Week 2 Lecture Fatty Acid Catabolism, Ketogenesis

Medical Biochemistry
Semester II
Week 2
FATTY ACID CATABOLISM
KETOGENESIS
Assistant prof. DACE REIHMANE
Medicine 2.0, Medical Biochemistry

1
(CFUBK_072), Assist. Prof. Dace Reihmane
Lipid metabolism
Learning goals:
● Describe the pathway for activation and transport of fatty acids to

the mitochondria for catabolism
● Outline the sequence of reactions involved in the oxidation of fatty

acids in the mitochondria; view differences between β, α, and ω
oxidation
● Describe the general features of pathways for the oxidation of

unsaturated, odd-chain, and branched-chain fatty acids
● Explain the rationale for the pathway of ketogenesis, and identify

the major intermediates and products of this pathway

2
Corresponding literature:
17| Fatty acid catabolism
© 2017 W. H. Freeman and Company

3
Additional literature
Medical Biochemistry Principles of Medical Biochemistry

HERE HERE

4
Sources of fatty acids
• Fats consumed in the diet
• Fats stored in cells as lipid droplets
• Fats synthesized in one organ for export to another
• Fats obtained by autophagy (which degrades the cell’s own organelles)
Humans obtain fats in the diet, mobilze fats stored in adipose tissue, and, in the liver,
convert excess dietary carboxydrates to fats for export to other tissues, while during
the starvation recycle lipids by autophagy
Resting
Dietary TAGs > 40% TAGs ~ 80% TAGs > 50%

of energy requirements
of energy requirements

5
Fats Provide Efficient Fuel Storage
The advantage of fats over polysaccharides:

● Fatty acids carry more energy per carbon
because they are more reduced
● Fatty acids complex or carry less water
because they are nonpolar
Glucose and glycogen are for short-term energy
needs and quick delivery
Fats are for long-term (months) energy needs, good
storage, and slow delivery

6
Fat Storage in White Adipose Tissue

7
Lipolysis - place of action
• TAGs are stored in adipocytes (in steroid-synthesizing cells of the

adrenal cortex, ovary, and testis, liver, muscle and other cells)
• Cytosol, lipid droplets

8
Hormones (glucagon; epinephrine) Trigger
Mobilization of Stored TAGs
Lipid droplets are

surrounded by the
phospholipid monolayer,
coated with perilipins –
proteins that prevent
untimely lipolysis
Albumin ~1/2 of serum

proteins
Noncovalentyl binds ~ 10 FA
PKA – protein kinase A

HSL – hormone sensitive lipase
ATGL – adipose triacylglycerol lipase Heart,
MGL – monoacylglycerol lipase Renal cortex

9
Lipases Cleave Fatty Acids from Glycerol Backbone
of Triacylglycerides

10
Glycerol metabolism - place of action
5% of TAG energy
• Primarily in liver, but enzymes expressed in all tissues
• Cytosol

11
Glycerol from Fats Enters
Gluconeogenesis or Glycolysis
• Glycerol kinase activates glycerol

at the expense of ATP
• Subsequent reactions
recover more than enough
ATP to cover this cost
• Allows limited anaerobic

catabolism of fats
• Passive diffusion through

aquaporins ensure tranport from
periferal cells to the liver
or gluconeogenesis

12
Transport in Cell Requires Conversion to Fatty Acyl-CoA
Fatty acyl-CoA synthetase

isoenzymes for:
• Short
• Intermediate
• Long
carbon chains
Oxidation or
synthesis of
membrane lipids
13
Acyl-Carnitine/Carnitine Transport
for fatty acids with > 12 C + AMP PPi
Than fatty acid+ATP+HSCoA----------------> AcylSCoA
Fatty acid enters cytosol as free fatty acid.
Acel-SCoA reaction produces 2 ADP, because AMP
should be converted to ADP first and
only then into ATP. In this reaction 1
ATP is used. AMP+ATP--->2ADP
Of the cell
------------
Passive
Acyl-carnitine/carnitine
transporter
CoenzymeA must stay at cytoplasm, membrane is unpermeable for Coa
Rate limiting step

for fatty acid oxidation!
14
Acyl means unknown amount of -CH2- groups. More than 14 -CH2- can not pass imitochondrial barier.
Olive oil

15
Summarize your knowledge about
Acycarnitine/carnitine shuttle
HOMEWORK
TASK 1:
Take a time to fill it out!

16
The  - Oxidation - place of action
• Every cell with mitochondria, but with different rate

• Mitochondria execp brain cells

17
Stages of Fatty Acid
Oxidation
Stage 1 consists of oxidative conversion of

two-carbon units into acetyl-CoA via  -
oxidation with concomitant generation of
NADH+H+ and FADH2
● involves oxidation of  carbon to
thioester of fatty acyl-CoA
Stage 2 involves oxidation of acetyl-CoA into
CO2 via citric acid cycle with concomitant
generation NADH+H+ and FADH2
Stage 3 generates ATP from NADH+H+ and
FADH2 via the respiratory chain
18
The  - Oxidation may be acyl-CoA
Each pass removes

one acetyl moiety
in the form of
acetyl-CoA
Single bond
between
methylene (-CH2-) if here O2, C-C bond is weaker
is relatively stable
in comparison to
C-C between two
carbonyl carbons

19
Long Fatty Acid Oxidation is Performed by a
Single Trifunctional Protein
• Hetero-octamer (α4β4 subunits)

• May allow substrate channeling between enzymes
• Associated with inner-mitochondrial membrane
• Processes fatty acid chains with 12 or more carbons
• Shorter chains processed by soluble enzymes in the matrix

20
Step 1:
Dehydrogenation
Catalyzed by isoforms of acyl-CoA

dehydrogenase (AD) on the inner-
mitochondrial membrane
● very-long-chain AD (12–18 carbons)
● medium-chain AD (4–14 carbons)
● short-chain AD (4–8 carbons)
Results in trans double bond, different from naturally

occurring unsaturated fatty acids
Analogous to succinate dehydrogenase reaction in the
citric acid cycle
● electrons from bound FAD (phrostetic group) transferred directly to the
electron- transport chain via electron-transferring flavoprotein (ETF)

21
Step 2:
Hydration
Catalyzed by two isoforms of

enoyl-CoA hydratase:
● soluble short-chain hydratase (crotonase)
● membrane-bound long-chain hydratase,
as first part of trifunctional complex
Water adds across the double bond yielding alcohol

on  carbon (typical reaction)
Analogous to fumarase reaction in the citric acid cycle
● same stereospecificity

22
Step 3:
Dehydrogenation
Catalyzed by -hydroxyacyl-CoA
dehydrogenase
The enzyme uses NAD cofactor as electron

acceptor (always for secondary alcohol)
Only L-isomers of hydroxyacyl-CoA act as substrates

Analogous to malate dehydrogenase reaction in the citric acid
cycle

23
Step 4:
Transfer of Fatty Acid
Chain and Release of
Acetyl-CoA
Catalyzed by acyl-CoA
acetyltransferase (thiolase)
via covalent mechanism
● The carbonyl carbon in -ketoacyl-CoA is electrophilic

● Active-site thiolate acts as a nucleophile and releases acetyl-CoA
● Terminal sulfur in CoA-SH acts as a nucleophile and picks up the fatty acid chain
from the enzyme
The net reaction is thiolysis of the carbon-carbon bond

24
Each Round Produces an Acetyl-CoA and Shortens
the Chain by Two Carbons
If You have C16- you need 7cycles
C14-6cycles
C20-9 cycles

25
NADH+H+ and FADH2 Serve as
Sources of ATP
TABLE 17-1 Yield of ATP during Oxidation of One Molecule of Palmitoyl-CoA to CO2
and H2O
Number of NADH+H+ Number of ATP
Enzyme catalyzing the oxidation step or FADH2 formed ultimately formeda
β Oxidation
Acyl-CoA dehydrogenase 7 FADH2 10.5
β-Hydroxyacyl-CoA dehydrogenase 7 NADH+H+ 17.5
Citric acid cycle
Isocitrate dehydrogenase 8 NADH+H+ 20
α-Ketoglutarate dehydrogenase 8 NADH+H+ 20
Succinyl-CoA synthetase 8b
Succinate dehydrogenase 8 FADH2 12
Malate dehydrogenase 8 NADH+H+ 20
Total 108 - 2ATP
aThese calculations assume that mitochondrial oxidative phosphorylation produces 1.5 ATP per FADH 2 oxidized and 2.5 ATP
per NADH+H+ oxidized.
bGTP produced directly in this step yields ATP in the reaction catalyzed by nucleoside diphosphate kinase (p. 516).

26
Similar Mechanisms Introduce Carbonyls in
Other Metabolic Pathways

27
Genetic Defects in Fatty Acyl-CoA
Dehydrogenases Cause Serious Disease
Most common is the mutation in gene coding medium-chain
(6 – 12C) Acyl-CoA dehydrogenase
• Carriers: 1 in 40
• Disease: 1 in 10’000
• Recurring episodes of syndrome include fat accumulation in
liver, high blood levels of octanoic acid, low blood glucose
levels, sleepiness, vomiting and coma
• Episodes are very serious, despite no symptoms in between
• Mortality 25 – 60% in early childhood
• With early detection and careful management of the diet the
prognosis is good

28
 - Oxidation in Mitochondria
versus Peroxisomes
Peroxisomal acyl-CoA dehydrogenase

passes electrons directly to molecular
oxygen
● energy released as heat
● hydrogen peroxide eliminated by catalase
Much more active on very-long-chain (≥22C)

fatty acids (such as hexacosanoic acid (26C)) and
branched-chain fatty acids such as phytanic acid
and pristanic acid (obtained from dairy
products, the fat of ruminant animals, meat and
fish)
TASK IN COLLO, compare b-oxy with one of these.
29
X-linked adrenoleukodystrophy
XALD affects young boys before the age of 10 years:

• Loss of vision, behaviroal disturbances and death within a
few years
https://www.spandidos-publications.com/10.3892/etm.2019.7804

30
The  - Oxidation
HOMEWORK
TASK 2:

31
Oxidation of Unsaturated Fatty Acids
Naturally occurring unsaturated fatty acids contain cis double

bonds
● are NOT a substrate for enoyl-CoA hydratase
Two additional enzymes are required
● isomerase: converts cis double bonds starting at carbon 3 to
trans double bonds
● reductase: reduces cis double bonds not at carbon 3
Monounsaturated fatty acids require the isomerase
Polyunsaturated fatty acids require both enzymes

32
Oxidation of Monounsaturated Fatty Acids
At this point acyl-CoA

dehydrogenase step is
skipped, resulting in 1
FADH2 less
33
Oxidation of
Polyunsaturated Fatty
Acids
At this point acyl-CoA

dehydrogenase step is
skipped, resulting in 1
FADH2 less
NADPH +H+ reduces the

remaining unsaturated
bond, resulting in no
further loss of FADH2

34
Oxidation of Odd-
Numbered Fatty Acids
• Most dietary fatty acids are even-

numbered
• Many plants and some marine
organisms also synthesize odd-
numbered fatty acids
• Propionyl-CoA (3-carbon compound)
forms during final cycle of  oxidation
of odd-numbered fatty acids
• During fermentation the rumen of
ruminants also produces propionyl-CoA
from carbohydrates

35
The ω oxidation of fatty acids
in the endoplasmic reticulum
• In liver and kidney

• Preferred substrates are fatty acids with 10 or 12 C
• Important when β-oxidation is defective (e.g. In
case of mutation or carnitine deficiency)
• Product: fatty acid with carboxyl group at each end
• Either end can be attached to CoASH
• Molecule enter mitochondria for further β-
oxidation
• The final 4C molecule (succinate) can enter CAC

36
The α oxidation of a
branched-chain fatty
acid (phytanic acid) in
peroxisomes
• More pronounced in liver and brain
• Long chain fatty acid with methyl branches –
derived from phytol side chain of chlorophyll
• Methyl group at β carbon
• Obtained from dairy products, the fat of
ruminant animals; microorganisms in the
rumen produce phytanic acid as they digest
plant chlorophyll
• Typical western diet includes 50-100 mg of
phytanic acid per day
• Removes single C atom from carboxyl end

37
38
Ketogenesis - place of action
• Liver
• Mitochondria

39
Ketone «Bodies» - historical artifact
Entry of acetyl-CoA into citric acid cycle

requires oxaloacetate
When oxaloacetate is depleted, acetyl-

CoA is converted into ketone bodies
● frees coenzyme A for continued  oxidation
Three forms of ketone bodies can leave

the liver: acetone, acetoacetate, and -
hydroxybutyrate

40
Ketone Bodies are Water Soluble
• Volatilized through lungs
• Energy for skeletal and heart

muscle, brain, renal cortex
• Most prominent

41
Formation of Ketone Bodies: Generating Free CoASH
The first step is reverse of the

last step in the  oxidation:
thiolase reaction joins two
acetate units
A third acetyl-CoA is
incorporated in the second step
Together, two CoASH are freed

from three acetyl-CoA

42
Formation of Ketone Bodies:
Degradation of HMG-CoA
• In order to traffic to other
tissues, CoASH must be
removed. Acetone,
acetoacetate, and
-hydroxybutyrate can
then travel through the
blood
• Acetone is removed as a
gas and exhaled, but
acetoacetate and
-hydroxybutyrate can
traffic to the brain for use
in energy production
43
Utilization of Ketone
Bodies: Fuel in
extrahepatic tissues
Note: it is not the

reversible process of CAC
synthesis
Liver lacks β-ketoacyl-CoA

transferase

44
The Liver Is the Source of Ketone Bodies
pyruvate
Medicine 2.0, Medical Biochemistry 45

Diabetic Ketoacidosis vs Hyperosmolar
hyperglycemic state
https://spectrum.diabetesjournals.org/content/diaspect/15/1/28/T1.large.jpg
46
The Ketogenesis
HOMEWORK
TASK 3:

47
Summary of lipid catabolism
• Unlike carbohydrate fuels, which enter the body primarily as
glucose or sugars that are converted to glucose, lipid fuels are
heterogeneous with respect to chain length, branching, and
unsaturation
• The catabolism of fats is primarily a mitochondrial process but
also occurs in peroxisomes (both beta and alpha oxidation) and
omega oxidation in ER
• During peroxisomal oxidation, fats can be oxidized to generate
heat, however, also produces H2O2
• Using a variety of chain length–specific transport processes and
catabolic enzymes, the primary pathways of catabolism of fatty
acids involve their oxidative degradation in two-carbon units, a
process known as β-oxidation, which produces acetyl-CoA
• In the process, a lot of NADH+H+ and FADH2 forms; these can
yield a lot of ATP in the electron-transport chain

48
Summary of lipid catabolism
• In most tissues, the acetyl-CoA units are oxidized further

and used for ATP production in the mitochondrion
• In liver, acetyl-CoA is catabolized to ketone bodies, primarily
acetoacetate and β-hydroxybutyrate, by a mitochondrial
pathway termed ketogenesis
• The ketone bodies are exported from the liver for energy
metabolism in peripheral tissue
• Ketonemia and ketonuria develop gradually during fasting,
whereas ketoacidosis may develop during poorly controlled
diabetes, when fat metabolism is increased to high levels
for support of gluconeogenesis

49
Thank you! 

50

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Medical Biochemistry

Assistant prof. DACE REIHMANE

Medicine 2.0, Medical Biochemistry

● Describe the pathway for activation and transport of fatty acids to

● Outline the sequence of reactions involved in the oxidation of fatty

● Describe the general features of pathways for the oxidation of

● Explain the rationale for the pathway of ketogenesis, and identify

Medicine 2.0, Medical Biochemistry

17| Fatty acid catabolism

© 2017 W. H. Freeman and Company

Medicine 2.0, Medical Biochemistry

Medical Biochemistry Principles of Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Dietary TAGs > 40% TAGs ~ 80% TAGs > 50%

Medicine 2.0, Medical Biochemistry

The advantage of fats over polysaccharides:

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

• TAGs are stored in adipocytes (in steroid-synthesizing cells of the

Medicine 2.0, Medical Biochemistry

Lipid droplets are

Albumin ~1/2 of serum

PKA – protein kinase A

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

• Glycerol kinase activates glycerol

• Allows limited anaerobic

• Passive diffusion through

Medicine 2.0, Medical Biochemistry

Fatty acyl-CoA synthetase

CoenzymeA must stay at cytoplasm, membrane is unpermeable for Coa

Rate limiting step

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

• Every cell with mitochondria, but with different rate

Medicine 2.0, Medical Biochemistry

Stage 1 consists of oxidative conversion of

Each pass removes

Medicine 2.0, Medical Biochemistry

• Hetero-octamer (α4β4 subunits)

Medicine 2.0, Medical Biochemistry

Catalyzed by isoforms of acyl-CoA

Results in trans double bond, different from naturally

Medicine 2.0, Medical Biochemistry

Catalyzed by two isoforms of

Water adds across the double bond yielding alcohol

Medicine 2.0, Medical Biochemistry

The enzyme uses NAD cofactor as electron

Only L-isomers of hydroxyacyl-CoA act as substrates

Medicine 2.0, Medical Biochemistry

● The carbonyl carbon in -ketoacyl-CoA is electrophilic

The net reaction is thiolysis of the carbon-carbon bond

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Medicine 2.0, Medical Biochemistry

Peroxisomal acyl-CoA dehydrogenase

Much more active on very-long-chain (≥22C)

TASK IN COLLO, compare b-oxy with one of these.

XALD affects young boys before the age of 10 years:

Medicine 2.0, Medical Biochemistry