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B I OCHEMI CAL STUD I ES ON OV I N E CERO I D- L I POFUSC I NOSI S
A the s i s presen ted i n parti a l fu l fi l ment of the
req u i rements for the degree of
Doctor o f Phi l osophy
i n
Veteri n a ry Patho l ogy
a t
Ma s sey Un i vers i ty
Dav i d No rri s Pa l mer
1987
i i
ABSTRACT
The ceroi d-l i pofu s c i noses a re a group of i nheri ted d i seases of humans and a n i ma l s c h a racteri sed by bra i n a trophy and the storage of a
fl uorescent l i popi gment i n a vari ety of t i ssues . Cl i n i ca l s i gn s i nc l ude l os s of v i s i on , sei zures , mental retardati on and dementi a , wi th the d i sease c u l mi nati ng i n premature death . Defects i n l i p i d metabol i sm o r the control o f l i pi d peroxi dati on have been postul a ted to expl a i n the i r pathogenesi s . Spe c i f i c defects i n pe roxi dases , fatty a c i d metabo l i sm , dol i c hol metabol i sm , ret i nol metabol i sm and i ron metabol i sm have been proposed . Evi dence for these mec han i sms ari ses from the appa re n t l i po i d fl uorescent Sch i ff base n ature of the
l i pop i gmen t .
Th i s study reports the analys i s of total ti s sue l i pi ds a nd l i po p i gment i sol ated from ti s sues of sheep affected wi th cero i d- l i pofusc i nos i s . Bra i n grey matter phosphol i pi d fatty a c i ds of di seased s heep we re compared wi th those of norma l s heep . Phosphati dyl ethanol ami ne of d i sea sed s heep conta i ned more 18 : 1 ( n- 9 ) and l ess 2 2 : 6 ( n- 3 ) than norma l and the i r phosphati dyl chol i ne l es s 16 : 0 . Other d i fferences were
mi nor . No d i ffe rences were found between the l i ver l i pi ds nor t he fatty ac i d profi l es of the i r phosphati dyl chol i ne ,
phosphati dyl ethanol ami ne or tri gl yceri de s .
Li popi gment from the l i ver of affected s heep wa s 70% prote i n aceous , the rest be i ng ma i n l y l i pi ds . These we re on l y one si xth a s
fl uorescent a s tota l l i ver l i pi ds , but contai ned a number of
fl uorophors . None were major component s of the l i popi gment o r the postul ated fl uorescent product of l i pi d peroxi dati on . Li pop i gment l i pi ds i nc l uded the l ysosoma l marker bi s ( monoacyl gl yceryl ) phosphate that conta i ned 4 2 . 9% l i nol eate and 16 . 5% l i nol enate . L i popi gment neutral l i p i d s were dol i chol , dol i c hyl esters , u b i qui none , free fatty aci d s and chol e sterol , i nd i cati ve of a l ysosoma l ori g i n of the
l i popi gment . P hosphati dyl chol i ne , pho s phati dyl i nosi tol ,
phospha t i dyl seri ne and phosphati dyl e thanol ami ne were present i n proporti ons and wi th fatty a c i d profi l es typ i c a l of l ysosome s .
i i i Li popi gment i sol a ted from l i ve r , k i dney, pancreas and bra i n of
affected s heep wi thout the use of proteol yt i c enzyme s wa s two-th i rd s prote i n . Si l ve r stai n i ng after sod i um dodecyl s u l phate pol yacryl ami de gel el ectrophores i s showed a major band of Mr 14 , 800 , heterogeneous materi a l between 5 , 000 - 9 , 000 Mr and a major band of Mr<3 , 500. These components d i d not sta i n for RNA o r ca rbo hydrate , and were d i ge sted by a nucl ease free protease . They a re not normal l ysosoma l prote i n s . The presence of the 3 , 500 Mr prote i ns i n whol e affected ti s sue homogenates d i sti ngui s hed them from homo genates of norma l t i s sue . Li pop i gment l evel s of dol i chol , u b i q u i none and chol e sterol we re cons i stent wi th t he l i popi gment be i ng prote i n en ri c hed l yso some deri ved cyto some s .
The concentrat i o n of meta l s i n l i po p i gment from the four t i s s ues wa s a l so anal ysed . Li ver l i po p i gment had a h i gh copper conten t , 1 . 3% , ki dney l i po p i gment a h i gh i ron content and bra i n l i popi gment s howed an accumul a t i o n of some trace e l ements . These data i nd i cate that the l i popi gment cytosomes have a hi story of be i ng funct i on a l l yso somes as far a s meta l metabol i sm i s concerned . Metal anal yses of cerebrospi n a l fl u i d reve a l ed no i nd i ca t i on of a defect i n meta l metabol i sm .
I t i s con cl uded that ovi ne cero i d- l i pofusci nosi s i s not a l i pi do s i s , nor does the l i popi gment ari se from the abnorma l peroxi dat i on of l i pi ds . There i s no evi dence of a ny di s turbance i n meta l metabo l i sm . Low mo l ec u l a r we i ght prote i n s are s tored i n l yso some deri ved
organel l e s and on these grounds the cero i d- l i pofusci noses shoul d be regarded a s l yso somal protei noses . The se may re s u l t from defects i n l ysosoma l proteol ysi s or i ts contro l .
i v
ACKNOWLEDGEMENTS
I wo ul d l i ke to a c knowl edge my debt to my fi rst s upervi sor , P rofe s s o r R . D . Jol l y , who made th i s project pos s i bl e and organ i sed t h e resources req u i red to pursue i t . He a l so provi ded hel pful advi ce and
encouragement t h roug hout and educated me i n the pri nc i p l es of
b i oc hemi cal pathol ogy and storage di sease s . Hi s energy , commi tment a n d mental acu i ty have been i nva l uabl e . I wo ul d a l so l i ke to
a c knowl edge the hel p of my second superv i so r , Dr . Da v i d Husbands who encouraged my educati on i n l i p i d and comparati ve b i o c hemi stry and e n s u red that facets of the work of bi ochemi c a l i nterest we re not u nderval ued .
Much of the wo rk i n th i s thes i s woul d not have been pos s i bl e wi thout the a s s i stance of a number of peopl e . Mrs . Gi l l Barns wa s respons i bl e fo r runn i ng the pol yacryl ami de gel s . He r cons i derabl e profe s s i onal d i l i gence and s ki l l ensured the success of th i s work in often
d i ff i c u l t and tryi n g c i rc umstances . She a l so provi ded techn i ca l hel p wi th a numbe r of other experiments a s d i d Mr . Peter Wi nter , Mrs . Ma ureen Anderson and Mi s s Brenda Hobman . I wou l d a l so l i ke to thank Mr . Ru s sel l Graydon , Ms . Jayne Chapman , Mrs . Ro sa l i nd Da l efi el d and Dr. Susan Cooper for the post mortems and the el ectron mi crographs . Ms . Jayne Chapman and Dr . Bruce Fa rquharson performed the l i ve r bi o p s i e s a n d Mr . Boyd Jones col l ected t h e cerebro s p i nal fl u i ds . Mr . Fa ri s Sha rpe hel ped wi th the po st mortems and i n obta i n i ng control t i s s ues . Profe ssor Jol l y oversaw th i s wor k and ensured a con ti nued supply of accurate l y di agnosed anima l s in a s good a phys i c a l cond i t i o n a s practi cabl e .
I wou l d a l so l i ke to tha n k the numerous other peopl e i n th i s Fac u l ty ; and other fac u l t i e s and research establ i s hments who gave hel pful advi ce and techn i ca l a s s i stance on numerous occa s i ons . Parti c u l a r thanks go t o Dr . Roger Reeves o f t h e Chemi stry a n d Bi ochemi stry Depa rtment who o rgani sed and performed the meta l e s t i mati ons , Dr . Chri stopher Moore and Mr . J u l i an Re i d of the same department who suppl i ed the quan t i tati ve ami no aci d anal yses , Pro fe s sor R . E . Munford of the Depa rtment of Phys i o l ogy and Anatomy who hel ped wi th the
equ i va l ent c ha i n l ength c a l cul ations .
v D r . John Bl unt of the Chemi s try Depa rtment , Un i versi ty of Cante rbury;
ran the NMR spectra and D r . Nevi l l e Grace of the Depa rtment of S c i enti fi c and I ndustri a l Research provi ded the control ti s sue meta l concentrations .
I wo ul d a l so l i ke to than k Mr . Tom Law for the photogra phy and Mrs . E l i z a beth Wa ke and e spec i a l l y Mrs . Al l a i n Scott for the i r hel p i n typi ng the man u scri pt and ensuri ng i ts producti on .
The work wa s s uppo rted by the Un i ted States Nati onal I n sti tute o f Neuro l og i ca l and Commun i cati ve Di sorders and Stro ke Grant NS 1 1 2 38 a n d the New Zea l a n d Med i ca l Re sea rc h Counc i l g rant 83/ 1 .
Abstract
Ac knowl edgemen ts Tabl e of Con te n ts L i st of Fi gure s Li st o f Ta b l e s
TABLE OF CONTENTS
Chapter I : The biochemi stry of ceroi d-l i pofusci nosi s : A revi ew Page
i i i v v i ix x
I n troduct i on 1 1
Li pop i gments and the i r termi nol ogy 1 3
Proposed mec hani sms o f l i popi gment fo rmat i o n 14 Fatty acid d i s tu rbances in cero i d- l i pofu s c i nosi s 16
( a ) Li p i d s in bl ood 16
( b ) Li p i d s i n bra i n 17
Peroxi dase defi c i enci e s i n ceroi d- l i pofu s c i nos i s 18 I ron and copper i mbal ances i n ceroi d-l i pofu sci no s i s 20
The chemi cal n a ture of the l i popi gment 20
A criti que of the peroxi dati on theory 2 2
Other postu l ates o n the pathogenesi s o f ceroi d-l i pofusci nos i s 24
Conc l ud i ng rema rks 25
Chapter II : t�ateri al s and methods An i mal s and ti s s ue s
Li pi d extra c t i ons a n d characteri sati ons ( a ) Total l i pi ds
( b ) P hosphol i pi d s ( c ) Neutra l l i pi d s
( d ) 11e thano l ysi s of bands 1 and 2 ( e ) NMR a n a l yses of bands 1 and 2
Prepara t i on and a n a l ysi s of fatty a c i d met hyl esters ( a ) Transmethyl ati on
( b ) Gas- l i qui d c h romatography ( c ) Equ i va l ent c ha i n l engths ( d ) Argentati on ch romatography ( e ) Hydrogenati on
28 28 28 29 29 30 30 30 30 3 1 3 1 3 1 3 2
I so l ati on and c h a ra cteri sati on of l i popi gment bod i es from l i ver u s i ng pronase
( a ) I sol ati on ( b ) Mi c roscopy ( c ) Den s i ti es
( d ) Fl uorescence of l i popi gment l i p i d s
( e ) Determi nation of the non- l i p i d content of l i popi gment bod i e s .
I s ol ation and c h a racteri sati on of l i pop i gment from l i ver , ki dney , pancreas and bra i n wi thout u s i ng pronase .
( a ) I sol a t i o n from l i ver or ki dney ( b ) I sol a t i on from pancreas
( c ) I sol a t i o n from bra i n ( d ) Characteri sati on
( e ) I n h i bi t i on of l ysosoma l proteases
( f ) Quanti tati ve a na l yses of l i popi gment components ( g ) Quanti tati ve ami no ac i d ana l ys e s
Sod i um dodecyl s u l phate pol yacryl ami de gel e l ectrophores i s ( SDS-PAGE )
( a ) SDS-PAGE ( b ) S i l ve r s tai ns ( c ) Other stai ns
P rotease d i ge s t i on of l i popi gment bod i es t1e tal ana l yses
C hemi cal s
Chapter III : Neutral l i pi ds , phosphol i pi ds and fatty aci ds i n l i ver and brai n.
Introduction Re sul ts
( a ) Li ver total l i pi d contents ( b ) Neutra l l i p i d s
( c ) Phospho l i pi ds ( d ) Fatty a c i d s
( i ) Fatty aci ds o f l i ver l i pi d s ( i i ) Fatty aci d s o f bra i n l i pi ds D i scuss i on
Add i ti onal comments on the fatty a c i d composi ti on of rumi nant phosphol i pi ds
Page 3 2 3 2 3 2 3 2 3 2 3 2
3 4 3 4 34 34 34 3 5 3 5 35
3 5 35 36 36 37 37 37
39 39 39 4 1 4 1 4 2 42 42 46 48
vi i
Chapter IV : li ve r l i popi gment l i pi d s and fl uorescence
v i i i Page
I n troducti on 51
Res u l ts 51
( a ) I sol a t i on 51
( b ) Fl uore s cence 53
( c ) Phosphol i p i d s 55
( d ) Neutra l l i pi ds 55
( e ) Fa tty aci d compo s i ti ons ( f ) Non- l i pi d components D i scussi on
58 58 58
Chapter V : li popi gment protei ns, dol i chol and ubi qui none I ntroducti on
Re s u l ts
( a ) I sol a t i o n
( b ) Li popi gment compo s i t i on ( c ) Li popi gment prot e i n s ( d ) Li popi gment i soprenoi d s D i scussi on
Chapter VI The rol e of metal s in l i popi gment formation
6 3 63 6 3 68 68 7 1 7 3
I ntroduc t i on 7 6
Re sul ts 7 6
( a ) Me ta l s i n l i ve r , ki dney , pancreas and bra i n and
i sol a ted l i pop i gments 7 6
( b ) Meta l s i n cerebrospi nal fl ui d 7 9
D i scus s i on 8 1
Chapter VII General di scussion 84
References 94
Publ i cati ons 1 1 3
Fi gure I.
2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . 1 0 . ll . 1 2 . 1 3 . 1 4 . 15 .
LIST OF FIGURES
Page Proposed mec h an i sm of pol ymeri c Schi ff ba se forma t i on 15 Proposed structure of IIpolyma l ona l de hydell 2 1
TLC o f n eutra l l i p i ds 40
Gas-l i qu i d c h romatography of fatty a c i d methyl esters 43
Li popi gment i sol a t i on 52
I sol a te d l i popi gment bod i es 52
Fl uore s cence spect ra 54
Heterogenei ty of l i pi d fl uorescence 54
TLC of p h o s p hol i p i ds
(
aci d i c s o l vent)
56TLC of phosphol i pi ds
(
ba se sol vent)
561H NMR spectra of bands 1 and 2 57
1 3C NMR s pectra of bands 1 and 2 57
Morphol ogy o f l i popi gment bod i es. 65-6 6 SDS-PAGE of l i popi gment bod i es a n d total ti s s ue
homogenate 69
Protease d i gestions of l i popi gment bodi es . 7 1 i x
Ta b l e 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 .
LIST OF TABLES
Nomencl a tu re of the ceroi d-l i pofus c i noses Total l i ve r l i pi d s
L i ver phosphol i p i d compo s i ti on
Total l i ve r phosphol i pi d fatty a c i d s
Fatty a c i d s i n bra i n grey matter phosphol i pi d s I sol ated l i popi gmen t pho sphol i p i d fatty a c i d s Li popi gmen t dens i t i e s
The yi e l d , a n d prote i n and l i p i d proportions of i sol ated l i popi gment bod i es
Chol esterol , dol i c hol and ubi q u i none l evel s i n l i popi gment bod i e s
10 . The metal content of l i popi gmen t i so l ated from s heep affected wi t h cero i d- l i pofusc i no s i s
1 1 . Metal concentrati ons i n cerebro s p i n a l fl u i d
x
Page 1 2 40 4 1 4 4 4 5 5 9 6 4 6 7 7 2 77-78 80
