- Email: [email protected]
A study of MER-29 in experimental animals
Study of MER-29 in Expe
men
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[~?] O. X.V. PORTMAN, JEA'-; M2~'i'I-;R AND D. M . HEGSTEi) UR INTER~T in t e s t i n g M E R - 2 9 in e x ~ r i m e n t a l a r d m a t s h a s been twofold. T h e r e is ! he p o ~ i b i l i t y t h a t inLormation a b o u t the :=~rdm cholesternt lowering potel,ey, the toxicolog3-, a n d the effect~ on the histc, genesis of experim e n t a l athcrosclero.~i~ m a y | ~ helpful fin evall~ating its u~dulne~s for m a n . P e r h a p s , m o r e i ~ l p o r t a n t t y f r o m our p o i n t of view, a c o m p a s s e s of t h e relative effect i v e r l e ~ in loweri~ng t~m ~ e n l m cholesterol |evel of different species a n d of a n i m a l s fi~l diffot~nt diets, a s well as d a t a at;~)u~ t h e s y n t h e s i s of cholesterol az,d t h e a c c u m u l a t i o n of i n t e r m e d i a t e s , m a y help c
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F i g . I . - - T h e e t T e e t a f f c ~ J l i n g ,~IEIt:-2~t {If| m g . / k g , o f I ~ l y ~ ' e i l ~ h t ) ot~ t h , " ~ , t ' r ~ , ~ ehot~teroi levd of C~.btis ~onkeys. T i a t e zert~ r ~ v ~ e n l s t h e a p p ~ t t c l ~ I s l c a d y s t a I tvalu~ o n d i e t s ( s l e r o t - f r e e ) s u p p t y i ~ s g 45 i w r c e n t o f (.altsrir~a a s ¢-o~'~t~ttt ,til o ¢ t , ttn~
oil. to. m e a s u r e l o t a l digit~min preeipitaifle step,is were nc, l m a d e r ~ u t i m q y d~riz,g ~hi;e x p e r i m e n t , b u t a iimitc~_l n u m b e r of such mc.,tsur~meJit~ pen'orntc~t dtlrh~g .~I I~R~9 t r e a t r a e n t ,~uggested t h a t t|u.. reducti(ms tff t~tal ~ , r u m ~-b::droxy s h - r . i s w~-r,not. ~J gTeat a s tho~- m e u t i o n e d abt~ve for vh~h(.s/er.I. A c e ~ t e - l - C " was a d m i n i s t e r e d to two m o n k e y s (o~m tin ~.ovn ~fiI di{.t a ~ d {n.* on t h e ec,c~unut oil diet), a n d m e v a i e m a ~ e . 2 - C " to t h e o t h e r two m o n k e y s d u r i n g t h e t r e a t m e n t p~.ric.d. T h e v.ame mot~kc-ys were s i m i l a r l y i n j ~ v t ~ t d u r i t | g the control ~ ( ~ . ~ r u m lipids were o b t a i n e d t¢t two hour.~, f o u r hour.% one d a y , four da:r~% arid ~ d a y s a f t e r a d m i n i s t r a t i o n of the bx~e|ed ceanp~mfids, m i d w~.rt. .~parat~.wJ on silieie tteid e o l m n n s u~to Olrc-c fractions, col~tainit~g reSl~,Ctive.ly ( t ) ehole~-terol , (2) triglycerides, free eholes~terM, arid f r ~ fi~tty acid.% a n d (3) phosph~iipidu. R a d ~ff 0 m ll~m-.~4tlxmifiabh" a n d safnmifiai)h" corn s of Oae t h r e e f r a c t i o n s ~ a s t~a (,~. F i g u r e 2 indiczlt~:m t h e r u ~ tff uppear~tnce a n d di~tp~:ara~t¢~, of tim non~ p o n i f i a b l e e o m p o n e n t ~ im~lated trfuzr alkali~,e h y d r o l y s i s f r . m t h e fracti.~,s c o n f i n i n g *~t~r c h o l ~ t e r o l a n d fr~-e ch~jteatero! for onv monk~,y g l w m me~'ahma t e2-C~' durlttg t h e eo~,tro! ~ r i r g J . T h e o r d i n a ~ expresses "appa~'enU' specitie activities. T h e r e wa~ no s t r i k i n g difftrrenct, itt the ~ n a x i m u m h e i g h t s ¢~r sI-pvs . f t h e i r eu.rVc,~ bct,ween tho",¢~ o b ~ i n e d d u v h | g t h e t r e a t m e n t i~:~4txl a n d t h t ~ , ~f tim eo~trol l-reri~ fi~r a n y ~ff the four m o n k e y s . " S i n c e it was l i k e l y t h a t t h e n o n - ~ p o t d f i a b l e s isolaivd from t h e ~iliei¢, . r i d e h r o n m t o g r a p h i e fraetiotm e o n t a i n ~ t la!~h:d ~mtterlats o t h e r that~ cholesterol,
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t h e ~mn-saponifiable fraetiCms wern r e e h r o m a l o g r a p h e d on mineral oil imp r e g u a l e d p a p e r . T h i s tatl.er pre~:edur~: h a s p r o v e d zzseful ix, ~-oparating chole~terol from ~,t,her s~erols. ! u figure 3 are s h o w n t|m rikdi~ehrr~matographs ~)0 per cent e0~anol a,~ ~he movi~tg p h a ~ , ) of a u t h e n t i c ( hotesterol-4-G a n d t h e non-saF~'~mfia~|e e , ) m p o n e n t s i:~bttx.d f r o m ~.he free a n d e s t e r cholesterol fraetirms rff ~ r u m t w e n t y - f o u r h o u r s rifler admini.~tn~li~m of m e v a l o n a t e - 2 - C ~* a t I5 u moles per K g . of l~?~dy weight. Chr~m~lr}gnm, s tare shown f(,r one m o n k e y d u r i n g the t r e a t m e n t ~ r i ¢ × t and d u r i n g the r~,~drol period. T h e labeled n o n - s a w m i f i a b t e s from F r a c t i o n 2 - - t h e free ehoh:slerol fratrti(n~-o~apF~x~red to t~e a u t h e n t i c eholesterol, atthc, ugh t h e r e w:,s a e~msistent p,u~k_, in a n ar,::i of t,.s~.r pr~laNw d u N n g . M E I I - 2 9 t r e a t m e n t . "I]~e ~,~n~-sap~mifiaI)te p o r t i(m elf t h e cImleslerot e:4ter fraction c o n t a i n e d a large :~m(m~! of n(m-p(~lar m a t e r i a l M~ieh dM not m o v e on t h e paper. D u r i n g 5 1 E ~ - ' 2 9 t r ( ' ~ l m ( ' n | the,re w:~s :;1~ ~ t'~ak ~ff i n t e r m e d i a t e gm|ari~y w h i c h corrvspcmdM te~ tho les~ p~tar peak of F r a e t i o n 2 d u r i n g t h e t r e a t m e n ! perinc]. T h i s e o m p o u n d was, l heref(~re, p r e s u m a b l y i~artiatiy esterifie(t in I g. At longer periods a f t e r m e v a l f m a l ( , admini.*tratitm, the prrq)ortion of m a t e r i a l otlier t h a n ('tmles~erol i~ lho.~, fra~etions h a d d e e l i n ~ t , a l t h o u g h the e x t r a p e a k s ~r~i.ated :z~ te~ d a y s a f l , , r nv'vM(~n:zt~* a d m i n i . ~ r a t i~m T h e e x t r a t)eak.~ (not cholesterol) r(,pr~..~(,l~te[l a fa~-ger pr~p(~rli~'m ~d l h e Iab~,le,d non.m~ponifiableu in l h e s e r u m
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Fig. 3.~.The ehromatographle behavior of lhe no~t-~nl~+nifiubte eon+i~rt|t.., of Fraello+l 2 (Free ~te~| ~m~nent) a r i d F r n e t ; o n I ( F . ~ t e r i f i ~ l ster+-~! e+,r+ll~S~:tt-nt + f r o t h t h e s e r a o f ~ Celmua m o n k e y t n j ~ t M with ~ne~'alonate-2-(2 +dr;rio 8 tile e,,tltml ~iM I n d d t ~ r i n g ~|ER,,'.~ a d m i n i , t r ~ t t i o n . ( L h r o m n t o i ~ r a p h } wn,~ l ~ e . r f ~ r l t ~ l on ntineral oil inlpretgnatM p~r w i t h ~ ~ r c c i t t elht~+l+d a~ +rnn~itt~ p h a ~ e + " ' F ' * ind|eat~ the ,oh-~t front.
during M ER-29 t r e a t m e n t d m n during the eol+trol ~ r i + ~ . "~te eo~lpound~ inv o l v ~ have zmt yet helm characiefiz~| or ide~tifi~'~J+ In t h ~ - v-:t~-zrin+e++~s mevalonat~ a p ~ a r e d to t ~ about fifteen thnea mo~t efficient ly u.~ed .~ a choh..s. t e ~ l preeur~'~r that+ wa~ acetate. The t ol M E l t - 2 9 on the relative hylx:r+:hc~lesteremia prt~.~+t i+~ the obe~se-hyt.~rglyeemie s t ~ i n of n m ~ v:tL,~al~J evaluated. Eight oI~..~~. mie+, {lq,ur male, four fema|e) w e ~ fed Purlieu Lak~r~ratory Chow, a , d an app~tr(,ntly idtmtie~d ~ o t i p wa.s fed Ole ~ m e diet p;u8 ~ rag+ of M ER-~3 ~,r Kg. of Ixxly woight per day. At the end of four weeks the mere3 ~2nlm eho|e~terot of the treut~=~l'grol~p ",v~s 107 rag, I~-r eI,+tR e0n.~pt~red ~) t5(~ rag. |o~reent for thz: e(mlro| group (p < . 0 0 l ). A ¢~erie.-~ of adulL
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arid 8t1 rag. per Kg. body w¢,ight~. There was no effe~;t on Ihe ~.rum ehoi<-sloroI lev~,l. In Ihe la~.ter exr~.'rinwn~s ~olal serum ~erols wer~ n¢~ e.~ima~e(|. i.r,~men~ ~r ~.~m|rol [~*rie~ds for !he m~mkeys ~r eaF~mS. The obr.so mice which X[|. |e 3~| tI'~*I||I~IIPII| gailmd le~,s weight lhan th~~ u ~ I r e a w , t e
ME|{-29 w~is ~*v:duat,~.d in three ~peeios: lhc (>bu.~ m~mkey, the ob~,:~-,-hyp.of glyeelni~, .-tr:ii~ of m~m~,, told the capon. "l'|~e serum eim|~-'t~rol was |~wered in lhe nm~lkey m~d tl~>~s~- hul noI i1~ t|t~" a~lmr~. Studi~,s of the app(,aran¢'(, and di.~app~:~ralw,, of |a})e|ed :~l~,rol~ fr(m~ the .~-o~l of ('aims mo~keys inj(.e~ed wilh r;Mi,~-tn(,V~,irmalO dtlrt~)~g thf, ee~ltr~)] and 5IEP,-29 trealmetti p¢,ri(~t were tm,:~,l~led.
'l'J~: ('~C~l!t.~laX: Thank you, 1~. Porunan.
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[~?] O. X.V. PORTMAN, JEA'-; M2~'i'I-;R AND D. M . HEGSTEi) UR INTER~T in t e s t i n g M E R - 2 9 in e x ~ r i m e n t a l a r d m a t s h a s been twofold. T h e r e is ! he p o ~ i b i l i t y t h a t inLormation a b o u t the :=~rdm cholesternt lowering potel,ey, the toxicolog3-, a n d the effect~ on the histc, genesis of experim e n t a l athcrosclero.~i~ m a y | ~ helpful fin evall~ating its u~dulne~s for m a n . P e r h a p s , m o r e i ~ l p o r t a n t t y f r o m our p o i n t of view, a c o m p a s s e s of t h e relative effect i v e r l e ~ in loweri~ng t~m ~ e n l m cholesterol |evel of different species a n d of a n i m a l s fi~l diffot~nt diets, a s well as d a t a at;~)u~ t h e s y n t h e s i s of cholesterol az,d t h e a c c u m u l a t i o n of i n t e r m e d i a t e s , m a y help c
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F i g . I . - - T h e e t T e e t a f f c ~ J l i n g ,~IEIt:-2~t {If| m g . / k g , o f I ~ l y ~ ' e i l ~ h t ) ot~ t h , " ~ , t ' r ~ , ~ ehot~teroi levd of C~.btis ~onkeys. T i a t e zert~ r ~ v ~ e n l s t h e a p p ~ t t c l ~ I s l c a d y s t a I tvalu~ o n d i e t s ( s l e r o t - f r e e ) s u p p t y i ~ s g 45 i w r c e n t o f (.altsrir~a a s ¢-o~'~t~ttt ,til o ¢ t , ttn~
oil. to. m e a s u r e l o t a l digit~min preeipitaifle step,is were nc, l m a d e r ~ u t i m q y d~riz,g ~hi;e x p e r i m e n t , b u t a iimitc~_l n u m b e r of such mc.,tsur~meJit~ pen'orntc~t dtlrh~g .~I I~R~9 t r e a t r a e n t ,~uggested t h a t t|u.. reducti(ms tff t~tal ~ , r u m ~-b::droxy s h - r . i s w~-r,not. ~J gTeat a s tho~- m e u t i o n e d abt~ve for vh~h(.s/er.I. A c e ~ t e - l - C " was a d m i n i s t e r e d to two m o n k e y s (o~m tin ~.ovn ~fiI di{.t a ~ d {n.* on t h e ec,c~unut oil diet), a n d m e v a i e m a ~ e . 2 - C " to t h e o t h e r two m o n k e y s d u r i n g t h e t r e a t m e n t p~.ric.d. T h e v.ame mot~kc-ys were s i m i l a r l y i n j ~ v t ~ t d u r i t | g the control ~ ( ~ . ~ r u m lipids were o b t a i n e d t¢t two hour.~, f o u r hour.% one d a y , four da:r~% arid ~ d a y s a f t e r a d m i n i s t r a t i o n of the bx~e|ed ceanp~mfids, m i d w~.rt. .~parat~.wJ on silieie tteid e o l m n n s u~to Olrc-c fractions, col~tainit~g reSl~,Ctive.ly ( t ) ehole~-terol , (2) triglycerides, free eholes~terM, arid f r ~ fi~tty acid.% a n d (3) phosph~iipidu. R a d ~ff 0 m ll~m-.~4tlxmifiabh" a n d safnmifiai)h" corn s of Oae t h r e e f r a c t i o n s ~ a s t~a (,~. F i g u r e 2 indiczlt~:m t h e r u ~ tff uppear~tnce a n d di~tp~:ara~t¢~, of tim non~ p o n i f i a b l e e o m p o n e n t ~ im~lated trfuzr alkali~,e h y d r o l y s i s f r . m t h e fracti.~,s c o n f i n i n g *~t~r c h o l ~ t e r o l a n d fr~-e ch~jteatero! for onv monk~,y g l w m me~'ahma t e2-C~' durlttg t h e eo~,tro! ~ r i r g J . T h e o r d i n a ~ expresses "appa~'enU' specitie activities. T h e r e wa~ no s t r i k i n g difftrrenct, itt the ~ n a x i m u m h e i g h t s ¢~r sI-pvs . f t h e i r eu.rVc,~ bct,ween tho",¢~ o b ~ i n e d d u v h | g t h e t r e a t m e n t i~:~4txl a n d t h t ~ , ~f tim eo~trol l-reri~ fi~r a n y ~ff the four m o n k e y s . " S i n c e it was l i k e l y t h a t t h e n o n - ~ p o t d f i a b l e s isolaivd from t h e ~iliei¢, . r i d e h r o n m t o g r a p h i e fraetiotm e o n t a i n ~ t la!~h:d ~mtterlats o t h e r that~ cholesterol,
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t h e ~mn-saponifiable fraetiCms wern r e e h r o m a l o g r a p h e d on mineral oil imp r e g u a l e d p a p e r . T h i s tatl.er pre~:edur~: h a s p r o v e d zzseful ix, ~-oparating chole~terol from ~,t,her s~erols. ! u figure 3 are s h o w n t|m rikdi~ehrr~matographs ~)0 per cent e0~anol a,~ ~he movi~tg p h a ~ , ) of a u t h e n t i c ( hotesterol-4-G a n d t h e non-saF~'~mfia~|e e , ) m p o n e n t s i:~bttx.d f r o m ~.he free a n d e s t e r cholesterol fraetirms rff ~ r u m t w e n t y - f o u r h o u r s rifler admini.~tn~li~m of m e v a l o n a t e - 2 - C ~* a t I5 u moles per K g . of l~?~dy weight. Chr~m~lr}gnm, s tare shown f(,r one m o n k e y d u r i n g the t r e a t m e n t ~ r i ¢ × t and d u r i n g the r~,~drol period. T h e labeled n o n - s a w m i f i a b t e s from F r a c t i o n 2 - - t h e free ehoh:slerol fratrti(n~-o~apF~x~red to t~e a u t h e n t i c eholesterol, atthc, ugh t h e r e w:,s a e~msistent p,u~k_, in a n ar,::i of t,.s~.r pr~laNw d u N n g . M E I I - 2 9 t r e a t m e n t . "I]~e ~,~n~-sap~mifiaI)te p o r t i(m elf t h e cImleslerot e:4ter fraction c o n t a i n e d a large :~m(m~! of n(m-p(~lar m a t e r i a l M~ieh dM not m o v e on t h e paper. D u r i n g 5 1 E ~ - ' 2 9 t r ( ' ~ l m ( ' n | the,re w:~s :;1~ ~ t'~ak ~ff i n t e r m e d i a t e gm|ari~y w h i c h corrvspcmdM te~ tho les~ p~tar peak of F r a e t i o n 2 d u r i n g t h e t r e a t m e n ! perinc]. T h i s e o m p o u n d was, l heref(~re, p r e s u m a b l y i~artiatiy esterifie(t in I g. At longer periods a f t e r m e v a l f m a l ( , admini.*tratitm, the prrq)ortion of m a t e r i a l otlier t h a n ('tmles~erol i~ lho.~, fra~etions h a d d e e l i n ~ t , a l t h o u g h the e x t r a p e a k s ~r~i.ated :z~ te~ d a y s a f l , , r nv'vM(~n:zt~* a d m i n i . ~ r a t i~m T h e e x t r a t)eak.~ (not cholesterol) r(,pr~..~(,l~te[l a fa~-ger pr~p(~rli~'m ~d l h e Iab~,le,d non.m~ponifiableu in l h e s e r u m
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Fig. 3.~.The ehromatographle behavior of lhe no~t-~nl~+nifiubte eon+i~rt|t.., of Fraello+l 2 (Free ~te~| ~m~nent) a r i d F r n e t ; o n I ( F . ~ t e r i f i ~ l ster+-~! e+,r+ll~S~:tt-nt + f r o t h t h e s e r a o f ~ Celmua m o n k e y t n j ~ t M with ~ne~'alonate-2-(2 +dr;rio 8 tile e,,tltml ~iM I n d d t ~ r i n g ~|ER,,'.~ a d m i n i , t r ~ t t i o n . ( L h r o m n t o i ~ r a p h } wn,~ l ~ e . r f ~ r l t ~ l on ntineral oil inlpretgnatM p~r w i t h ~ ~ r c c i t t elht~+l+d a~ +rnn~itt~ p h a ~ e + " ' F ' * ind|eat~ the ,oh-~t front.
during M ER-29 t r e a t m e n t d m n during the eol+trol ~ r i + ~ . "~te eo~lpound~ inv o l v ~ have zmt yet helm characiefiz~| or ide~tifi~'~J+ In t h ~ - v-:t~-zrin+e++~s mevalonat~ a p ~ a r e d to t ~ about fifteen thnea mo~t efficient ly u.~ed .~ a choh..s. t e ~ l preeur~'~r that+ wa~ acetate. The t ol M E l t - 2 9 on the relative hylx:r+:hc~lesteremia prt~.~+t i+~ the obe~se-hyt.~rglyeemie s t ~ i n of n m ~ v:tL,~al~J evaluated. Eight oI~..~~. mie+, {lq,ur male, four fema|e) w e ~ fed Purlieu Lak~r~ratory Chow, a , d an app~tr(,ntly idtmtie~d ~ o t i p wa.s fed Ole ~ m e diet p;u8 ~ rag+ of M ER-~3 ~,r Kg. of Ixxly woight per day. At the end of four weeks the mere3 ~2nlm eho|e~terot of the treut~=~l'grol~p ",v~s 107 rag, I~-r eI,+tR e0n.~pt~red ~) t5(~ rag. |o~reent for thz: e(mlro| group (p < . 0 0 l ). A ¢~erie.-~ of adulL
A . I ! li~
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ENi'EIIIMENTAI,
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5:~5
arid 8t1 rag. per Kg. body w¢,ight~. There was no effe~;t on Ihe ~.rum ehoi<-sloroI lev~,l. In Ihe la~.ter exr~.'rinwn~s ~olal serum ~erols wer~ n¢~ e.~ima~e(|. i.r,~men~ ~r ~.~m|rol [~*rie~ds for !he m~mkeys ~r eaF~mS. The obr.so mice which X[|. |e 3~| tI'~*I||I~IIPII| gailmd le~,s weight lhan th~~ u ~ I r e a w , t e
ME|{-29 w~is ~*v:duat,~.d in three ~peeios: lhc (>bu.~ m~mkey, the ob~,:~-,-hyp.of glyeelni~, .-tr:ii~ of m~m~,, told the capon. "l'|~e serum eim|~-'t~rol was |~wered in lhe nm~lkey m~d tl~>~s~- hul noI i1~ t|t~" a~lmr~. Studi~,s of the app(,aran¢'(, and di.~app~:~ralw,, of |a})e|ed :~l~,rol~ fr(m~ the .~-o~l of ('aims mo~keys inj(.e~ed wilh r;Mi,~-tn(,V~,irmalO dtlrt~)~g thf, ee~ltr~)] and 5IEP,-29 trealmetti p¢,ri(~t were tm,:~,l~led.
'l'J~: ('~C~l!t.~laX: Thank you, 1~. Porunan.