1. 1
1

2. Biosynthesis of steroid
hormones
© Department of Biochemistry (V.P.),
Faculty of Medicine, MU Brno 2011
2

3. The origin of hydroxymethylglutaryl-CoA :
CO S CoA COOH
H CH2 acetyl-CoA CH2
H2O CoA SH
O C CH3 HO C CH3
CH2 CH2
CO S CoA CO S CoA
acetoacetyl-CoA hydroxy.methyl.glutaryl-CoA
HMG-CoA
(cytosol)
3

4. HMG-CoA reductase
COOH COOH
2 NADPH + 2H+ CoA-SH
CH CH
2 2
4H
HO C CH3 HO C CH3
CH2 CH2
CO S CoA CH2 OH
hydroxymethylglutaryl-CoA mevalonic acid
● the crucial reaction of cholesterol synthesis
● the place of physiologic regulation of cholesterol synthesis in a cell
● the place of therapeutic influence upon hypercholesterolemia
with so called „statins“ 4
● the place of treatment of osteoporosis with „bisphosphonates“

5. COOH
- CO2
CH 2 - H2O CH 2
HO C CH3 C CH3
CH2 CH2
2 ATP
CH2 OH CH2 O P P
mevalonic acid active isoprene unit
dihydroxy- (here: one from its forms -
methyl- valeric (= pentanoic) acid isopentenyl diphosphate)
5

6. Synthesis of isoprenoids :
All isoprenoids are synthesized from acetyl-CoA
by way of isopentenyl diphosphate
and its isomer dimethylallyl diphosphate.
isopentenyl diphosphate
dimethylallyl diphosphate
6

7. SQUALENE C30
( triterpene, 6 isoprene units, symmetry )
7

8. ● steroid skeleton is synthesized from squalene (C30)
● the biosynthesis of steroids originates in cholesterol (C27)
and includes gradual breakdown of the side chain
● cells are able to synthesize required cholesterol
● the exception is placenta:
cholesterol for the synthesis of steroid hormons by placenta
must be delivered from the maternal blood
8

9. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
9

10. cholesterol
3 5
HO
( cholest-5-en-3-ol )
10

11. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
11

12. pregnenolone :
„ 3-hydroxy-pregn-5-en-20-on “
„ pregn-5-en-3-ol-20-on “ 20
O
3
HO 5
(metabolite of cholesterol, splitting off the part of side
chain on C-20)
enzyme: mitochondrial P-450SCC monooxygenase (NADPH)
SCC = side chain cleavage
12

13. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
13

14. progesterone :
20
O
34
O
( 4-pregnen-3,20-dion )
14

15. Progesterone in woman :
origin: ovary - corpus luteum liver: conjugation with GlcUA
(placenta) (pregnanediol-20-glucosiduronate)
excretion: urine
blood plasma: binding on transcortin*) = CBG = corticosteroid binding
(+ albumin) globulin
What is less common in progesterone:
in comparison with other sex hormones 1/ in plasma it does not bind on SHBG
2/ it does not form the 3-glucosiduronate,
however 20- …
and probably it is not conjugated with sulfates
Metabolic remark:
progesterone inhibits the influence of aldosterone in the kidneys  increased excretion of NaCl
*) Transcortin (= CBG) is α1-globulin of blood plasma (about 37 mg/l). P.o. contraception and pregnancy incrises its
P-concentration up to twice. It is synthesized in the liver, Mr cca 52.000, it binds roughly 75 % of P-cortisol.
15

16. O
pregnenolone
HO
O
O progesterone
16

17. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
17

18. cortisol (hydrocortisone) :
( 11,17,21-trihydroxy-4-pregnen-3,20-dion )
21
CH 2 OH
HO
O
11 17
OH
O
„complete“ hydroxylation: 17  21  11
18

19. O
progesterone
O
21 CH
2OH
HO
11 17 O
OH
cortisol
O
19

20. O
progesterone
O
21 CH
2OH
HO
11 O
corticosterone
O
20

21. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
21

22. aldosterone :
O
CH2OH
C
HO H
11 O
O
( 11,21-dihydroxy-3,20-dioxo-4-pregnen-18-al ) 22

23. aldosterone (hemiacetal) :
OH CH2OH
C
O H
11 O
O
( 11,18-epoxy-18,21-dihydroxypregn-4-en-3,20-dion ) 23

24. The transformation of „angular“ methyl C18
(  aldosterone )
O
CH 3 CH2OH C
H
hydroxylase dehydrogenase
(18-) (18-)
M I T O CH O N D R I A L E N Z Y M E S „aldosterone
(also the 11-hydroxylase is a mitochodrial enzyme) synthase complex“
24

25. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
25

26. testosterone
(„TST“)
OH
17
34
O
( 17-hydroxy-4-androsten-3-on )
26

27. 17 O
OH
O
17α-hydroxyprogesterone
O
17
androstenedione
O
27

28. O
17
androstenedione
O
OH
17
testosterone (TST)
O
28

29. Testosterone (TST) :
 origin: the Leydig cells of testes ~ 95 %
adrenal gland ~ 5 %
 plasma: ~ 3 % free testosterone
~ 97 % binding: SHBG = sex hormone binding globulin
(+ albumin)
 free testosterone  target cell  5-reductase (NADPH)
 5-dihydrotestosterone  higher affinity to responsive
elements of cell nucleus
 Sertoli cells.: 1/ ABP = androgen binding protein (it is not a receptor,
by binding of testosterone it obtains its high concentration
necessary for spermatogenesis)
2/ inhibin (negative influence on hypothalamus + pituitary)
3/ antiMüller hormone (suppresses the evolution of female
sex organs) 29

30. Testosterone cca 5 mg / d, adult man
O
(TST) : 17
OH
oxidation 17
~ 90 %
17-ketosteroids
aromatization ~ 1-5 %
androsterone
reduction etiocholanolone
~ 4 (- 8) %
reduction ~ 2 %
5-dihydrotestosterone
estradiol (E2) androstanediol
What is less common in testosterone:
1/ majority of hormones is transformed by the reduction into inactive substances,
however the testosterone obtains effectiveness by the reduction to 5-dihydro-
testosteron
2/ on the main metabolic way (~ 90 %) of testosterone is a reduction in
conjugated bonds in the A-ring only. At C-17 is an oxidation to 17-ketosteroids).
30

31. Testosterone (TST) :
the origin of 17-ketosteroids from testosterone comprises the reduction of conjugated
double bonds in the A-ring and the oxidation of 17-OH group to the 17-keto- (17-oxo-).
The resulting connection of A and B rings may be trans- and cis- :
O
17
A B A B
O HO
„androsterone“ (A/B trans)
testosterone „etiocholanolone“ (A/B cis)
The determination of 17-ketosteroids (right „17-oxosteroids“) in the urine gives
overall picture of androgenes: in healthy man the fraction from the adrenal cortex
comprises from 2/3 to 3/4, the rest is from testes.
In woman the whole excreted quantum of androgens comes from the adrenal cortex.
31

32. Androgens in man :
OH O OH
17
A B
O HO O
TST androsterone TST
birth puberty
A/B trans (no = on C-4),
„mutual change“ of functional
groups in the position 3 and 17,
it belongs to 17-ketosteroids
32

33. OH
17
testosterone
3
O
O
17
androsterone
3
HO
substituents are „interchanged“
in the positions 3 and 17
double bond is not present
33

34. Testosterone (TST) :
cholesterol
A B A B
HO O
pregnenolone progesterone
steroid skeleton (A/B) steroid skeleton (A/B)
like cholesterol has conjugated bonds
androstenediol TESTOSTERONE (TST)
„prohormone“
E2 5-dihydrotestosterone
The time changes: foetus + newborn  testosterone, child  androsterone, adult  testosterone 34

35. cholesterol (C27)
pregnenolone (C21)
progesterone (C21)
glucocorticoids androgens (C19)
(C21)
mineralocorticoids
(C21)
estrogens (C18)
35

36. estradiol
(„E2“)
OH
x 17
3
HO
( 1,3,5(10)-estratrien-3,17-diol )
36

37. The elimination of „angular“ methyl C19
(  estrogens )
O
O H C
CH 3 CH2OH C OH
H
hydroxylase dehydrogenase lyase
(19-) (19-) (19-)
ENZYMES OF SMOOTH ENDOPLASMIC RETICULUM
( in the complex „aromatase“ )
37

38. (for rough orientation only !!)
Consecutive reactions of „aromatase“ :
NADPH
altogether 3 „monooxidase“ reactions
(required NADPH + O2)
androstenedione
enolisation
19-oxo
NADPH
NADPH
formic acid
estrone („E1“)
38

39. „Aromatase“ :
● reaction: aromatization of A ring and C-19 demethylation
● enzyme: P450arom (= aromatase), CYP 19
EC 1.14.14.1
● occurence: in estrogene producing cells:
ovaries testes (!!)
placenta adipose tisssue
adrenal skin
brain
 inhibitors of aromatase: ● sometimes in estrogen-dependent
tumors (breast ca),
● misused for anabolic effect too
(they increase the concentration
of testosterone) 39

40. Estrogens in woman :
aromatase: ovaries liver: conjugation with GlcUA
(placenta) with PAPS
liver
adipose tissue
skin excretion: the urine, the bile
blood plasma: binding on SHBG
40

41. OH
testosterone (TST)
O
OH
estradiol (E2)
HO
41

42. O
17
androstenedione
O
O
estrone (E1)
HO
42

43. The enzymes of main metabolic ways of steroids:
1/ hydroxylases (monooxygenases)
2/ dehydrogenases (desaturases
and dehydrogenases of hydroxysteroids)
3/ lyases (desmolases, SCC)
The others enzymes of steroids metabolism :
hydrogenases, ...
43

44. Monooxygenases
RH + O2 + NADPH + H+ 
 ROH + H2O + NADP+
Monooxygenases = „oxygenases with mixed function“
Mixed function: the oxygenation of substrate RH
the oxidation of NADPH
(Monooxygenases take place in steroids hydroxylation
and in the first stage of xenobiotic metabolism). 44

45. Monooxygenases and CYP (1) :
The hydroxylation requires the dioxygene activation.
It is mediated by cytochrome P450.
● the name: cytochromes P450 have the maximum of absorbance
at 450 nm, when is bonded CO on them, „P“ = pigment,
abbreviated as „CYP“
● CYP are enzymes that use iron to oxidize some substrates
● CYP catalyses a variety of reactions
- (including: epoxidation, N-dealkylation, O-dealkylation,
S-oxidation and hydroxylation !)
- fundamental metabolic way is the oxidative biotransformation
of xenobiotics (the first phase of it is hydroxylation too !).
● located: 1/ in membrane of smooth ER
2/ in the inner membrane of mitochondria
45

46. Monooxygenases and CYP (2) :
● of CYP accepts electrone from an „electron transfer chain“,
the last proteine in this chain is a relevant „reductase CYP“
● in endoplasmic reticulum (ER) the chain is:
NADPH → FAD → FMN → CYP
the last protein of the chain is „NADPH cytochrome P450
reductase“
● in mitochodria is in „electron transfer chain“ involved an additional
component, the iron-sulfur protein adrenodoxin (located between
the reductase and the cytochrome)
46

47. Monooxygenases and CYP (3) :
● the bond between the two atoms in an oxygen molecule is rather
strong
● substantial amount of energy is required to break the bond
● energy is supplied by addition of electrons to the iron atom of
heme (other substrates were oxidized by removing of electrone)
● the reception of electrone by cyt P450 evokes the change
Fe3+  Fe2+ .
This iron oxidation state (Fe2+) is able to bond dioxygen
(identically as in Hb !!)
● the second transfered electrone makes releasing of double
bond of bonded oxygen
● radicals are formed : R• from substrate RH (by removing of
hydrogen) and •OH from previous dioxygene.
Then –OH group is created from both radicals.
47

48. 48

49. Mitochondrial enzymes :
hydroxylase
dehydrogenase
side chain
(-) hydroxylase 18 cleavage
„SCC“
20
11
49

50. Enzymes of (smooth) endoplasmatic reticulum :
hydroxylase
aromatase 21
(aromatizing complex)
20
} lyase
17
A (-) hydroxylase
3
(-) dehydrogenase
50

51. Side chain cleavage „SCC“) :
1/ the bond is breaked between two carbons, each
of them has bonding oxygen*)
(The carbon nearest to steroid skeleton has the bonding
-OH group, the more father carbon has oxygen in the
form of -OH or =O (oxo-) group)
2/ the result of shortening reaction is the oxo- (keto-)
derivative of steroid
3/ reactions are situated in mitochondria
*) the mechanism: the more electronegative oxygens attract electrones from both carbons.
In turn is decreasing of electrone density of bond between two neighbouring
carbons and the bond is enzymaticaly disrupted.
It is non-hydrolytic splitting, so without water
 therefore enzymes are „lyases“ and not „hydrolases“ !
From transient derivatives to side chain cleveage is worth remembering
17α-hydroxyprogesterone only …
51

52. Side chain cleavage „SCC“) :
20 OH
OH 20
22 O
20,22-dihydroxycholesterol pregnenolone
52

53. Side chain cleavage „SCC“) :
20
O
O
17 OH 17
17-hydroxypregnenolonone dehydroepiandrosterone
17-hydroxyprogesterone androstendione
53

54. 17
cholesterol
HO
O
17
DHEA
HO = dehydroepiandrosterone
54

55. CH3
The relationship among C O
main steroid hormones:
O
progesteron
OH
CH2OH
C O
HO
O
testosteron
O
kortikosteron
CH2OH CH2OH
O
C O C O OH
H C
HO HO OH
O O HO
aldosteron kortisol estradiol 55

56. Adrenal cortex – the places of corticoids synthesis
zona glomerulosa  aldosterone
zona fasciculata  cortisole
zona (fasciculata a) reticularis  androgens
56

57. Adrenal cortex (1) :
● three zones in histological picture
● cells with two functional units,
with different enzymatic equipment → different products,
that are controlled independently
1/ the cells of the outer layer - zona glomerulosa
• do not express 17-hydroxylase, so that they do not produce
precursors of glucocorticoids and adrenal androgens
• on the other hand they secrete aldosterone, because the gene for
aldosteronsyntase is expressed (in that zone only)
• the synthesis and secretion of aldosterone is controlled by
renin-angiotensin system and by concentration of K+ in plasma
• (the influence of ACTH is very weak and transient)
57

58. Adrenal cortex (2) :
2/ both inner zones - zona fasciculata and zona reticularis
• produce glucocorticoids, androgens (minimum
of testosterone) and small amounts of estrogens
• the production of less effective mineralocorticoids (DOC and
corticosterone) is not very important
• the synthesis and secretion controlled by ACTH
58

59. Common structure of corticoids :
21
CH2 OH
O
O
( DOC = deoxycorticosterone )
59

60. 21-hydroxylation and its deficiency :
• the hydroxyl in the position 21 is the structural characteristic
of corticoids
• so in deficiency of 21-hydroxylase cannot be formed gluco- and
mineralocorticoids
• absent glucocorticoids cause the secretion of ACTH by feedback
and so a hypertrophy of adrenal glands
• ACTH stimulates the transfomation: cholesterol
 pregnenolon by cAMP, consequently: progesterone 
17-hydroxyprogesterone  androstendione  testosterone
• increased concentrations of testosterone cause a virilism in girls
(visible at birth),
in boys is sexual precocity apparent several months later
60

61. Order of hydroxylations in corticoids :
21
11 17
61

62. „Complete“
hydroxylation
pathway in 21
corticoids :
11 17
Hydroxyl in the position 21 is always present
62
(the structural characteristic of corticoids)

63. Hydroxylation pathway
of corticoids beginning
in the position 21 :
21
11 17
"-steron"
„-sterone“
63

64. Hydroxylation of corticoids
keeps always in metabolic pathways an adumbrated direction
17 21  11 (the direction of arrows in schemes „anti-clockwise“).
The C-17-hydroxylation can be avoid, whereas the C-21-hydroxylation
is obligatory (the presence of hydroxyl represents here the structural
characteristic of corticoids, the difference from progesterone).
The presence/absence of oxygen at C-11 dictates the class of glucocorticoids
or mineralocorticoids (the exception: aldosterone, see there).
The derivatives of pregnane, with the absent C-17-hydroxyl have the ending
„-sterone“ in their name.
(This nomenclature aid may be used in steroid substances with 21 carbon
atomes i.e. in derivatives of pregnane only. It is not valid elsewhere !)
[ The hydroxylations C-17 and C-21 take place in smooth endoplasmatic
reticulum, hydroxylation at C-11 in mitochondria.
Mitochondrial hydroxylation is substantially slower. ]
64

65. cortisol (hydrocortisone) :
( 11,17,21-trihydroxy-4-pregnen-3,20-dion )
21
CH 2 OH
HO
O
11 17
OH
O
„complete“ hydroxylation: 17  21  11
65

66. Cell location of origin of corticoids :
11-OH SCC
dehydrogenace, izomerace
17-OH
21-OH
66

67. CORTICOIDS :
1/ mineralocorticoids
act on the kidney to increase the reabsorption of Na+ and the excretion of K+.
The charge of Na+, which is over a simple substitution for K+, is balanced with
retention of Cl- .
NaCl increases the osmotic pressure, water is absorbed for its adjustement.
It leads to an increase in blood volume and blood pressure.
Main representative: aldosterone
Structure: C21, mineralocorticoids do not have oxygen in the position 11 .
(Aldosterone is the exception, its C-11-hydroxyl is „camouflaged“
by forming of hemiacetal.)
67

68. 2/ glucocorticoids
have a catabolic effect. They enhance the degradation of proteins and fat.
Glucogenic amino-acids from degradated proteins are substrates of
gluconeogenesis (i.e. production of glucose from non-sugar substances).
Glucocorticoids increase glycemia and inhibit the inflamatory response and
immune reaction (immunosuppressive effect).
Main representative: cortisol, a typical hormone of chronic stress.
Structure: C21, in glucocorticoids there is always present oxygen at C-11
(hydroxy- or oxo- group).
68

69. Remember !
In majority of cases we cannot distinguish completely „pure“ glucocorticoids
and „pure“ mineralocorticoids.
It is valued mainly in drugs, where practically every glucocorticoid
has a small mineralocorticoid effects too.
69

70. Biological effect - glucocorticoids:
- increase of liver gluconeogenesis from amino-acids
- increase of protein catabolism in skeletal muscle
- „stress hormone“
- suppression of immune reaction
(immunosuppressive effect)
- antiinflamatory effect (non-infective inflamation)
Biological effect - mineralocorticoids:
- Na+ retention in distal tubule of kidney
- increase excretion of K+
70

71. Control of biosynthesis :
- glucocorticoids:
1/ diurnal rhythm
2/ negative feedback at cortisol (ACTH)
3/ stress
- mineralocortikoids:
1/ [K+]
2/ systeme renin – angiotensin - aldosterone
71

72. 72