Thin-layer Chromatography of Steroid Hormones

Thin-layer Chromatography
of Steroid Hormones
Biology 231
Supplement
#7
OBJECTIVES
1. Identify the major classes of steroid
hormones and the glands that secrete them.
2. Describe the primary differences between
different functional classes of steroid
hormones.
3. Demonstrate the technique of thin-layer
chromatography, and explain how this
procedure works.
MATERIALS
Thin-layer plates (silica gel, F–254),
chromatography developing chambers,
capillary tubes
Driers (chromatography or hair driers),
ultraviolet viewing box (short wavelength),
rulers or spotting template (optional)
Steroid solutions, 1.0 mg/ml in absolute
methanol of testosterone, hydrocortisone,
cortisone, corticosterone, and
deoxycorticosterone; 5 mg/ml of estradiol
Unknown steroid solution containing any two of
the steroids previously described
Developing solvent: 60 ml benzene plus 10 ml
ethyl acetate plus 10 ml acetone, or a
volume containing a comparable 6:1:1 ratio
of solvents
Slight differences in steroid structure produce
significant differences in biological effects.
Differences in structure and thus solubility can be
used to separate a mixture of steroids and to
identify unknown molecules.
STEROID HORMONES
The steroid hormones, secreted by the adrenal
cortex and the gonads, are characterized by a
common four-ring structure. The carbon atoms
in this structure are numbered as follows:
Seemingly slight modifications in chemical
structure result in very great differences in
biological activity. On the basis of their activity
and their structure, the steroid hormones can be
grouped into the following functional categories:
(1) androgenic hormones; (2) estrogenic
hormones; (3) progestational hormones; and
(4) corticosteroid hormones, which are further
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divided into the subcategories of glucocorticoids
and mineralocorticoids.
The androgenic hormones are characterized
structurally by the fact that they are nineteencarbon
steroids and functionally by the fact that
they promote the development of secondary male
sex characteristics. The most potent androgenic
hormone secreted by the testes is testosterone.
Although the primary source of androgens is the
testes, the adrenal cortex also secretes small
amounts. Large amounts of androgens are present
in the plasma of persons suffering from tumors of
the testes. Adrenal hyperplasia (Cushing’s
syndrome) and tumors of the adrenal cortex can
also cause excessive androgen levels, which can
have a masculinizing effect in females.
Testosterone and the other androgens are secreted
in the testes by the interstitial Leydig cells. This
secretion is stimulated by a gonadotrophic
hormone of the anterior pituitary, interstitial cellstimulating
hormone (ICSH), which is identical to
luteinizing hormone (LH).
Although the structural difference between the
androgens and the estrogens is seemingly
slight—the estrogens are eighteen-carbon steroids
with three points of unsaturation (double bonds,
see appendix 1) in the A ring—the difference in
biological effects is pleasantly pronounced. The
chief estrogenic hormone is estradiol.
The estrogens are normally secreted in cyclically
increasing and decreasing amounts by the
ovaries, reaching a peak at about the time of
ovulation. The cyclical secretion of estrogens is
stimulated by the cyclical secretion of a
gonadotrophic hormone of the anterior pituitary,
follicle-stimulating hormone (FSH).
Abnormally high concentrations of circulating
estrogenic hormones may be due to tumors of the
adrenal cortex or the gonads. This can have a
feminizing effect in males.
In the normal female cycle, the estrogens
stimulate growth and development of the inner
lining of the uterus (the endometrium). The final
maturation of the endometrium is under the
control of the hormone progesterone, secreted in
the phase of the cycle after ovulation (luteal
phase) by the corpus luteum of the ovaries. The
cyclical secretion of progesterone is stimulated
by the cyclical secretion of luteinizing hormone
(LH) from the anterior pituitary. (LH and ICSH
are two names for the same hormone, which has
different effects in the two sexes.)
During pregnancy, the placenta secretes
increasing amounts of progesterone, which is
correlated with the development of the fetus.
Progesterone is a twenty-one-carbon steroid.
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The glucocorticoids, secreted by the zona
fasciculata and the zona reticularis, stimulate the
breakdown of muscle proteins and the conversion
of amino acids into glucose (gluconeogenesis).
The secretions of the z. fasciculata and the
z. reticularis are stimulated by the anterior
pituitary hormone, adrenocorticotrophin
(ACTH). The most potent glucocorticoids are
corticosterone, hydrocortisone (cortisol), and
cortisone.
The steroid hormones of the adrenal cortex
(corticosteroids) also contain twenty-one carbons
but differ from progesterone by the presence of
three or more oxygen groups. These hormones
are divided into two functional classes and are
secreted by two functionally distinct regions of the
cortex.
The mineralocorticoids, secreted by the zona
glomerulosa, are involved in the regulation of
sodium and potassium balance. The secretion of
aldosterone (a mineralocorticoid) is stimulated by
angiotensin II and is thus regulated by the
secretion of renin from the kidneys. The most
potent mineralocorticoids are aldosterone and, to
a lesser degree, deoxycorticosterone (DOC).
An abnormal secretion of the mineralocorticoids
is usually associated with hypertension and may
be produced by primary aldosteronism or by
secondary aldosteronism due to low blood
sodium, high blood potassium, hypovolemia,
cardiac failure, kidney failure, or cirrhosis of the
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liver. An increased secretion of the
glucocorticoids is found in Cushing’s syndrome
(adrenal hyperplasia), pregnancy, and stress due to
disease, surgery, and burns.
carry others. If the process is halted before all
the steroids have been washed off the top of the
plate, some will have migrated farther from the
origin than others.
THIN-LAYER
CHROMATOGRAPHY
In this exercise, an attempt will be made to
identify two unknown steroids that are present in
the same solution. To do this, you must
(1) separate and (2) identify these steroids by
comparing their behavior with that of known
steroids.
Since each steroid has a different structure, each
will have a different solubility (ability to be
dissolved) in a given solvent. These differences
will be used to separate and identify the steroids
on a thin-layer plate.
The thin-layer plate consists of a thin layer of
porous material (in this procedure, silica gel) that
is coated on one side of a plastic glass, or
aluminum plate. The solutions of steroids are
applied on different spots of the plate (a procedure
called “spotting”), and the plate is placed in a
solvent bath with the spots above the solvent.
If this chromatography were repeated using the
same steroids and the same solvent, the final
pattern (chromatogram) would be the same as
obtained previously. In other words, the distance
that a given steroid migrates in a given solvent,
relative to the solvent front, can be used as an
identifying characteristic of that steroid. We can
give this identity a numerical value by calculating
the distance the steroid traveled relative to the
front (the R value) follows.
f
We can identify the unknown steroid by
comparing its R f value in a given solvent with the
Rf values of known steroids in the same solvent.
As the solvent creeps up the plate by capillary
action, it will wash the steroids off their original
spots (the origin) and carry them upward toward
the other end of the plate. Since the solubility of
each steroid is different, it takes longer for the
solvent to wash and carry some than to wash and
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CLINICAL SIGNIFICANCE
The chromatographic separation and
identification of steroid hormones has revealed
much about endocrine physiology that is clinically
useful. It was learned, for example, that the
placenta secretes estrogens that are more polar
(water soluble) than the predominant ovarian
estrogen, estradiol. These polar placental
estrogens—estriol and estetrol—are now
measured clinically during pregnancy to assess the
health of the placenta.
Chromatography of androgens recovered from
their target tissues (such as the prostate) has
revealed that these tissues convert testosterone
into other products. Further, these products
appear to be more biologically active (more
androgenic) than testosterone itself. Testosterone
secreted by the testes is thus a prehormone, which
is enzymatically converted in the target tissue into
more active products — dihydrotestosterone
(DHT), in many tissues. Males who have a
congenital deficiency in 5-reductase, the enzyme
responsible for this conversion, therefore, show
many symptoms of androgen deficiency even
though their testes secrete large amounts of
testosterone.
Procedure
1. Using a pencil, make a tiny notch on the left margin of the thin-layer plate, approximately
1½ inches from the bottom. The origin of all the spots will lie on an imaginary line extending
across the plate from this notch.
2. Using a capillary pipette, carefully spot steroid solution 1 (estradiol) about ½ inch in from the lefthand
margin of the plate, along the imaginary line. Repeat this procedure, using the same steroid at
the same spot, two more times. Allow the spot to dry between applications.
3. Repeat step 2 with each of the remaining steroid solutions (2, testosterone; 3, hydrocortisone;
4, cortisone; 5, corticosterone; 6, deoxycorticosterone; 7, unknown), spotting each steroid
approximately ½ inch to the right of the previous steroid, along the imaginary line.
4. Observe the steroid spots at the origin under an ultraviolet lamp. (Note: Do not look directly at the
UV light.)
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5. Place the thin-layer plates in a developing chamber filled with solvent (benzene/ethyl
acetate/acetone, 6:1:1), and allow the chromatogram to develop for 1 hour.
6. Remove the thin-layer plate, dry, and observe it under the UV light. Using a pencil, outline the
spots observed under the UV light.
7. In the laboratory report below, record the R f values of the known steroids, and determine the
steroids present in the unknown solution.
DATA FROM EXERCISE 7
Record your data in the table below and calculate the R f value of each spot.
Steroid
Distance
to Front
Distance
to Spot
Rf
1. Estradiol
2. Testosterone
3. Hydrocortisone
4. Cortisone
5. Corticosterone same
6. Deoxycorticosterone
7. Unknown 1
Unknown 2
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