Metabolism of lipids

AMU, School of Medicine

Department of Biomedical Sciences
  Introduction

 Lipids are a heterogeneous group of water- insoluble

(hydrophobic) Organic molecules. Hydrophobicity is due to the
long – hydrocarbon chains.
 Because they are insoluble in aqueous solutions, body lipids are
generally found either:
A- Compartmentalized: e.g., Membrane- associated lipids (provide
the hydrophobic barriers that permit partitioning of aqueous
contents of cells and sub cellular structures )or as droplets of fat
(Triacylglycerols) in adipose tissue.
B- Transported in plasma in association with protein as lipoprotein
particles (chylomicrons, VLDL, LDL, HDL ).

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Metabolism of Dietary Lipids
Digestion, Absorption, Secretion and Utilization of dietary lipids

Dietary lipids consists of:

 More than 90% are triglycerides (TAG), the other are
 Cholesterol (most of dietary is in free form)
 Cholesteryl esters,
 Phospholipids, and
 Unesterified fatty acids/non-esterified fatty acids (NEFAs).

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1. Digestion of dietary lipids

A. Processing of dietary lipids in the stomach

 In infants, digestion begins in stomach catalyzed by the
acid-stable lingual lipase.
 That originates from glands at the back of the tongue.
 TAG molecules containing short or medium chain fatty acids
such as those found in milk fat are the target of this enzyme.
 However, the rate of hydrolysis is slow because the lipid is not yet
emulsified.
 These TAG molecules can be degraded by a separate gastric acid
lipase that is secreted in small quantities.

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Digestion of dietary lipids…

This enzyme is active only at neutral pH and therefore, of

little use in the adult stomach where, the pH is low, but is
active in neonates whose stomach pH is nearer to neutrality and
whose diets contain mainly milk lipids.
They also become important digestive enzymes in individuals
with pancreatic insufficiency, such as those with cystic fibrosis
Overall in adults , dietary lipids are not digested to any
extent in the mouth or the stomach but rather progress more or
less intact to the small intestine.

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 Digestion of dietary lipids…

B. Digestion in small intestine (duodenum)

 Effective digestion due to the presence of Pancreatic lipase and bile
salts (emulsifying agent).
 Emulsification increases the surface area of the hydrophobic
lipid droplets so that the digestive enzymes which work at the
interface of the lipid droplet and the surrounding aqueous
duodenal contents can act effectively.
 Emulsification is accomplished by two complementary
mechanisms namely, the use of the detergent (amphipathic )
surface active properties of bile salts synthesized in the liver and
stored in the gall bladder and mechanical mixing of peristalsis.

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Digestion of dietary lipids…

Bile salts : Bile salts are derivatives of cholesterol and consist of

a sterol ring to the side chain of which a molecule of glycine or
taurine is covalently attached by an amide linkage.
These emulsifying agents interact with both the lipid particles and
the aqueous duodenal contents, thereby stabilizing the particles
as they become smaller and preventing them from coalesing.

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Fig. Micellar solubilization of fat

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 Digestion of dietary lipids…

C. Degradation of dietary lipids by pancreatic enzymes

 The dietary triacylglycerol, cholesteryl esters, and
phospholipids are enzymatically degraded ("digested") by
pancreatic enzymes, whose secretion is hormonally
controlled.
I. TAG degradation by Pancreatic Lipase:
 TAG molecules are too large to be taken up efficiently
by the mucosal cells of the intestinal villi.
 They are, therefore, acted upon by an esterase, pancreatic
lipase, which preferentially removes the fatty acids at
carbons 1 and 3.
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 The primary products of hydrolysis are thus a mixture of
2-monoacylglycerol and free fatty acids .
 Note: This enzyme is found in high concentrations in
pancreatic secretions (2–3% of the total protein present),
and it is highly efficient catalytically, thus insuring that
only severe pancreatic deficiency, such as that seen in
cystic fibrosis, results in significant malabsorption of fat.
 A second protein, colipase, also secreted by the pancreas,
binds the lipase at a ratio of 1:1, and anchors it at the
lipid-aqueous interface.

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 Colipase is secreted as the zymogen, procolipase, which is
activated in the intestine by trypsin.
 Orlistat, an anti-obesity drug, inhibits gastric and pancreatic
lipases, thereby decreasing fat absorption, resulting in loss of
weight.
II. Cholesteryl ester degradation by Cholesterol esterase:
 Most dietary cholesterol is present in the free (nonesterified)
form, with 10–15% present in the esterified form.
 Cholesteryl esters are hydrolyzed by pancreatic cholesteryl
ester hydrolase (cholesterol esterase), which produces
cholesterol plus free fatty acids .
 Cholesteryl ester hydrolase activity is greatly increased in the
presence of bile salts.

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III. Phospholipid degradation by Phospholipase A2:
 Pancreatic juice is rich in the proenzyme of phospholipase
A2 that, like procolipase, is activated by trypsin and, like
cholesteryl ester hydrolase, requires bile salts for optimum
activity.
 Phospholipase A2 removes one fatty acid from carbon 2 of a
phospholipid, leaving a lysophospholipid.
 For example, phosphatidylcholine (the predominant
phospholipid during digestion) becomes
lysophosphatidylcholine.
 The remaining fatty acid at carbon 1 can be removed by
lysophospholipase, leaving a glycerylphosphoryl base (for
example, glycerylphosphorylcholine, that may be excreted in
the feces, further degraded, or absorbed.
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 Overview of lipid
digestion,
absorption &
Secretion

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Hormonal control of lipid metabolism in the small intestine

 Secretion of the pancreatic lipid degradative-enzymes is

hormonally controlled.
 Cells in the mucosa of the jejunum and lower duodenum secrete a
small peptide hormone , cholecystokinin (chole=bile, cyto=sac &
kinin=move) in response to the presence of lipids and partially
digested proteins entering these upper regions of small Intestine
(chyme).
 This hormone causes the gall bladder to contract and release bile
and causes the exocrine cells of the pancreas to release digestive
enzymes.

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Hormonal control of lipid metabolism in
the small intestine…
 Other intestinal cells produce another small
peptide hormone, Secretin in response to the
low pH of chyme entering the intestine , it
causes the pancreas to secrete a watery solution
rich in bicarbonate that helps to neutralize the
pH of the intestinal contents bringing it to the
appropriate pH for the activity of digestive
enzymes.

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2. Absorption of lipids by intestinal mucosal cells
a. Mixed micelles = Bile salts, together with free fatty acids, free
cholesterol and 2- monoacylglycerol form clusters of
amphipathic lipids that coalesce with their hydrophobic groups
on the inside and their hydrophilic groups on the outside of the
cluster, and which are soluble in the aqueous environment of the
intestinal lumen.
 The micelles approach the brush border membrane of the enterocytes
 It is the monoglycerides and fatty acids that are free in solution that
are absorbed, not the micelles.
 Because of their nonpolar nature, monoglycerides, cholesterol and
fatty acids can just diffuse across the plasma membrane of the
enterocyte

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 Absorption of lipids by intestinal mucosal
cells…
b. Short and medium chain-length fatty acids do not require
the assistance of a micelle for absorption by the intestinal
mucosa.

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Fig. Absorption of lipids by intestinal mucosal cells

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Absorption of lipids by intestinal mucosal cells…

 Cholesterol and other sterols are poorly absorbed.

Overall, about 50% of dietary cholesterol is absorbed.
 Dietary fat increases cholesterol absorption.
 Fiber (especially soluble fiber) and phytosterols
decrease cholesterol absorption

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Fig. The effect of soluble fibbers on cholesterol and bile salt
absorption

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Inhibitors of lipid digestion and absorption

Orlistat:
 Non hydrolysable analog of triacylglycerol. Powerful inhibitor of pancreatic
lipase. Blocks lipid absorption and hence results in lipid excretion
Olestra:
 Commercial lipid (artificial fat) produced by esterification of natural fatty
acids with sucrose instead glycerol. 6 to 8 fatty acids are covalently coupled
with sucrose
 They taste like lipid but not hydrolyzed to absorbable constituents and hence
excreted.
Ezetimibe:
The drug that blocks a protein that specifically mediates cholesterol
transport across the apical plasma membrane of enterocytes.

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 Lipid mal-absorption (steatorrhea or fatty
stool)
 It is the loss of > 6g of fat together with fat soluble
vitamins (A,D,E,K) and essential fatty acids.
Causes of steatorrhea:
1. defective digestion due to deficiency of pancreatic
lipase as a result of chronic pancreatits, cystic fibrosis
and severe gastric hyperacidity (Zollinger- Ellison
syndrome), obstruction of pancreatic duct by tumours
and pancreatectomy. Feacal fat is mostly undigested
TAGs.

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Lipid mal-absorption (steatorrhea)

2. Defective absorption due to deficiency of bile salts

as a result of bile duct obstruction as in tumors or
stones of the bile duct and in some cases of hepatitis
and liver cirrhosis.
Feacal fat is in the form of 2-monoacylglycerol. Also
absorption may be due to defective intestinal mucosal
cells as in shortened bowel (by surgical removal) and
coeliac diseases such as sprue.

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 Secretion of lipids from intestinal mucosal cells

 After solubilization and lipase digestion, monoacylglycerol and free

fatty acids are taken up by epithelial cells in the mucous membrane of
the small intestine. What happens to them once inside is somewhat
surprising: they are immediately converted back to triacylglycerol.
This involves the transient activation of fatty acids to acyl-CoA at the
expense of ATP.
 The newly resynthesized TAGs and cholesteryl esters are very
hydrophobic, and aggregate in an aqueous environment.
 It is, therefore, necessary that they be packaged as particles of lipid
droplets surrounded by a thin layer composed of phospholipids,
unesterified cholesterol, and a molecule of the characteristic protein,
apolipoprotein B-48

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Compositions of chylomicrons

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Secretion of lipids from intestinal mucosal cells…

 Assembly of chylomicrons ( lipids are surrounded by a thin layer of

protein, phospholipid and unesterified cholesterol.
 This layer stabilizes the particle and increases its solubility in aqueous
solutions. Secretion of chylomicrons from intestinal mucosal cells into
lymph (after a lipid- rich meal ( gives lymph a milky appearance =
chyle).
 Then chylomicrons are conveyed to the thoracic duct, then to the left
subclavian vein, where they enter the blood.
 The apolipoprotein B- 48 (the major apolipoprotein in chylomicrons) is
synthesized in intestinal mucosal cells.
 If not synthesized, chylomicrons will not be assembled and
triacylglycerols accumulate in these cells leading to a genetic disorder,
congenital abetalipoproteinemia.

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Abetalipoproteinemia
 The assembly of chylomicrons within the endoplasmic reticulum of
the enterocyte requires the activity of microsomal triglyceride transfer
protein (MTP).
 Thus, it appears as though MTP activity is necessary to transfer
triacylglycerol formed within the SER to the ApoB protein.
 MTP also may be required for the transfer of triacylglycerol from the
cytoplasm to the lumen of the ER to form this lipid droplet.
 The lack of triglyceride transfer activity leads to the disease
abetalipoproteinemia.
 The symptoms of abetalipoproteinemia include lipid malabsorption
(and its accompanying symptoms, such as steatorrhea and vomiting),
which can result in caloric deficiencies and weight loss. Because lipid-
soluble vitamin distribution occurs through chylomicron circulation,
signs and symptoms of deficiencies in the lipid-soluble vitamins may
be seen in these patients.
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The apolipoprotein B- 48

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 Use of dietary lipids by the tissues
 Once the chylomicrons have entered the circulation, the capillary
wall barrier must again be overcome in the delivery of
triacylglycerol to extravascular cells.
 This is accomplished with the help of lipoprotein lipase, which is
located on the endothelial surface.
 It binds the chylomicrons and extracts triacylglycerol from them,
which it then cleaves again to fatty acids and glycerol.
 These small molecules can cross the endothelial barrier by
diffusion and reach the cells in the surrounding tissue.
 Lipoprotein lipase is found on the luminal surface of endothelial
cells of the capillary beds of the peripheral tissues (skeletal muscle
and adipose tissue).
 Its deficiency in familial lipoprotein lipase deficiency results in
massive chylomicronemia.
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Capillary walls contain an enzyme called lipoprotein-lipase that
dismantles the triacylglycerols in the lipoproteins into fatty acids
and glycerol, thus enabling these to enter into the adipose cells &
muscle cells.
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Fates of free-fatty acids

1. Directly enter adjacent muscle cells or adipocytes.

2. Alternatively, free fatty acids may be transported in blood in
association with serum albumin, until they are taken by cells.
3. Most cells can oxidize fatty acids to obtain energy (ATP).
However, the brain and other nervous tissues, erythrocytes
and the adrenal medulla cannot use plasma free- fatty acids
for fuel, regardless of the blood levels of fatty acids.
4. Adipocytes can also re-esterify free-fatty acids to produce
triacylglycerol molecules, which are stored until the fatty
acids are needed by the body.

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 Fates of glycerol

 Glycerol is used by the liver to produce glycerol-3-phosphate, which can

enter glycolysis or gluconeogenesis by oxidation to dihydroxyacetone
phosphate.

Fate of chylomicron remnants

 After removal of most of its triacylglycerol (TAG) content are taken up by
the liver where , they are hydrolyzed to their component parts.
 This is due to the presence of apolipoprotein E on the surface of chylomicrons
which has apolipoprotein E receptors in hepatic cells plasma membranes.
 Deficiency of apolipoprotein E leads to familial type III
hyperlipoproteinemia ( familial - dys-betalipoproteinemia) , which leads to
defective removal of chylomicron -remnants from the plasma, and they
accumulate in plasma.

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Fig. Major fates of Dietary lipids

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Oxidation of Fatty Acids and
Ketogenesis
 Biomedical importance
 The fatty acids are both oxidized to acetyl-CoA and synthesized
from acetyl-CoA,.
 However the fatty acid oxidation is not the simple reverse of fatty
acid biosynthesis but an entirely different process taking place in a
separate compartment of the cell.
 The separation of fatty acid oxidation in mitochondria from
biosynthesis in the cytosol allows each process to be individually
controlled and integrated with tissue requirements.
 Each step in fatty acid oxidation involves acyl-CoA derivatives
catalyzed by separate enzymes, utilizes NAD+ and FAD as
coenzymes, and generates ATP. It is an aerobic process, requiring
the presence of oxygen.

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 Biomedical importance…
 Liver, Kidney, skeletal and heart muscle are the major location of fatty
acid oxidation.
 Increased fatty acid oxidation is a characteristic of starvation and of
diabetes mellitus, leading to ketone body production by the liver
(ketosis).
 Ketone bodies are acidic and when produced in excess over long
periods, as in diabetes, cause ketoacidosis, which is ultimately fatal.
 Because gluconeogenesis is dependent upon fatty acid oxidation, any
impairment in fatty acid oxidation leads to hypoglycemia.
 This occurs in various states of carnitine deficiency or deficiency
of essential enzymes in fatty acid oxidation, eg, carnitine
palmitoyltransferase, or inhibition of fatty acid oxidation by
poisons, eg, hypoglycin.

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 β-oxidation of fatty acids
 The major pathway for catabolism of saturated fatty acids
is a mitochondrial pathway called β- oxidation, in which
two- carbon fragments are successively removed from the
carboxyl end of the fatty acyl CoA, producing acetyl CoA.

Steps of β- oxidation of fatty acids:

1. Tansport of fatty acids into the mitochondria:

 After a fatty acid is taken up by a cell, it is activated to the
fatty acyl CoA derivative by fatty acyl CoA synthetase
(thiokinase) in the cytosol, with the consumption of 2 ATP
molecules.

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β-oxidation of fatty acids…
 In the presence of ATP and coenzyme A, the enzyme acyl-CoA
synthetase catalyzes the conversion of a fatty acid (or free
fatty acid) to an “active fatty acid” or acyl-CoA, which uses
one high-energy phosphate with the formation of AMP and PPi
2. Because β- oxidation occurs in the mitochondria, the fatty
acyl CoA must be transported across the inner
mitochondrial membrane , which is impermeable to the
bulky, polar molecules such as coenzyme A.
 Therefore a specialized carrier in this membrane
transports the acyl group from the cytosol into the
mitochondrial matrix. This carrier is carnitine, and the
transport process is called the carnitine shuttle
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Carnitine

 Carnitine (-hydroxy-γ-trimethylammonium butyrate),

(CH3)3N+-CH2-CH(OH)-CH2-COO−, is widely distributed and is
particularly abundant in muscle.
 The skeletal muscle contains 97% of all carnitine in the body.
 Carnitine is synthesized enzymatically in the liver and kidney
from the amino acids lysine and methionine, but is not
synthesized in the skeletal or cardiac muscles , therefore, these
tissues are totally dependent on carnitine produced by
hepatocytes or taken in dietary meat products.
 Additional functions of the carnitine system includes allowing
the export of branched chain acyl groups produced during
catabolism of branched chain amino acids (valine, leucine and
isoleucine) from the mitochondria.
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 Genetic defects in carnitine shuttle

1. The congenital absence of a carnitine ( acyl)transferase

in the skeletal muscle, or low concentrations of carnitine
due to defective synthesis result in an inability to use
long- chain fatty acids as a metabolic fuel, causing
myoglobinemia and weakness following exercise.
2. Genetic carnitine palmitoyl transferase I deficiency
affects the liver, where an inability to use long –chain
fatty acids for obtaining energy, greatly impairs the
liver’s ability to synthesize glucose during a fast.
This can lead to severe hypoglycemia, coma and death
(fasting hypoglycemia).

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 Genetic defects in carnitine shuttle…
3. Carnitine palmitoyl transferase II deficiency occurs primarily in
the cardiac and skeletal muscles , where, symptoms of carnitine
deficiency range from cardiomyopathy to skeletal muscles
weakness with myoglobinemia following prolonged exercise.
Treatment
 Treatment of these cases involves the avoidance of prolonged fasts
and adopting diets high in carbohydrate content and low in long
chain fatty acids, but supplemented with medium chain fatty acids
and in cases of carnitine deficiency , supplementation with carnitine.

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Secondary carnitine deficiency occurs in the following situations:

1. In patients with liver disease causing decreased synthesis of

carnitine.
2. In individuals suffering from mal-nutrition or those on
strictly vegeterian by diets.
3. In those individuals with an increased requirement for
carnitine a result of pregnancy, severe infections, burns and
trauma.
4. In renal failure patients undergoing hemodialysis which
removes carnitine from blood.

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Malonyl CoA is an inhibitor of the carnitine shuttle
 Malonyl CoA inhibits carnitine acyltransferase I, thus
inhibiting the entry of acyl groups into the mitochondrial
matrix. Therefore, when fatty acid synthesis is occurring
in the cytosol ( as indicated by the presence of malonyl
CoA, the building block of fatty acids), the newly made
fatty acid chains cannot be transferred into the
mitochondria and degraded.

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 Entry of short- and medium-chain fatty acids into
the mitochondria:
 Fatty acids shorter than 12 carbons can cross the inner
mitochondrial membrane without the aid of carnitine or
the CPT system.
 Once inside the mitochondria, they are activated to their
CoA derivatives by matrix enzymes, and are oxidized.
 Note: Medium-chain fatty acids are plentiful in human
milk. Because their oxidation is not dependent on CPT-I,
it is not subject to inhibition by malonyl CoA.

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 Reactions of β-oxidation:
 It consists of a sequence of four reactions involving the β-carbon
(carbon 3) that results in shortening the fatty acid chain by two
carbons. The steps include an oxidation that produces FADH2 and
unsaturation of fatty acyl CoA, a hydration step, a second
oxidation that produces NADH, and a thiolytic cleavage that
releases a molecule of acetyl CoA.
 Each step is catalyzed by enzymes with chain-length specificity.
These four steps are repeated for saturated fatty acids of even-
numbered carbon chains (n/2) – 1 times (where n is the number of
carbons), each cycle producing an acetyl group plus one NADH
and one FADH2.
 The final thiolytic cleavage produces two acetyl groups.
Note: Acetyl CoA is a positive allosteric effector of pyruvate
carboxylase, thus linking fatty acid oxidation and gluconeogenesis.
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Medium- chain length fatty acyl CoAdehydrogenase deficiency

There are three fatty acyl CoA dehydrogenase species in

mitochondria that have specificities for short, medium or
long chain length fatty acids.
 Medium chain length acyl CoA dehydrogenase
deficiency is found in approximately 1 in 10,000 births
and is therefore more prevalent than phenylketonuria.
It causes a decrease in fatty acid oxidation and severe
hypoglycemia , and is the cause of up to 10% of cases of
sudden infant death syndrome (SIDS).

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Oxidation of saturated fatty acids with odd number of carbons.

Proceeds by the same reaction

steps as that of fatty acids with
even number of carbons , until the
final three carbons are reached , a
compound called propionyl CoA.
 Propionyl CoA is the end
product of β- oxidation of fatty
acids with an odd number of
carbons.
Propionyl CoA is metabolized by
a two step pathway as shown in the
figure

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 Methylmalonic acidemia and aciduria

 Two types of inheritable methylmalonic acidemia and

aciduria have been described : one in which the methyl
malonyl CoA mutase is missing, and the other in which the
patient is unable to convert vitamin B12 into its active
coenzyme form. Either defect results in metabolic acidosis
and developmental retardation

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 Oxidation of unsaturated fatty acids
 The oxidation of unsaturated fatty acids provides less energy
than that of saturated fatty acids because unsaturated fatty acids
are less highly reduced and, therefore, fewer reducing
equivalents can be produced from these structures.
 Oxidation of monounsaturated fatty acids, such as oleic acid
requires one additional enzyme, 3,2-enoyl CoA isomerase,
which converts the 3-trans derivative obtained after three rounds
of β-oxidation to the 2-trans derivative required as a substrate by
the enoyl CoA hydratase. Oxidation of polyunsaturated fatty
acids, such as 18:2(9,12) (linoleic acid), requires an NADPH-
dependent 2,4-dienoyl CoA reductase in addition to the
isomerase.

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 β-Oxidation in the peroxisome:

 Very-long-chain fatty acids (VLCFA), or those 22 carbons long or longer,

undergo a preliminary β-oxidation in peroxisomes. The shortened fatty acid
(linked to carnitine) diffuses to a mitochondrion for further oxidation. In
contrast to mitochondrial β-oxidation, the initial dehydrogenation in
peroxisomes is catalyzed by an FAD-containing acyl CoA oxidase.
 The FADH2 produced is oxidized by molecular oxygen, which is reduced
to H2O2; thus, no ATP is generated by this step. The H2O2 is reduced to H2O
by catalase
 Note: Genetic defects either in the ability to target matrix proteins to
peroxisomes (resulting in Zellweger syndrome —a peroxisomal biogenesis
disorder) or in the ability to transport VLCFA across the peroxisomal
membrane (resulting in X-linked adrenoleukodystrophy), lead to
accumulation of VLCFA in the blood and tissues.

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 α-Oxidation of fatty acids
 Branched-chain, 20 carbon fatty acid, phytanic acid:
(3,7,11,15 tetramethyl palmitic acid).
 This is not a substrate for acyl CoA dehydrogenase because of
the methyl group on its β carbon. Instead, it is hydroxylated at
the α-carbon by phytanoyl CoA α-hydroxylase (PhyH), carbon
1 is released as CO2, and the product, 19 carbon pristanic acid,
is activated to its CoA derivative and undergoes β-oxidation.
 Refsum disease is a rare, autosomal recessive disorder caused
by a deficiency of peroxisomal PhyH. This results in the
accumulation of phytanic acid in the plasma and tissues. The
symptoms are primarily neurologic, and the treatment involves
dietary restriction to halt disease progression.

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ω-oxidation

 It is the oxidation of the terminal CH3 group(ω carbon) of

fatty acid. This produce dicarboxylic fatty acids
 By beta-oxidation they are converted in to adipic acid (6
carbons) and suberic acid (8 carbons)
 ω-Oxidation is a minor pathway for fatty acid oxidation
and catalyzed by hydrolase enzyme of cytochrome P450.

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Ketogenesis and Ketolysis
 Ketone bodies : an alternative fuel for cells.

 Under metabolic conditions associated with a high rate of fatty acid

oxidation, the liver produces considerable quantities of acetoacetate and (β)
or 3-hydroxybutyrate. Acetoacetate continually undergoes spontaneous
decarboxylation to yield acetone.
 These three substances are collectively known as the ketone bodies (also
called acetone bodies or incorrectly “ketones”)
 They are water soluble & transported in the blood to peripheral tissues,
where they can be reconverted to acetyl CoA and oxidized by the TCA
cycle.
 They are produced in the liver during periods when the amount of acetyl
CoA present exceeds the oxidative capacity of the liver.

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Fig. Formation, utilization, and excretion of ketone bodies. (The main pathway is
indicated by the solid arrows.)
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Ketogenesis/synthesis of ketone bodies by liver

1. Formation of acetoacetyl-CoA from two molecules of

acetyl-CoA by thiolase. This step is the reversal of the
final step in β-oxidation;
2. Formation of hydroxymethylglutaryl-CoA (HMG-CoA)
by HMG-CoA synthase;
3. Release of acetoacetate by HMG-CoA lyase; and
4. Reduction of acetoacetate to β-hydroxybutyrate by β-
hydroxybutyrate dehydrogenase.

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Fig. Pathway of ketogenesis in liver

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 Synthesis of ketone bodies by the liver…

 Mitochondrial HMG CoA synthase combines a third molecule of acetyl

CoA with acetoacetyl CoA to produce HMG CoA.
 Note: HMG CoA is also a precursor of cholesterol
 Note that HMG CoA synthase , is the rate- limiting step in the synthesis
of ketone bodies and is present in significant quantities only in the liver.
 This enzyme is exclusively present in liver mitochondria.
 There are two isoforms of this enzyme-cytosolic and mitochondrial. 
 The mitochondrial enzyme is needed for ketogenesis while the
cytosolic form is associated with cholesterol biosynthesis.

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Why are three enzymes required to synthesize
acetoacetate?
 An enzyme that cleaves the thioester bond of the thiolase 
product  acetoacetyl-CoA  would  also produce
acetoacetate, but such a thioesterase does not seem to exist.
 However, the pathway that does exist is not especially
wasteful; the third acetyl-CoA used merely acts
catalytically.
 It is possible that having two mitochondrial enzymes
(HMG-CoA synthase and HMG-CoA lyase) required for
ketone body synthesis assists in controlling the pathway.

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Formation of acetone
 Acetone is formed by decarboxylation in the presence of
decarboxylase enzyme and, because it is a beta-keto acid,
acetoacetate also undergoes a slow, spontaneous decarboxylation to
acetone.
 The odor of acetone may be detected in the breath of a person who
has a high level of acetoacetate in the blood.  
 “Acetone-breath” has been used as a crude  method of
diagnosing  individuals  with  untreated Type I diabetes mellitus.

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 Formation of β-Hydroxy Butyrate
3-Hydroxybutyrate (β-Hydroxy Butyrate) is formed by the
reduction of acetoacetate in the mitochondrial matrix by D(-)3-
hydroxybutyrate dehydrogenase.
 3-Hydroxybutyrate is quantitatively the predominant ketone body
present in the blood and urine in ketosis.
The β-hydroxybutyrate dehydrogenase reaction has two functions:
1) it stores energy equivalent to an NADH in the ketone body for
export to the tissues, and
2) it  produces  a  more  stable molecule.
The ratio of β hydroxybutyrate to acetoacetate depends on the
NADH/NAD+ ratio inside mitochondria. If NADH concentration is
high, the liver releases a higher proportion of β-hydroxybutyrate.

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Fig. Formation of β-Hydroxy Butyrate & Acetone

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Regulation of ketogenesis

 During fasting anti-insulin hormones are secreted. This stimulates

lipolysis & in turn stimulates ketogenesis.
 By lipolysis the liver is flooded with fatty acids mobilized from
adipose tissue.
 The resulting elevated hepatic acetyl CoA produced primarily by
fatty acid degradation inhibits pyruvate dehydrogenase , and
activates pyruvate carboxylase .
 The OAA thus produced is used by the liver for gluconeogenesis
rather than for the TCA cycle.
 Therefore, acetyl CoA is channeled into ketone body synthesis.
 Note: Fatty acid oxidation decreases the NAD+ to NADH ratio, and
the rise in NADH shifts OAA to malate. This pushes acetyl CoA
away from TCA-cycle and into ketogenesis

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 Insulin= inhibits
Glucagon= Stimulates

Malonyl-CoA=
inhibits

Fig. Regulation of ketogenesis

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Utilization of ketone bodies/ketolysis/
 Ketone bodies serve as a fuel for extra hepatic tissues
 The ketone bodies are water soluble and are transported across the
inner mitochondrial membrane as well as across the blood-brain barrier
and cell membranes.
 They can be used as a fuel source by a variety of tissues including the
CNS.
 They are preferred substrates for aerobic muscle and heart, thus
sparing glucose when they are available.
 Tissues that can use fatty acids can generally use ketone bodies in
addition to other energy sources.
 The exceptions are the liver and the brain. Ketolysis does not occur in
Liver because it does not contain Thiophorase enzyme.

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Liver Vs Peripheral tissues for Ketolysis

 The enzyme, Succniyl Co A: Acetoacetate Co A transferase,

also known as Thiophorase, is present at high levels in most
tissues except the liver.
 Importantly, very low level of enzyme expression in the liver
allows the liver to produce ketone bodies but not to utilize
them.
 This ensures that extra hepatic tissues have access to ketone
bodies as a fuel source during prolonged fasting and
starvation, and
 Also, lack of this enzyme in the liver prevents the futile cycle of
synthesis and breakdown of acetoacetate.
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Fig. Overview of organ relationships in fat utilization. Triacylglycerol is cleaved
in fat tissue to fatty acids and glycerol. Fatty acids are utilized by –oxidation
in heart and skeletal muscle, or first converted to ketone bodies in the liver.
Ketone bodies can be consumed by many organs including the brain.
Glycerol is utilized for gluconeogenesis.
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Ketone body synthesis in the liver and use in peripheral tissues. Note:
Thiophorase is also known as succinyl CoA: acetoacetate CoA transferase.
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 Biological significance of ketone bodies
 Acetoacetate and β-hydroxybutyrate are normal fuels of cellular
respiration and are quantitatively important as sources of energy.
 Heart muscle and the renal cortex use acetoacetate in preference to glucose.

 In contrast, the brain adapts to the utilization of acetoacetate during

starvation and diabetes.
 In prolonged starvation,75% of the fuel needs of the brain are met by
ketone bodies.
 Individuals eating diets extremely high in fat and low in carbohydrate, or
starving, or suffering  from  a severe lack of insulin (Type I diabetes 
mellitus) therefore increase the synthesis and utilization of ketone bodies

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Ketonemia
Ketonemia - increased concentration of ketone bodies in blood
 It is due to increased production of ketone bodies by the liver
rather than to a deficiency in their utilization by extra hepatic tissues.
The production of ketone bodies occurs at a relatively low rate
during normal feeding and under conditions of normal physiological
status. 
Normal physiological responses to carbohydrate shortages cause the
liver to increase the production of ketone bodies from the acetyl-CoA
generated from fatty acid oxidation.
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 Causes of Ketosis
Uncontrolled diabetes mellitus
 Starvation
 Chronic alcoholism
 Von- Gierke’s disease
 Heavy exercise
 Low carbohydrate diet- For weight loss
 Glycogen storage disease type 6(Due to phosphorylase kinase deficiency)
 Pyruvate carboxylase deficiency

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Salient Features of Ketosis

1. Metabolic acidosis. Acetoacetate and betahydroxy

butyrate are acids. When they accumulate, metabolic
acidosis results
2. Reduced buffers. The plasma bicarbonate is used up
for buffering of these acids.
3. Kussmaul's respiration. Patients will have typical
acidotic breathing due to compensatory hyperventilation.
4. Smell of acetone in patient's breath

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 Salient Features of Ketosis…

5. Osmotic diuresis induced by ketonuria may lead to

dehydration.
6. Sodium loss. The ketone bodies are excreted in urine as
their sodium salt, leading to loss of cations from the body.
7. Dehydration. The sodium loss further aggravates the
dehydration.
8. Coma. Dehydration and acidosis contribute to the lethal
effect of ketosis.

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