Cholesterol Metabolism Clinical Implications

1. “Cholesterol” the most feared among Lipids, speaks:
“ Consumed through the diet and produced in the body,
participate in innumerable cellular functions.
Implicated in several Health complications,
And blamed I am, for no fault of Mine.”

2. Cholesterol
Synthesis,Transport and
Excretion

3. Objectives:
 Cholesterol: Basic Structure, Functions
 Details of Biosynthesis of Cholesterol
 Regulation of the Cholesterol Synthesis
 Absorption of Cholesterol, Transport
 Excretion of Cholesterol
 Bile Acids and Bile Salts

4.  Cholesterol is an Alicyclic Compound, widely
distributed in Free and Esterified forms.
 Member of sterol derivative.
 Solid Alcohol of high molecular weight.
 Molecular formula C27H45OH.
Introduction

5. History
 Isolated from Gall stones in 1784.
 Thirteen Nobel prizes has ben awarded to the scientists who
devoted most of their career in important discovery related to
Cholesterol.

6. Structure
Includes:
 Perhydrocyclopentano Phenanthrene Nucleus with four
fused rings.
Perhydrocyclo
pentanophenanthrene(STERANE) skeleton
Cholesterol

7.  A single Hydroxyl group a C-3.
 An Unsaturated center between C5-C6 atoms.
 An Eight membered branched Hydrocarbon chain attached
to the D-ring at position 17.
 Methyl Group (Designated C-19) attached at position 10 and
another methyl group (Designated C-18) at position 13.

8.  Actual concentration of Cholesterol in Plasma of healthy
people 150-200 mg/dl
 This value is almost Twice the Normal Concentration of
Blood Glucose.
 This accounts for high solubility of Cholesterol in blood
which is due to Plasma Lipoproteins (LDL and VLDL) that
have the ability to bind and thereby solubilize large
amounts of Cholesterol.

9.  30% of Total Plasma Cholesterol –Free
70% - Cholesterol Esters ; where some long chain fatty
acid Linoleic Acid attached by an Ester Bond to
Hydroxyl (-OH group) on C3 of the A-Ring.

10. Cholesterol synthesis
• Synthesized in all tissues.
• Major sites for synthesis- liver, adrenal cortex, testes,
ovaries and intestine.
• All carbon atoms are derived from acetyl CoA.
• Enzymes involved in biosynthesis are partly located in ER
and partly in cytoplasm.

11.  All of the carbon atoms of cholesterol are derived from
acetate.
Cholesterol Biosynthesis

12.  Observation of their pattern of incorporation into cholesterol
led Konrad Bloch to propose that acetate was first converted to
isoprene units, C5 units that have the carbon skeleton of
isoprene.
 Isoprene units are condensed to form a linear
precursor to cholesterol, and then cyclized.

13.  Outline of Major Stages of Cholesterol Biosynthesis was :
Acetate Isoprenoid Intermediate Squalene
Cholesterol Cyclization
Product

14. Mevalonate is a Key Intermediate in
Cholesterol Biosynthesis
 Cholesterol Biosynthesis begins with
condensation of two molecules of Acetyl
CoA
Catalysed by Thiolase
 Next Step requires enzyme HMG CoA Synthase
Catalyses the condensation of a Third Acetyl CoA to
yield HMG CoA.

15. Synthesis of HMG CoA
 HMG CoA Synthase is
present in both cytosol and
mitochondria of liver.
 Mitochondrial-
ketogenesis
 Cytosolic – cholesterol
synthesis

16.  HMG CoA then reduced to Mevalonate by HMG CoA
reductase.
 Activity of this Reductase Control of Rate of
Cholesterol synthesis.

17.  HMG-CoA An important Intermediate for the
biosynthesis of both Cholesterol and
Ketone Bodies.
 Biosynthesis of Cholesterol catalyzed by enzymes in
the Cytosol and
Endoplasmic reticulum.
 Synthesis of Ketone Bodies Restricted to Mitochondrial
Matrix.

18. The Rate of Mevalonate Synthesis determines
the rate of cholesterol Biosynthesis
 Primary regulation of Cholesterol Biosynthesis centred on the
HMG-CoA Reductase Reaction.
 HMG-CoA Found in ER
 887 AA in a single Polypeptide chain.
 Structure of the enzyme deduced by
Michael Brown and Joseph Goldstein.
 Has Two Domains.

19.  Amino Terminal Domain Mol Wt. 35,000
Seven Hydrophobic
Segments
Thought to cross the
membrane.
 Senses signals that lead to its degradation when cholesterol
levels are high.
 Carboxyl-Terminal Domain Mol Wt. 62,000
Contains the Catalytic site of
the Enzyme.
Thought to protrude into the
Cytosol.

20. HMG CoA Reductase Regulation
• Sterol-dependent regulation of gene expression
• Sterol-accelarated enzyme degradation
• Sterol-independent
phosphorylation/dephosphorylation
• Hormonal regulation

21.  Rate of Synthesis of Reductase mRNA is controlled by the
Sterol Regulatory Element Binding Protein (SREBP).
SREBP( Transcription Factor) binds to the short DNA
Sequence Sterol Regulatory Element(SRE) on the 5’ side
of the Reductase gene When Cholesterol levels
are low.
Sterol Dependent Regulation of Gene
Expression

22.  In its Inactive state, the SREBP resides in the
Endoplasmic Reticulum membrane, where it is
associated with SREBP Cleavage Activating Protein
(SCAP) An Integral Membrane Protein
 When Cholesterol levels Fall SCAP escorts SREBP in
small membrane vesicles
to Golgi Complex.
Where it is released from the membrane by two
specific Proteolytic Cleavages.

23.  First Cleavage frees a fragment of SREBP from SCAP.
 The Second Cleavage releases the Regulatory Domain from
the membrane.
Released Protein migrates to the Nucleus and binds the SRE of
the HMG- CoA Reductase Gene as well as other genes in the
Cholesterol Biosynthetic Pathway to enhance Transcription.

24. From Stryer 7th Edition

25.  High Levels of Cholesterol:
Proteolytic Release of SREBP BLOCKED
 SREBP in the Nucleus is Rapidly degraded.
These two events halt the transcription of genes
of the cholesterol biosynthetic pathways.

26.  When Cholesterol concentration is Low
SCAP binds to Vesicular Proteins that facilitate the
transport of SCAP-SREBP to the golgi apparatus as
already described.
 When Cholesterol is Present SCAP binds Cholesterol
Structural Change in
SCAP
It binds to another ER Protein called Insig.

27.  Insig: Anchor that retains SCAP and thus SREBP in the ER in
the presence of Cholseterol.
 Interactions between SCAP and Insig can also be forged when
Insig binds 25, hydroxycholesterol Metabolite of
Cholesterol.
 Thus, two distinct Steroid Protein Interactions serve to prevent
the inappropriate amount of SCAP-SREBP to the Golgi
Complex.

28. From Stryer 7th Edition

29. Sterol-dependent regulation
Cholesterol High
• SCAP binds to insigs
(ER membrane proteins)
• SCAP-SREBP is
retained in the ER
• Downregulation of
cholesterol synthesis
Cholesterol Low
• SCAP escorts SREBP to
Golgi bodies
• Two proteases cleave
SREBP to a soluble
fragment that enters the
nucleus and binds SRE
• HMG CoA gene
transcription is activated

30. Sterol Accelerated Regulation
 The enzyme is bipartite: its cytoplasmic domain carries out
catalysis and its membrane domain senses signals that lead to
its degradation.
 The membrane domain may undergo structural changes in
response to increasing concentrations of sterols such as
lanosterol and 25-hydroxycholesterol.
 Under these conditions, the reductase appears to bind to a
subset of Insigs that are also associated with the ubiquitinating
enzymes.

31.  The reductase is polyubiquitinated
Subsequently extracted from the membrane in a process
that requires Gerenylgeraniol.
The extracted reductase is then degraded by the proteasome.

32. From Stryer 7th Edition

33. Enzyme phosphorylation and
dephosphorylation
 AMP- activated protein kinase
(AMPK) for phosphorylation
switches off the enzyme.
 Phosphoprotein phosphatase for
dephosphorylation.
 Phosphorylated form of enzyme is
inactive.
 Dephosphorylated form – active
 Thus, Cholesterol Synthesis
ceases when ATP level is low.

34. Hormonal Regulation
• Insulin and thyroxine favor upregulation of enzyme
expression
• Glucagon and cortisol have opposite effect

35. From Harper’s 28th Edition

36.  The statin drugs are structural analogs of
HMG CoA, and are (or are metabolized to)
reversible, competitive inhibitors of HMG
CoA reductase.
 Used to decrease
Plasma Cholesterol
Levels.
Inhibition by drugs

37.  First stage in this sequence of this reactions :
 Synthesis of the Five-Carbon Isoprenoid Intermediates
Isopentenyl Pyrophosphate and Dimethylallyl Pyrophosphate.
 Requires ATP.
It takes Six Mevalonates and Ten Steps to make
Lanosterol, the First Tetracyclic Intermediate

38. 1. The CoA group of HMG-CoA is reduced to an alcohol in
an NADPH-dependent reduction catalyzed by
HMG- CoA reductase, yielding Mevalonate.
2. New OH group is phosphorylated by mevalonate-5
phosphotransferase.
3. The phosphate group is converted to a pyrophosphate
by phosphomevalonate kinase.
4. The molecule is decarboxylated and the resulting alcohol
dehydrated by pyrophosphomevalonate decarboxylase.
The formation of isopentenyl pyrophosphate
involves four reactions:

40. Squalene (C30) is synthesized from six molecules
of isopentenyl pyrophosphate (C5)
 Squalene is synthesized from isopentenyl pyrophosphate by the
reaction sequence.
C5 C10 C15 C30
 This stage in the synthesis of cholesterol starts with the
isomerization of isopentenyl pyrophosphate to dimethylallyl
pyrophosphate.

41.  Isopentenyl pyrophosphate and dimethylallyl pyrophosphate
undergo a Head- to-Tail condensation, in which one
pyrophosphate group is displaced and a 10-carbon chain,
Geranyl pyrophosphate, is formed.
 Geranyl pyrophosphate undergoes another Head-to-tail
condensation with isopentenyl pyrophosphate, yielding the
15-carbon intermediate Farnesyl pyrophosphate.
 Finally, two molecules of farnesyl pyrophosphate join head to
head, with the elimination of both pyrophosphate groups, to
form squalene.

42. From MN Chatterjee
Textbook of
Biochemistry 8th edition

43.  Geraniol, a component of rose oil, has the aroma of geraniums.
 Farnesol is an aromatic compound found in the flowers of the
Farnese acacia tree.
 Squalene , first isolated from the liver of sharks
(genus Squalus), has 30 carbons, 24 in the main
chain and 6 in the form of methyl group branches.

45.  The formation of lanosterol from squalene takes place in two
steps:
 Squalene-2,3-epoxide is formed first catalysed by the
enzyme squalene mono-oxygenase; requires NADPH and
molecular O2.
 In the next step, an enzyme cyclase brings about the
cyclisation of squalene to form lanosterol.
Cyclisation of squalene to form lanosterol

46. From Lanosterol to Cholesterol Takes Another
Twenty Steps
 Approximately, Twenty Enzymatic Steps, Starting with
Lanosterol.
 Comprise a series of Double Bond Reductions and
Demethylations.
 Final Reaction : Reduction of the ∆7 double bond in 7-
dehydrocholesterol.
 Alternative Pathway from Lanosterol to Cholesterol: Involves
Three Demethylations to give Zymosterol and then
Isomerization of ∆8 double bond to the ∆5 to produce
Desmosterol.

47.  Enzymes Involved in the Transformation of Lanosterol to
Cholesterol are all located in the Endoplasmic reticulum.

48. Cholesterol Absorption
 Cholesterol enters the Intestinal Lumen from Three Sources
I. Diet
II. Bile
III. Intestine
 Almost all Cholesterol is present in Unesterified (Free Form)
 For absorption Cholesterol must be solubilized.
Solubilised by the formation of
Micelles
Then, absorbed by an Active process involving
the Enterocyte protein NPC1L1
Drug Target for Cholesterol absorption
inhibitor EZETIMIBE

49.  Absorption of Cholesterol and other sterols (Plant Sterols) is
limited by the presence of ABCG5/G8 transporter on
Enterocytes.
Pumps Excess Sterols back into the Lumen
for Excretion.
 Most Cholesterol absorption occurs from the Jejunum to the
terminal Ileum of the small intestine.

50. Cholesterol Esterification
 Why??
 Enhance the Lipid carrying capacity of the lipoprotein
in the plasma.
 Prevents Intracelleular toxicity of Free Cholesterol.
 How??
 By Enzymes Lecithin Cholesterol Acyl Transferase(LCAT) in
Plasma
 ACAT ( Acylcholesterol Acyltransferase) within the Cell.

52. Cholesterol Esters
• Acyl-CoA:cholesterol acyl
transferase (ACAT) is an ER
membrane protein
• ACAT transfers fatty acid of CoA to
C3 hydroxyl group of cholesterol
• Excess cholesterol is stored as
cholesterol esters in cytosolic
lipid droplets.
Fig. 8

53. Many Other Factors Influence the Cholesterol
Balance in tissues
Cell cholesterol increase is due to
 Uptake of cholesterol-containing lipoproteins by receptors,
eg, the LDL receptor or the scavenger receptor.
 Uptake of free cholesterol from cholesterol-rich lipoproteins
to the cell membrane.
 Cholesterol synthesis, and
 Hydrolysis of cholesteryl esters by the enzyme cholesteryl
ester hydrolase.

54. Decrease is due to
 Efflux of cholesterol from the membrane to HDL via the
ATP-binding cassette transporters A1 (ABCA1) and G1
(ABCG1), or SR-B1 class B scavenger receptor B1 ;
 Esterification of cholesterol by ACAT (acyl-
CoA:cholesterol acyltransferase); and

 Utilization of cholesterol for synthesis of other steroids,
such as hormones, or bile acids in the liver.

56. Transport of Cholesterol
 After Absorption Cholesterol together with Triglycerides,
Phospholipids,and a number of
specific Apoproteins is assembled into
a large Lipoprotein called
CHYLOMICRON.
 One Apoprotein Component (Apo B-48) Vital for the
formation and Secretion of Chylomicrons.
 Patients with the deficiency of this apoprotein leads to a
condition called as Chylomicron Retention Disorder
Characterised by Excess lipid in Enterocytes and Fat
Malabsorption.

57.  The liver is a major site of cholesterol synthesis.
 Cholesterol and Triacylglycerols in excess of the liver's own
needs are exported into the blood in the form of very low
density lipoproteins.
 Triacylglycerols in very low density lipoproteins are hydrolyzed
by lipases on capillary surfaces.
 The resulting remnants, which are rich in cholesteryl esters, are
called intermediate -density lipoproteins.
 Half of them are taken up by the liver for processing, and half
are converted into low-density lipoprotein by the removal of
more triacylglycerol.

59.  Low-density lipoprotein is the major carrier of cholesterol in
blood
 The shell also contains a single copy of apo B-100 which
 Is recognized by target cells.
 They solubilize hydrophobic lipids
 The role of LDL is to transport cholesterol to peripheral
tissues and regulate de novo cholesterol synthesis at these
sites.
 The process of LDL uptake is Receptor-mediated
endocytosis.
Low-Density Lipoproteins Plays a Central
Role in Cholesterol Metabolism

60.  It contains a core of some 1500 cholesterol molecules
esterified to fatty acids most commonly linoleate.
 A shell of phospholipids and unesterified cholesterol
molecules surrounds this highly hydrophobic core.

61.  Apo B-100 on the surface of an LDL
particle binds to a specific receptor
protein on the plasma membrane of
nonhepatic cells.
 The receptors for LDL are localized
in specialized regions called coated
pits contain a specialized
protein called Clathrin.
 The receptor- LDL complex is
internalized by Endocytosis; that is,
the plasma membrane in the vicinity
of the complex invaginates and then
fuses to form an Endocytic vesicle.
Receptor-mediated endocytosis

62.  These vesicles, containing LDL, subsequently fuse with
Lysosomes, acidic vesicles that carry a wide array of degradative
enzymes.
 The protein component of LDL is hydrolyzed to free amino acids.
 The cholesteryl esters in LDL are hydrolyzed by a Lysosomal acid
Lipase.
 The LDL receptor itself usually returns unharmed to the plasma
membrane.

64.  The round-trip time for a receptor is about 10 minutes; in its lifetime
of about a day, it may bring many LDL particles into the cell.
 The released unesterified cholesterol can then be used for
membrane biosynthesis.
 Alternatively, it can be reesterified for storage inside the cell.
 In fact, free cholesterol activates acyl CoA:cholesterol
acyltransferase (ACAT), the enzyme catalyzing this reaction.

65.  Reesterified cholesterol contains mainly oleate and
palmitoleate,, in contrast with the cholesterol esters in LDL,
which are rich in linoleate, a polyunsaturated fatty acid.
 It is imperative that the cholesterol be reesterified.
 High concentrations of unesterified cholesterol disrupt the
integrity of cell membranes.

66.  The amino acid sequence of the human LDL receptor
reveals the mosaic structure of this 115-kd protein,839
Amino acids which is composed of five different types of
domains.
 The amino-terminal region of the mature receptor is the site
of LDL binding.
 Michael S. Brown and Joseph L. Goldstein were awarded the
1985 Nobel Prize for Physiology and Medicine for their
identification of the Low Density Lipoprotein (LDL)
Receptor[5] and its relation to Choletserol metabolism
and Familial Hypercholesterolemia.
The LDL Receptor Is a Transmembrane Protein
Having Five Different
Functional Regions

67.  It consists of a cysteine-rich sequence of about 40 residues that is
repeated, with some variation, seven times.
 A cluster of negatively charged side chains in this LDL-binding
domain interacts with a positively charged site on an apo B-100
molecule on the surface of an LDL particle.
 The third domain, which is very rich in
serine and threonine residues,
contains O-linked sugars.

68.  These oligosaccharides may function as struts to keep the
receptor extended from the membrane so that the LDL-
binding domain is accessible to LDL.
 The fourth type of domain consists of 22 hydrophobic
residues that span the plasma membrane.
 The fifth domain consists of 50 residues and emerges on the
cytoplasmic side of the membrane, where it controls the
interaction of the receptor with coated pits and participates in
endocytosis.

69.  In addition to the highly specific and regulated receptor-
mediated pathway for LDL uptake, macrophages possess
high levels of scavenger receptor activity known as
scavenger receptor class A (SR-A).
 Of particular concern is the oxidation of the excess blood
LDL to form oxidized LDL in which the lipid components or
apo B have been oxidized.
 The scavenger receptor is not down-regulated in response
to increased intracellular cholesterol.
Uptake of chemically modified LDL by
macrophage scavenger receptors

70. • The oxLDL when taken up macrophages become engorged to
form foam cells.
• These foam cells become trapped in the walls of the blood
vessels and contribute to the formation of atherosclerotic
plaques that cause arterial narrowing and lead to heart
attacks.

72. Excretion of cholesterol
 By conversion into bile acids and bile salts- excreted in the
feces.
 By secretion of cholesterol in bile- transported to intestine
for elimination.
 In the intestine cholesterol is converted by bacteria into
coprostanol and cholestanol before excretion.

73. Bile Salts
• Bile acids & salts are effective detergents.
• Synthesized in the liver.
• Stored & concentrated in the gallbladder.
• Discharged into gut and aides in absorption of intraluminal
lipids, cholesterol, & fat soluble vitamins.
• Bile acid refers to the protonated form while bile salts refers to
the ionized form
– The pH of the intestine is 7 and the pKa of bile salts is 6,
which means that 50% are protonated.

74. Synthesis of Bile Salts
• Rate-limiting step performed by the 7α-hydroxylase and is
regulated by bile salt concentration
• End product: Cholic acid & Chenocholic acid
Fig. 9 Fig. 10

76.  The primary bile acids enter the bile as glycine or taurine
conjugates.
 Conjugation takes place in peroxisomes.
 In humans, the ratio of the glycine to the taurine conjugates
is normally 3:1.
 In the alkaline bile, the bile acids and their conjugates are
assumed to be in a salt form—hence the term“bile salts.”
 A portion of the primary bile acids in the intestine is
subjected to further changes by the activity of the intestinal
bacteria.

78. • Products of fat digestion, including cholesterol absorbed
in the first 100 cm of small intestine, the primary and
secondary bile acids are absorbed almost exclusively in the
ileum, and 98–99% is returned to the liver via the portal
circulation.
• However, lithocholic acid, because of its insolubility, is not
reabsorbed to any significant extent.
• Only a small fraction of the bile salts escapes absorption and
is therefore eliminated in the feces.
Most Bile Acids Return to the Liver
in the Enterohepatic Circulation

80. Fate of Bile Salts
Fig. 12

81.  The activity of the enzyme is feedback-regulated via the nuclear
bile acid-binding receptor Farnesoid X receptor (FXR).
 When the size of the bile acid pool in the enterohepatic circulation
increases, FXR is activated and transcription of the cholesterol 7-
hydroxylase gene is suppressed.
 Chenodeoxycholic acid is particularly important .
 Also enhanced by cholesterol of endogenous and dietary origin
and regulated by insulin, glucagon, glucocorticoids, and thyroid
hormone.
Bile Acid Synthesis Is Regulated at the 7-α
Hydroxylase Step

82. • The movement of cholesterol from the liver
into the bile must be accompanied by the
simultaneous secretion of phospholipid and
bile salts.
• If this dual process is disrupted and more
cholesterol enters the bile than can be
solubilized by the bile salts and lecithin
present, the cholesterol may precipitate in
the gallbladder, initiating the occurrence of
cholesterol gallstone disease—cholelithiasis
Bile salt deficiency: Cholelithiasis

83. References:
 Lehninger’s Principles of Biochemistry- 5th Edition
 Biochemistry 7th Edition : Jeremy M berg,John L. Tymoczko,
Lubert Stryer
 Harper’s Illustrated Biochemistry- 28th Edition
 Lippincott’s Illustrated Reviews Biochemistry: Fifth Edition
 Teitz Textbook of Clinical Chemistry and Molecular Diagnostics-
Fifth Edition

84. Thank You