Fundamentals of

Biomedical
Sciences III
DR . EKATERINE MIKAUTADZE
Biochemistry
Cholesterol Metabolism. Regulation of HMG-CoA reductase.

Conversion of Cholesterol to Cholic Acid and Chenocholic Acid. Fate of the Bile Salts. Blood

Lipoproteins – Chylomicrons, Very Low-Density Lipoproteins, Intermediate-Density Lipoproteins

and LowDensity Lipoproteins, High-Density Lipoproteins. Lipoprotein Receptors.

Clinical Correlation: Biochemical Aspects of Atherosclerosis.

- Marks’ basic medical biochemistry, Fourth edition, 2013 Lippincott

Williams & Wilkins, a Wolters Kluwer business. pp. 630-649.
I. INTESTINAL ABSORPTION OF
CHOLESTEROL
Cholesterol absorption by intestinal cells is a key regulatory point
in human sterol metabolism because it ultimately determines
what percentage of the 1,000 mg of biliary cholesterol produced
by the liver each day, and what percentage of the 300 mg of
dietary cholesterol entering the gut per day is eventually absorbed
into the blood. In normal subjects, approximately 55% of this
intestinal pool enters the blood through the enterocyte each day.
I. INTESTINAL ABSORPTION OF
CHOLESTEROL
Although the absorption of cholesterol from the intestinal lumen is a diffusion
controlled process, there is also a mechanism to remove unwanted or
excessive cholesterol and plant sterols from the enterocyte. The transport of
sterols out of the enterocyte and into the gut lumen is related to the products
of genes that code for the adenosine triphosphate (ATP)-binding cassette
(ABC) protein family, specifically ABCG5 and ABCG8. These proteins couple
ATP hydrolysis to the transport of unwanted or excessive cholesterol and
plant sterols (phytosterols) from the enterocyte back into the gut lumen.
Another member of the ABC family, ABCA1, is required for reverse cholesterol
transport and the biogenesis of HDL.
I. INTESTINAL ABSORPTION OF
CHOLESTEROL
Cholesterol cannot be metabolized to carbon dioxide and water and is,
therefore, eliminated from the body principally in the feces as unreabsorbed
sterols and bile acids. ABC protein expression increases the amount of sterols
present in the gut lumen, with the potential to increase elimination of the
sterols into the feces. Patients with a condition known as phytos terolemia (a
rare autosomal recessive disease, also known as sitosterolemia) have a defect
in the function of either ABCG5 or ABCG8 in the enterocytes, which leads to
accumulation of cholesterol and phytosterols within these cells. These
eventually reach the bloodstream, markedly elevating the level of cholesterol
and phytosterol in the blood. This accounts for the increased cardiovascular
morbidity in individuals with this disorder.
I. INTESTINAL ABSORPTION OF
CHOLESTEROL
From these experiments of nature, it is clear that agents that either amplify the
expression of the ABC proteins within enterocytes or that block choles terol
absorption from the lumen have therapeutic potential in the treatment of patients
with hypercholesterolemia. Ezetimibe is a compound that is structurally different
from the sterols. Its primary action in lowering serum cholesterol levels is to block
cholesterol absorption through a specific but as yet poorly characterized cholesterol
absorption mechanism in the brush border of enterocytes. The target of ezetimibe
is the Niemann–Pick C1-like 1 protein (NPC1L1), which is believed to transport
cholesterol into cells via an absorptive endocytotic mechanism, involving the
protein clathrin. The reduction of cholesterol absorption from the intestinal lumen
has been shown to reduce blood levels of LDL cholesterol, particularly when used
with a drug that also blocks endogenous cholesterol synthesis.
 II. CHOLESTEROL SYNTHESIS
Cholesterol is an alicyclic compound whose basic structure
includes the perhydrocyclopentanophenanthrene nucleus
containing four fused rings (Fig. 34.1).
In its “free” form, the cholesterol molecule contains 27
carbon atoms, a simple hydroxyl group at C3, a double
bond between C5 and C6, an eight-membered hydrocarbon
chain attached to carbon 17 in the D-ring, a methyl group
(carbon 19) attached to carbon 10, and a second methyl
group (carbon 18) attached to carbon 13 (Fig. 34.2).
 II. CHOLESTEROL SYNTHESIS
Approximately one-third of plasma cholesterol exists in the
free (or unesterified) form. The remaining two-thirds exist
as cholesterol esters in which a long chain fatty acid
(usually linoleic acid) is attached by ester linkage to the
hydroxyl group at C3 of the A-ring. The proportions of free
and esterified cholesterol in the blood can be measured
using methods such as high-performance liquid
chromatography (HPLC).
 II. CHOLESTEROL SYNTHESIS
The structure of cholesterol suggests that its synthesis involves
multimolecular interactions and significant reducing power. All 27
carbons are derived from one precursor, acetyl-CoA. AcetylCoA
can be obtained from several sources, including the oxidation of
fatty acids, the oxidation of ketogenic amino acids such as leucine
and lysine, and the pyruvate dehydrogenase reaction. The
reducing power for cholesterol synthesis is supplied in the form
of NADPH. The latter is provided by glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase of the
hexose monophosphate shunt pathway. Cholesterol synthesis
occurs in the cytosol, requiring hydrolysis of high-energy
thioester bonds of acetyl-CoA and phosphoanhydride bonds of
ATP. Its synthesis occurs in four stages.
 II. CHOLESTEROL SYNTHESIS
A. Stage 1: Synthesis of Mevalonate from Acetyl-CoA
The first stage of cholesterol synthesis leads to the production
of the intermediate mevalonate (Fig. 34.3). The synthesis of
mevalonate is the committed, rate-limiting step in cholesterol
formation. In this cytoplasmic pathway, two molecules of
acetyl CoA condense, forming acetoacetyl-CoA, which then
condenses with a third molecule of acetyl-CoA to yield the six-
carbon compound β-hydroxy-β-methylglutaryl-CoA (HMG-
CoA). The HMG-CoA synthase in this reaction is present in the
cytosol and is distinct from the mitochondrial HMG-CoA
synthase that catalyses HMG-CoA synthesis involved in
production of ketone bodies.
 II. CHOLESTEROL SYNTHESIS
A. Stage 1: Synthesis of Mevalonate from Acetyl-CoA
The committed step and major point of regulation of
cholesterol synthesis in stage 1 involves reduction of HMG-CoA
to mevalonate, a reaction that is catalyzed by HMG-CoA
reductase, an enzyme embedded in the membrane of the
endoplasmic reticulum. HMG-CoA reductase contains eight
membrane-spanning domains, and the amino-terminal
domain, which faces the cytoplasm, contains the enzymatic
activity. The reducing equivalents for this reaction are donated
by two molecules of NADPH. The regulation of the activity of
HMG CoA reductase is controlled in multiple ways.
II. CHOLESTEROL SYNTHESIS
1. TRANSCRIPTIONAL CONTROL

The rate of synthesis of HMG-CoA reductase messenger RNA (mRNA) is con trolled
by one of the family of sterol-regulatory element-binding proteins (SREBPs) (Fig.
34.4A). These transcription factors belong to the basic helix–loop–helix leucine
zipper (bHLH-Zip) family of transcription factors that directly activate the
expression of more than 30 genes dedicated to the synthesis and uptake of
cholesterol, fatty acids, triacylglycerols, and phospholipids as well as the
production of the NADPH cofactors required to synthesize these molecules.
II. CHOLESTEROL SYNTHESIS
II. CHOLESTEROL SYNTHESIS
1. TRANSCRIPTIONAL CONTROL
SREBPs specifically enhance transcription of the HMG-CoA reductase gene by
binding to the sterol-regulatory element (SRE) upstream of the gene. When bound,
the rate of transcription is increased. SREBPs, after synthesis, are integral proteins of
the endoplasmic reticulum (ER). The SREBP is bound to SCAP (SREBP cleavage-
activating protein) in the ER membrane when cholesterol levels are high.
II. CHOLESTEROL SYNTHESIS
1. TRANSCRIPTIONAL CONTROL
When cholesterol levels drop, the sterol leaves its SCAP-binding site, and the
SREBP:SCAP complex is transported to the Golgi apparatus. Within the Golgi, two
proteolytic cleavages occur (via the site 1 [S1P] and site 2 [S2P] proteases), which
release the N-terminal transcription factor domain from the Golgi membrane. Once
released, the active amino terminal component travels to the nucleus to bind to
SREs. The soluble SREBPs are rapidly turned over and need to be continuously
produced to stimulate reductase mRNA transcription effectively. When cytoplasmic
sterol levels rise, the sterols bind to SCAP and prevent translocation of the complex
to the Golgi, leading to a decrease in transcription of the reductase gene and thus
less reductase protein being produced.
 II. CHOLESTEROL SYNTHESIS
2. PROTEOLYTIC DEGRADATION OF HMG-COA REDUCTASE
Rising levels of cholesterol and bile salts in cells that synthesize
these molecules also may cause a change in the oligomerization
state of the membrane domain of HMG-CoA reductase,
rendering the enzyme more susceptible to proteolysis (see Fig.
34.4B). This, in turn, decreases its activity. The membrane
domains of HMG-CoA reductase contain sterol-sensing regions,
which are similar to those in SCAP.
 II. CHOLESTEROL SYNTHESIS
3. REGULATION BY COVALENT MODIFICATION
In addition to the inductive and repressive influences we have
mentioned, the activity of the reductase is also regulated by
phosphorylation and dephosphorylation (see Fig. 34.4C).
Elevated glucagon levels increase phosphorylation of the
enzyme, thereby inactivating it, whereas hyperinsulinemia
increases the activity of the reductase by activating
phosphatases, which dephosphorylate the reductase. Increased
levels of intracellular sterols also may increase phosphorylation
of HMG-CoA reductase, thereby reducing its activity as well
(feedback suppression). Thyroid hormone also increases
enzyme activity, whereas glucocorticoids decrease its activity.
 II. CHOLESTEROL SYNTHESIS
3. REGULATION BY COVALENT MODIFICATION
The enzyme that phosphorylates HMG-CoA reductase is the
adenosine monophos phate (AMP)-activated protein kinase,
which itself is regulated by phosphorylation by one of several
AMP-activated protein kinase kinases (one of which is LKB1,
described further in the following text). Thus, cholesterol
synthesis decreases when ATP levels are low and increases
when ATP levels are high, similar to what occurs with fatty acid
synthesis (recall that acetyl-CoA carboxylase is also
phosphorylated and inhibited by the AMP-activated protein
kinase). The need for ATP in cholesterol biosynthesis will be
evident as the further reactions of the pathway are discussed.
 II. CHOLESTEROL SYNTHESIS
B. Stage 2: Conversion of Mevalonate to Two Activated
Isoprenes
In the second stage of cholesterol synthesis, three phosphate
groups are transferred from three molecules of ATP to
mevalonate (Fig. 34.5).
The purpose of these phosphate transfers is to activate both
carbon 5 and the hydroxyl group on carbon 3 for further
reactions in which these groups will participate. The phosphate
group attached to the C3 hydroxyl group of mevalonate in the 3-
phospho-5-pyrophosphomevalonate intermediate is removed
along with the carboxyl group on C1.
 II. CHOLESTEROL SYNTHESIS
B. Stage 2: Conversion of Mevalonate to Two
Activated Isoprenes
This produces a double bond in the five-carbon
product, Δ3-isopentenyl pyrophosphate, the first of
two activated isoprenes that are necessary for the
synthesis of cholesterol. The second activated isoprene
is formed when Δ3-isopentenyl pyrophosphate is
isomerized to dimethylallyl pyrophosphate (see Fig.
34.5). Isoprenes, in addition to being used for
cholesterol biosynthesis, are also used in the synthesis
of coenzyme Q and dolichol.
 II. CHOLESTEROL SYNTHESIS
C. Stage 3: Condensation of Six Activated Five-Carbon
Isoprenes to Form the 30-Carbon Squalene
The next stage in the biosynthesis of cholesterol
involves the head-to-tail condensation of isopentenyl
pyrophosphate and dimethylallyl pyrophosphate. The
“head” in this case refers to the end of the molecule to
which pyrophosphate is linked. In this reaction, the
pyrophosphate group of dimethylallyl pyrophosphate
is displaced, and a 10-carbon chain, known as geranyl
pyrophosphate, is generated (Fig. 34.6).
 II. CHOLESTEROL SYNTHESIS
C. Stage 3: Condensation of Six Activated Five-Carbon
Isoprenes to Form the 30-Carbon Squalene
Geranyl pyrophosphate then undergoes another head-
to-tail condensation with isopentenyl pyrophosphate,
resulting in the formation of the 15-carbon
intermediate, farnesyl pyrophosphate. After this, two
molecules of farnesyl pyrophosphate undergo a head-
to-head fusion, and both pyrophosphate groups are
removed to form squalene, a compound that was first
isolated from the liver of sharks (genus Squalus).
Squalene contains 30 carbons (24 in the main chain
and 6 in the methyl group branches; see Fig. 34.6).
 II. CHOLESTEROL SYNTHESIS
C. Stage 3: Condensation of Six Activated Five-Carbon
Isoprenes to Form the 30-Carbon Squalene
Geranyl pyrophosphate and farnesyl pyrophosphate
are key components in cholesterol biosynthesis, and
both farnesyl and geranyl groups can form covalent
bonds with proteins, particularly the G proteins and
certain proto-oncogene products involved in signal
transduction. These hydrophobic groups anchor the
proteins in the cell membrane.
 II. CHOLESTEROL SYNTHESIS
D. Stage 4: Conversion of Squalene to the Four-Ring
Steroid Nucleus
The enzyme squalene monooxygenase adds a single oxygen atom
from O2 to the end of the squalene molecule, forming an epoxide.
NADPH then reduces the other oxygen atom of O2 to H2O. The
unsaturated carbons of the squalene 2,3-epoxide are aligned in a
way that allows conversion of the linear squalene epoxide into a
cyclic structure. The cyclization leads to the formation of
lanosterol, a sterol with the four ring structure characteristic of
the steroid nucleus. A series of complex reactions, containing
many steps and elucidated in the late 1950s, leads to the
formation of cholesterol (Fig. 34.7).
III. SEVERAL FATES OF
CHOLESTEROL
Almost all mammalian cells are capable of producing cholesterol.
Most of the biosynthesis of cholesterol occurs within liver cells,
although the gut, the adrenal cortex, and the gonads (as well as
the placenta in pregnant women) also produce significant
quantities of the sterol. A fraction of hepatic cholesterol is used
for the synthesis of hepatic membranes, but the bulk of
synthesized cholesterol is secreted from the hepatocyte as one of
three moieties: cholesterol esters, biliary cholesterol (cholesterol
found in the bile), or bile acids. Cholesterol ester production in
the liver is catalyzed by acyl-CoA–cholesterol acyl transferase
(ACAT). ACAT catalyzes the transfer of a fatty acid from coenzyme
A to the hydroxyl group on carbon 3 of cholesterol (Fig. 34.8).
III. SEVERAL FATES OF
CHOLESTEROL
Cholesterol esters are more hydrophobic than is free cholesterol.
The liver packages some of the esterified cholesterol into the
hollow core of lipoproteins, primarily VLDL. VLDL is secreted from
the hepatocyte into the blood and transports the cholesterol
esters (triacylglycerols, phospholipids, apoproteins, etc.) to the
tissues that require greater amounts of cholesterol than they can
synthesize de novo. These tissues then use the cholesterol for the
synthesis of membranes, for the formation of steroid hormones,
and for the biosynthesis of vitamin D. The residual cholesterol
esters not used in these ways are stored in the liver for later use.
III. SEVERAL FATES OF
CHOLESTEROL
The hepatic cholesterol pool serves as a source of cholesterol
for the synthesis of the relatively hydrophilic bile acids and
their salts. These derivatives of cholesterol are very effective
detergents because they contain both polar and nonpolar
regions. They are introduced into the biliary ducts of the liver.
They are stored and concentrated in the gallbladder and later
discharged into the gut in response to the ingestion of food.
They aid in the digestion of intraluminal lipids by forming
micelles with them, which increases the surface area of lipids
exposed to the digestive action of intraluminal lipases.
III. SEVERAL FATES OF
CHOLESTEROL
Free cholesterol also enters the gut lumen via the biliary tract
(approximately 1,000 mg daily, which mixes with 300 mg of
dietary cholesterol to form an intestinal pool, roughly 55% of
which is resorbed by the enterocytes and enters the
bloodstream daily). On a low-cholesterol diet, the liver
synthesizes approximately 800 mg of cholesterol per day to
replace bile salts and cholesterol lost from the enterohepatic
circulation into the feces. Conversely, a greater intake of dietary
cholesterol suppresses the rate of hepatic cholesterol synthesis
(feedback repression).
 IV. SYNTHESIS OF BILE SALTS
A. Conversion of Cholesterol to Cholic Acid and
Chenocholic Acid
Bile salts are synthesized in the liver from
cholesterol by reactions that hydroxylate the
steroid nucleus and cleave the side chain. In the
first and rate-limiting reaction, an -hydroxyl group
is added to carbon 7 (on the side of the B-ring).
The activity of the 7-α-hydroxylase that catalyzes
this step is decreased by an increase in bile salt
concentration (Fig. 34.9).
 IV. SYNTHESIS OF BILE SALTS
A. Conversion of Cholesterol to Cholic Acid and
Chenocholic Acid
In subsequent steps, the double bond in the B-ring
is reduced, and an additional hydroxylation may
occur. Two different sets of compounds are
produced. One set has α-hydroxyl groups at
positions 3, 7, and 12 and produces the cholic acid
series of bile salts. The other set has α-hydroxyl
groups only at positions 3 and 7 and produces the
chenodeoxycholic acid series (also known as the
chenocholic acid series, Fig. 34.10).
 IV. SYNTHESIS OF BILE SALTS
A. Conversion of Cholesterol to Cholic Acid and
Chenocholic Acid
Three carbons are removed from the side chain by an
oxidation reaction. The remaining five-carbon
fragment attached to the ring structure contains a
carboxyl group (see Fig. 34.10). The pKa of the bile
acids is approximately 6. Therefore, in the contents of
the intestinal lumen, which normally have a pH of 6,
approximately 50% of the molecules are present in
the protonated form and 50% are ionized, which form
bile salts. (The terms bile acids and bile salts are often
used interchangeably, but bile salts actually refers to
the ionized form of the molecules.)
 IV. SYNTHESIS OF BILE SALTS
B. Conjugation of Bile Salts
The carboxyl group at the end of the side chain
of the bile salts is activated by a reaction that
requires ATP and coenzyme A.

The CoA derivatives can react with either

glycine or taurine (which is derived from
cysteine), forming amides that are known as
conjugated bile salts (Fig. 34.11).
IV. SYNTHESIS OF BILE SALTS
 IV. SYNTHESIS OF BILE SALTS
B. Conjugation of Bile Salts
In glycocholic acid and glycocheno deoxycholic acid, the bile acids are conjugated with
glycine. These compounds have a pKa of approximately 4, so compared to their unconjugated
forms, a higher percentage of the molecules is present in the ionized form at the pH of the
intestine. The taurine conjugates, taurocholic and taurochenodeoxycholic acid, all have a pKa
of approximately 2.
Therefore, compared with the
glycoconjugates, an even greater
percentage of the molecules of
these conjugates are ionized in the
lumen of the gut.
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