Cortisol metabolism in the sheep (Romney breed) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science

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Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

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CORTISOL f1E-[1ABOLISM

nr

^'I'HE ^SHEEP

(Rom11ey breed)

A thesis presented in partial fulfilment of the requirements for the c1ec,ree of f1aster of Science

by

Alan Keith LANGlt-,OHD

Massey University Ne'ir Zealand

1968

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rhe author wishes ^:~o :;hank ^~..;cJ.l..J... ):) supervisor,

GreenHay' ior hi~3 c~e~J.ero1;.s help extended d.urin;; the

course of this study, with appreciation for ~he sur~ical

preparation.J uhicb. B.1loiJec!_ TYLH'Sl1_it of sori~e interesting facets of this i~~estigation.

·N;.anks are due L·O .,_Jrs • . , " S:.;r-ic:clc'. 1cl for collection of the rl:yt:hm ;=;a:,lples; to the r'hysioloc!;y :o:.t~d ~~~-·El. L;o!:ty

Department of ~assey University {or the use of their fa.cili ties an.d a1Lnals; and to Prof. ~. A. Hodges for determining the mass spectra of several steroids isoJuted in this study.

Spec iZJ.l aypreclG a on i.s exte~1<1ed to ;·-tr>s. J. L'a;ylor ,_or typing chi::; tbesis.

·_Co tllo:c:e who asf:j_sted in a.n;y otl1er ;,-;·ay during the cot¹rse of this s t;udy, the a.u·l;hor e~:l:;en.ds his grati~-;ude.

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ABS'~Rl-tC'J:

ri'he metabolism of cortisol in -che normal Romney ewe i/Jas investigated by an:::~.lyeis. of the radioac liive

J0_etabolites excreted in the urine following intravenous (I. V.) e.di-Jinistratic,n of 4-c 14cortieol. '!:he metabolite glucuronides ~·rere hy-~lr~lYf>ed ~!ith b-glucuronicU'lse &'1d extracted fro1r, tho aqu_eouc; mec>._ium \·:ith etLyl acetate.

Eh.e neutral fraction 'Has divided into c--'19 2.nct. c-21 metabolites by sequential elution fro~ a florisil colu:cnn. Extensive use vms made of ·~.J.J.C. for ·che separai.Jion a.Yld analysis of each fraction before the quanti tat ion of ind.ividual components.

i i i

A series of experiments was perfor~ed with sur~icnlly

mocl.ified sheep invol'irin.-: collec cion of bile ancl urine

,, !J

b~th e.f ~~er I. 1/, irqec tion of 4-c ¹-cortisol, and af c(::r intraduodenal infusion of r~dioactive biliary rnet~boli~es

'bt • d f T --- l . • t t . " ¹ 14 t . 1 Tlb

o -alne rom ~.v. acm1n1s ra lon oi ~-c· cor-lso • ~ e metabolites c0llected at each stage were analysed both qualitatively and quantitatiYely.

I'he urine collected each ':lour for 18 consecutive days from a normal sheep, was subjected to colorimetric de-termination for a-ketol and 17-ketogenic steroid

content. I'he data obtained was analysed for diurnal variation in chromogen output, and the daily secretion rate of cortisol· \'Tas estimated.

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...

Fig 0.1 a

CORTISOL

C,3 t7.c

CORTISOL (side view)

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I~-0 ^~\J~;I,T

c;

T.1i~ ·1'ffi~l~

~ .... ..---... ^--~·._ ..._ ... ,

__

.

Nor:1e:.:1clatur~·; 1;~sed t;hroughout; this script is l)a;:wd on tr•.at ovcl 'l;;d fr~v~· the ;::iba Con f e L .. nce llJ :t~onclon ( 1·)50) a;:1d. is gener3.1ly in accordance '!Ti t;b_ ch<} ~~ c-.~_;provecl by ·the

:\.Co ^-E'-()8 EiocheLli:coc;ry (\.·it:1sh.)

§,

2227 ('19CS) o

~.¹he steroid s~celet;on is !Tumbered as ;:;hov.m in ::"if.~. 0. ~ .

1~'or co;npr,ctne;:.;s the torm;: ADcJ.rostan- c.nd 1'rc:;n.::,n- <~re

reducod to A-· ::'l-oci :~-)-, res:t>:.:ctively. Double bonds i:n tho parent ~~ructure are uositioned b~ the suffix -ene-, eg.

P·-¹'-:-ene~-. '':¹he COJJ..Ci:~urc ·(;j_on oJ che hydrogen :~t

c-5

^L.~

Ll.dice.ted by :"i prefix 5a-· or 5b-. '1_¹b.e prefi~: :,i.llo-- ^·:T;lr:n

w=;.::;c~, refers to a

:Ja-

conn_g'.J.r[c·sion ( fi:_-,·. 0.2).

3ubstituents are prefixed by numerical position oncJ.

Gret-;k letter ( v.rhen pertinent)~ t,ut nuc!c:;r;<t;ing :o;:rtic.les

:~he same sicl e :J_f:; the G.ngular methy1 ;sroups r::d; c-1 0 c1nd

iv

c _..-,

3'

are der;i~:;nated - ·-· :)jld rr2.1'f:O ^1'¹i ·~h ;_:; he.s.v-;r line; t:;}"Jose f:0J levi :::he 7.Jlane cf lle rj_ng a.:ce a.·- ^c'X:(l clrc:.· ... ;·~- '· i :. :•.

: ·ro:_ ¹.... -<=><' ~._._1 -·-l l. ·,1'' - \ j ( l' :j_ :~~ 0 0.1 ) .

.,... ··n ,. ..

Jj.'J..i.Jo

c ·--'19 c-2'1

Conjur:;ate

C.R.:I!'o EtAc

EtCm

adrenocorticocrorhic ^~ornone

adrenal cor~ez, usu~lly c-21.

blue tetrnzoliu8 (chloride)

steroid with a two carbon side chain.

formed by esterification with a small

E10lecule eg. (_;lucuronic acid (;,lucu:r.·o _.id.e).

corticotrophin rel?Bsing factor.

ethyl acetate ethanol

Glycerol side chain - =C, OH-CH, OH-CIL)OH

~

a-::etol side chain - -CO-CH 20H

17 ketogenic - c-21 corticosteroid attacked by mild oxidJ..sing agents to give a 17-ketosteroid.

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17 ketosteroid - steroid vlith a ketone on carbon-17.

Kg ;;:iesl hur

8G silica eel

.Gteroid ^.:mcleus ^-~ struc l::;ure b<we,"L on c:'J.e cyclopcntano- -Dhe.nanthrene ^si~:elet;on ^wit~hc-10 and c-13

methyl ^@TOv.ps

·~retrahyc~ro dertvo.ti ve by GOli1plete h;.{dl'Oge:o.ation of ring-A

I'.~'i.A.H. te cral;wti:;.yl s.ramoniua hydroxide .

.1\~)brevic.tion J:lol. fornula

v

cortisol urocortisol

F P-4-ene-11b,17a,21-ol-3,20-one 'TIH'F -'-···

5b

-.t'-__,a, ^·~· ^A 1 "' ' 10~ .·a,~ ^/j

7

r•1 -o 1 --::-one .~ 0 c:.llotetrahydrocortisol ello¹¹ 58.-P-3a, 11 b, 17D., 21-ol-20-one

cortisone E P-4-ene-17a,21-ol-3,11 520-one

urocort:isone ''~HE 5b-P-3a, 17a, 2..-i -ol--;~), 20-one cortol 5b-P-3a, ^'1^'ib, 17a, 20a, 2'l-oJ.

b-cortol 5b-P-3a,11b,17a,20b,21-ol

cortolone 5b-P·-3o., 17a} 20a, 21-ol-11 ~one

b-cortolone 5b-P-3a,17a,2Ch,21-ol-11-o~s

11-0H-Androsterone 11 OEL )a·-Lc-38., ·1·1 b-ol-17-one 11-keto-Lnd.rof:'terone 'i 1KA 5a--!\-3a-ol-11 ₅17-one 11-0H-Etiocholanolono ... \ 1 OEE

5b-A-3a,

11b-ol-17-one 11-keto<:I:tiocholanolone 11l\E 5b-A-3a.-cl---¹1, 17-one

corticoeterone B 1-'-'+--Gne-/l 1 t, ;-::1--ol--3, 20-one deoxycorticosterone 11DOC P-4-ene-21-ol-3,20-one dehyc1rocpiandrosterone DHEA A-5-ene-3a-ol-17-one

testosterone A-4-ene-17b-ol-3-one

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Fig 0. 2

(a)

Androstane

c

^d)

5J3- Androstane

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Chapter

Abs-cract Nomenclature

J~ABLE OF CONTENTS

Terminolo:-;y e_;j_d Abbrevis.cions

page iii

iv iv

1 Review of Literature

1.1 Metabolic effects of cortisol

1.1.1 2lucocorticoid properties of cortisol 1 .1. 2 r·-[ineralocorc:i_coid properties of

corcisol

1.2 ·!}he 'oh~!.:t'811<:A.l cor:~-::..:x

1.2.1 CL,·:mistry and_ ane:::omy

1.2.2 ?~ture of cdrenocorcicsl secretion 1.2.3 ?ate of adrLnal hormone secretion 1.2.4 Siosynthosic of corticost2roids 1.2.5 Cyclic socrGtio~ fro3 the adrenGl

1.3

Chemistry of ~he corticostGroids

1 • 3.1 Dc-riv c,tion of_' Lhe cor tic o~~ ceroid s·cruct·ure

1.3.2 Structural festur8S of the adrenal steroicts

~.3.3 Stcreoc~cuistry of biologicJl .Jc tlvity

1.4 ~he metabolism of cortisol 1 .'-!-.1 Rcduc tion in ring-A 1.ll.2 Si~0 chain cleavage

-1. !-+-.

3

R:::duc t;ion of the :20-ke tone

1.4.4 ~ydroxylation

1.4

.5

Cr:.c cortisol-cor:~isonc equilibrium

1.4.£ R3duction of 17-~cconos

1.5

Steroid conjugation

1.6 ':>teroicl-prote:in ;~:-,sociation in blood

1.7

Ultimate distribu~ion

2 I-latoriRls and Expcri:ncnto_l I';a chods 2 .1 Ilatierials

1 1

rJ

(

17

22 23 27 28 2(3 30 32

33 3G

Lt.Q

42 2.2 Preparation Dnd cc.rc:- oi' experimcYJ.tal shc;.:;p lJ-2

2.3

Collection and preservation of samples ~3

2.4 :extraction of steroid conjugates

43

2.

5

Hydrolysis of steroid con.jugates 44

2.5.1

Assay of b-glucuronidase 44 2

.5.2

Optimt:m pH of paua b-glucuronid.ase 1-1-5

2.5.3

Protein precipitation from paua

stomach

45

2.5.4 Partial purification of paua

b-glucuronid.ase 46

2.5.5

Partial purification of limpet

b-glucuron.idase

47

2.5.6

Enzymic hydrolysis of coajugates

47 2.5.7

Acid hydrolysis of conjuzates

49

•

vi

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2. 6 Extraction of neutr:.:tl free steroid from aqueous solution

2.

7

:~Jtcroid sepc:ration techniques 2.

7.

^'1 Colunm chron.J.cography

2. _,-~

7. :7

;~ ₇ ;rhin _Oe_'-o,-.ti1~'1.-yer _~o-.., o"'' c· chrmns.tor:::ra.Dhy ('T'·~-"1 coc,·'~e""o""'

-

^~- ::; ~ L • o _) . • G -:>" •• L .l J - ' - u ~ '-'' u , ^..1. _,_c_,_,::>

thin layer plates

;:: • fi _,_ ~ethor::l.s o t: corticoste:coicJ. c:1nal ,_~.sis

on 2.E3.1 :lccordinc; of I.\. 2.nd

U.\r.

::.:_::;cctra

~.~.2 Chemical 00difica~ion of ~he

c ortic os teroic.r·

? • >: .• 3 Colorir:1etric ;:r1e i_:ho·: s

:~

• 9

n.adiOChE~i<JiCal :;:'rocedFres

~2. 9.1 ':?nclJ.oo.c t;i vo il<:d7.?ri-.lP

2.' • :? :.~uen0iccYt:ive deter·;in-Tt;ion of r._·_c'1_tocheidc c:.ls

') • ...-! -;_:_:_<.e -:_), 1 ?-ol~11· one '·,rou:p

3.'1.'l Ch8.r';,ci~eriF;::~cion of c-.. '19-C, c-1~:·-._.)

3 • -·;.

² C'~~L-rC'C tc-:ri at ion Of C-1 9-C,

3

.1. :~ Cb:=;.ro.ct~,r~L~-:::.-I; ion of c-·19-·7

3 ..

2 ·:~J1e

-?.5,

?1 '1 ? ~17-ol :~~roU.};'J

) . -:·' .1 C~-~.rcc tcrLJs.tion of c-1 ')-2, c-1

S-3

3.2.2 ChPracterisation of c-19-1, c-19-4

~5. :~. :-; Chc:,ractc::ri::_;.s.tion of c-1 ~:-5

_:::.. -~ ClJRr~'1.ct-:;ri::-;c_:;:;_on of ,_:lw c-.~'1--.i\. seri22 ::: ,-, -'u~---,-~l· ^·i-;~-l· ^o·1 o-<· c111·-;:1-•·.-el'---:,oo1l' :·es

_/ • ( \ .~ LA~l - U C~ U ..l .L ·-· -J. J (.... _!_ 'J

InYGB~i~ation into ~he oath••ay~ of cor !Ji c:.'ol :-netcl.~)Q lite excre t;\on

50 51 51 53 57 59 59

6C)

G4

66 -·,-·- .~:-0

/ . - . ...)\.)

,-:;o

\.._) .)

T' 70 73 74- 75

7C:

77 77

?C 70

f""l C:)

r:'5

( )

3.c.1

:~~X:)·IJ.-1 Urino.ry excretion of c ¹L!--2

~et2bolites j_n ~hole sheep 86 -, ') ._, ' 2 ·••-,,- ·'-- • f 14 ;_._,.•. ) C · : - • d :;_,.,_;.,__ _:~x:fYG.- .. ~ ... creLJ~on o c - .. c<:-v( • • • _,erol _

in bile c:.nd urine 37

3. c;. 3

h:d;b.".'ays of cortisol excretion 88

_ 3. 9

Investi0atio:£J. of diurnal V8riations in

urin2ry metabolite excretion

90

3.9.1

_:(.uantitation of B.;_(l.,,. chromoz:en

91 3.

<). 2 Quanti cation of 'l-'il1.PElrman

chromogen

92

4 Discussion

4.1 Hydrolysis of steroid conjuza:;es 4.2 Excretion of steroid conjugates

93 95

vii

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viii 4. 3 J~xtraction of free steroid 95

'-!-. 4 Ghrorcts. coc;raJ)hic analysis of

corticosteroids

96

4.5

aoute of disposal of cortisol

97

4.6 ~iecc:.bolis"l of cortisol in ul:wle .sheep

99

L~. 7 .·i~ecai:Jolism of cortisol ijl surc;ic~?,lly

modified sheep '1 02

4.8 t~thods of colorimetric estimation of

corticosteroids 106

L~.

9

Colorime !Jric estimation of 17-o~;-c;J in

_,_, Gne r•, ufi80p . iD :;he sheep - 1 OJ

4.10 Cortisol production rate in the shee; 111 4.11 Diurnal variations of corticos~eroid

netaboli-Ges in sheerJ urine 1 'I 1

.!umnary

Literature cited

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LIST OF 1'AJ3LES

1-1 Cortisol secretion rates

page 14-a 1-2 Plasma corticosteroid binding capacity . 37~

1-3

Distribution of total plasma cortisol Extraction of conjugates from urine Elution characteristics of florisil 2-2

3-1 3-2 3-3

IJist of c-19 metc.bolit;e,s ('\'I hole sheep) •

Digi~onin prscipitntion of c-19-6,~

]3oro1_13t(1_ri0.e recilict;ion of c-'1 s~-()' 'j

7-4

:3orohyclriCLe re(L"l,ction of c-'1 ::::··8, C)

£\RHr values i'or 1'1;::0 rc<~1.1.c\:iion •

3-6

Iloro~1ydrid.e rorJuc '~ion of c~-19-7

3-7 •.

¹.L.C. of c-1S-2,::J.

3-S

^Cr07 oxidation of c-19-~,3 :::;

3-10 Compe,ri3o:n of c-19-'? ,,~ v:i::;h c-19-~~,3

3-11 ·2.L.C. of c-19--1,'-!- •

•

3-"12 3-'1)

n.

C:·-~'lcl 8;(_-:~c va.lues for 1'(1.;-17:'. ^c~·iLlorisE, ::;ion

1'·1 • J.

.!~.L.C. of 17·-ko;;o::oi.:;eroids •

•

'-!-5a

52

^a

S9a

728.

738

7)o.

73a

76a

7-.-.., ^vr.!..

77a

r?~-oa

7:

^{• ... )C--}^~

' 7 _{~-I ~}/1/_ n vr,_;., OXlo.a·clOll OJ. r· . , ' . -"

17 -

·-·.:e cos

'

.:;erOlCi.f:

' . ,

o')'"'-'o ( __ ,

)

3-15

J~orohydride reduc ·cion of ·17-Le co'~ tieroidr; '(\_ J~,

3-16

3-17

^!..^l^{•..J. ..}^'^:.\.i.^.-,

of redt_-._cecl_ 17-l.:etoD teroid 3-'1 3 ·~uantit2.tion of c-2'l E1808bolites 3-19 T.L.C. of acetyl~tcd corbols 3-20 Mild oxidation of c-21 fraction

:J-c:)1 AR~

1

v<,lucs for 11 ==0 reduction • • 3-22 Urinary excre cion of I. ~-T. --cortL'1ol • 3-23 Excretion of ketosteroid

3-24 I~xcrotion of F in modified .sheep

3-25 r~uantitatiive .:;,nalysis of experimenti-3 4-1 Diurnal excret;ion of ketonic chromogen

in t;he cow • •

• •

'79b

8LJ-a

;jll.a

87a 87a 89a

• 112

ix

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LIWP O:B' :B'IGURES

- - - -

0.1 ~~;truccu.re of cortisol _• • • • i i i a

0. 2 Eo::Je:.lclilture of s l::;eroicJs V2

1 .1 /-iosyn.-che sis of cortisol _• • 15a

2.:; He i~hanolic fr::;.c t;ion-~tion of b-c,lucl.'.J.'()l)i(: ,_, se Lkia

;__:. LJ- E:)Tclroly;';is ^of (Jtc:roid co21 jtF:ate ·- buffer ^LJ.(}a

~:.

5

IIydrol ~Tsis of ~-'tcre:id con,ju.: ::'-ce -- nrine

2.7

!i'lorir;il colu .. m fr,_c .:;irm;<;ion ( .. I<e:..::E).

If II

l '

If (2.5;~ eOH)

(5~~ .leOH).

;::.-10 ::;'roctio:1a.tion of -':--c ¹⁴--J.:, met: bolites

3.--,

^Sc:m ^ofc-'19 :.1~.c. cccnar2.tion • --.., /) _{)• 1a} ;'-.:JC<:-1D 0 ~- f "'7 I - .. eGOSuC;:::'OlO. ¹ ^' ^L- ^• ^.,

•

'+8b

50

a

,- ')1

/ __ ()

52c

5 '),

_~a 52e 63a

=.;.

2 _.;_u GOrc.clio2r,:ph of c-'1

S

-~. L. C. sepc:LrD. jion G~l~.L

3.':;· -~~epnrc;::;ion of c-19 .frc"'.Ccion :Ln~o C>:.i.'}:'OEGll\;s 7C)::-, ).4- :Scan of c-21 scr::~.racion. by ·r.L.C. C_\1o.

3.

4a .i\.ucor:::,diogrG.~•h of c--·21 ·.'.I:. C. sup::crc..,_ c5.on

3. 5

.-2uan :;i ta·cion of c-21 ;;wtr-_:)oli·~~es

3.6

a,b,c,d,- scc:m of IVB, I'!U, J.?E, IFU 'i',J.J.C.

chroi,lato~:·:r2::.1S res pee t i vely

3.7

Diurnal excre~ion of J-~etol

3.8

Diurnal excretion of 17-~GS •

3.9

Urine volume variations _• _•

• 92a.

92b 4 .. 1 Com::c'"~arison of a.-l::e·:,ol and 17-ICGS excretion • 1'1L1-a 4.2 Diurnal excretion of reducing corticoids

in

rams • •

114b

Overall Eletabolism of corG"isol in the sheep 118a

X