The reaction of glutaraldehyde with tissue lipids

Chem Ph)s Llplds 3 (1969) 292-295 v, North-Holland Publ Co, Amsterdam

THE REACTION OF GLUTARALDEHYDE WITH TISSUE LIPIDS ROY GIGG and SHEILA PAYNE ~atumal ht~tttute [or ~lerh~al R~ ~at~h ,thll Hill London ,~ H 7

Received Februarl 4, 1969 Portions of mouse brain, ktdneF and h~er were fixed wuh g[utaraldeh~de and the hp~ds were then extracted with chloroform methanol Comparison of the extracted hplds with those from stmdar porhons of the same unfixed hssues by thin la~,er chromatography sho~ed the absence of phosphatldyl ethanolamme in the extract from the fixed tissues Actd hydrolFs~s of the fixed, extracted hssue hberated free fatt~ acMs suggesting that the phosphattdyl ethanolamme had been fixed to the tissue proteins

Introduction Since 1963, g l u t a r a l d e h y d e has been mcreaslngly used as a fixative for electron m~croscopy and cytochem~stD and for the rater-molecular crosslinking of crystalhne enzymes ~) and it has been shown that the reagent crosshnks proteins w a free a m m o g r o u p s to g~ve a stable insoluble matrix The chemistry o f the reaction has been &scussed briefly by Rlchards and K nowles-') who suggest that the properties o f the cross-hnked materials can be explained by assuming that the free a m i n o - g r o u p s o f proteins a d d across the d o u b l e bonds o f :qJ'-unsaturated a l d e h y d e groupings o f polymerlsed glutaraldebyde The presence o f free a m i n o - g r o u p s m p h o s p h a t M y l e t h a n o l a m m e a n d p h o s p h a t l d y l serme suggested that these llpMs should also interact with g l u t a r a l d e h y d e w~th the posslblhty that these hplds could be cross-hnked to protein m tissues These hxed 1,plds could affect the i n t e r p r e t a t i o n o f electron microscopy results on fixed tissues m which other hpMs have been extracted by the washing and d e h y d r a t i n g procedures The p r o b l e m s o f the extraction o f hpMs during fixation o f ussues for electron mtcroscopy has been discussed pre,,lously 3) K o r n and W e l s m a n n ~) have stu&ed the loss ot hplds from A c a n t h a m o e b a during p r e p a r a t i o n for electron m~croscopy They concluded that the llpMs were unaffected by g l u t a r a l d e h y d e fixation and were totally extractable The authors did not investigate the c o m p o s i t i o n of the hplds extracted and no d a t a on the c o m p o s l h o n o f the p h o s p h o l l p M s o f A c a n t h a m o e b a have been pubhshed although a loss of hpMs r e p o r t e d by Korn and W e l s m a n n may well have been due to the hxatton o f phosphat~dyl ethanolamme 292

ACTION OF GLUTARALDEHYDE ON LIPIDS

293

We therefore decided to investigate the effect of the fixation of brain, kidney and hver of mouse with glutaraldehyde on the extraction of hplds from these tissues

Experimental The brain, kidney and liver were removed from a mouse and one half of each hssue was cut Into 1 2 mm slices and placed in a 2 5°o solution of glutaraldehyde in 0 1 M phosphate buffer at pH 7 and kept at room temperature for 2 hr The fixed tissue was then washed with 0 1 M phosphate buffer to remove excess of glutaraldehyde and then homogemsed with chloroform-methanol (2 1) The unfixed half of the tissue was homogemsed directly with chloroform-methanol (2 1) A lipid extract of unfixed brain tissue was reacted with 2 5°~ glutaraldehyde in 0 1 M phosphate buffer at pH 7 for 30 mm and the product was isolated by dilution with water and extraction with chloroform Phosphatidyl ethanolamme isolated from ox-heart by the method of Rhodes and Lea ~) was reacted with glutaraldehyde and the product isolated in the same way Thin layer chromatography of the extracted hplds was carried out on Kleselgel G (Merck) on 2 0 x 2 0 cm plates Samples were first chromatographed in ether to 17 cm to remove neutral hplds, air-dried and then re-run in ether to 14 cm in order to remove free glutaraldehyde from the phosphohpids The plate was then developed with chloroform-methanol-water (14 6 1) followed by n-propanol-ammoma according to the method of Jatzke~ltz 6)

Results Fig 1 shows that in extracts of the three fixed hssues the spot (3) due to phosphaUdyl ethanolamme has disappeared and that in the extracts from fixed kidney and hver no new product has appeared However m the fixed brain extract a new spot (l) has appeared This new materla[ has the same R/value as the chloroform soluble product obtained when glutaraldehyde is reacted wnh phosphatldyl ethanolamme or with the phosphatldyl ethanolamme present in a total lipid extract of brain The absence of this material in the extracts of fixed kidney and liver indicates that the phosphatldyl ethanolamme of these tissues has been cross-hnked by the glutaraldehyde to tissue protein and thus remained insoluble The presence of firmly bound lipid in the fixed, extracted tissues was confirmed by treating the residual tissue with 6N hydrochloric acid at 100 for 2 hr followed by extraction with ether and t i c All three fixed and extracted tissues gave strong spots for free fatty acids whereas the unfixed and extracted tissues treated tinder the same conditions gave only traces of fatty acids This result

294

RO'~ (,IGG AND SH[ILA PAYN[

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Fig I a - cerebrosldes, b, d a n d f - hpld extracts of unfixed brain hver a n d kidney respectively, c, e a n d g - hplds extracted from glutaratdehyde fixed brain, hver a n d kldne~ respect~el~ 1-pos~t~on of product from reaction of phosphat]d~l e t h a n o l a m m e w~th glutaraldehyde, 2-cerebrostdes, 3*phosphat~dyl e t h a n o l a m m e 4-sulphatldes, 5-phosphatld~[ c h o h n e , 6 - s p h m g o m y e h n , 7-phosphat~dyl mosJtol indicates that m brain some of the phosphatldyl

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Discussion The

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A C I - I O N ,~)F ( , L U T A R A L D E H ~ D E

O N LIPID'q

295

krdney fixed with glutaraldehyde suggests that this hp~d has completely cross-hnked with protein during the fixation procedure In the case o f brain only partial c r o s s - h n k m g has occurred This fi,~atlon o f hptds to proteins m tissues treated w~th glutaraldehyde may affect the interpretation o f electron microscopy results and may be o f Interest m the cr o ss- h n k m g o f hplds to enzymes in the cases where hptds are known to be essentml for enzymic reactions 7) F m ther investigations o f the fixed tissues may reveal the relatwe positions of the polar end g r o u p o f phosphatldyl e t h a n o l a m m e and free a m i n o - g r o u p s o f speclhc proteins in cell m e m b r a n e s and h p o p r o t e m s

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