2 steroid metabolism


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2 steroid metabolism

  1. 1. 1 1
  2. 2. Biosynthesis of steroid hormones © Department of Biochemistry (V.P.), Faculty of Medicine, MU Brno 2011 2
  3. 3. The origin of hydroxymethylglutaryl-CoA : CO S CoA COOHH CH2 acetyl-CoA CH2 H2O CoA SHO C CH3 HO C CH3 CH2 CH2 CO S CoA CO S CoA acetoacetyl-CoA hydroxy.methyl.glutaryl-CoA HMG-CoA (cytosol) 3
  4. 4. HMG-CoA reductase COOH COOH 2 NADPH + 2H+ CoA-SH CH CH 2 2 4HHO C CH3 HO C CH3 CH2 CH2 CO S CoA CH2 OHhydroxymethylglutaryl-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. 5. COOH - CO2 CH 2 - H2O CH 2HO 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. 6. Synthesis of isoprenoids :All isoprenoids are synthesized from acetyl-CoAby way of isopentenyl diphosphateand its isomer dimethylallyl diphosphate. isopentenyl diphosphate dimethylallyl diphosphate 6
  7. 7. SQUALENE C30( triterpene, 6 isoprene units, symmetry ) 7
  8. 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. 9. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 9
  10. 10. cholesterol 3 5 HO ( cholest-5-en-3-ol ) 10
  11. 11. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 11
  12. 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. 13. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 13
  14. 14. progesterone : 20 O 34O ( 4-pregnen-3,20-dion ) 14
  15. 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. 16. O pregnenoloneHO OO progesterone 16
  17. 17. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 17
  18. 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. 19. O progesteroneO 21 CH 2OH HO 11 17 O OH cortisolO 19
  20. 20. O progesteroneO 21 CH 2OH HO 11 O corticosteroneO 20
  21. 21. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 21
  22. 22. aldosterone : O CH2OH C HO H 11 OO( 11,21-dihydroxy-3,20-dioxo-4-pregnen-18-al ) 22
  23. 23. aldosterone (hemiacetal) : OH CH2OH C O H 11 OO( 11,18-epoxy-18,21-dihydroxypregn-4-en-3,20-dion ) 23
  24. 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. 25. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 25
  26. 26. testosterone(„TST“) OH 17 34O ( 17-hydroxy-4-androsten-3-on ) 26
  27. 27. 17 O OHO 17α-hydroxyprogesterone O 17 androstenedioneO 27
  28. 28. O 17 androstenedioneO OH 17 testosterone (TST)O 28
  29. 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. 30. Testosterone cca 5 mg / d, adult man O(TST) : 17 OH oxidation 17 ~ 90 % 17-ketosteroidsaromatization ~ 1-5 % androsterone reduction etiocholanolone ~ 4 (- 8) % reduction ~ 2 % 5-dihydrotestosterone estradiol (E2) androstanediolWhat 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- testosteron2/ 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. 31. Testosterone (TST) :the origin of 17-ketosteroids from testosterone comprises the reduction of conjugateddouble 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. 32. Androgens in man : OH O OH 17 A BO 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. 33. OH 17 testosterone 3O O 17 androsterone 3HO substituents are „interchanged“ in the positions 3 and 17 double bond is not present 33
  34. 34. Testosterone (TST) : cholesterol A B A B HO O pregnenolone progesteronesteroid skeleton (A/B) steroid skeleton (A/B)like cholesterol has conjugated bonds androstenediol TESTOSTERONE (TST) „prohormone“ E2 5-dihydrotestosteroneThe time changes: foetus + newborn  testosterone, child  androsterone, adult  testosterone 34
  35. 35. cholesterol (C27) pregnenolone (C21) progesterone (C21)glucocorticoids androgens (C19) (C21) mineralocorticoids (C21) estrogens (C18) 35
  36. 36. estradiol(„E2“) OH x 17 3 HO ( 1,3,5(10)-estratrien-3,17-diol ) 36
  37. 37. The elimination of „angular“ methyl C19 (  estrogens ) O O H CCH 3 CH2OH C OH H hydroxylase dehydrogenase lyase (19-) (19-) (19-) ENZYMES OF SMOOTH ENDOPLASMIC RETICULUM ( in the complex „aromatase“ ) 37
  38. 38. (for rough orientation only !!)Consecutive reactions of „aromatase“ : NADPHaltogether 3 „monooxidase“ reactions(required NADPH + O2) androstenedione enolisation 19-oxo NADPH NADPH formic acid estrone („E1“) 38
  39. 39. „Aromatase“ :● reaction: aromatization of A ring and C-19 demethylation● enzyme: P450arom (= aromatase), CYP 19 EC● 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. 40. Estrogens in woman :aromatase: ovaries liver: conjugation with GlcUA (placenta) with PAPS liver adipose tissue skin excretion: the urine, the bileblood plasma: binding on SHBG 40
  41. 41. OH testosterone (TST)O OH estradiol (E2)HO 41
  42. 42. O 17 androstenedioneO O estrone (E1)HO 42
  43. 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. 44. MonooxygenasesRH + 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. 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. 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. 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. 48
  49. 49. Mitochondrial enzymes : hydroxylase dehydrogenase side chain(-) hydroxylase 18 cleavage „SCC“ 20 11 49
  50. 50. Enzymes of (smooth) endoplasmatic reticulum : hydroxylasearomatase 21(aromatizing complex) 20 } lyase 17 A (-) hydroxylase 3 (-) dehydrogenase 50
  51. 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. 52. Side chain cleavage „SCC“) : 20 OH OH 20 22 O20,22-dihydroxycholesterol pregnenolone 52
  53. 53. Side chain cleavage „SCC“) : 20 O O 17 OH 1717-hydroxypregnenolonone dehydroepiandrosterone17-hydroxyprogesterone androstendione 53
  54. 54. 17 cholesterolHO O 17 DHEAHO = dehydroepiandrosterone 54
  55. 55. CH3The relationship among C Omain 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. 56. Adrenal cortex – the places of corticoids synthesiszona glomerulosa  aldosteronezona fasciculata  cortisolezona (fasciculata a) reticularis  androgens 56
  57. 57. Adrenal cortex (1) :● three zones in histological picture● cells with two functional units, with different enzymatic equipment → different products, that are controlled independently1/ 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. 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. 59. Common structure of corticoids : 21 CH2 OH O O ( DOC = deoxycorticosterone ) 59
  60. 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. 61. Order of hydroxylations in corticoids : 21 11 17 61
  62. 62. „Complete“hydroxylationpathway in 21corticoids : 11 17Hydroxyl in the position 21 is always present 62(the structural characteristic of corticoids)
  63. 63. Hydroxylation pathwayof corticoids beginningin the position 21 : 21 11 17 "-steron" „-sterone“ 63
  64. 64. 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 endoplasmatic reticulum, hydroxylation at C-11 in mitochondria. Mitochondrial hydroxylation is substantially slower. ] 64
  65. 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. 66. Cell location of origin of corticoids : 11-OH SCC dehydrogenace, izomerace 17-OH 21-OH 66
  67. 67. 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.) 67
  68. 68. 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). 68
  69. 69. 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. 69
  70. 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. 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. 72