Lipid metabolism I
Biochemistry I
Lecture 8                             2013 (E.T.)

2
2
Major classes of lipids
triacylglycerols
phospholipids
sphingofosfolipids
glycerophospholipids
steroids
prostanoids
leukotriens
Mainly structural components of  membranes
Energy nutrients
Derived lipids

3
Metabolisms of lipids
metabolism of TG a FA
100 g/day
Source of energy
metabolism of structural lipids
2 g/day
Triacylaglycerols are the most  effective form of energy deposition.
compound
Heat of combustion (kJ/g)
Glykogen
TG
17
38

4
Triacylglycerols, fatty acids and esterified cholesterol
are very hydrophobic
they are not soluble in water
unless they are emulsified or included in micelles in the presence of tensides.

5
oil on water
water
lipids make the upper phase

6
Four natural tensides work in fat digestion
Tenside
Type
Origin
Bile acids
2-Acylglycerol
FA anions
Phospholipids
anionic
non-ionic
anionic
amphoteric
from cholesterol in liver
TAG hydrolysis in gut
TAG hydrolysis in gut
food

7
Emulsification of lipids in the intestine – formation of micelles
colipase
The main tensides are fatty bile acids

8
A:\micela.bmp
Intestinal lumen Mucosal cell (enterocyte)
Lipid adsorption in the intestine
Formation of mixed micelles from products of digestion
Mixed micelles are composed of fatty acids, mono/diacylglycerols, bile acids, phospholipids and
fat-soluble vitamins
Bile acids, phospholipids and fatty acids function as tensides

9
In the extracellular fluids
hydrophobic lipids are transported in the form of lipoprotein particles

10
monolayer
Hydrophobic core
Superficial layer
(hydrophilic surface)
Lipoprotein particles transport triacylglycerols               and cholesterol in body fluids

11
11
Types of  lipoproteins
VLDL (very low density)
LDL (low density
Chylomikron CM
HDL (high density)
TG
Proteiny
CH
PL

12
12
Metabolism of  triacylglycerols
1. Hydrolytic cleavage of ester bonds
2. Metabolism of fatty acids and glycerol

13
O
CH2–O–C–
O
CH2–O–C–
O
–C–O–CH
are enzymes that catalyse hydrolysis of ester bonds of triacylglycerols releasing free fatty acids.
Extracellular lipases
Pancreatic lipase secreted into the duodenum,
Lipoprotein lipase on the surface of the endothelium lining the capillaries
Intracellular lipases
Hormone-sensitive lipase of adipocytes mobilizing fat stores
Lysosomal lipase
Lipases

14
14
Transport of fatty acids in ECT
FA in blood – carried by albumin
(1 mmol/l, half-life 2 min)
FA are released mainly from TG in adipocytes by the action of hormon-sensitive lipase (hormonal
regulation) or from lipoprotein particles
Transport of FA in cells
• special membrane proteins facilitate the transport of FA across cytoplasmatic membrane
• fatty acid binding proteins facilitate the intracellular transport
• carnitine facilitates the transport across mitochondrial membrane

15
Degradation of lipids in the body
TG
HS-lipase
FA
Binding to albumin
FA
Binding to FABP
b-oxidation
acetylCoA
ER
mitochondrie
Binding to  carnitin
adipocytes
liver, muscle
Hormone-sensitive lipase in adipocytes
is an intracellular lipase that through hydrolysis of triacylglycerols  mobilizes the fat energy
reserves.
The activity of this lipase is controlled by hormones:
Glucagon (at low blood glucose)
and adrenaline/noradrenaline (in stress)
Chylomicron, VLDL
LP-lipase

16
Degradation of fatty acids: β-oxidation
Fatty acids serve as an energy source for most of the cells
(not for the nervous system and for red blood cells).
The tissues gain fatty acids
   - either from lipoprotein particles after the triacylglycerols have been hydrolysed by
lipoprotein lipase,
   - or as fatty acids mobilized by the action of hormones on the fat stores in adipose tissue and
supplied bound onto albumin.
Location: matrix of mitochondria

17
1.  Activation of FA by linking to coenzyme A
2.  Transport of acyl CoA into the mitochondrial matrix
3.  β-Oxidation of acyl CoA in the mitochondrial matrix to acetyl CoA that enters the citrate
cycle.
The utilization of fatty acids in the cells requires three stages of processing

18
1.  Activation of a fatty acid – synthesis of acyl coenzyme A
Acyls can be attached to the sulfanyl group by means of a thioester bond.
~
O
O
H
C
H
2
O
P
O
O
O
N
N
N
N
N
H
2
O
P
O
O
O
H
O
P
O
O
O
C
H
2
C
HS
C
H
2
C
H
2
H
N
O
C
C
H
2
C
H
2
H
N
O
C
C
H
C
H
3
C
H
3
Cysteamine
β-Alanine       Pantoic acid
Pantothenic acid
3´–phospho ADP
Coenzyme A

19
The synthesis of the high-energy acyl-CoA thioester is catalysed by acyl-CoA synthetases
R–COO–  +  CoA–SH                           R–CO–S-CoA
Acyl-CoA synthetases are located on the outer mitochondrial membrane.
There is a loss of energy equivalent to 2 molecules of ATP, because the reaction is made
irreversible by the hydrolysis of inorganic diphosphate (AMP + ATP « 2 ADP).
ATP
AMP + 2 Pi
Synthesis of acyl-CoA

20
Acyl-CoA itself cannot cross the inner mitochondrial membrane;
instead, acyl groups are transferred to carnitine, transported across the membrane as
acylcarnitine, and transferred back to CoA within the mitochondrial matrix.
Short-chain fatty acids (4 – 10 carbon atoms) do not require the carnitine shuttle, they can cross
the inner mitochondrial membrane.
Trimethyl(2-hydroxy-3-carboxypropyl)ammonium
2Carnitine carries long-chain activated fatty acids                              into the
mitochondrial matrix
CH3
CH3
H3C
N
–
CH2–CH–CH2–COO
OH
Carnitine

21
The transfers of acyls from acyl-CoA to carnitine and from
acylcarnitines to CoA are catalysed by
carnitine acyltransferases I and II.
(also named  carnitinpalmitoyltransferase CPT1 and 2)
N
C
H
3
C
H
3
C
H
2
H
3
C
C
H
C
H
2
O
C
O
O
H
O
C
Ester bond

22
Carnitinacyltransferase I or II
C
H
2
C
H
C
H
2
N
O
H
C
O
O
H
(
C
H
3
)
3
+
+
C
O
S
C
o
A
H
3
C
C
H
2
C
H
O
C
C
H
2
N
C
O
O
H
(
C
H
3
)
3
+
O
C
H
3
+
C
o
A
S
H
Inhibition by malonyl-CoA
Formation of acylcarnitine
Intermembrane space

>

23
Transport of fatty acid into mitochondria – carnitine shuttle
Two forms of carnitinacyltransferase
(also named  carnitinpalmitoyltransferase CPT)
RCO-S-CoA
CoA-SH
RCO-S-CoA
       intermembrane space                 inner mitoch. membrane          matrix
Carnitin/acylkarnitin
translocase
Cn-OH
RCO-OCn
Cn-OH
RCO-OCn
CoA-SH
CPT1
CPT2
acylCoA
acylCoA
acylcarnitine
acylcarnitine

24
Sources and need of carnitine
Protein-CH2CH2CH2CH2NH3                               protein-CH2CH2CH2CH2N(CH3)3
Side chain of lysine
proteolysis
trimethyllysine
carnitine
SAM
 Intake in food: cca 100 mg/day ( meat, milk, also plant sources). Bioavailability  - ~ 75%
Liver, kidney
Transport in blood
 Synthesis in organism (10-20 mg/day)
+
+
Resorption in kidneys – 98-99% is resorbed in tubuli
Carnitine pool ~ 20g
Ascorbate is needed

25
Carnitine deficiences
•Liver diseases  ® decreased synthesis
•Malnutrition, vegetarian diet
•Increased requirements for carnitine (pregnancy, burns, trauma)
•Enzyme deficiency (transferases, translocase)
Carnitine supplementation is necessary
Decreased ability of tissues to use long chain fatty acids  as a metabolic fuel.

26
Consequences of carnitine deficiency
The ability to use fatty acids as a source of energy is reduced
•Deficiency in liver – nonketotic hypoykemia during fasting
during fasting b-oxidation is necessary for provision of acetylCoA for ketogenesis and ATP
production in citric acid cycle
•
•Deficiency in liver – muscle weakness, cramps during work

27
27
Inborn deficiency in carnitine transport
Autosomal recesive deficiency of Na+-dependent carnitine transporter  in muscle and kidney
•Carnitine deficiency in muscle and heart
•typically appear during infancy or early childhood and can include severe brain dysfunction
(encephalopathy), a weakened and enlarged heart (cardiomyopathy), confusion, vomiting, muscle
weakness, and low blood sugar (hypoglycemia). All individuals with this disorder are at risk for
heart failure, liver problems, coma, and sudden death.
Can be detected by expanded newborn screening by tandem mass spectrometry.
Therapy: lifelong use of  L-carnitine

28
FA-transport enzyme deficiency
•CPT-I deficiency — affects the liver; severe hypoglycemia, total carnitine 150–200 % of normal
value.
•CPT-II deficiency— cardiac and skeletal muscle, carnitine cca 25–50 %
mild (adult form) — rhabdomyolysis after prolonged exercise,starvation or at exposure to cold;
severe (neonatal form) — cardiomyopathy, muscle weakness, congenital malformation.
•Carnitin acylkarnitin translocase deficiency — hypoketotic hypoglycemia at fasting, arythmia,
apnoe. Often death in infancy.
Inborn errors in fatty acids metabolism are components of newborn screening

29
•The available research on L-carnitine supplementation does not appear to support claims of
enhanced aerobic or anaerobic exercise performance.
•Carnitine supplementation with supraphysiological doses above and beyond that which the body
requires, does not result in increased fat oxidation at rest or during exercise in well-nourished
individuals;
• thus, it appears that we can synthesize the necessary amounts from a diet adequate in its
precursors.
•Athletes wishing to explore carnitine's purported benefits must be aware that the dietary
supplement industry is not regulated and, therefore, product safety is not guaranteed. The
bioavailability is 5-10%
•High doses (5 or more grams per day) may cause diarrhea. Other rare side effects include increased
appetite, body odor, and rash.
Carnitine as dietary supplement ?
j0339768[1]

30
Transport of fatty acids with the short chain
Fatty acids with the chains shorter than 12 carbons do not require carnitine for their transport
into the mitochondria.
They freely cross the mitochondrial membrane.

31
31
3. b-Oxidation of fatty acids
•Main way of  FA degradation
•Fatty acid is enters the process in form of  acyl-CoA
•b-carbon is oxidized (C-3)
•repetition of four reactions :
dehydrogenation ® hydration ® dehydrogenation ® thiolysis by CoA (fatty acid is shortened by two
carbons and acetyl-CoA is released)

32
32
(1) First dehydrogenation
configuration trans
Saturated acyl CoA
α,β-Unsaturated acyl CoA
  (2,3-unsaturated)

33
33
(2) Hydration of double bond
Hydration is not a redox reaction, by addition of water to a double bond the sum of the oxidation
numbers of both carbon atoms remain the same.
α,β-Unsaturated acyl CoA
β-Hydroxyacyl CoA
  (L-3-Hydroxy)

34
34
(3) Dehydrogenation of hydroxyacyl

35
35
(4) The final step:
the thiolysis of 3-oxoacyl-CoA by CoA-SH
     ACYL CoA
Shortened by two carbons
CC
thiolase
Acetyl CoA
Substrate for the citrate cycle

36
Distinguish: three types of lysis
Hydrolysis
cleavage of substrate with water:
sucrose + H2O ® glucose + fructose
(starch)n + H2O  ®  maltose + (starch)n-2
Phosphorolysis
cleavage of O-glycosidic bond by phosphate:
(glycogen)n  + Pi  ® (glycogen)n-1 + glucose-1-P
Thiolysis
cleavage of C-C bond by sulfur of CoA–SH
β-oxidation of FA or utilization of KB,
RCH2COCH2CO-SCoA + CoA-SH ® RCH2CO-SCoA + CH3CO-SCoA
!

37
37
b-oxidation
• 1.dehydrogenation
(FAD)
• 2.hydration
• 3 dehydrogenation
(NAD+)
• 4 thiolytic cleavage and transfer of acyl to  CoASH
FA oxidase
acyl-CoA dehydrogenase
D2-enoyl-CoA hydratase
3-hydroxyacyl-CoA dehydrogenase
thiolase

38
38
Acyl-CoA dehydrogenases (first reaction in β-o.)
4 main types
for FA with short chain (SCAD)
  mediate chain (MCAD)
                          long chain (LCAD)
 very long chain (VLCAD)
Examination of MCAD, LCAD and  VLCAD deficiency is a component of newborn screening.
MCAD deficiency
One of the most common inborn errors of fatty acid metabolism. Under conditions of health this may
not cause significant problems. However, when such individuals do not eat for prolonged periods or
have increased energy requirements, the impairment of fatty acid oxidation may lead to fatty acid
buildup, hypoglycemia, hyperammonemia and, possibly, sudden death.

39
Palmitoyl CoA  +  7 FAD  +  7 NAD+  +  7 H2O  +  7 CoA
    8 acetyl CoA  +  7 FADH2  +  7 NADH + 7 H+
8 ´ 12 ATP =  96 ATP
14 ATP + 21 ATP – 2 ATP + 96 ATP = 129 ATP/palmitate
The energetic yield of β-oxidation of palmitate
– to 8 acetyl coenzymes A
– and 8 acetyl CoA in the citrate cycle
Net yield of complete palmitate oxidation to CO2
14 ATP
21 ATP  –  2 ATP (activation of
                                      palmitate)
+

40
Net yield of the aerobic breakdown of glucose is
38 mol ATP / mol glucose  (M = 180 g / mol; 6 mol C),
   i.e. 0.21 mol ATP / g glucose,  or
           6.3 mol ATP / mol C.
Net yield of complete oxidation of palmitate is
           129 mol ATP / mol palmitate (M = 256 g / mol; 16 mol C),
   i.e.  0.50 mol ATP / g palmitate, or
            8.1 mol ATP / mol C.

41
Oxidation of unsaturated FA
Oleic acid: cis D9-C18
cis D7-C16
cis D5-C14
cis D3-C12
trans D2-C12
isomerase
Loss of  FADH2
Analogous process with b-oxidation

42
FA with odd number of  C provide propionyl-CoA
propionyl-CoA
C
O
2
+ H2O
racemase
D-methylmalonyl-CoA
L-methylmalonyl-CoA
succinyl-CoA
C
H
3
C
H
2
C
O
 -
S
-
C
oA
A
T
P
A
D
P
bi
ot
i
n
C
H
C
O
O
-
C
O
-
S
-
C
oA
C
H
3
C
H
C
O
O
-
C
O
-
S
-
C
oA
C
H
3
C
H
2
-
C
H
2
C
O
O
-
C
O
-
S
-
C
oA
B12
It is formed also by metabolism of some AA

43
43
b-oxidation in the peroxisome
Very-long-chain fatty acids (20 C or longer)
•preliminary b-oxidation in peroxisomes
•shortening of the chain
•shortened FA is transferred to a mitochondrion
FAD is the cofactor of b-oxidation in peroxisome
It is oxidized by molecular oxygen
FADH2  +  O2  →  FAD  +  H2O2
Energy is not obtained

44
44
b-oxidation of FA is powerfull source of energy
When does it take place?
When the cells requires energy and availability of glucose is limited
 b-oxidation is initiated by hormones in post-absorptive state or starvation, particularly in
liver, muscle and myocardium

45
Lipids in postresorption phase (glucagon)
liver
Acetyl-CoA
Muscle, myocard
FA
Adipose tissue
FA  +  glycerol
TAG
FA-albumin
Acetyl-CoA
Effect of glucagon
FA
HSL

46
Mobilization of fat stores in post-absorptive phase (glucagon)
Glucagon (or adrenaline) activate hormone sensitive lipase in adipose tissue
•HSL cleaves triacylglycerols to fatty acids and glycerol
•Fatty acids are released into the blood
•the plasma level of free fatty acids increases
•FA are taken up by the liver and other peripheral tissues (esp. muscle, myocard and kidney) at the
rates proportional to the plasma concentration.

47
Formation of ketone bodies - ketogenesis
Ketone bodies are formed in the liver mitochondria and released into blood plasma.
The two acids are detectable in plasma at any time, the usual ratio β-hydroxybutyrate to
acetoacetate is 3 – 6 (it reflects the intramitochondrial NADH/NAD+ ratio).
There are always traces of ketone bodies in urine, since there is no renal threshold for the two
acids.
Ketone bodies are readily metabolised in non-hepatic tissues.
– CO2
- 2 H
+ 2 H
Acetone
Acetoacetic acid
O
O
O–H
CH3–C–CH2–C
b
-Hydroxybutyric acid
CH3–CH–CH2–C
O
OH
O
CH3–C–CH3

48
Ketogenesis in liver mitochondria
Acetoacetyl-CoA
Acetyl-CoA
3-Hydroxy-3-methylglutaryl-CoA   (HMG-CoA)
Acetoacetate (free)     Acetyl-CoA
Acetone
β-Hydroxybutyrate
H2O
4 C
2 C
6 C
2 C
4 C
2 C
2 C
3 C
4 C

49
During fasting fatty acids are mobilized from adipose tissue and part of them is transported into
the liver
®increased production of acetyl-CoA by  b-oxidation
® capacity of citric cycle is overloaded (lack of oxalacetate)
® synthesis of keton bodies
The causes of increased keton bodies formation

50
Utilization of ketone bodies in non-hepatic tissues
β-Hydroxybutyrate
are broken down in the citrate cycle
β-Hydroxybutyrate and acetoacetate are important in providing energy for peripheral tissues.
Acetone is a waste product,
eliminated by the kidney or expired, it can be smelt on the breath.
Acetoacetate is reactivated to acetoacetyl-CoA through the transfer of CoA from succinyl-CoA.
(thioforase)

51
Formation and utilization of keton bodies
liver
Acetyl-CoA
Keton bodies
Keton bodies in blood
CNS
CO2
muscle
FA
Adipose tissue
FA  +  glycerol-P
TAG
FA-albumin
Acetyl-CoA
Lack of  oxaloacetate
Synthesis of thioforase is induced  in brain after several days of starvation

52
The production of ketone bodies increases at high ratio
glucagon/insulin, when fat stores are mobilized (prolonged
fasting, starvation, uncontrolled diabetes mellitus type I).
An extreme production of ketone bodies (ketosis) is very dangerous, because ketogenesis is a
proton-producing process that disturbs acid-base balance (evoking ketoacidosis) and, through
excretion of the two acids into urine, is a cause of serious loss of cations.
Acetoacetic acid                pKa = 3.52
β-Hydroxybutyric acid      pKa = 4.70

53
Can be triacylglycerols formed de novo in the body?
In human:
fatty acids (except the essential)
triacylglycerols
can  be synthesized

54
Fatty acids synthesis
 Location:
Mainly liver, lactating mammary gland, in lesser extent adipocytes,brain

When?
sufficient amounts of acetylCoA, that need not be utilized for production of energy
After the meal, when sufficient amounts of glucose are available for production of acetyl CoA,
?

55
1. Transport of acetyl-CoA from matrix to cytoplasma
2. Malonyl-CoA formation
3. Serie of reactions on fatty acid synthase enzyme complex
(cytoplasma)
Steps in fatty acid synthesis

56
Transfer of acetyl CoA to the cytosol
acetyl-CoA is formed in matrix of mitochondria mainly by oxidative decarboxylation of pyruvate
(from glucose, amino acids)
• acetyl-CoA cannot freely penetrate  the mitochondrial membrane
• it is transported in form of citrate
When it does occur?
In case that citrate is not necessary for citric acid cycle

57
When the citrate is not necessary for citric acid cycle?
-the well fed state
– sufficient amounts of glucose are available producing acetyl CoA,
– low energy expenditure – high ATP concentrations within the cells inhibit degradation of acetyl
CoA in the citrate cycle,
– absence of stress that activates mobilization of fat stores, free fatty acids released through
the action of catecholamines inhibit fatty acid synthesis.

58
Transfer of acetyl CoA to the cytosol
58
CYTOPLAZMA
MATRIX
acetyl-CoA + oxalacetate
ADP + Pi
ATP
oxalacetate        acetyl-CoA
CoA
citrate
citrate
pyruvate, AK
CoA
isocitrate
malate
malate
NADH
NAD+ + H+
Blocked by ATP

59
2. Synthesis of malonyl CoA
AcetylCoA does not have energy enough to enter the synthesis of fatty acids
Principle of carboxylation and decarboxylation
Formation of malonylCoA by carboxylation and its decarboxylation in the next step

Synthesis of malonyl CoA
is the rate-limiting step in fatty acid synthesis, catalysed by
acetyl-CoA carboxylase:
S
H
N
N
H
O
C
O
–Enzyme
S
H
N
N
O
C
O
–Enzyme
–COOH
+ HCO3–
ATP                 ADP + Pi
CH2–CO–S–CoA
COO–
CH3–CO–S–CoA
Malonyl CoA
Acetyl CoA
Biotinyl–E
Carboxybiotinyl–E

61
Regulation of acetyl-CoA carboxylase
Activation by citrate
Inhibition by acyl-CoA with long chains (palmitate)
Hormonal regulation:
insulin
glucagon, adrenalin ¯

62
The fatty acyl synthase complex
ACP domaine with
phosphopantethein arm
Seven enzyme activities:
AT
Acetyl/acyl-CoA transacylase
MT
Malonyl transacylase
CE
Condensing enzyme
(Oxoacyl-PPt synthase)
KR
Oxoacyl reductase
DH
Hydroxyacyl dehydratase
ER
Enoyl reductase
TE
Palmitoyl thioesterase
One of the two functional units
Two proteins with –SH group bind intermediates of the synthesis
In mammals, the complex is a homodimer
Each monomer is arranged in three domains carrying the seven catalytic activities.
One domain in both  monomers includes the acyl carrier protein (ACP) area to which the
phosphopantethein "arm" is attached
Both monomers cooperate so that each of them takes part on the synthesis of two fatty acids
processed simultaneously,

63
The flexible phosphopantethein "arm" of the synthase
linked to a serine residue of acyl carrier protein ACP
is found also in coenzyme A
(as just one half of the coenzyme A molecule):
~
O
H
O
P
O
O
O
C
H
2
C
HS
C
H
2
C
H
2
H
N
O
C
C
H
2
C
H
2
H
N
O
C
C
H
C
H
3
C
H
3
Cysteamine
β-Alanine       Pantoic acid
Pantothenic acid
         NH
CH2–CH
         CO
ACP
The processed acyls attached to the sulfanyl group are
carried from one active site of the synthase complex to another.

64
1
Transfer of the acetyl group of acetyl CoA to the sulfur
of a cystein residue of the condensing enzyme.
The reaction is catalysed by acetyl transacylase.
Reactions of fatty acid synthesis
ACP
SH
Cys
S
CO–CH3

65
2
The malonyl group is transferred to the sulphur atom of the phosphopantetheine attached to ACP.
The reaction is catalysed by malonyl transacylase.
ACP
S
Cys
S
CO–CH3
COOH
CH2
CO

66
3
Condensation
joining acetyl unit to a two-carbon part of the malonyl unit on phosphopantetheine.
CO2 is released.
An acetoacetyl unit is formed.
The reaction is catalysed by condensing enzyme (3-oxoacyl synthase).
ACP
S
Cys
SH
+ CO2
CH3
C=O
CH2
CO
ACP
S
Cys
S
CO–CH3
COOH
CH2
CO

67
4
The first reduction
catalysed by β-ketoacyl reductase with NADPH.
The product is 3-hydroxyacyl unit.
ACP
S
Cys
SH
CH3
C=O
CH2
CO
+ NADPH+H+
ACP
S
Cys
SH
CH3
CH–OH
CH2
CO
+ NADP+

68
ACP
S
Cys
SH
CH3
CH–OH
CH2
CO
ACP
S
Cys
SH
CH3
CH
CH
CO
+ H2O
5
Dehydration
catalysed by 3-hydroxyacyl dehydratase.
The product is trans–2–enoyl (named crotonyl) unit.

69
ACP
S
Cys
SH
CH3
CH
CH
CO
+ NADPH+H+
ACP
S
Cys
SH
CH3
CH2
CH2
CO
+ NADP+
6
The second reduction
catalysed by enoyl reductase with NADPH.
The product is saturated acyl (now butyryl) unit. Initial acetyl was elongated by two carbon atoms.

70
ACP
S
Cys
SH
CH3
CH2
CH2
CO
ACP
SH
Cys
S
CH3
CH2
CH2
CO
7
The saturated acyl is transferred to the cysteine sulfur atom
on the condensing enzyme.
The synthase is now ready for another round of elongation

71
After the completion of the first elongating cycle, new malonyl
is "loaded“ on the sulfanyl group of PPt.
In the second round of fatty acid synthesis, butyryl unit condenses with malonyl to form a C6-acyl,
……
The elongation cycles continue until C16-acyl unit (palmitoyl) is formed.
Palmitoyl unit is a good substrate for thioesterase that hydrolyses palmitoyl-PPt to yield
palmitate (16:0).

72
In mammals, palmitate is the major product of FA synthesis.
A minor saturated product is stearate (18:0).
Further elongation of fatty acids is provided by similar mechanisms, but the elongating system is
located on the membranes of endoplasmic reticulum

73
NADPH is required in the reductive steps of FA synthesis
The main source of NADPH is the pentose phosphate pathway
.
A certain part of NADPH is supplied by the reaction catalysed by   NADP+–linked malate enzyme
("malic enzyme“):
            Malate  +  NADP+  ®  pyruvate  + CO2  +  NADPH
The reaction takes part on the transport of acetyl-CoA (in the form of citrate) across the inner
mitochondrial membrane.

74
The stoichiometry of fatty acid synthesis
The synthesis of palmitate (C16):
The synthesis of malonyl CoA
7 Acetyl CoA + 7 CO2 + 7 ATP  ®  7 malonyl CoA + 7 ADP + 7 Pi + 14 H+
The synthesis catalysed by the fatty acid synthase complex
Acetyl CoA + 7 malonyl CoA + 14 NADPH + 20 H+ ®
®  palmitate + 7 CO2 + 14 NADP+ + 8 CoA + 6 H2O
The overall stoichiometry for the synthesis of palmitate is
8 Acetyl CoA + 7 ATP + 14 NADPH + 6 H+  ®
                ®  palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP + 7 Pi

75
Compare
Feature
FA b-oxidation
FA synthesis
Localization
mitochondria
cytoplasm
Acyl attached to
CoA-SH
ACP
Basic unit
acetyl (C2)
acetyl (C2)
Redox cofactors
NAD+, FAD
NADPH
Enzymes
separated
dimer / complex
Stimulated by
glucagon
insulin

76
Elongation of fatty acids
Although palmitate (C16) is the major product of the fatty acid synthase complex, and is the chief
saturated fatty acid in human fat,
stearate and oleate (C18) are common and longer-chain fatty acids, arachidate  (C20),
behenate     (C22) and
  lignocerate (C24) occur in phospholipids.
Elongation by enzymes bound to the endoplasmic reticulum:
– Activation of palmitate by conversion to palmitoyl CoA,
– activation of acetyl CoA by its carboxylation to malonyl CoA,
– elongation similar to synthesis catalysed by FA synthase complex,    but the intermediates are
CoA-thioesters, not enzyme-bound  acyls. The reductant is also NADPH.
Elongation process in mitochondria (for the synthesis of fatty acids incorporated into
mitochondrial lipids):
– Reversal of the β-oxidation.

77
Desaturation of fatty acids
Unsaturated fatty acids of the series n-6 are comprised in all plant oils (olive oil, sunflower oil
etc.).
15-Desaturase is present predominantly in plants growing in cold water (algae, phytoplankton), then
a high concentration of polyunsaturated fatty acyls of the series n-3 is in fish oils (fish feeds
phytoplankton).
In higher animals, only the desaturases are known which generate double bonds at carbons 9, 6, 5,
and 4.
Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9.
Fatty acids containing double bonds beyond C-9 are synthesized by plants, they contain also 12- and
15-desaturase.
9
12
15

78
Polyunsaturated fatty acids n-3 and n-6 are essential for animals
They serve as precursors of eicosanoids (prostanoids and leukotrienes).
If food intake is sufficient (vegetable oils,
fish),                                                        linoleate (linoleic acid) and
α-linolenate (linolenic ac.) are precursors of other PUFA as arachidonate (n-6) and
eicosapentaenoate (n-3), from which eicosanoids are formed.
Linoleate  18:2 (9,12)
γ-Linolenate  18:3 (6,9,12)
Eicosatrienoate  20:3 (8,11,14)
Arachidonate   20:4 (5,8,11,14)
6-desaturation
elongation
5-desaturation
α-Linolenate  18:3 (9,12,15)
Octadecatetraenoate  18:4 (6,9,12,15)
Eicosatetraenoate   18:4 (8,11,14,17)
Eicosapentaenoate  18:5 (5,8,11,14,17)
6-desaturation
elongation
5-desaturation

79
 18:0            18:1 (9)                18:2 (9,12)                           18:3 (9,12,15)
n-9 series
18:2 (6,9)
20:2 (8.11)
20:3 (5,8,11)
22:3 (7,10,13)
18:3 (6,9,12)
18:4 (6,9,12,15)
20:3 (8,11,14)
20:4(5,8,11,14)
22:4 (7,10,13,16)
20:4 (8,11,14,17)
20:5(5,8,11,14,17)
22:5 (7,10,13,16,19)
plants
phytoplankton
6-desaturase
6-desaturase
5-desaturase
5-desaturase
n-6 series
n-3 series
elongation
elongation
elongation
elongation
Elongation and desaturation of FA
j0437615[1] j0430031[1]

80
Mechanism of long-chain fatty acyl-CoAs desaturation
Location: smooth endoplasmic reticulum of liver cells.
Desaturases are hydroxylating monooxygenases. The substrate is hydroxylated and after it water is
eliminated from the hydroxylated product with the formation of the double bond.
The reductant is NADH+H+, from which the electrons are carried by the flavine enzyme and the
cytochrome b5 to a desaturase.

81
Example:
9
10
1
S
CoA
O
H
H
HO
H
H
H
1
S
CoA
O
1
S
CoA
O
O=O  + NADH+H+
+ H2O + NAD+
+ H2O
Stearoyl–CoA
Oleoyl–CoA
Mechanism of long-chain fatty acyl-CoA
desaturation

82
Synthesis of triacylglycerols
glycerol-3P
lysophosphatidate
P
C
O
C
H
2
O
C
H
2
O
C
O
R
H
S
C
oA
A
D
P
A
T
P
C
H
O
H
C
H
2
O
H
P
C
H
2
O
C
H
O
H
C
H
2
O
H
C
H
2
O
H
R
C
O
S
C
oA
H
S
C
oA
glycerol
1. Synthesis of lysophosphatidate
N
A
D
+

P
C
H
2
O
H
C
O
C
H
2
O
C
H
2
O
P
C
H
2
O
C
O
R
C
H
O
H
ER –liver, adipocytes, enterocytes
NADH + H+
NADPH + H+
NADP
*

83
83
2. Synthesis of  phosphatidate
lysophosphatidate
phosphatidate
Usually unsaturated

84
84
P
C
H
2
O
C
O
R
C
H
O
C
O
R
C
H
2
O
P
i
R
R
C
O
S
C
oA
H
S
C
oA
C
H
2
O
C
O
R
C
H
O
C
O
R
C
H
2
O
C
O
R
PC,PE,PS
PI, kardiolipin
triacylglycerol
 3. Synthesis of triacylglycerols
Intestine ® CM
Lier ® VLDL
Adipocytes ® deposition
hydrolase
C
H
2
O
C
O
C
H
O
C
O
R
C
H
2
O
H
ER