CHOLESTEROL: BIOSYNTHESIS,UTILIZATION,DETECTION

METHOD & CLINICAL SIGNIFICANCE

Cholesterol- Biochemical Importance

• Cholesterol is the major sterol in the animal tissues.
• Cholesterol is present in tissues and in plasma either as free cholesterol or as a storage form,
combined with a long-chain fatty acid as cholesteryl ester.
• In plasma, both forms are transported in lipoproteins.
• Plasma low-density lipoprotein (LDL) is the vehicle of uptake of cholesterol and cholesterol
into many tissues.
• Free cholesterol is removed from tissues by plasma high-density lipoprotein (HDL) and
transported to the liver, where it is eliminated from the body either unchanged or after
conversion to bile acids in the process known as reverse cholesterol transport.

Sources
• Cholesterol is derived from Diet De novo synthesis and From the hydrolysis of cholesteryl
esters.
• More than half of the cholesterol of the body arises by synthesis (about 700 mg/d), and the
remainder is provided by the average diet.
• The liver and intestine account for approximately 10% each of
total synthesis in humans.
• All tissues containing nucleated cells are capable of cholesterol synthesis, which occurs in the
endoplasmic reticulum and the cytosol.
❖ Biosynthesis of Cholesterol
➢ The biosynthesis of cholesterol may be divided into five steps:
■ Biosynthesis of mevalonate occurs from acetyl-CoA.
■ Formation of Isoprenoid units formed from mevalonate by loss of CO2.
■ Condensation of six isoprenoid units form squalene.
■ Cyclization of squalene gives rise to the parent steroid,lanosterol.
■ Formation of Cholesterol from lanosterol.

❖ Step1: Biosynthesis of Mevalonate

➢ The first stage in the synthesis of cholesterol is the formation of mevalonate from
acetyl CoA.
➢ This set of reactions takes place in the cytosol.
➢ Initially, two molecules of acetyl-CoA condense to form Acetoacetyl-CoA
catalyzed by cytosolic thiolase.

➢ Acetoacetyl-CoA condenses with a further molecule of acetyl-CoA catalyzed by

HMGCoA synthase to form HMG-CoA, that is reduced to mevalonate by
NADPH catalyzed by 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA
reductase
➢ The synthesis of mevalonate is the principal regulatory step in the pathway of
cholesterol synthesis.
➢ The enzyme catalyzing this irreversible step, (HMG-CoA reductase), is an
important control site in cholesterol biosynthesis,and is the site of action of the
most effective class of cholesterol lowering drugs, the HMG-CoA reductase
inhibitors (Statins).
➢ In stage 1, HMG-CoA, a C6 intermediate, is formed by sequential condensation
of 3 acetyl-CoA molecules. This cytoplasmic process follows the same reaction
sequence described for the biosynthesis of ketone bodies in mitochondria
➢ Next, HMG-CoA is converted to mevalonate via reduction by 2 NADPH
catalyzed by the microsomal enzyme, HMG-CoA reductase. This rate-limiting
step in cholesterol biosynthesis is inhibited by mevalonate, the immediate
product, by CH, the main product of the pathway, by certain fungal metabolites
(i.e., the cholesterol-lowering "statin" drugs), and by starvation.
➢ Glucagon and cortisol, hormones typically elevated during starvation, depress
hepatic CH biosynthesis,whereas insulin and thyroid hormones elevate it.
Opposing effects of insulin and glucagon in the liver are mediated by the
dephosphorylation and phosphorylation of HMG-CoA reductase, respectively,
whereas the effects of thyroxine and cortisol are mediated by the induction and
repression of HMG-CoA reductase biosynthesis. Although CH feeding decreases
hepatic CH biosynthesis, increased dietary carbohydrate or triglyceride intake
augments it primarily by increasing availability of acetyl-CoA and NADPH. The
pathways that supply these substrates from carbohydrate in the liver, particularly
glycolysis , the pyruvate dehydrogenase reaction and the NADPH-generating
steps of the hexose monophosphate shunt, are activated by insulin
 .

❖ Step2: Formation of Isoprenoid Units:

➢ Mevalonate is phosphorylated sequentially by ATP by three Kinases.
➢ After decarboxylation the active isoprenoid unit,isopentenyl diphosphate,is
formed.
➢ In stage 2, formation of activated isoprenoid units from mevalonate requires 3
ATP, and results in the loss of H2O and CO2. These isoprenoid units now become
building blocks not only of the basic steroid skeleton, but also of other
derivatives, such as dolichol and ubiquinone.
❖ Step3: Condensation of six isoprenoid units form squalene
➢ Squalene (C30) is synthesized from six molecules of Isopentenyl Pyrophosphate
(C5) and the reaction sequence is:C5 → C10 → C15 → C30
➢ Isopentenyl diphosphate is isomerized by a shift of the double bond to form
dimethylallyl diphosphate, then condensed with another molecule of isopentenyl
diphosphate to form the ten-carbon (C10) intermediate geranyl diphosphate.
➢ A further condensation with isopentenyl diphosphate forms farnesyl diphosphate.
➢ Two molecules of farnesyl diphosphate condense at the diphosphate end to form
squalene.
➢ Initially, inorganic pyrophosphate is eliminated, forming presqualene
diphosphate, which is then reduced by NADPH with elimination of a further
inorganic pyrophosphate molecule.
➢ The newly formed cyclized structure is Lanosterol.In stage 3, six isoprenoid units
condense in a series of 3 reactions to form the intermediate, squalene. NADPH is
required in the final condensation reaction. Plant sterols (like the phytoestrogens,
the auxins and gibberellins (steroid-like plant hormones), and the fat soluble
vitamins), are polyprenoids derived from mevalonate and/or squalene, but not
 CH.The CH biosynthetic scheme in plants seems to end at squalene. Plant sterols
differ from animal sterols in the nature of their side chains, and generally cannot
be utilized by animal cells. Less than 5% of dietary plant sterols are normally
absorbed by the mammalian digestive tract (compared to 30% of available CH).
The liver normally clears plant sterols efficiently from portal blood, and excretes
them into bile.

❖ Step4: Formation of Lanosterol

➢ Squalene can fold into a structure that closely resembles the steroid nucleus .
➢ Before ring closure occurs, squalene is converted to squalene 2,3-epoxide by a
mixed-function oxidase (i.e.) squalene epoxidase in the endoplasmic reticulum.
➢ The methyl group on C14 is transferred to C13 and that on C8 to C14 as
cyclization occurs, catalyzed by oxidosqualene: lanosterol cyclase.
➢ The newly formed cyclized structure is Lanosterol.
➢ In stage 4, squalene is first converted to an oxide, and then to lanosterol, which
possesses the C17 cyclopentanoperhydrophenanthrene nucleus common to all
animal steroids.Formation of CH from lanosterol takes place in the endoplasmic
reticulum, and involves changes in the steroid nucleus and side chain.Five steps
are thought to be involved, in which three methyl groups are released from the
ring as CO2, and a double bond in the side chain is reduced. The exact order in
which these steps occur is not known with certainty.Hepatic intermediates from
squalene to CH are attached to a sterol carrier protein, which binds sterols and
other insoluble lipids, allowing them to react in the soluble cell interior. This
protein is also thought to act in the conversion of CH to bile acids, and in the
formation of membranes and lipoproteins (e.g., VLDL and high-density
lipoprotein (HDL)).
➢ Following movement of CH into bile, and then into the intestine, coprostanol is
formed via bacterial reduction of the double bond of CH between C5 and C6.
Cholestanol, a precursor to coprostanol, is formed in the liver as well as in the
intestinal tract, and these two stanols,together with CH, normally become primary
fecal sterols.
❖ Step5: Formation of Cholesterol
➢ The formation of cholesterol from lanosterol takes place in the membranes of the
endoplasmic reticulum and involves changes in the steroid nucleus and side
chain .
➢ The methyl groups on C14 and C4 are removed to form 14- desmethyl lanosterol
and then zymosterol. The double bond at C8–C9 is subsequently moved to C5–C6
in two steps, forming desmosterol.
➢ Finally, the double bond of the side chain is reduced,producing cholesterol.

❖ CHOLESTEROL METABOLISM
➢ Cholesterol metabolism refers to a series of biochemical reactions that occur
when cholesterol is synthesized and broken down in the body.
❖ FUNCTION OF CHOLESTEROL METABOLISM
➢ Normal cholesterol metabolism keeps cholesterol levels in balance, ensuring the
survival and normal functioning of the organism.
❖ ANABOLISM OF CHOLESTEROL
➢ Cholesterol in the human body mainly comes from endogenous biosynthesis.
Cholesterol is primarily synthesized in the endoplasmic reticulum (liver) from
acetyl-CoA, one of the metabolites of glucose, fatty acids, and certain amino
acids. Endogenous synthetic cholesterol starts from a series of enzymatic
reactions in which acetyl-CoA is mediated by HMG-CoA reductase (HMGCR),
squalene synthase, squalene monooxygenase, lanosterol synthase, and farnesyl-
diphosphate synthase (FPPS).
 ➢ The cholesterol synthesis pathway is a multi-stage process.
➢ First, two molecules of acetyl-CoA are catalyzed to one molecule of acetoacetyl-
CoA by thiolase.
➢ Acetyl-CoA and acetoacetyl-CoA forms HMG-CoA in the action of HMG-CoA
synthetase.
➢ The HMG-CoA reductase catalyzes HMG-CoA to generate mevalonic acid
(MVA). The process is irreversible. HMG reductase is a rate-limiting enzyme for
cholesterol synthesis.
➢ Upon phosphorylation, decarboxylation, and dehydroxylation, MVA is condensed
to form squalene.
➢ Squalene produces lanosterol through the catalysis of endoplasmic reticulum
cyclase and oxygenase.
➢ Finally, lanosterol is converted to cholesterol through multiple redox reactions.
➢ In addition to the body's synthesis, cholesterol can also be absorbed from the
small intestine. The main sources of cholesterol in the intestine are food, bile and
shed intestinal epithelial cells. Triglycerides and phospholipids are gradually
broken down and digested after the lipids in food are processed by various
digestive enzymes in the small intestine, releasing free cholesterol. There is also a
large amount of free cholesterol in the bile, which is excreted into the bile by
sterol transporter ABCG5 or ABCG8 on the liver membrane. In humans, NPC1L1
(Niemann-pick type C1 like 1), a transmembrane protein, is also expressed on the
bile duct membrane and is responsible for reabsorbing cholesterol secreted into
bile by the liver.
❖ CATABOLISM OF CHOLESTEROL
➢ The catabolism of cholesterol is also mainly carried out in the liver. Cholesterol
cannot be completely oxidized and decomposed into CO2 and H2O in the body. It
is transformed into other compounds containing cyclopentane poly-hydro
phenanthrene parent nuclei through oxidation and reduction. And these
compounds continue to enter the metabolism process in vivo or are expelled from
the body.
 ➢ Cholesterol is an important component of the cell membrane in the body. Besides,
it can also be converted into a variety of substances with important physiological
functions. For example, cholesterol can be converted into adrenal cortical
hormones and sex hormones such as androgen, estrogen, and progesterone. In the
skin, cholesterol can be oxidized to 7-dehydrogenated cholesterol, which is often
converted to vitamin D3 by ultraviolet radiation. In the liver, cholesterol can be
oxidized into bile acids. And then bile acids flow into the duodenum where they
promote the digestion of lipids and the absorption of lipid-soluble vitamins.
➢ In addition to the body's conversion and use of cholesterol, some excess
cholesterol is catalyzed to form the cholesterol ester by acetyl cholesterol
transferase (ACAT) and then stored in the cell. Cholesterol ester transport was
mainly mediated by ABCA1 (ATP-binding cassette sub-family A) and ABCG1
(ATP-binding cassette sub-family G member1). Some other cholesterol is directly
secreted into the intestinal cavity. And partial cholesterol is assembled as high-
density lipoprotein, which later enters the liver for catabolism. In the lower part of
the small intestine, most bile acids are reabsorbed into the liver through the
hepatic circulation. The process contributes to the hepatic intestinal circulation of
bile. A small amount of bile acid is excreted by intestinal bacteria. Besides, the
liver can also drain cholesterol directly into the intestine. Cholesterol can also be
reduced to sterol fecal by intestinal bacteria. The sterol fecal subsequently is
transported out of the body.

❖ DETECTION OF CHOLESTROL
 ➢ A complete cholesterol test is also called a lipid panel or lipid profile. Your doctor
can use it to measure the amount of “good” and “bad” cholesterol and
triglycerides, a type of fat, in your blood.
➢ Cholesterol is a soft, waxy fat that your body needs to function properly.
However, too much cholesterol can lead to:
■ heart disease
■ Stroke
■ atherosclerosis, a clogging or hardening of your arteries

➢ If you’re a man, you should get your cholesterol levels checked regularly, starting by age
35 or younger. If you’re a woman, you should begin routine cholesterol screening by age
45 or younger. To be on the safe side, you may want to get your cholesterol tested every
five years beginning as early as age 20. If you’ve been diagnosed with diabetes, heart
disease, stroke, or high blood pressure, or if you’re taking medication to control your
cholesterol levels, you should check your cholesterol every year.

❖ CLINICAL SIGNIFICANCE OF CHOLESTEROL ESTIMATION

➢ Normal Serum Cholesterol levels=150-200 mg/dl (adult )
➢ Normal Serum Cholesterol levels in Newborn=100 mg /dl
➢ Women < men ( low level of serum estrogen low cholesterol)
➢ Estimation of Serum Cholesterol levels by Liebermann Bur chard reactions
CHOLESTEROL + ACETIC ANHYDRIDE -------H2SO4 GREEN COMPLEX
➢ TOTAL CHOLESTEROL = HDL LDL+VLDL
➢ TG /5= VLDL
➢ AFTER THE Precipitation of LDL & VLDL BY Polyethylene glycol (PEG )
➢ LDL CHOLESTEROL = Total cholesterol –( HDL + VLDL)
= Total cholesterol –( HDL + TG/5)
➢ LDL CHOLESTEROL=70-200 mg/dl
➢ Serum HDL CHOLESTEROL = 30-60 mg/dl(increase in HDL cholesterol is
beneficial /decrease in HDL harmful & leads to Coronary Heart Disease (CHD )
ATHEROSCLEROSIS
 ➢ Increase in Serum Cholesterol levels Coronary Heart Disease (CHD )
ATHEROSCLEROSIS.

❖ CLINICAL SIGNIFICANCE

A) HYPERCHOLESTEROLEMIA - Increase in plasma cholesterol (> 200 mg/dl)

concentration is known as hypercholesterolemia and is observed in many disorders.

1. DIABETES MELLITUS: Due to increased cholesterol synthesis since the

availability of acetyl CoA is increased.

2. HYPOTHYROIDISM: this is believed to be due to decrease in the HDL

RECEPTORS on hepatocytes .

3. OBSTRUCTIVE JAUNDICE: due to ab obstruction in the excretion of cholesterol

through bile

4. NEPHROTIC SYNDROME : increase in plasma globulin concentration is the

characteristics feature of nephrotic syndrome .
Hypercholesterolemia is associated with atherosclerosis and coronary heart disease.
Atherosclerosis is characterized by deposition of cholesteryl esters and other lipids in the
intima of the arterial walls often leading to hardening of coronary arteries and cerebral
blood vessels. A positive correlation between raised plasma lipids with atherosclerosis on
one h4nd and coronary heart disease on the other has been established. More specifically,
LDL-cholesterol is positively correlated, whereas HDL-cholesterol i s negatively
correlated with cardiovascular diseases.

❖ BAD AND GOOD CHOLESTEROL : Cholesterol is a natural metabolite performing a

wide range of functions (membrane structure, precursor for steroid hormones, bile acids).
The usages good and bad to cholesterol, although inappropriate, are still in use. The
cholesterol in high concentration, present in LDL, is considered bad due to its
involvement in atherosclerosis and related complications. Thus, LDL may be regarded as
lethally dangerous Lipoprotein.
SUBMITTED BY: KARAN KAMBOJ
ADMISSION NO.2017V137B-IIVER
ROLL NO.-BA-2088