2 steroid metabolism

1. 1
1

2. 2
Biosynthesisofsteroid
hormones
© DepartmentofBiochemistry (V.P.),
FacultyofMedicine,MUBrno2011

3. 3
Theoriginofhydroxymethylglutaryl-CoA:
H
O
CH2
CoA
CH3
CH
2
CO
CO
S
S
C
CoA
acetyl-CoA
acetoacetyl-CoA hydroxy.methyl.glutaryl-CoA
HMG-CoA
COOH
CoA
COH
SCO
CH2
CH3
CH2
H2OCoASH
(cytosol)

4. 4
2NADPH+2H
+
CoA-SH
4H
COOH
CH
2
C CH3HO
CH2
CO S CoA
COOH
CH
2
CH3CHO
CH2
CH2 OH
HMG-CoAreductase
hydroxymethylglutaryl-CoA mevalonicacid
●the crucialreactionof cholesterolsynthesis
●theplaceofphysiologicregulationofcholesterolsynthesisina cell
●theplaceoftherapeuticinfluenceuponhypercholesterolemia
withsocalled„statins“
●theplaceoftreatmentofosteoporosiswith„bisphosphonates“

5. 5
mevalonicacid activeisopreneunit
OHCH2
CH2
C CH3
2
CH
COOH
HO
CH
2
CH3C
CH2
CH2OP P
-CO2
-H2O
2ATP
(here:onefromitsforms-
isopentenyldiphosphate)
dihydroxy-
methyl-valeric (= pentanoic) acid

6. 6
isopentenyl diphosphate
dimethylallyl diphosphate
Allisoprenoidsaresynthesizedfromacetyl-CoA
bywayofisopentenyldiphosphate
anditsisomerdimethylallyldiphosphate.
Synthesisofisoprenoids:

7. 7
SQUALENE C
30
(triterpene, 6isopreneunits, symmetry)

8. 8
●steroidskeletonis synthesized fromsqualene(C
30)
●thebiosynthesisofsteroidsoriginatesincholesterol(C
27)
andincludesgradualbreakdownofthesidechain
●cellsareabletosynthesizerequiredcholesterol
●theexceptionisplacenta:
cholesterolforthesynthesis ofsteroidhormonsbyplacenta
mustbedeliveredfromthematernalblood

9. 9
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

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

11. 11
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

12. 12
pregnenolone:
„3-hydroxy-pregn-5-en-20-on“
„pregn-5-en-3-ol-20-on“
(metaboliteofcholesterol,splittingoffthepartofside
chainonC-20)
enzyme: mitochondrial P-450
SCCmonooxygenase (NADPH)
SCC=sidechaincleavage
3
5
20
O
HO

13. 13
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

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

15. 15
Progesteroneinwoman:
origin:ovary-corpusluteum
(placenta)
bloodplasma:bindingontranscortin
*
)= CBG = corticosteroidbinding
(+albumin) globulin
liver:conjugationwith GlcUA
(pregnanediol-20-glucosiduronate)
excretion:urine
Metabolic remark:
progesterone inhibits the influence of aldosterone in the kidneysincreased excretion of NaCl
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 the3-glucosiduronate,
however20-…
and probably it is not conjugated with sulfates
*)Transcortin(= CBG) isα
1-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, M
rcca 52.000, it binds roughly 75 % of P-cortisol.

16. 16
O
O
O
HO
pregnenolone
progesterone

17. 17
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

18. 18
„complete“ hydroxylation: 172111
cortisol (hydrocortisone):
( 11,17,21-trihydroxy-4-pregnen-3,20-dion)
OH
O
HO
O
O
2
CHOH
1711
21

19. 19
O
O
O
O
OH
HO
CH2OH
11
21
17
progesterone
cortisol

20. 20
O
O
O
O
HO
CH2OH
11
21
progesterone
corticosterone

21. 21
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

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

23. 23
CH2OH
O
O11
C
O
H
H
O
aldosterone(hemiacetal):
(11,18-epoxy-18,21-dihydroxypregn-4-en-3,20-dion)

24. 24
3
CH
2
OH C
O
CH
H
Thetransformationof„angular“methylC
18
(aldosterone)
hydroxylase
(18-)
dehydrogenase
(18-)
MITOCHONDRIAL ENZYMES „aldosterone
(alsothe11-hydroxylaseisa mitochodrialenzyme)synthasecomplex“

25. 25
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

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

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

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

29. 29
Testosterone(TST):
origin: theLeydig cellsoftestes~95%
adrenalgland~5%
plasma: ~3%freetestosterone
~ 97%binding:SHBG=sexhormonebindingglobulin
(+ albumin)
freetestosteronetargetcell5-reductase (NADPH)
5-dihydrotestosteronehigheraffinitytoresponsive
elementsofcellnucleus
Sertolicells.: 1/ ABP= androgenbindingprotein(itisnota receptor,
bybindingoftestosteroneitobtainsitshighconcentration
necessaryforspermatogenesis)
2/ inhibin(negativeinfluenceonhypothalamus+pituitary)
3/ antiMüllerhormone(suppressestheevolutionoffemale
sexorgans)

30. 30
O
17
OH
17
Testosterone
(TST): oxidation
~90%
17-ketosteroids
aromatization~1-5%
estradiol (E2)
5-dihydrotestosterone
reduction~4(-8)%
reduction~2%
androstanediol
androsterone
etiocholanolone
Whatislesscommonin testosterone:
1/ majorityof hormonesis transformedbythereductionintoinactivesubstances,
howeverthetestosteroneobtainseffectivenessbythereductionto5-dihydro-
testosteron
2/ onthemain metabolicway(~90%)oftestosteroneisareductionin
conjugatedbondsintheA-ringonly.AtC-17isanoxidationto17-ketosteroids).
cca5mg /d,adultman

31. 31
O
HO
A B
17
Testosterone(TST):
O
A B
the origin of 17-ketosteroidsfrom testosterone comprises the reduction of conjugated
double bonds in theA-ring and the oxidation of 17-OH group to the 17-keto-(17-oxo-).
The resulting connection of Aand B rings may betrans-andcis-:
„androsterone“ (A/B trans)
„etiocholanolone“ (A/B cis)
testosterone
The determination of 17-ketosteroids (right „17-oxosteroids“) in the urinegives
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.

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

33. 33
testosterone
androsterone
doublebondisnotpresent
substituentsare„interchanged“
inthepositions3and17
O
HO
O
OH
3
17
3
17

34. 34
Testosterone(TST):
HO
A B
O
A B
pregnenolone progesterone
cholesterol
steroid skeleton (A/B)
like cholesterol
steroid skeleton (A/B)
has conjugated bonds
androstenediol TESTOSTERONE (TST)
„prohormone“
5-dihydrotestosteroneE2
The time changes:foetus + newborntestosterone, childandrosterone, adulttestosterone

35. 35
cholesterol(C
27)
pregnenolone(C
21)
progesterone(C
21)
glucocorticoids
(C
21)
mineralocorticoids
(C
21)
androgens(C
19)
estrogens (C
18)

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

37. 37
Theeliminationof„angular“methylC
19
(estrogens)
hydroxylase
(19-)
dehydrogenase
(19-)
ENZYMES OF SMOOTH ENDOPLASMIC RETICULUM
(inthe complex„aromatase“)
lyase
(19-)
3
CH
2
OH C
O
CH
H
H
OH
O
C

38. 38
formic acid
NADPH
androstenedione
enolisation
NADPH
19-oxo
estrone („E1“)
NADPH
Consecutivereactionsof„aromatase“:
altogether 3 „monooxidase“ reactions
(required NADPH + O
2)
(for rough orientation only !!)

39. 39
„Aromatase“:
●reaction:aromatizationofAringandC-19demethylation
●enzyme:P450arom(=aromatase),CYP19
EC1.14.14.1
●occurence:inestrogeneproducingcells:
ovaries testes (!!)
placenta adiposetisssue
adrenal skin
brain
inhibitorsofaromatase:●sometimesinestrogen-dependent
tumors(breastca),
●misusedforanaboliceffecttoo
(theyincreasetheconcentration
oftestosterone)

40. 40
Estrogensinwoman:
aromatase:ovaries
(placenta)
liver
adiposetissue
skin
bloodplasma:bindingonSHBG
liver:conjugationwith GlcUA
with PAPS
excretion:theurine, thebile

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

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

43. 43
Theenzymesofmainmetabolicwaysofsteroids:
1/ hydroxylases (monooxygenases)
2/ dehydrogenases (desaturases
anddehydrogenasesofhydroxysteroids)
3/ lyases (desmolases, SCC)
Theothersenzymesofsteroidsmetabolism:
hydrogenases, ...

44. 44
Monooxygenases
RH+O
2+ NADPH+H
+

ROH+H
2O+ NADP
+
Monooxygenases=„oxygenaseswithmixedfunction“
Mixedfunction: theoxygenationofsubstrate RH
theoxidationofNADPH
(Monooxygenasestakeplacein steroidshydroxylation
andinthefirststageofxenobioticmetabolism).

45. 45
Thehydroxylationrequiresthedioxygeneactivation.
ItismediatedbycytochromeP450.
●thename:cytochromesP450havethemaximumofabsorbance
at450nm, whenisbondedCOonthem, „P“=pigment,
abbreviatedas„CYP“
●CYPareenzymesthatuseirontooxidizesomesubstrates
●CYPcatalysesavarietyofreactions
-(including:epoxidation,N-dealkylation,O-dealkylation,
S-oxidationandhydroxylation!)
-fundamentalmetabolicwayistheoxidativebiotransformation
ofxenobiotics (thefirstphaseofitishydroxylationtoo!).
●located: 1/inmembraneofsmoothER
2/intheinnermembraneofmitochondria
MonooxygenasesandCYP(1):

46. 46
●ofCYPacceptselectronefroman„electrontransferchain“,
thelastproteineinthischainisarelevant„reductaseCYP“
●inendoplasmicreticulum(ER)thechainis:
NADPH → FAD → FMN → CYP
thelastproteinofthechainis„NADPHcytochromeP450
reductase“
●inmitochodriaisin„electrontransferchain“involvedanadditional
component, theiron-sulfurproteinadrenodoxin(locatedbetween
thereductaseandthecytochrome)
MonooxygenasesandCYP(2):

47. 47
●thebondbetweenthetwoatomsinanoxygenmoleculeisrather
strong
●substantialamountofenergyisrequiredtobreakthebond
●energyissuppliedbyadditionofelectronstotheironatomof
heme (othersubstrateswereoxidizedbyremovingofelectrone)
●thereceptionofelectronebycytP450evokesthechange
Fe
3+
Fe
2+
.
Thisironoxidationstate (Fe
2+
) isabletobonddioxygen
(identicallyasinHb!!)
●thesecondtransferedelectronemakesreleasingofdouble
bondofbondedoxygen
●radicalsareformed: R•fromsubstrateRH(byremovingof
hydrogen)and •OH frompreviousdioxygene.
Then–OHgroupiscreatedfrombothradicals.
MonooxygenasesandCYP(3):

48. 48

49. 49
11
18
20
sidechain
cleavage
„SCC“
hydroxylase
dehydrogenase
(-)hydroxylase
Mitochondrialenzymes:

50. 50
3
A
17
20
21
Enzymesof(smooth)endoplasmaticreticulum:
aromatase
(aromatizingcomplex)
(-)dehydrogenase
hydroxylase
}lyase
(-)hydroxylase

51. 51
Sidechaincleavage „SCC“):
1/ thebondisbreakedbetweentwocarbons,each
ofthemhasbondingoxygen
*
)
(Thecarbonnearesttosteroidskeletonhasthebonding
-OHgroup,themorefathercarbonhasoxygeninthe
formof-OH or =O (oxo-)group)
2/ theresultofshorteningreactionistheoxo-(keto-)
derivativeofsteroid
3/ reactionsaresituatedinmitochondria
*)themechanism: 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-hydrolyticsplitting, so without water
therefore enzymes are „lyases“ and not „hydrolases“ !
From transient derivatives to side chain cleveage is worth remembering
17α-hydroxyprogesterone only …

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

53. 53
17-hydroxypregnenolonone
17-hydroxyprogesterone
dehydroepiandrosterone
androstendione
Sidechaincleavage „SCC“):
1717
20
O
OH
O

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

55. 55
Therelationshipamong
mainsteroidhormones:
kortikosteron
O
C
CH2OH
O
HO
O
C
CH3
O
progesteron
C
O
C
CH2OH
O
HO
O
H
O
C
CH2OH
O
HO
OH
HO
OH
O
OH
testosteron
aldosteron kortisol estradiol

56. 56
Adrenalcortex–theplacesofcorticoidssynthesis
zonaglomerulosaaldosterone
zonafasciculatacortisole
zona(fasciculata a) reticularisandrogens

57. 57
Adrenalcortex(1):
●threezonesinhistologicalpicture
●cellswithtwofunctionalunits,
withdifferentenzymaticequipment→ different products,
thatarecontrolledindependently
1/thecellsoftheouterlayer-zonaglomerulosa
•donotexpress17-hydroxylase,sothat theydonotproduce
precursorsofglucocorticoidsandadrenalandrogens
• ontheotherhandtheysecretealdosterone,becausethegenefor
aldosteronsyntaseisexpressed (inthatzoneonly)
• thesynthesisandsecretionofaldosteroneiscontrolledby
renin-angiotensinsystemandbyconcentrationofK
+
inplasma
•(theinfluenceofACTHisveryweakandtransient)

58. 58
Adrenalcortex(2):
2/bothinnerzones-zonafasciculataandzonareticularis
• produceglucocorticoids,androgens(minimum
oftestosterone)andsmallamountsofestrogens
• theproductionoflesseffectivemineralocorticoids(DOCand
corticosterone)isnotveryimportant
• thesynthesisandsecretioncontrolledbyACTH

59. 59
Commonstructureofcorticoids:
21
O
OHCH
2
O
( DOC = deoxycorticosterone )

60. 60
21-hydroxylationanditsdeficiency:
•thehydroxylintheposition21isthestructuralcharacteristic
ofcorticoids
•soindeficiencyof21-hydroxylasecannot beformedgluco-and
mineralocorticoids
•absentglucocorticoidscausethesecretionofACTHbyfeedback
andsoahypertrophyofadrenalglands
•ACTHstimulatesthetransfomation: cholesterol
pregnenolonbycAMP, consequently: progesterone
17-hydroxyprogesteroneandrostendionetestosterone
•increasedconcentrationsoftestosteronecauseavirilismingirls
(visibleatbirth),
inboysissexualprecocityapparentseveralmonthslater

61. 61
Orderofhydroxylationsincorticoids:
11
21
17

62. 62
11
21
17
„Complete“
hydroxylation
pathwayin
corticoids:
Hydroxylintheposition21isalwayspresent
(thestructuralcharacteristicofcorticoids)

63. 63
Hydroxylationpathway
ofcorticoidsbeginning
intheposition21:
11
21
17
"-steron"„-sterone“

64. 64
Hydroxylationofcorticoids
keepsalwaysin metabolicpathwaysanadumbrateddirection
172111(thedirectionofarrowsin schemes„anti-clockwise“).
TheC-17-hydroxylationcanbeavoid,whereastheC-21-hydroxylation
isobligatory(thepresenceofhydroxylrepresentsherethestructural
characteristicofcorticoids,thedifferencefromprogesterone).
Thepresence/absenceofoxygenatC-11dictatestheclassofglucocorticoids
ormineralocorticoids (theexception:aldosterone,seethere).
Thederivativesofpregnane, withtheabsentC-17-hydroxylhavetheending
„-sterone“intheirname.
(Thisnomenclatureaidmaybeusedinsteroidsubstanceswith21carbon
atomesi.e.in derivativesofpregnaneonly. Itisnotvalidelsewhere!)
[The hydroxylations C-17and C-21 take place in smooth endoplasmatic
reticulum, hydroxylation at C-11 in mitochondria.
Mitochondrial hydroxylation is substantiallyslower.]

65. 65
„complete“ hydroxylation: 172111
cortisol (hydrocortisone):
( 11,17,21-trihydroxy-4-pregnen-3,20-dion)
OH
O
HO
O
O
2
CHOH
1711
21

66. 66
Celllocationoforiginofcorticoids:
17-OH
21-OH
11-OH SCC
dehydrogenace,izomerace

67. 67
CORTICOIDS:
1/ mineralocorticoids
actonthekidneytoincreasethereabsorptionofNa
+
andtheexcretionofK
+
.
Thechargeof Na
+
,whichisoverasimplesubstitutionforK
+
,isbalancedwith
retentionof Cl
-
.
NaCl increasestheosmoticpressure,wateris absorbedforitsadjustement.
Itleadstoanincreasein bloodvolumeandbloodpressure.
Mainrepresentative:aldosterone
Structure:C
21,mineralocorticoidsdonothaveoxygenintheposition 11.
(Aldosteroneistheexception,itsC-11-hydroxyl is„camouflaged“
byformingofhemiacetal.)

68. 68
2/ glucocorticoids
haveacataboliceffect.Theyenhancethedegradationofproteinsandfat.
Glucogenicamino-acidsfromdegradatedproteinsaresubstratesof
gluconeogenesis(i.e.productionofglucosefromnon-sugarsubstances).
Glucocorticoidsincreaseglycemiaandinhibittheinflamatoryresponseand
immunereaction(immunosuppressiveeffect).
Mainrepresentative:cortisol,atypicalhormoneofchronicstress.
Structure:C
21,inglucocorticoidsthereisalwayspresentoxygenat C-11
(hydroxy-oroxo-group).

69. 69
Remember !
Inmajorityofcaseswe cannotdistinguishcompletely„pure“glucocorticoids
and„pure“mineralocorticoids.
Itisvaluedmainlyindrugs,wherepracticallyeveryglucocorticoid
hasasmallmineralocorticoideffectstoo.

70. 70
Biologicaleffect-glucocorticoids:
-increaseoflivergluconeogenesisfromamino-acids
-increaseofproteincatabolisminskeletalmuscle
-„stresshormone“
-suppressionofimmunereaction
(immunosuppressiveeffect)
-antiinflamatoryeffect (non-infectiveinflamation)
Biologicaleffect-mineralocorticoids:
-Na
+
retentionindistaltubuleofkidney
-increaseexcretionofK
+

71. 71
Controlofbiosynthesis:
-glucocorticoids:
1/ diurnalrhythm
2/ negativefeedbackatcortisol(ACTH)
3/ stress
-mineralocortikoids:
1/[K
+
]
2/ systeme renin–angiotensin-aldosterone

72. 72