolipidA sphingophospholipid
The "head“ group
Ceramide
N-Acylsphingosine
25
Sphingosine contains 18 carbons atoms, trans-double bond in position 4,
amino group at position 2, and two hydroxyls at position 1 and 3.
Its alternative name is 4-sphingenine (syst. 2-aminooctadec-4-ene-1,3-diol.
CH–CH–CH2–OH
NH2
OH
Ceramides are N-acylated sphingosines. The acyl residue is attached
to the amino group of sphingosine by an amide link:
CH–CH–CH2–OH
NH
OH
C
O
The acyl residue has often 24 carbon atoms (lignoceric acid and its derivatives.
26
Glycolipids
are ceramides to which a saccharidic component is attached by glycosidic
bond: monoglycosylceramides – cerebrosides, oligoglycosylceramides,
acidic sulphoglycosylceramides, and sialoglycosylceramides – gangliosides.
Sphingolipids Ceramide is the lipidic part of all types of sphingolipids.
CH–CH–CH2–O–P–O–CH2–CH2–N–CH3
NH
OH
C
O
+
CH3
CH3O–
O
β-D-Glucopyranosyl
NH
OH
C
O
CH–CH–CH2–O
Sphingophospholipids
are esters of ceramide-1-phosphate and ethanolamine or (mostly) choline.
Ceramidephosphocholines are called sphingomyelins.
Phosphocholine
Cerebroside:
27
Saccharidic components of glycolipids - examples:
Cerebroside Ceramide–(1←1β)Glc
Oligoglycosylceramide Ceramide–(1←1β)Glc (4←1β)Gal
Sulphoglycosphingolipid Ceramide–(1←1β)Glc-3´-sulphate
Gangliosides
GM3 (monosialo ganglioside type III)
Ceramide–(1←1β)Glc-(4←1β)Gal
(3←2α)
NeuAc
GM2 Ceramide–(1←1β)Glc-(4←1β)Gal-(4←1β)GlcNAc
(3←2α)
NeuAc
GM1 Ceramide–(1←1β)Glc-(4←1β)Gal-(4←1β)GlcNAc-(3←1β)Gal
(3←2α)
NeuAc
28
Ganglioside GM2
Ceramide–(1←1β)Glc-(4←1β)Gal-(4←1β)GlcNAc
(3←2α)
NeuAc
29
Biosynthesis of sphinganine and N-acylsphingosine (ceramide)
The carbon chain of sphingosine is formed by condensations between
acyl-CoA – usually palmitoyl-CoA – and serine:
Palmitoyl-CoA Serine+
NADPH+H+
CoA-SH + NADP+
+ CO2
3-Ketosphinganine
NADPH+H+
NADP+
Sphinganine
Acyl-CoA (24 C)
CoA-SH
Dihydroceramide
Desaturation
(FAD-enzyme)
2 H
Ceramide
30
Biosynthesis of sphingomyelin and glycolipids
All sphingolipids are formed by attachment of an activated group
to the free 1-hydroxyl of a ceramide.
Synthesis of sphingomyelin
CDP acts as a carrier of phosphoryl choline:
Ceramide + CDP-choline Sphingomyelin + CMP
(Ceramide-P-choline)
Synthesis of glycolipids
A glycosyl is supplied by the transfer from UDP-monosaccharide:
Ceramide + UDP-Gal Cerebroside + UDP
Attachment of further glycosyls proceeds in a similar way.
Sialyl group (NeuAc in gangliosides) is transferred from CMP-NeuAc.
Oligoglycosylceramide + CMP-NeuAc Ganglioside + CMP
Sulphosphingolipids are formed by transfer of sulphate from
3´-phosphoadenosine-5´-phosphosulphate (abbr. PAPS).
31
Degradation of sphingolipids in lysosomes
In lysosomes, a number of specific enzymes catalyse hydrolysis
of ester and glycosidic linkages of sphingolipids.
Sphingomyelins loose phosphocholine to give ceramide.
Glycolipids due to the action of various specific glycosidases get
rid of the saccharidic component to give ceramide, too.
Ceramide is hydrolysed (ceramidase)
to fatty acid and sphingosine.
Sphingosine is decomposed in the pathway that looks nearly
like the reversal of its biosynthesis from palmitoyl-CoA and
serine. After phosphorylation, sphingosine is broken down to
phosphoethanolamine (decarboxylated serine) and
palmitaldehyde, that is oxidized to palmitate.
32
Phosphocholine
FATTY ACID
CERAMIDE
(N-Acylsphingosine)
SPHINGOSINE
Sphingosine-1-P
Phosphoethanolamine
Palmitaldehyde
PALMITIC ACID
Ceramide Glc Gal GalNAc Gal
NeuNAc
Ceramide Gal
Ceramide Glc
Ceramide Gal–O-SO3
–
Ceramide–P–-choline
SPHINGOMYELIN
CEREBROSIDE
SULPHATIDE
GANGLIOSIDE GM1
ATP
NAD+
Degradation of sphingolipids
33
In general, the turnover of sphingolipids is very slow, particularly in brain.
Sphingolipidosis
Inherited defects in production of the enzymes that catabolize
sphingolipids result in accumulation of their substrates in lysosomes,
leading to lysosomal damage and to disruption of the cell as new
lysosomes continue to be formed and their large number interferes with
other cellular functions.
In the sphingolipidosis mainly the cells of the central nervous system
(including brain and retina) are affected.
34
Sphingolipidoses – genetic defects (deficiency of lysosomal enzymes)
Phosphocholine
FATTY ACID
CERAMIDE
Sphingosine
Sphingosine-1-P
Phosphoethanolamine
Palmitaldehyde
PALMITIC ACID
Ceramide Gal
Ceramide Glc
Ceramide Gal–O–SO3
–
SPHINGOMYELIN
CEREBROSIDE
SULPHATIDE
ATP
NAD+
GANGLIOSIDE
Ceramide Glc Gal GalNAc Gal
NeuNAcNiemann-Pick disease
Farber´s lipogranulomatosis
Metachromatic leukodystrophy
Krabbe´s disease
Gaucher´s disease
Tay-Sachs disease
GM1 gangliosidosis
35
Eicosanoids
36
Eicosanoids
are a family of polyunsaturated C20 fatty acid derivatives,
(Greek eikosi – “twenty”),
which act as local hormones and have a wide range of biological functions.
The major precursors are essential polyunsaturated fatty acids
– arachidonic acid (eicosatetraenoic, abbr. ETE)
20:4 (5,8,11,14) from the n-6 series,
– eicosapentaenoic acid (abbr. EPE)
20:5 (5,8,11,14,17) from the n-3 series,
and, in part, non-essential
– eicosatrienoic acid 20:3 (5,8,11) from the n-9 series.
COO–
37
Although the intracellular concentration of free precursors is very low, they
can be released from C-2 of membrane phospholipids by the action of
phospholipase A2 and also by the degradation of diacylglycerol
generated in the PI cycle.
The activity of phospholipase A2 is a process closely regulated by extracellular mediators
(adrenaline, thrombin, angiotensin II, bradykinin).
On the other hand, corticosteroids through induction of lipocortin inhibit the activity of
phospholipase A2.
Cyclooxygenase pathway leads to the synthesis of prostaglandin H,
an endoperoxide, the precursor of cyclic prostaglandins,
prostacyclins, and
thromboxanes.
Lipoxygenase pathway converts precursor acids to acyclic
hydroperoxyacids (HETEs), from which
either leukotrienes (action of 5-lipoxygenase)
or lipoxins (action of 15- and 12-lipoxygenase) are formed.
38
39
Cyclooxygenase pathway
Synthesis of cyclic eicosanoids - prostanoids
Cyclooxygenase (COX, prostaglandin endoperoxide synthase)
is a membrane-bound enzyme, which has cyclooxygenase and
peroxidase activities. It exists in two forms:
COX-1 is a constitutive enzyme, expressed in almost all tissue;
COX-2 is inducible – its synthesis is induced by cytokines in
inflamed tissue.
COX catalyses the conversion of arachidonate to PGH2 – the common
precursor of all the prostanoids of the 2-series (diene prostanoids): after
formation of the ring, from four double bonds of arachidonate there will
remain only two double bonds in the side chains.
Similarly, COX catalyses conversion of eicosapentaenoate to PGH3, the precursor of the
prostanoids of the 3-series (triene prostanoids), and conversion of eicosatrienoate to
PGH1
40
Precursor of all prostanoids
of the 2-series
Prostaglandin H2
41
PGE synthase
Prostaglandin H2
Thromboxane TXA2
Prostacyclin PGI2
Prostaglandin PGE2
Prostaglandin PGF2α
PGE 9-keto reductase
TXA synthase
PGI synthase
42
Inhibition of cyclooxygenase blocks prostanoid production
Prostanoids mediate, at least partly, the inflammatory response.
Advisable effects of supressed prostanoid production:
the anti-inflammatory effect,
relief of pain, mitigation of fever.
On the contrary, there may be some undesirable effects of blocked prostanoid
production, e.g. decline in blood platelet aggregation, decreased protection of
endothelial cells and of gastric mucosa.
Inhibitors of cyclooxygenase act as nonsteroidal anti-inflammatory drugs
(NSAIDs, analgetics-antipyretics):
- acetylsalicylic acid (aspirin) – inhibits both COX-1 and COX-2 irreversibly
by acetylation the enzyme at its
active site.,
- acetaminophen and ibuprofen – reversible COX inhibitors.
Drugs are being developed which will act as selective inhibitors of COX-2
(named coxibs, e.g. celecoxib, rofecoxib) without the adverse gastrointestinal
and anti-platelet side effects of non-specific inhibitors of COX.
43
Lipoxygenase pathway
Synthesis of leukotrienes
Precursor of all leukotrienes
of the 4-series
5-Lipoxygenase
COO–
OOH
COO–
O
COO–
Arachidonate
5-HydroperoxyETE
Leukotriene LTA4
O2
44
Leukotrienes are produced primarily in leukocytes and mast cells and all of them have
three conjugated double bonds (trienes), the position of which may be different and the
configuration either trans or cis..
The classes of LTs are designated by letters A – E), the subscript denotes the total
number of double bonds.
COO–
O
LTA4
LTB4
OH
S
Cys→Gly
LTD4
Slow-reacting substance
of anaphylaxis (SRS-A)
Peptidoleukotrienes (leukotrienes C, D, E)
– carbon atom 6 binds the sulfur atom
of glutathione (γ-Glu→Cys →Gly) in the class LTC,
of cysteinyl-glycine in the class LTD, and
of only cysteine in the class LTE.
LTB4
12-Lipoxygenase
GSH
Glu
45
Leukotrienes are the most effective eicosanoids, e.g. their vasodilating effect
is about 5 000 times more intensive than that of the same amount of histamine.
Eicosanoids are produced in various types of tissue.
The site of their synthesis depends on expression of genes for the
enzymes which take part in the synthetic pathways.
E.g., in the lung and the spleen, the enzyme equipment enables
biosynthesis of all eicosanoid types.
In blood platelets, only thromboxan synthase is present.
The endothelial cells of blood vessels synthesize only prostacyclins.
Catabolism of eicosanoids is rapid.
The biological half-life of prostanoids t½ was found to be in the range
from seconds to few minutes.
46
Examples Structural group Synthesized in
The most remarkable
effect:
PGE2 prostaglandin E nearly all cell types
inflammatory reaction,
vasodilation,
inhibition of HCl secretion
PGF2α prostaglandin F nearly all cell types vasoconstriction
increase of body temp.
PGI2 prostacyclin
endothelial cells,
smooth muscle cells
of blood vessels
vasodilation,
inhibition of platelet
aggregation
TXA2 thromboxane blood platelets
platelet aggregation,
vasoconstriction
LTD4 leukotriene leukocytes, mast cells bronchoconstriction,
vasoconstriction
LXA4 lipoxin various cell types bronchoconstriction,
vasodilation
Eicosanoids
47
Cholesterol and bile acids
48
Constituent of all animal membranes which modulates the fluidity of
cell membranes. It also occurs in trace amounts in plants.
Necessary precursor of the synthesis of bile acids, steroid hormones
and calciols (vitamin D).
Although much cholesterol is obtained from the diet, the animal body
can synthesize all the cholesterol it requires.
Cholesterol is synthesized in all nucleated cells.
Biosynthesis: approx. 800-1000 mg per day.
Dietary intake: approx. 500 mg per day
(egg yolk, animal fat and meat, fat dairy products).
Cholesterol
(Cholest-5-ene-3β-ol)
49
Biosynthesis of cholesterol
Cholesterol is synthesized from acetyl coenzyme A, all 27 carbon
atoms of cholesterol are derived from acetyl-CoA.
The synthesis is localized in the cytosol and on the membranes of
endoplasmic reticulum (some enzymes catalysing the synthesis are
integral membrane proteins of ER).
About 1/3 of cholesterol is formed in the liver, substantial amounts are
also formed in the gut and skin. High rates of the synthesis are observed in the
adrenal cortex and gonades.
The synthesis is a four-stage proces:
1 The synthesis of mevalonate from acetyl-CoA.
2 The conversion of two mevalonates to two activated isoprene units
that are the key building blocks of cholesterol.
3 The condensation of six molecules of activated isoprenes to form
squalene.
4 The cyclization of squalene in an astonishing reaction and the
conversion of the four-ring steroid nucleus into cholesterol.
50
15 Acetyl-CoA (tens of reactions) cholesterol
The result of isotope-labeling experiment show the source of carbon atoms.
Cholesterol was synthesized from acetate labeled in its methyl (blue)
or carboxylate (red) atom:
CH3–CO–
51
3-Hydroxy-3-methylglutaryl-CoA
( HMG-CoA )
Cytosol, ER membrane
1 The synthesis of mevalonate from acetyl-CoA.
Acetoacetyl-CoA
CH3–CO–CH2–CO–CoA
CoA
Acetyl–CoA
2 CH3CO-CoA
CH3CO-CoA
CoA
-
–
OOC–CH2–C–CH2–CO–CoA
CH3
OH
Compare with the first steps
of ketogenesis in the matrix
of mitochondria!
HMG-CoA synthase
52
3-Hydroxy-3-methylglutaryl-CoA is then reduced in the 4-electron reaction
to mevalonate (3,5-dihydroxy-3-methylvalerate)::
This reduction of MHG-CoA to mevalonate catalysed by
HMG-CoA reductase is the rate-limiting step in the pathway of
cholesterol synthesis. Both the amount of the enzyme and its activity is strictly
controlled
HMG-CoA reductase
HMG-CoA
-
–
OOC–CH2–C–CH2–CO–CoA
CH3
OH
2 NADPH + 2 H+
2 NADP+
CoA
Mevalonic acid
-
–
OOC–CH2–C–CH2–CH2-OH
CH3
OH
Cytosol
The fate of HMG-CoA synthesized in the mitochondrial matrix is different –
HMG-CoA is split into free acetoacetate and coenzyme A (ketogenesis).
53
Control of cholesterol biosynthesis
by regulating the activity of HMG-CoA reductase:
Inhibition
– by cytosolic free cholesterol
(feed-back control; Brown and Goldstein)
– by reversible phosphorylation of the enzyme
– by drugs called statins.
Statins are competitive inhibitors of
HMG-CoA reductase, either fungal
products (e.g. lovastatin), or quite
synthetic compounds (3rd
generation of
statins, e.g. cerivastatin).
The highlighted part of the lovastatin
molecule resembles the HMG-moiety. Lovastatin
54
Hormonal control of the HMG-CoA reductase activity
through reversible phosphorylation:
GLUCAGON
ADRENALINE
cAMP
HMG- CoA reductase kinase
(phosphorylated ACTIVE)
HMG- CoA reductase
kinase kinase
(phosphorylated ACTIVE)
HMG-CoA reductase
HMG-CoA Mevalonate → → → → Cholesterol
Protein kinase A
(phosphorylated ACTIVE)
Phosphoprotein phosphatase
inhibitor
(phosphorylated ACTIVE)
Phosphoprotein phosphatase
phosphorylated INACTIVE
Activates HMG-CoA reductase
by dephosphorylation
INSULIN
(activates phosphoprotein
phosphatase)
55
2 The conversion of mevalonate to activated isoprene units
Mevalonate 5-diphosphate
H2O
2 ATP 2 ADP
ATP
ADP + Pi
Mevalonate
-
–
OOC–CH2–C–CH2–CH2-OH
CH3
OH
CO2
–
OOC–CH2–C–CH2–CH2-O–P–O–P–O
CH3
OH
O–
O O
O–
3,3-Dimethylallyl diphosphate
CH3
CH3
C
CH
CH2
O –P–O–P–O–
O
O–
O–
O
Isopentenyl diphosphate
CH2
CH3
–P–O–P–O–
O
O–
O–
O
C
CH2
CH2
O
56
3 The condensation of molecules of activated isoprenes
to form squalene (30 C):
+ 5 C + 5 C
10 C + 5 C
15 C + 15 C
+ diphosphate
+ diphosphate
+ 2 diphosphate + NADP+
SQUALENE
+ NADPH + H+
30 C
57
4 The cyclization of squalene and the conversion of the
steroid nucleus into cholesterol.
Due to free rotation round single covalent bonds, the „stretched“ form of squalene
may take also the conformation that suggests the interactions causing the subsequent
closure of the four-ring steroid nucleus:
Squalene (30 C)
Lanosterol (30 C)
Lanosterol is merely an intermediate in man, but occurs free in wool fat.
NADPH+H+
+ O2
H2O
Monooxygenase
2,3-Oxidosqualene cyclase
and spontaneous rearrangement
58
Squalene (30 C) → → → lanosterol (30 C) → → → cholesterol (27 C)
The final conversion of lanosterol to cholesterol
involves more than 5 steps:
-oxidative removal of three –CH3 groups
(catalysed by a monooxygenase) as 2 CO2 and HCOO–
,
- rearrangement of double bonds,
- reduction (saturation) of one of the two double bonds.
Lanosterol (30 C)
Cholesterol
(27 C; cholest-5-ene-3β-ol)
Almost all the reactions in cholesterol synthesis take place on the endoplasmic
reticulum. The products become successively less water-soluble, a carrier protein
(SCP, steroid carrier protein) is required to transport the intermediates from one
enzyme site to another.
3 NADPH+H+ + 3 O2
H2O
NADPH+H+
2 CO2
HCOO–
59
In higher animals, the steroid nucleus of cholesterol is neither decomposed to simple
products nor oxidized to CO2 a H2O.
The liver is the organ which excretes most of the cholesterol, either directly or as bile acids.
Cholesterol utilization and elimination from the body
Free CHOLESTEROL
(a constituent of cytoplasmic membranes)
LIPOPROTEINS
transport in blood plasma
Cholesterol esters
(Intracellular pool)
LIVER
CHOLESTEROL
in sebaceous glands
secretion
CHOLESTEROL and BILE ACIDS
in the bile – in feces
"neutral“ sterols and bile acids
CHOLESTEROL
in secluded enterocytes
SKIN
CALCIOL
ADRENAL CORTEX
and GONADES
METABOLITES
of STEROID HORMONES
in the urine
Pregnenolone
CORTICOIDS
PROGESTINS
ANDROGENS
ESTROGENS
BILE ACIDS
UV light
Esterases
ACAT, in plasma LCAT
60
Cholesterol in the gut
Coprostanol
5β-Cholestan-3β-olCholesterol
In the small intestine, dietary cholesterol as well as cholesterol secreted
in the bile is not absorbed completely (only about 40-50 %).
Most of the cholesterol that escapes absorption and enters the large intestine
undergoes reduction to coprostanol. The reaction is catalysed by the
enzymes of intestinal microflora.
Bacterial
reductases
61
Synthesis of bile acids in the liver 500 mg / d
Secretion into bile:
Cholesterol 1 000 – 2 000 mg / d
Bile acids 5 000 – 10 000 mg / d
Lacteals
Cholesterol
Portal vein
Bile acids
Reabsorption
The balance of cholesterol intake and elimination
DIETARY INTAKE of cholesterol 80 – 500 mg per day
BIOSYNTHESIS 800 – 1000 mg per day
Other Steroid hormones, sebaceous secretion,
cholesterol of intestinal cells 200 mg / d
Feces Cholesterol and other neutral sterols 800 mg / d
Bile acids 500 mg / d
ELIMINATION
1000-1500 mg / d :
BODY POOL
Cholesterol 150 g
Bile acids 3 – 5 g
62
Phytosterols - sterols of plant origin
are structurally related to cholesterol; only the side chain on C-17 is changed.
Phytosterols are not resorbed in the gut.
On the contrary, consumption of phytosterols reduces the resorption of cholesterol.
Plant oils (corn, rapeseed, soya, sunflower, walnut) contain up to 0.9 % phytosterols.
Average intake of phytosterols in Czech republic - about 240 mg per day,
in Finland (some foods are enriched with phytosterols) - 350 mg per day.
β-Sitosterol
– predominant in the sterol fraction of plant oils
An example:
63
3
12
7
Bile acids
Structure of the major bile acids
Primary acids
Secondary acids
(reabsorbed from
the intestine)
CHOLATE
3α
,7α
,12α
-trihydroxy-5β-cholan-24-oate
CHENODEOXYCHOLATE
3α,7α-dihydroxy-
DEOXYCHOLATE
3α,12α-dihydroxy-
LITHOCHOLATE
3α-hydroxy-
64
H2N–CH2-CH2–SO3
–
Taurine
(2-Aminoethanesulphonic acid)
H2N–CH2–COO–
Glycine
(Aminoacetic acid)
GLYCOCHOLATE
(N–Choloylglycine)
TAUROCHOLATE
(N–Choloyltaurine)
The primary bile acids, cholate and chenodeoxycholate, are conjugated
within endoplasmic reticulum of the liver cells with glycine or taurine.
Those amides called conjugated bile acids (or bile „salts“, resp.) are then
secreted into bile ductules:
The structure of conjugated chenodeoxycholate is analogous to glyco- and taurocholate.
Conjugated acids are more acidic (pKa 2-4) than the unconjugated acids (pKa 6), therefor
they are more efficient emulgators than the unconjugated ones.
65
7α-Hydroxycholesterol
Cholesterol
cyt P450
O2 NADPH+H+
OH
Biosynthesis of the bile acids
occurs only in the liver cells:
The first and rate-limiting step of the conversion to bile acids is
the hydroxylation of cholesterol at C-7 catalysed by 7α-hydroxylase.
The enzyme is a monooxygenase of the cytochrome P450 class, bound in the membrane
of endoplasmic reticulum and its activity is supported by the presence of L-ascorbate.
The second hydroxylation at C-12 in the synthesis of cholate is connected
with rearranging of the ring A and B.
(In the synthesis of chenodeoxycholate (not shown) the second hydroxylation
is omitted.)
66
7α-Hydroxycholesterol
5β-Cholestane-3α,7α,12α-triol
Secretion into bile ductules
Cholate
Choloyl-CoA
Coenzyme A, ATP
Glycine
(or taurine)
GLYCOCHOLATE
(or taurocholate)
Propionyl-CoA, ADP
Coenzyme A, ATP
ADP
Co-A
MITOCHONDRION
26-Hydroxylation
Oxidation to C-26 carboxyl
Activation to acyl-CoA
Propionyl-CoA released
ON THE MEMBRANE OF ER
CONJUGATION
WITHIN ENDOPLASMIC RETICULUM
Dehydrogenation to 3-oxoIsomerization
of the double bond
Hydrogenation of 3-oxo and of double bond at C-4
O2 + NADPH+H+
Cyt P 450
67
Cholate and chenodeoxycholate are called primary bile acids.
They are the direct products of cholesterol degradation in the liver
and are secreted in the bile.
In the intestine they may be modified by bacterial action – they are
dehydroxylated to give the secondary bile acids, deoxycholate and
lithocholate.
Bile acids are efficiently reabsorbed and returned to the liver via portal
vein and secreted again –
bile acid undergo the enterohepatic circulation.