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Tissue distribution and aortic binding of dl -glyceraldehyde-C14
Tissue 1)isl:ril)ution m
It has been demonstraled in previous communications in this series thal slereospecific aliphatic aldehydes which obey n described three-carbon rule and exis~ in solmion in the hydrated (gem-dihyda',xy or aldol) form ;ire capable of acting in vitro as ~anning or cross~ linking agents (br collagen tissue prepmario:is, ~-: l)iiia have been recorded, also. io suggest thai these compounds can induce arteriosclerosis-like lesions in isolated arterial segments in vivoJ Since it is possible that simiiar or related interchain crossdinking reactions may generally characterize aging processes in the fibrous proteil~s of connective tissues,~'viI was of considerable additional interest to determine whether or no~ known cross-linking aldehydes mighl selectively localize in and bind to such tissues. The presently reported study was accordingly conducted using an aldehyde of potential dietary and metabolic importance s in both an in vivo and an in vitro model system.
E X P E FiI ), IE N T A I,,
Material Formyl-labeled DL-gtyceraldehyde-CV' (tot no, 86-195-1) was custom symhesized by New England Nuclear Corp., Boslon, Mass. The material was run direcdy withou! flwther purification after dilution in 0.15 M saline for in vivo studies and in 0. I5 M phosphate buffer (pH 7.3-7.4) for the vitro studies.
From the Division of Orlhopaedic Surgery, Deparlmem of Surgery, The .Iohns Hopkins University School of Medicine. Baltimore, Marykmd. Aided by grants from lhc National Insfilele of Arlhfifix amt Metabolic Diseases (AM-02642 Met), "]['he Maryla|~d Heart Association, Inc.. and t}~eAmerican Cm:cer SocieW (IN-I 1VL Submitted t~r pubficaliog November 2. I964. 121t
3lethod:~ ]It V:i",IL ll1 vj',(l! ~ t l R l i ~ ~A~cl'e ,co~tdil¢led v. hh scxtmlJy m,:tturc n~alc ~,,.VJsl;tr l':ils ~AcigJlillg approxmlately 300 ~,,m, Vach animal x~as lightly imeslhetizcd by hl)laiaIhma) tiber ;rod (he superlicial femoral vein in one hind limI~ Menlifled near ~he inguinal ligamen{. ()no ml. aliquols of the glyceraldch>de-( "~ solution conlaining apfuoxim:lh.'ly t /~c, of radioactivit:, were i~:iected direcdy in one group of eight rals. A similar volume bul conlaining unhtbeled i~iglyce|iddehyde t,> make a final concentration of O, I M in O, t 5 M saline was added to tl'~e radioaclive milleriat in a second scrim, of eight animals,
All animals were sacriliced by an overdose of inhalalional elhcr 24 hours ;filer injection. Represemalivc tissues (e.g,, brain, exlernM ear, xiphoM cartilage, lung, heart, rectus abdominis muscle, liver, spleen, kidney, llolqa, tail skin and tail lendon) were removed quickly, rinsed briefly in running cold lap water and dried to co|lslkltlt weight in ;: vacuum desiccator over silica gel and Cat;In fbr lwo I,o ftmr days. Aliquots contah-~ing less than 400 rag. of each of the dried tissues were placed in Pyrex test tubes arid overlaid wilh 1 ml. of cone. ltN()a for 8 to 12 hours ;.It 70" C, Two ml. of p-dioxane was then added to the warm solulion. The residuaI volume was carefully measured and 1.0 mI. added to 1 x Y4 inch stainless steel phmchets which had been previous)y cleaned by boiling in Haemosol and distilled waler. The solution was permiltcd to remain at room temperature for 24 hours and finally heated to dryness at 7(F C, for lwo (o four days, The dried material was coumed on a NuclearChicago model CIIOB automatic gasdtow counter, which was kindly made available by Dr. Joseph Colbourn. I)ata were expressed in lerms of the percenlage of lhe administered dose counted per gram of dry tissue weight. JSR ~ Vol. V. No, 3 ~ March 1965
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(XJXI~IENT
Signilic:ml a l d e h y d e upi:ikc, in c o n s i d e r a b l e e x c e s s of thai h o l e d in all o t h e r l i s s u e s , was f o u n d prinmrily M the aorla and .-,econdarily in the hlng in each o f the in v i v o studies. Smaller a m o t m l s w e r e o b s e r v e d in lt~e a b d o m i n a l viscera, lnlercslmgly, only iathct small alllOtlnlS were fotmd in lhc r e m a i n i n g connective l i s s u t s w h i c h w e r e e x a m i n e d ( T a b l e 1). A p p r c c i a M e tiptal,;c by intact {torI{l ~tod ~iortic elastin w a s also o b s e r v e d in the in vilFo sttidics. T h i s w a s i n v a r i a b l y g r e a t e r a f t e r !he longer illler;.iclion lirncs I\~r b o t h lissttc p r e p a r a l i o n s and for both (,f Ihe l w o g l y c e r n l d e h y d e sohitiorls {'1 able 2). T h u s , it s e e m s quite certain that the rmlive aor!ic wall, b o l h in r i v e and in viiro, a n d aortic e l a s d n in vitro a r e c a p a b l e o f r a l h c r s e l e c t i v e l y binding I l l - g t y c e r a l d c h y d e . (Olher s t r u c l u r a t l y similar c r o s s - l i n k i n g a l d e h y d e s p r e s u m a b l y b e h a v e in an identical manner.) T h e dose or l he a d m i i l i s l e r c d material does |lot a p p e a r tO be particularly critical so ftlF ~iS p e r c e n t a g e o f a d s o r p t i o n is c o n c e r n e d , though.
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ax d e m o n s l r a l c d cb4ewhece, ~~" ~ c o n c e n l r i l t i o n cfl',2cls life o|" ilTlportancc ill dctcrminiR~ b o t h the tensile alld e l e c l r o p h o r c / i c p r o p e r t i e s o f lhe wall. I..Inl;.)rtunalely, il is not k n o w n }!t p r e s e n l e x a c t l y lo w h i c h antllomic site or lo w h i c h c h e m i c a l g r o u p s oil elasfin a n d / o r c o l l a g e n chah~s lhc a l d e h y d e p r e % r e n t i a l l y bhRIs, if i n d e e d t h e r e is a specific s/le (e.g.. the s o - c a | l e d "'internal elastic m e r n b r a i m " ) . M o r e p r o b a b l y , o n the b a s i s b o t h o f the p r e s e m o b s e r v a t i o n s and oF e a , i i e r m o r p h o l o g i c and s h r i n k a g e
122
KINCAID
JSR -
ET AL,
data, ~-:~the process is diffuse,rather than specifically localized and involves both collagen and elastin chains. The important consideration in either circumstance is that cellular activity evidently is not necessarily required for localization. A freely circulating aldehyde apparently can be readily adsorbed and more or less preferentially bound to the matrix structure-of the aortic wall. Preliminary observations ctlrrentty in progress suggest that flee aldehydes may occur under certain circumstance in human sera, as implied by previous data reported by Landau and Merlevede. ~ A theoretical mechanism is therefore provided by the present observations whereby certain of the heretofore ~trtificially segregated mechanical and dietary Nctors in clinical and experimental atherogenesis can be imerrelated) :~-~
2,
3.
4.
5, 6.
SUMMARY 7.
A study has been conducted of the in vivo tissue distribution of intravenously administered DL-glyceraldehyde-C ~ i n male Wistar rats and of the in vitro gtyceraldehyde-binding properties of segments of native bovine aortas and of aortic elastin. The data indicate that the aortic wall can selectiveIy bind glyceraldehyde, which is a potent cross-linking (Le., tanning) agent of both metabolic and dietary origin. The possibility is raised that such binding may provide a means toward a unified explanation for the pathogenesis of certain of the properties of the arteriosclerotic aortic wall. T REFERENCES
l . M i l c h , R. A.: (a)Thermal shrinkage of aldehydetreated goatskir~s. ~1. Am. Leather Chem, Assn., 57: 581-588, 1 9 6 2 . lb') Studies of collagen tissue ~ i n g ;
8. 9.
I(L
t~. 12.
! 3. ~4,
Vol. V, No. 3 -
March 1965
h,,leractiol~ of certain imcrmediary metaholites with collagen, Gerontologia, 7:t2~J-152. 1963, (c) Viscometric h~rdening of ge]alin sols ~n the presence of certain h~lermcdiary melahoiites+ .I. Surg. Res,, 3; 254-262, 1963. (d) Aqucc~us solution infra-red spectra of collagen-reactive aldehydes, l]h~chim, hliophys. Ac~a, 93:45-53, ~964, (e) Hydrotherm~Jt shrinkage of metabolite-treated a~r~ae. J. AIherosc. Rcs., in press, 1964. Milch. R, A,, and Murray. R. A.: (:l} S1ud~cs of collagen tissue aging: Thermal shrinkage of melabo[ite-treated collagenous tissues. P~oc, Soc. l~xper, I]iol, Med.. I 11 : 551.554, 1962. (b) Infra-red spectra of aqueous dialdehyde s~arch t~oludons, ,1, Am. l.eather Chem. A~sn,, 59:311)-316. 1964, Milch, R, A.. Jude, J. R., and Knm~ck, J : Effecls of collagen-reactive aldehyde me/abolilCs tm ~he s~ructm'e of the canine ac~rtlc wall and their possible role in atherog:ene sis. Surgery, 54: 1(14~123, 196 3. Gus~avsom K. H.: "t'he Chemis[ry of T~mning Pr(~esses. Academic Press. New York. 1956, Verzar, l-L: The aging of collagen, UO Helvet. Physiol. pha~macoL Attn. !4:207-221, 1956. (b) Geron[o!ogia. 2:81-~03. 1958, (c) Geromologh~. 4:105-1 ] I. 196(1, Bjorksten, L: A common molecular basis for the ~ging syndrome. ,1. Am. Geriatrics Soc.. 6:740-748, 1958. Borns~cin P_ arid Picz~ K. A.: A bk~chemical sltldy of htm]an skin co~tage~ and the reh~[ion betwee1~ inmland ~ntermoiecutar cross-linki~g. J. Clim Invesl.. 43:1813-1823. 1964. Landau. B, R., and Mertevede. W.: Initia! re~lctions in the metnbolism of I)- and i-glyceraldehyde by ra[ liver. J. Biol. Chem.. 238:861-867. I963. Lansing, A. 1.. Rosen~hal. T. B.. Alex. M., and Dcmpsey, E, W.: The structure m~d chemical characterization of elastic fibers a:s revealed by elas~ase and by eleclmn microscopy. Anal, Rec., 114:555-575, !952. Go~te~ L.. Slern, P . Elsden, D. F,. and Partridge, S. M,: The chemistry of connective tissues. 8, The composition of elastin from three bovine tissues. giochem. ,1., 87:344-35 t. 1963. Mendoza, S. A., and Milch. R A.: Tensile strength of skin collagen, Sm\m Forum, 15:433-434, I964. Milch, R. A., Murray, R, A., Jones, P. D., and Stuart, D, C., Jr.: Tensile electrophorelic and morphologic properties o f native and ~ddehyde-crossfinked bovine aortae, Submitled for publication, Texon.. M,: The hemodynamic concept of athero~clerosis, Bali. N.Y, Acad. Med., 36:263-274, ~960. Adams, C. W, M.: Multiple factors in lhe palhogenesis of atheroselerosis. Guy's Hospital Rep., I I2:222-253, 1963,
It has been demonstraled in previous communications in this series thal slereospecific aliphatic aldehydes which obey n described three-carbon rule and exis~ in solmion in the hydrated (gem-dihyda',xy or aldol) form ;ire capable of acting in vitro as ~anning or cross~ linking agents (br collagen tissue prepmario:is, ~-: l)iiia have been recorded, also. io suggest thai these compounds can induce arteriosclerosis-like lesions in isolated arterial segments in vivoJ Since it is possible that simiiar or related interchain crossdinking reactions may generally characterize aging processes in the fibrous proteil~s of connective tissues,~'viI was of considerable additional interest to determine whether or no~ known cross-linking aldehydes mighl selectively localize in and bind to such tissues. The presently reported study was accordingly conducted using an aldehyde of potential dietary and metabolic importance s in both an in vivo and an in vitro model system.
E X P E FiI ), IE N T A I,,
Material Formyl-labeled DL-gtyceraldehyde-CV' (tot no, 86-195-1) was custom symhesized by New England Nuclear Corp., Boslon, Mass. The material was run direcdy withou! flwther purification after dilution in 0.15 M saline for in vivo studies and in 0. I5 M phosphate buffer (pH 7.3-7.4) for the vitro studies.
From the Division of Orlhopaedic Surgery, Deparlmem of Surgery, The .Iohns Hopkins University School of Medicine. Baltimore, Marykmd. Aided by grants from lhc National Insfilele of Arlhfifix amt Metabolic Diseases (AM-02642 Met), "]['he Maryla|~d Heart Association, Inc.. and t}~eAmerican Cm:cer SocieW (IN-I 1VL Submitted t~r pubficaliog November 2. I964. 121t
3lethod:~ ]It V:i",IL ll1 vj',(l! ~ t l R l i ~ ~A~cl'e ,co~tdil¢led v. hh scxtmlJy m,:tturc n~alc ~,,.VJsl;tr l':ils ~AcigJlillg approxmlately 300 ~,,m, Vach animal x~as lightly imeslhetizcd by hl)laiaIhma) tiber ;rod (he superlicial femoral vein in one hind limI~ Menlifled near ~he inguinal ligamen{. ()no ml. aliquols of the glyceraldch>de-( "~ solution conlaining apfuoxim:lh.'ly t /~c, of radioactivit:, were i~:iected direcdy in one group of eight rals. A similar volume bul conlaining unhtbeled i~iglyce|iddehyde t,> make a final concentration of O, I M in O, t 5 M saline was added to tl'~e radioaclive milleriat in a second scrim, of eight animals,
All animals were sacriliced by an overdose of inhalalional elhcr 24 hours ;filer injection. Represemalivc tissues (e.g,, brain, exlernM ear, xiphoM cartilage, lung, heart, rectus abdominis muscle, liver, spleen, kidney, llolqa, tail skin and tail lendon) were removed quickly, rinsed briefly in running cold lap water and dried to co|lslkltlt weight in ;: vacuum desiccator over silica gel and Cat;In fbr lwo I,o ftmr days. Aliquots contah-~ing less than 400 rag. of each of the dried tissues were placed in Pyrex test tubes arid overlaid wilh 1 ml. of cone. ltN()a for 8 to 12 hours ;.It 70" C, Two ml. of p-dioxane was then added to the warm solulion. The residuaI volume was carefully measured and 1.0 mI. added to 1 x Y4 inch stainless steel phmchets which had been previous)y cleaned by boiling in Haemosol and distilled waler. The solution was permiltcd to remain at room temperature for 24 hours and finally heated to dryness at 7(F C, for lwo (o four days, The dried material was coumed on a NuclearChicago model CIIOB automatic gasdtow counter, which was kindly made available by Dr. Joseph Colbourn. I)ata were expressed in lerms of the percenlage of lhe administered dose counted per gram of dry tissue weight. JSR ~ Vol. V. No, 3 ~ March 1965
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(XJXI~IENT
Signilic:ml a l d e h y d e upi:ikc, in c o n s i d e r a b l e e x c e s s of thai h o l e d in all o t h e r l i s s u e s , was f o u n d prinmrily M the aorla and .-,econdarily in the hlng in each o f the in v i v o studies. Smaller a m o t m l s w e r e o b s e r v e d in lt~e a b d o m i n a l viscera, lnlercslmgly, only iathct small alllOtlnlS were fotmd in lhc r e m a i n i n g connective l i s s u t s w h i c h w e r e e x a m i n e d ( T a b l e 1). A p p r c c i a M e tiptal,;c by intact {torI{l ~tod ~iortic elastin w a s also o b s e r v e d in the in vilFo sttidics. T h i s w a s i n v a r i a b l y g r e a t e r a f t e r !he longer illler;.iclion lirncs I\~r b o t h lissttc p r e p a r a l i o n s and for both (,f Ihe l w o g l y c e r n l d e h y d e sohitiorls {'1 able 2). T h u s , it s e e m s quite certain that the rmlive aor!ic wall, b o l h in r i v e and in viiro, a n d aortic e l a s d n in vitro a r e c a p a b l e o f r a l h c r s e l e c t i v e l y binding I l l - g t y c e r a l d c h y d e . (Olher s t r u c l u r a t l y similar c r o s s - l i n k i n g a l d e h y d e s p r e s u m a b l y b e h a v e in an identical manner.) T h e dose or l he a d m i i l i s l e r c d material does |lot a p p e a r tO be particularly critical so ftlF ~iS p e r c e n t a g e o f a d s o r p t i o n is c o n c e r n e d , though.
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gram o{ dry /~s~m:" a~fie~ iu;,-ra,'litm fro- I a)ld 27 hotl
fs.
ax d e m o n s l r a l c d cb4ewhece, ~~" ~ c o n c e n l r i l t i o n cfl',2cls life o|" ilTlportancc ill dctcrminiR~ b o t h the tensile alld e l e c l r o p h o r c / i c p r o p e r t i e s o f lhe wall. I..Inl;.)rtunalely, il is not k n o w n }!t p r e s e n l e x a c t l y lo w h i c h antllomic site or lo w h i c h c h e m i c a l g r o u p s oil elasfin a n d / o r c o l l a g e n chah~s lhc a l d e h y d e p r e % r e n t i a l l y bhRIs, if i n d e e d t h e r e is a specific s/le (e.g.. the s o - c a | l e d "'internal elastic m e r n b r a i m " ) . M o r e p r o b a b l y , o n the b a s i s b o t h o f the p r e s e m o b s e r v a t i o n s and oF e a , i i e r m o r p h o l o g i c and s h r i n k a g e
122
KINCAID
JSR -
ET AL,
data, ~-:~the process is diffuse,rather than specifically localized and involves both collagen and elastin chains. The important consideration in either circumstance is that cellular activity evidently is not necessarily required for localization. A freely circulating aldehyde apparently can be readily adsorbed and more or less preferentially bound to the matrix structure-of the aortic wall. Preliminary observations ctlrrentty in progress suggest that flee aldehydes may occur under certain circumstance in human sera, as implied by previous data reported by Landau and Merlevede. ~ A theoretical mechanism is therefore provided by the present observations whereby certain of the heretofore ~trtificially segregated mechanical and dietary Nctors in clinical and experimental atherogenesis can be imerrelated) :~-~
2,
3.
4.
5, 6.
SUMMARY 7.
A study has been conducted of the in vivo tissue distribution of intravenously administered DL-glyceraldehyde-C ~ i n male Wistar rats and of the in vitro gtyceraldehyde-binding properties of segments of native bovine aortas and of aortic elastin. The data indicate that the aortic wall can selectiveIy bind glyceraldehyde, which is a potent cross-linking (Le., tanning) agent of both metabolic and dietary origin. The possibility is raised that such binding may provide a means toward a unified explanation for the pathogenesis of certain of the properties of the arteriosclerotic aortic wall. T REFERENCES
l . M i l c h , R. A.: (a)Thermal shrinkage of aldehydetreated goatskir~s. ~1. Am. Leather Chem, Assn., 57: 581-588, 1 9 6 2 . lb') Studies of collagen tissue ~ i n g ;
8. 9.
I(L
t~. 12.
! 3. ~4,
Vol. V, No. 3 -
March 1965
h,,leractiol~ of certain imcrmediary metaholites with collagen, Gerontologia, 7:t2~J-152. 1963, (c) Viscometric h~rdening of ge]alin sols ~n the presence of certain h~lermcdiary melahoiites+ .I. Surg. Res,, 3; 254-262, 1963. (d) Aqucc~us solution infra-red spectra of collagen-reactive aldehydes, l]h~chim, hliophys. Ac~a, 93:45-53, ~964, (e) Hydrotherm~Jt shrinkage of metabolite-treated a~r~ae. J. AIherosc. Rcs., in press, 1964. Milch. R, A,, and Murray. R. A.: (:l} S1ud~cs of collagen tissue aging: Thermal shrinkage of melabo[ite-treated collagenous tissues. P~oc, Soc. l~xper, I]iol, Med.. I 11 : 551.554, 1962. (b) Infra-red spectra of aqueous dialdehyde s~arch t~oludons, ,1, Am. l.eather Chem. A~sn,, 59:311)-316. 1964, Milch, R, A.. Jude, J. R., and Knm~ck, J : Effecls of collagen-reactive aldehyde me/abolilCs tm ~he s~ructm'e of the canine ac~rtlc wall and their possible role in atherog:ene sis. Surgery, 54: 1(14~123, 196 3. Gus~avsom K. H.: "t'he Chemis[ry of T~mning Pr(~esses. Academic Press. New York. 1956, Verzar, l-L: The aging of collagen, UO Helvet. Physiol. pha~macoL Attn. !4:207-221, 1956. (b) Geron[o!ogia. 2:81-~03. 1958, (c) Geromologh~. 4:105-1 ] I. 196(1, Bjorksten, L: A common molecular basis for the ~ging syndrome. ,1. Am. Geriatrics Soc.. 6:740-748, 1958. Borns~cin P_ arid Picz~ K. A.: A bk~chemical sltldy of htm]an skin co~tage~ and the reh~[ion betwee1~ inmland ~ntermoiecutar cross-linki~g. J. Clim Invesl.. 43:1813-1823. 1964. Landau. B, R., and Mertevede. W.: Initia! re~lctions in the metnbolism of I)- and i-glyceraldehyde by ra[ liver. J. Biol. Chem.. 238:861-867. I963. Lansing, A. 1.. Rosen~hal. T. B.. Alex. M., and Dcmpsey, E, W.: The structure m~d chemical characterization of elastic fibers a:s revealed by elas~ase and by eleclmn microscopy. Anal, Rec., 114:555-575, !952. Go~te~ L.. Slern, P . Elsden, D. F,. and Partridge, S. M,: The chemistry of connective tissues. 8, The composition of elastin from three bovine tissues. giochem. ,1., 87:344-35 t. 1963. Mendoza, S. A., and Milch. R A.: Tensile strength of skin collagen, Sm\m Forum, 15:433-434, I964. Milch, R. A., Murray, R, A., Jones, P. D., and Stuart, D, C., Jr.: Tensile electrophorelic and morphologic properties o f native and ~ddehyde-crossfinked bovine aortae, Submitled for publication, Texon.. M,: The hemodynamic concept of athero~clerosis, Bali. N.Y, Acad. Med., 36:263-274, ~960. Adams, C. W, M.: Multiple factors in lhe palhogenesis of atheroselerosis. Guy's Hospital Rep., I I2:222-253, 1963,