2 steroid metabolism

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Biosynthesis of steroidhormones

© Department of Biochemistry (V.P.),Faculty of Medicine, MU Brno 2011

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The origin of hydroxymethylglutaryl-CoA :

H

O

CH2

CoA

CH3

CH2

CO

CO

S

S

C

CoA

acetyl-CoA

acetoacetyl-CoA hydroxy.methyl.glutaryl-CoA

HMG-CoA

COOH

CoA

COH

SCO

CH2

CH3

CH2

H2O CoA SH

(cytosol)

4

2 NADPH + 2H+ CoA-SH

4H

COOH

CH2

C CH3HO

CH2

CO S CoA

COOH

CH2

CH3CHO

CH2

CH2 OH

HMG-CoA reductase

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“● the place of treatment of osteoporosis with „bisphosphonates“

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mevalonic acid active isoprene unit

OHCH2

CH2

C CH3

2CH

COOH

HO

CH2

CH3C

CH2

CH2 O P P

- CO2

- H2O

2 ATP

(here: one from its forms -isopentenyl diphosphate)

dihydroxy-methyl- valeric (= pentanoic) acid

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isopentenyl diphosphate

dimethylallyl diphosphate

All isoprenoids are synthesized from acetyl-CoAby way of isopentenyl diphosphateand its isomer dimethylallyl diphosphate.

Synthesis of isoprenoids :

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SQUALENE C30

( triterpene, 6 isoprene units, symmetry )

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● 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 placentamust be delivered from the maternal blood

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cholesterol (C27)

pregnenolone (C21)

progesterone (C21)

glucocorticoids(C21) mineralocorticoids

(C21)

androgens (C19)

estrogens (C18)

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cholesterol

( cholest-5-en-3-ol )

HO

3 5

11

cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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pregnenolone :„ 3-hydroxy-pregn-5-en-20-on “„ pregn-5-en-3-ol-20-on “

(metabolite of cholesterol, splitting off the part of side

chain on C-20)

enzyme: mitochondrial P-450SCC monooxygenase (NADPH)SCC = side chain cleavage

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20

O

HO

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cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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progesterone :

O

O

3 4

20

( 4-pregnen-3,20-dion )

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Progesterone in woman :

origin: ovary - corpus luteum(placenta)

blood plasma: binding on transcortin*) = CBG = corticosteroid binding(+ albumin) globulin

liver: conjugation with 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 the 3-glucosiduronate,however 20- …and probably it is not conjugated with sulfates

*) 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.

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O

O

O

HOpregnenolone

progesterone

17

cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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„complete“ hydroxylation: 17 21 11

cortisol (hydrocortisone) :( 11,17,21-trihydroxy-4-pregnen-3,20-dion )

OH

O

HO

O

O

2CH OH

1711

21

19

O

O

O

OOH

HO

CH2OH

11

21

17

progesterone

cortisol

20

O

O

O

OHO

CH2OH

11

21

progesterone

corticosterone

21

cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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CH2OH

O

OHO

11

C

O

H

aldosterone :

( 11,21-dihydroxy-3,20-dioxo-4-pregnen-18-al )

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CH2OH

O

O11

CO

H

H

O

aldosterone (hemiacetal) :

( 11,18-epoxy-18,21-dihydroxypregn-4-en-3,20-dion )

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3 CH2OH C

O

CHH

The transformation of „angular“ methyl C18

( aldosterone )

hydroxylase(18-)

dehydrogenase(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“

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cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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testosterone(„TST“)

3 4

17

O

OH

( 17-hydroxy-4-androsten-3-on )

27

OH

O

O17

17α-hydroxyprogesterone

O

17

Oandrostenedione

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O

17

O

OH

17

O

androstenedione

testosterone (TST)

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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 responsiveelements of cell nucleus

Sertoli cells.: 1/ ABP = androgen binding protein (it is not a receptor,

by binding of testosterone it obtains its high concentrationnecessary for spermatogenesis)

2/ inhibin (negative influence on hypothalamus + pituitary)3/ antiMüller hormone (suppresses the evolution of female

sex organs)

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O

17

OH

17

Testosterone(TST) : oxidation

~ 90 %

17-ketosteroidsaromatization ~ 1-5 %

estradiol (E2)

5-dihydrotestosterone

reduction ~ 4 (- 8) %

reduction ~ 2 %

androstanediol

androsteroneetiocholanolone

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 inconjugated bonds in the A-ring only. At C-17 is an oxidation to 17-ketosteroids).

cca 5 mg / d, adult man

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O

HO

A B

17


O

A B

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- :

„androsterone“ (A/B trans)„etiocholanolone“ (A/B cis)testosterone

The determination of 17-ketosteroids (right „17-oxosteroids“) in the urine givesoverall picture of androgenes: in healthy man the fraction from the adrenal cortexcomprises from 2/3 to 3/4, the rest is from testes.In woman the whole excreted quantum of androgens comes from the adrenal cortex.

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O

HO

A B

17

OH

O

OH

O

TST TSTandrosterone

birth puberty

Androgens in man :

A/B trans (no = on C-4),„mutual change“ of functionalgroups in the position 3 and 17,

it belongs to 17-ketosteroids

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testosterone

androsterone

double bond is not present

substituents are „interchanged“in the positions 3 and 17

O

HO

O

OH

3

17

3

17

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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

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cholesterol (C27)

pregnenolone (C21)

progesterone (C21)


(C21)

androgens (C19)

estrogens (C18)

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estradiol(„E2“)

OH

HO

x

3

17

( 1,3,5(10)-estratrien-3,17-diol )

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The elimination of „angular“ methyl C19

( estrogens )

hydroxylase(19-)

dehydrogenase(19-)

ENZYMES OF SMOOTH ENDOPLASMIC RETICULUM( in the complex „aromatase“ )

lyase(19-)

3 CH2OH C

O

CH

H

H

OH

OC

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formic acid

NADPH

androstenedione

enolisation

NADPH

19-oxo

estrone („E1“)

NADPH

Consecutive reactions of „aromatase“ :altogether 3 „monooxidase“ reactions(required NADPH + O2)

(for rough orientation only !!)

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„Aromatase“ :

● reaction: aromatization of A ring and C-19 demethylation

● enzyme: P450arom (= aromatase), CYP 19EC 1.14.14.1

● occurence: in estrogene producing cells:ovaries testes (!!)placenta adipose tisssueadrenal skin

brain

inhibitors of aromatase: ● sometimes in estrogen-dependenttumors (breast ca),

● misused for anabolic effect too(they increase the concentrationof testosterone)

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Estrogens in woman :

aromatase: ovaries(placenta)liveradipose tissueskin

blood plasma: binding on SHBG

liver: conjugation with GlcUAwith PAPS

excretion: the urine, the bile

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OH

O

HO

OH

testosterone (TST)

estradiol (E2)

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O

O

17

androstenedione

O

HO

estrone (E1)

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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, ...

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Monooxygenases

RH + O2 + NADPH + H+

ROH + H2O + NADP+

Monooxygenases = „oxygenases with mixed function“

Mixed function: the oxygenation of substrate RHthe oxidation of NADPH

(Monooxygenases take place in steroids hydroxylationand in the first stage of xenobiotic metabolism).

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The hydroxylation requires the dioxygene activation.It is mediated by cytochrome P450.

● the name: cytochromes P450 have the maximum of absorbanceat 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 biotransformationof xenobiotics (the first phase of it is hydroxylation too !).

● located: 1/ in membrane of smooth ER2/ in the inner membrane of mitochondria

Monooxygenases and CYP (1) :

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● 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 → CYPthe last protein of the chain is „NADPH cytochrome P450reductase“

● in mitochodria is in „electron transfer chain“ involved an additionalcomponent, the iron-sulfur protein adrenodoxin (located betweenthe reductase and the cytochrome)


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● the bond between the two atoms in an oxygen molecule is ratherstrong

● 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 doublebond of bonded oxygen

● radicals are formed : R• from substrate RH (by removing ofhydrogen) and •OH from previous dioxygene.Then –OH group is created from both radicals.


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11

18

20

side chaincleavage„SCC“

hydroxylasedehydrogenase

(-) hydroxylase

Mitochondrial enzymes :

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3A

17

20

21

Enzymes of (smooth) endoplasmatic reticulum :

aromatase(aromatizing complex)

(-) dehydrogenase

hydroxylase

} lyase

(-) hydroxylase

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Side chain cleavage „SCC“) :

1/ the bond is breaked between two carbons, eachof them has bonding oxygen*)

(The carbon nearest to steroid skeleton has the bonding

-OH group, the more father carbon has oxygen in theform 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 neighbouringcarbons 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 remembering17α-hydroxyprogesterone only …

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20,22-dihydroxycholesterol pregnenolone


20

22

20

OOH

OH

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17-hydroxypregnenolonone

17-hydroxyprogesterone

dehydroepiandrosterone

androstendione


1717

20O

OHO

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HO

O

17

HO

17

cholesterol

DHEA= dehydroepiandrosterone

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The relationship amongmain steroid hormones:

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

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Adrenal cortex – the places of corticoids synthesiszona glomerulosa aldosteronezona fasciculata cortisolezona (fasciculata a) reticularis androgens

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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 produceprecursors of glucocorticoids and adrenal androgens

• on the other hand they secrete aldosterone, because the gene foraldosteronsyntase is expressed (in that zone only)

• the synthesis and secretion of aldosterone is controlled byrenin-angiotensin system and by concentration of K+ in plasma

• (the influence of ACTH is very weak and transient)

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Adrenal cortex (2) :

2/ both inner zones - zona fasciculata and zona reticularis

• produce glucocorticoids, androgens (minimumof testosterone) and small amounts of estrogens

• the production of less effective mineralocorticoids (DOC andcorticosterone) is not very important

• the synthesis and secretion controlled by ACTH

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Common structure of corticoids :

21

O

OHCH2

O

( DOC = deoxycorticosterone )

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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

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Order of hydroxylations in corticoids :

11

21

17

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11

21

17

„Complete“hydroxylationpathway incorticoids :

Hydroxyl in the position 21 is always present(the structural characteristic of corticoids)

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Hydroxylation pathwayof corticoids beginningin the position 21 :

11

21

17"-steron"„-sterone“

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Hydroxylation of corticoidskeeps always in metabolic pathways an adumbrated direction17 21 11 (the direction of arrows in schemes „anti-clockwise“).The C-17-hydroxylation can be avoid, whereas the C-21-hydroxylationis obligatory (the presence of hydroxyl represents here the structuralcharacteristic of corticoids, the difference from progesterone).

The presence/absence of oxygen at C-11 dictates the class of glucocorticoidsor 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 carbonatomes i.e. in derivatives of pregnane only. It is not valid elsewhere !)

[ The hydroxylations C-17 and C-21 take place in smooth endoplasmaticreticulum, hydroxylation at C-11 in mitochondria.

Mitochondrial hydroxylation is substantially slower. ]

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„complete“ hydroxylation: 17 21 11

cortisol (hydrocortisone) :( 11,17,21-trihydroxy-4-pregnen-3,20-dion )

OH

O

HO

O

O

2CH OH

1711

21

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Cell location of origin of corticoids :

17-OH21-OH

11-OH SCC

dehydrogenace, izomerace

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CORTICOIDS :

1/ mineralocorticoidsact 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 withretention 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: aldosteroneStructure: C21, mineralocorticoids do not have oxygen in the position 11 .(Aldosterone is the exception, its C-11-hydroxyl is „camouflaged“by forming of hemiacetal.)

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2/ glucocorticoidshave a catabolic effect. They enhance the degradation of proteins and fat.Glucogenic amino-acids from degradated proteins are substrates ofgluconeogenesis (i.e. production of glucose from non-sugar substances).Glucocorticoids increase glycemia and inhibit the inflamatory response andimmune 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).

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

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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+

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Control of biosynthesis :

- glucocorticoids:

1/ diurnal rhythm2/ negative feedback at cortisol (ACTH)3/ stress

- mineralocortikoids:

1/ [K+]2/ systeme renin – angiotensin - aldosterone

2 steroid metabolism

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