.

Paper : 03 Structure and Function of Biomolecules -II

Module : 23 Steroids

Principal Investigator Dr. Sunil Kumar Khare,Professor

Dept. of Chemistry,
I.I.T. Delhi

Dr. Sunil Kumar Khare,Professor

Paper Co-ordinator
Dept. of Chemistry,
I.I.T. Delhi

Dr. M.N.Gupta, Emeritus Professor

Dept. of Biochemical Engg. and
Biotechnology, I.I.T. Delhi

Dr. M.N.Gupta, Emeritus Professor

Content Writer Dept. of Biochemical Engg. and
Biotechnology, I.I.T. Delhi

Dr. Prashant Mishra, Professor

Content Reviewer
Dept. of Biochemical Engg. and
Biotechnology, I.I.T. Delhi 1

Structure and Function of Biomolecules II

Biochemistry
Steroids
 Description of Module

Subject Name Biochemistry

Paper Name Structure and Function of Biomolecules II

Module Name/Title Steroids

Dr. Vijaya Khader

Dr. MC Varadaraj

Structure and Function of Biomolecules II

Biochemistry
Steroids
 1. Objectives

 To learn about cholesterol‟s structure, precursor and its role in biomolecules.

 To learn about various sex hormones.
 To understand the role of corticoid hormones.
 To learn about bile salts and their role in fat digestion.
 To understand how steroid hormones alter gene expression.

2. Concept Map

Structure and Function of Biomolecules II

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 3. Description

Steroids are structurally related and widely distributed compounds in animals and plants. Apart
from sterols (which includes cholesterol), vitamin D, bile acids, adrenal cortex hormones, the so
called sex hormones, sapogenins are included in the list.
At one time, a steroid was defined as any compound which gives Diel‟s hydrocarbon when
distilled along with selenium at 360 oC. At higher temperature of 420 oC, chrysene and picene are
obtained.

One way of classifying lipids is into two classes of saponifiable and nonsaponifiable. Most of the
lipids are saponifiable.
Saponifiable lipids upon hydrolysis by alkali give rise to products which become water soluble.
More strictly speaking, at least the products become more water soluble than reactants.
It was Chevreul who in 1812 observed that all lipids are not saponifiable. Colloid and Young in
1936 suggested to Royal Society of Chemistry, UK that compounds related to cholesterol should be
called steroids.
Earlier, the word sterol existed for crystalline alcohols (Greek Stereos: solid). Cholesterol was
recognized as a C27 sterol. Sterols were known to be unsaponifiable or non saponifiable lipids.

Cholesterol

Thus, cholesterol the most widely known steroid is actually responsible for this class of compounds
named after it. The cholesterol structure determination involved some well known scientists of that
era: Wieland, Windans, Diels, Rosenheim and King.
The fundamental C-skeleton in cholesterol is cyclopentanoperhydrophenanthrene.
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 Figure 1: Cholesterol structure

Quite a few steroids are synthesized by the mammals and this includes cholesterol. In fact in
humans, cholesterol is the steroid which is present in largest amount.
Human blood plasma contains total cholesterol of about 200 mg/ 100ml, about 25% of this as free
cholesterol. Almost all cholesterol, free and its esters are present as lipoproteins.
A standard test for cholesterol is Liebermann-Burchard reaction. In this test, acetic anhydride in
CHCl3 solution in added to cholesterol sample in conc. H2SO4. Formation of a green colour
indicates presence of cholesterol.
In fact, cholesterol and other sterols as well give this and few other tests.
Addition of conc. H2SO4 itself to cholesterol solution in CHCl3 turns the CHCl3 layer red. This is
called Salkowski reaction.

Structure and Function of Biomolecules II

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 Cholesterol can be estebilized in the form of its Digitonide which is formed when a saponin,
digitonin‟s ethanolic solution is added to its ethanolic solution. Cholesterol digitonide is a
molecular complex with 1 molecule of each and forms white precipitate.
It is curious that, the biosynthesis of this compound with a complex ring system is solely from a
single source for its carbon atoms. That precursor is acetate. The isoprene is a key intermediate in
this pathway.

Figure 2: Isoprene

In the 1940, Bloch fed isotopically labeled acetate and found that both carbons were part of
cholesterol molecules in liver tissue slices.

Structure and Function of Biomolecules II

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 Figure 3

The incorporation of methyl group and carboxyl group carbons of acetate was treated in the
synthesized cholesterol molecule and is indicated as „m‟ and „c‟ respectively.
This was one of the early triumphs of use of radioactive labels in determining biosynthetic and
catabolic pathways of biological important molecules. Before the simple precursors with
radioactive labels were available, scientists did not have this approach available.
Establishing the entire biosynthetic pathway involved extensive efforts by Bloch, Cornforth,
Popjak, Lynen. The importance of steroids was recognized by the industry very early. So, Foekev‟s
group at Merck also made some key contributions.
The determination of this pathway paved the way for many other important pathways as much of
the pathway is common to those pathways.

Structure and Function of Biomolecules II

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Steroids
 The common perceptions about “bad cholesterol” and “good cholesterol” are based upon over
simplified communications about the medical community. Cholesterol is same molecule in both
cases. It is a case of being judged by the company one keeps!
Cholesterol, like most of the lipids, moves between various tissues/ organs in the bound form as
part of lipoproteins. Lipoproteins are complexs of carrier apolipoproteins with cholesterol (and its
esters, phospholipids and triglycerides).
LDL (Low density lipoprotein) has highest % concentration of cholesterol + cholesterol esters and
is responsible for cholesterol associated with it to be termed as bad cholesterol.
Higher systems avoid biosynthesis of complex molecules like cholesterol. Cholesterol is a very
necessary molecule for several reasons. So, if enough cholesterol is available for its use in
membranes, bile salts, steroid hormones etc, activity of an enzyme HMGCoA reductase (the rate
limiting step in the biosynthetic pathway) is slowed down.
Most of the “cholesterol level lowering” drugs are designed to inhibit the activity of HMGCoA
reductase. The intermediate HMGCoA is also common precursor for some other compounds.

Structure and Function of Biomolecules II

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 If cholesterol biosynthesis regulation is defective, serious consequences ensure. LDL is high and
during transport it deposits cholesterol in blood vessels as arthosclerotic plaques. This causes
difficulties in blood flow and condition is known as atherosclerosis.
HDL (bound cholesterol) represents good cholesterol. HDL as it travels through in blood can pick
up additional cholesterol. Atherosclerosis correlates positively with high levels of LDL and
negatively with high levels of HDL in blood. Artherosclerosis leads to heart failure, as we
commonly call it. The cause is obstruction of blood flow in coronary arteries.
On the other hand, familial hypercholestemia is a human genetic disorder. Associated with high
levels of cholesterol in blood, it leads to severe atherosclerosis in childhood itself. The defect is in
the LDL receptor which mediates cholesterol uptake by the cells. As cholesterol is not taken up by
the cells, the cholesterol synthesis continues. In this case also, inhibitors of HMGCoA reductase
like Isovastatin, simvastatin, pravastatin and compactin are used in therapy. These drugs are
commonly referred to as statins.

Figure 4: Inhibitors of HMG-CoA synthase

Structure and Function of Biomolecules II

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 Cholesterol was initially isolated from human gall stones which mostly consist of this steroid. The
natural sources of cholesterol are fish liver oil, brain and spinal cords of animals. Wool,
essentially a protein, does contain some fat called lanoline. Lanoline is a mixture of palmitate,
stearate and oleate esters of cholesterol.
Sterols (the steroids with an alcohol group and the general class to which cholesterol belongs)
occur in animal and plant oils and fats. During the early days, sterols obtained from animal and
plant sources were called zoosterols and phytosterols. If isolated from yeast and fungi, these were
classified as mycosterols.
Thus, cholesterol, cholestanol and coprostanol were called zoosterols. Once the structure of
steroids and their characterization was carried out, it was realized that same compound could
occur in many kingdoms. So, this classification lost its meaning.
Sir R. Robinson and Woodward synthesized cholesterol chemically. Apart from its complex
structure, the challenge was the presence of light asymmetric C-atoms which can give rise to 256
optical isomers.
Bernal‟s work with x-ray diffraction showed steroid molecules to be flat. It also showed that H-
atom at C9 is trans to the group at C10.
Some steroids have allo- as a prefix in their names. Bernal‟s work established the way rings are
fused in steroids.

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 Figure 5

The compounds related to cholestane are referred to as allo- compounds. Those related to
coprostane were called normal compounds. For the sake of simplicity, normal prefix is dropped
and absence of any prefix means it is a “normal” steroid.
Callow‟s group (1936) used optical rotations to characterize steroids. It was found that all steroids
with a double bond at 5:6 position are laevorotatory.
Cholesterol is a white crystalline compound with a melting point of 149 oC and is laevorotatory. It
can be easily isolated from yellow portion of hen egg yolk. Many a times, nuts like almonds and
plant oils are described as “cholesterol free”. Plants do not contain cholesterol. So, such
advertisements are actually misleading.

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 The plasma membranes are rich in cholesterol. Cholesterol plays a major role in controlling the
fluidity of these membranes. It increases fluidity by disturbing the regular array of fatty acyl chains
in the membranes. At the same time, cholesterol prevents large movement of those chains. This
„Ying and Yang‟ effect of cholesterol makes it very important molecule in eukaryotes. Hence, to
perceive this as a “bad molecule” from health perspective is not correct.
Cholesterol is well suited to be a compound of biomembranes as it is an amphipathic molecule.
Brown and Goldstein in their Nobel lecture in 1985 described cholesterol as the “most highly
decorated small molecule in biology. Thirteen nobel prizes have been awarded to scientists who
devoted major parts of their carrier to cholesterol” Brown and Goldstein were given nobel prize for
establishing LDL pathway.

Bile salts

The metabolism of dietary lipids present a challenge as lipids are insoluble in water rich milieu of
digestive tract of animals. The problem is solved by dispersing lipid in emulsions.
Bile salts are more polar than cholesterol. This structural design makes these the designated
amphipathic molecules for emulsifying the lipid molecules.
Emulsions not only make lipids accessible to water soluble enzymes, these also increase the surface
area of the substrate lipids. This increased surface area also promotes intestinal absorption.

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 Figure 6: Synthesis of glycocholate, the major bile salt

Liver is the site of synthesis for bile salts. From there these molecules reach gall bladder and are
stored there. When needed, gall bladder releases bile salts in the small intestine. It is here that bile
salts emulsify dietary lipids like fats/oils.
Cholesterol itself is made in the liver. While small % of it is incorporated as components of the
membranes of hepatocytes. The other fates of cholesterol are export from the liver as biliary
cholesterol, bile acids and cholesterol esters. Thus, cholesterol is the precursor of bile acids.
Cholyl CoA is the intermediate in synthesis of many bile salts. Glycocholate, the product of
reaction of cholyl CoA with glycine is the most important bile salt. Tauro-cholate, another bile salt
is the product of cholyl CoA with taurine (NH2-CH2-SO3-).

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 Steroid hormones

Figure 7: Biosynthetic relations of steroid hormones

Hormones are secretions of ductless glands. Pituitary hormones are polypeptides. So is well known
hormone insulin which is secreted from islets of Langerhans in pancreases. Adrenal hormones
adrenalin is a catecholamine.
Gonads (testes in males, ovaries in the females) produce so called sex hormones which are steroids
in nature. Their activity is controlled by some pituitary hormones produced by anterior lobe of that
master gland. So, these hormones (Polypeptides) are called primary sex hormones, the gonad
hormones are called secondary sex hormones.

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 These hormones are called sex hormones as these are involved in the sexual processes and gender
determination.
The sex hormones are of three types:
 Androgens (male hormone)
 Oestrogens (female or follicular hormone)
 Progesterone (Corpus luteum hormone)
Cholesterol is the precursor of these and other steroid hormones:

The steroid hormones are hence either adrenocortical hormones or sex hormones. These are
synthesized in different endocrine tissues and reach the target cells through blood. The word
endocrine is desired from two Greek words: endon: within, krinein: to release.
This distinguishes these from paracrine (which affect neighboring cells) hormones and autocrine
(affects the same cell) hormones. All steroid hormones have nuclear receptors. Binding of
hormones to these receptors alters the gene expression.

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 Androgens

Figure 8

The word Androgens is derived from the Greek word „Andros‟ for male. Two important steroid
male hormones (Androgens) are testosterone and dihydro-testosterone. Dihydrotestosterone is
formed in prostatic tissue by reduction of testosterone by 5α-reductase.

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 The other hormones with androgenic activity are Adrenosterone, Androst-4-ene-3,17-dione, 11β-
Hydroxyandrost-4-ene-3,17-dione, dehydroepiandrosterone and 17α-Hydroxyprogesterone.
Castration of young animals results in failure of development of sex characteristics. In human
males, castration prior to puberty, inhibits ossification of long bones and reduces the ultimate
height as well.
Male sex hormones and female sex hormones have antagonistic physiological effects. The first
androgens isolated were androsterone and dehydroisoandrosterone by Butenandt from male urine.
Testosterone was first isolated in pure form from testes of bull.
Cholesterol is converted to androgens in testis, ovary, adrenals and placenta. Catabolic products of
the androgens have been isolated both from tissues and urine.

Estrogens

Involvement of hormone in controlling the uterine cycle was known very early. It was in 1929 that
Butenandt and Doisy independently isolated estrone from urines of pregnant women. Thereafter
two more female sex hormones Estriol and estradiol were isolated.
Esterogens have been isolated from adrenal glands, testes, placenta and urine. Secretion of these
hormones by the follicles of the ovary results in development of sex organs and other secondary
gender characteristics like hair distribution and texture, skin texture, voice and fat distribution. In
the absence of estrogens human female menstrual cycle is disturbed.
The estrogens are derived from androgens in liver and other steroid producing tissues. The
esterogens and their catabolites are excreted in urine as conjugates of glucuronic acid and sulfuric
acid.

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 Figure 9

While natural estrogens are active when given parenterally, synthetic estrogens 17α-
ethynylestradiol and stilbestrol can be given orally.

Figure 10: Synthesis of androgens and estrogens

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 Androgens are precursors of estrogens (estrone and estradiole) which are formed by aromatization
of a ring and loss of a –Me group. These enzymatic reactions depend upon both NADPH + O2.

Progesterones

Figure 11: Synthesis of progesterone and corticoids

As we saw, the androgens in turn are synthesized from progesterone. In fact, cholesterol is first
converted to pregnanolone. Pregnanolone in turn produces progesterone.
Cholesterol, a C27 steroid loses a C6 side chain to form pregnanolone. The initial reaction is
hydroxylation at C-20 and C-22. The next reaction is cleavage of the bond between C-20 and C-22.

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 This is catalysed by an enzyme desmolase. All the reactions are again dependent upon NADPH +
O2.
Pregnanolone is the precursor of all steroid hormones. So, its formation is a critical step in steroid
biochemistry. This is regulated by adenocorticotrophic hormone (ACTH), also called
corticotrophin. ACTH is synthesized by the anterior pituitary gland.

The conversion of pregnanolone to progesterone is a two step process. First is the oxidation of 3-
OH to a 3 keto group. The second is shifting of the double bond from 5 positions to 3.
Progesteron is also the precursor of cortisol and aldosterone which is a major glucocorticoid.
These conversions require enzyme catalysed hydroxylation reactions. Conversion to aldosterone
also requires oxidation of C-18 methyl group to –CHO group.
The deficiency of 21-hydroxylase (involved in the synthesis of glucocorticoids and
mineralocorticoids) constitutes most common inherited disorder. High levels of androgens results
in accelerated growth and short stature in males, in females, masculinization of external genital
organs may result.

Corticoids

The adrenal cortex produces corticoid steroid hormones. These are called mineralocorticoids
(example. Aldosterone) or glucocorticoids (Cortisone or hydroxycortisone). These have multiple
physiological effects.
Production of glucocorticoids is controlled by ACTH, a hormone of anterior pituitary. Decreased
production of glucocorticoid due to deficiency of 21-hydroxylase makes ACTH secretion go up via
feedback loop. Adrenal glands enlarge.

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 High ACTH level increases conversion of cholesterol to pregnanolone. In turn, levels of
progesterone and 17-hydroxy progesterone go up. The latter is converted to higher amount of
androgens.
Aldosterone‟s primary sphere of influence is electrolyte and water metabolism. Cortisol
administration results in increased glycogenesis and gluconeogenesis from amino acids. More
glycogen is stored in liver and peripheral utilization of glucose diminisher.
To a varying degree, adrenal steroids: cortisol, corticosterone and aldosterone influences multiple
biochemical processes such as carbohydrate, protein and lipid metabolism and inflammatory and
allergic processes.
A significant percentage (~30%) of humans deficient in 21-hydroxylase lose Na+ in urine
continuously. The adrenal steroids regulate the concentration of Na+ and K+ in extracellular fluids.
Aldosterone and deoxycorticosterone are known to result in increased reabsorption of Na+ , Cl-,
HCO3- in the kidney distal tubules, sweat glands, salivary glands and gastrointestinal mucosa.
Excess hormonal concentration causes rise in extracellular Na+ concentration and expansion of
extracellular volume and decline in K+ concentration in serum. Elevation of blood pressure and
deleterious effects on cardiac muscle activity are clinical manifestation.

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 Figure 12: Model of thyroxime

Signal transduction mechanisms of hormones follow diverse mechanism. This is understandable in

view of their diverse structure and physiological functions. Many hormones bind to cell surface
receptor to initiate signal transduction.

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 Steroid hormones, on the other hand directly affect gene expression. Hence, their target is cell
nucleus. A critical difference between steroid hormone effect is that while other hormones produce
results in minutes, steroid hormones take hours to reach full impact. This delay effect is due to the
time taken in gearing up the protein synthesis as a result of change in gene expression pattern.
The sequence of events is that a steroid hormone bind to a receptor, the receptor steroid complex
enters the cell nucleus. The complex binds to the DNA and alters gene expression.
Affinity chromatography with steroid as affinity ligands facilitates purification of hormone
receptors such as those corresponding to estrogenes, progesterone and glucocorticoids.
Antibodies against such receptors constituted valuable screening probes for proteins expressed by
library of cDNA clones. Such studies have revealed that receptors contain DNA binding sequences
which are rich in cysteine, arginine and lysine.
So, receptors act as homing devices to take steroids to the genes. The steroid binding regions on
gene are in proximity to the DNA binding region. Steroid binding promotes transcription.
Steroid hormones are not the only ones which operates in this fashion. Even, thyroxine (the thyroid
hormone) also form a complex with a receptor and the complex acts as a transcriptional enhancer.
In that system, the insight came from the fact that v-erb-A oncogene of avian erythroblastosis virus
binds c-erb-A gene in the host cell. That gene also happen to be the gene for thyroxine.
In both cases, the receptors contain DNA binding regions which have sequences typical of metal
binding fingers. Such metal binding finger domains are known to exist in wide variety of both
DNA binding and RNA binding proteins. Such domains may have an ancestral gene from where
these domains were incorporated in nucleic acid binding protein.

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 The specific DNA regions to which the hormone receptor complexes bind are called hormone
response elements (HREs). Hormone binding to the receptor itself induces a conformational change
in the receptor. This allows receptors to also interact with transcriptional factors.
HREs for various hormone receptor complexes are similar in length but their nucleotide sequences
vary with each hormone. This consensus sequence is thus specific to each hormone. Each sequence
has two (nucleotide)6 sequences either together or separated by a nucleotide trimer.
The hormones receptor complex binds as a dimer, each monomer‟s zinc fingers binding to
(nucleotide)6 sequence. Apart from HRE sequences, its number and position relative to the gene
also varies with each hormone.

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 Steroid glycosides

Figure 13

Many plants are rich in steroid glycosides. Most important among these are so called “neutral
saponins”. The carbohydrate component imparts polarity and hence saponins are very good surface
active agent. This property makes them useful in many industrial applications.
The aglycones are called sapogenins. The important sapogenins are digitogenin, gitogenin and
tigogenin. Sapogenins can hemolyse erythrocytes. The aglycones can be obtained by hydrolysis of
sapogenins.
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 Some other steroid glycosides, found along with saponins have effect on cardiac activity. Used in
adequate amount, these compounds stop heart function. Aborigines used to apply these on the tip of
their arrows to kill enemies!
The important aglycones of these glycosides are digitoxigenin, periplogenin and sarmatogenin.
Used in appropriate amounts, these cardiac glycosides can stimulate mammalian heart activity and
have medicinal value.
The venom of some toads are also steroid derivatives. An important toad steroid is gamabufotalin.
In these venoms, the steroid forms ester with suberylarginine.
Steroids find large amount of applications which includes their therapeutic action. Steroid
transformations are an active area of biocatalysis. Microbial cells in free and whole cell forms have
been extensively used in such steroid transformation. Hydroxylation and redox reactions are
important reaction types involved.

We have discussed steroid hormones in this module. A better appreciation of this hormonal action is
possible by integrating the knowledge about all hormones. There is lot of connectivity to be discovered
that way among various hormones and even within steroid hormone.

A case in point is that we exemplified cortisone as a glucorticoid. It does not merely affect
carbohydrate metabolism but nitrogen and even fat metabolism as well. Furthermore, deoxycortisone
also is involved in electrolyte balance so it functions like a mineralocorticoid as well.

Finally, steroid glycosides are fascinating molecules producing diverse physiological actions.

Summary

 Cholesterol is an important and essential constituent of biomembranes.

 All steroid hormones and bile salts are produced from cholesterol.
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  Androgen and esterogens are sex hormones. The adrenal hormones are glucocorticoids.
 Steroid hormones act by altering gene expression.

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