CAPÍTULO III: LA GUARDA Y CUSTODIA COMPARTIDA (II)
3.3 GUARDA Y CUSTODIA COMPARTIDA EN SUPUESTOS DE VIOLENCIA DOMÉSTICA
1
.. 3 2 •
0 L-3-Hydroxyacy\ CoA Dehydrogenase. cisA transA Enoyl CoA Isomerase.
CH3COSCoA1 'Thi&,.•
l
• Eooyl CoA Hy<Wtu.o:1
,
OH •• Enoyl CoA Hydratase. 'L3HydroxyacyiCoA Oehydrogel'lclse
OlH •
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• ,• 3-Hydroxyacyl CoA Epimerase. Thiolase.
CH3
p
CH3fl
\J\/'ft-SCoA (L)isomer. \IVV'c-SCoA.
OH • ,
1·
,
1
Classical 13- oxidation. Classical j3-oxidation.CH3COSCoA. CH3COSCoA.
P�tkway Pr�pc�aJ.. � Stom.l ,.. Cat,ar, MoM,� PatJtway c1 kunau"' Dcmme',
< 1 9 7 8 > ( 19 6 5 )Fig . 8 Oxidation o f l inoleic acid i n mitochondr i a .
Although i t i s genera lly accepted that the basic mechani sm for the oxidatio n o f unsaturated fatty acids i s e s sentia lly the same a s for s aturated fatty acids , the i ntroduct ion
o f c i s double b onds in the acyl chain leads to intermediate s n o t encountered i n the S -oxidation o f s a turated fatty acids . The above scheme i l lu strates the two proposed p athways for the oxidat ion o f l inoleic acid ( See section 1 . 1 0 ) .
LONG-CHAI N FATTY ACI DS.
n
iDATIONLonQ-ct'IClin Short-chain produ fatty acids. Transported to Non-Hepatic Tissues. KETONE BODIES
AcriJ!·CoA,
Cl CITRATE . +- "carnitine Acetyi-CoA - - - --
Ac�l-CDA,
OAA�
I
Cil
RATE. T.CA FADH2 CYCLE..
..
C02 .E lectron Tra n s p o rt Chain
( membrane associated pathway ) NAD+
FAD ( re oxi dised ) .
Fig . 9 Metabo l i sm of acetyl-CoA i n liver mitochond ri a . Acetyl-CoA synthes i zed within the mitochondri a l matrix c an e i ther be comp letely degraded to and H2 o wi thin the mitochondr i a or transported out of mitochondri a
( indicated b y broken l ine ) ei ther a s ke tone bodies ( Sugden and W i l liamson , 1 9 8 1 ; Middleton , 1 9 7 8 ; Mayes and Lake r , 1 9 8 1 ) , citrate ( Watson and Lowenstein , 1 9 7 0 ) , o r pos s i b ly as f ree acetyl-CoA v i a the c ar n i tine ace tyl -CoA tran s ferase reactio n ( Freidman and F raenke l , 1 9 5 5 ; F r i t z e t a l . , 1 9 6 3 ) . However , the involvement o f thi s last mechani sm i n the
export of acetyl -CoA from the mitochondria has yet to b e proven .
1 . 1 1 Perox i s omal oxidat i on
Laz arow and de Duve ( 1 9 7 6 ) have s hown that rat l iver perox i s omes are capable of oxid i z ing long-chain acyl-CoA ester s . The oxi dat ion of the s e long-cha in acyl subs trates proceeds by an a lmo s t identical ser i e s of reaction s as des c r i bed for mitochondr i a l B -ox i dat i on ( La z arow , 1 9 7 8 ) , wi th the generation of acetyl-CoA through succes s i ve s t ep s of dehydrogenat i on , hydrat ion , dehydrogenat ion , and
thiolyt i c c leavage . In contra s t to mi tochondr i a l B -oxidat i on , however , t h e f i rs t dehydrogenat i on step i nvolves the r eduction of 02 to H2 02 , wh i le the s econd dehydrogenat i on step , as in mi tochondr i a , reduces NAD+ to NADH . When mi tochondr i a l and perox i s oma l f atty acid
oxidat i on were compared i n whole l i ver homogenates
( Mannaerts et al . , 1 9 7 9 ) i t was observed that , in contr as t w i t h mi tochondr i a l oxidat i on , perox i s omal oxidat i on d i d not produce ketone bodies , was not dependent on carn i t ine , and rema ined unchanged dur ing s t arvat ion and d iabetes . In add i t i on to th i s , perox i s omal oxidat i on is not coupled to a phosphorylating system and is insen s i t ive to inhib i t ion by cyanide ( La zarow and de Duve , 1 9 7 6 ) .
The degr adation of the acyl chain by the en z ymes of B - ox i dat ion i n mi tochond r i a i s thought to be essent i a l l y complete with the format ion of acetyl-CoA , equi va l en t t o the number of acetyl un i t s contained in the fatty ac i d . T h i s , however , does not appear to be the s ame in peroxi s ome s . Laz arow ( 1 9 7 8 ) observed that pur i f i ed perox i s omes wou l d cataly z e t h e oxidat ion of palmitoyl-CoA with t h e gener a t i on of only 5 acetyl-CoA un i t s per equ ivalent of palmi toyl -CoA . Perox i s omes were les s act i ve with octanoyl-CoA than w i t h lauroyl-CoA or palmi toyl-CoA and wer e inact ive when s ho r t chain acyl-CoA esters s u c h as butyryl -CoA wer e u s e d a s substrates .
T he incomplete oxi dat ion of long-cha in fatty ac i d s and i nactivity towards shorter chain acyl-CoAs r a i s es ques t i ons as to the fate of thes e s hort cha i n der ivat ives . The
1 9 7 3 ) , although not needed for oxidat i on , sugge s ts that thes e s horter cha i n acyl-CoAs may be transported e l s ewher e i n t h e cell . T h e pos s i b i l i ty of t h e further oxidat ion o f the s e s h o r t cha i n sub strates b y mi tochond r i a i s s t i l l a top i c o f d i s cu s s ion .
Although the results , obta i ned thus far , sugges t that the perox i s omes are a ma j or s i te for the B-ox idat i on o f long chain fatty ac ids ( La z arow and de Duve , 1 9 7 6 ; Mannaert s et al . , 1 9 7 9 ) , the relat i ve contr i but i on of both the
perox i s omal and mitochondr i a l pathways to the overa l l ox ida t i on of fatty a c i d s in t h e cell i s uncerta i n .
When cons ider ing maxima l en z yme activity , the
peroxi s omal s-ox idat i on capac i ty in mous e l i ver is about the s ame as that observed in the mi tochond r i a l system ( Murphy et al . , 1 9 7 9 ) . In contrast , the hepat i c perox i s omal
B-ox idat ion capac i ty in rats account s for about 2 5 - 3 3 % of the total act ivity ( Krahling et a l . , 1 9 7 8 ) .
A role for perox i s omal oxidat ion has been propo s ed by Nea t et a l . , ( 1 9 81 ) a s regards the oxida t i on of fatty a c i d s dur i ng per i od s of sustai ned high hepa t i c i n f lux of thes e subs trates , and espec i a l ly in rela t i on to the metabo l i sm of fatty acids that are poorly oxid i z ed by mi tochondr i a l
s-ox idat i on .
The overa l l regula t i on of fatty acid oxida t ion i s a very comp lex p i cture with a wide var i ety o f contr i bu t i ng factors . However , as in many other met ab o l i c s chemes , var i ous en z yme steps seem to act as important control points .
level in th i s thes i s , us i ng the rat and the s heep as model an imal systems .
MATERIALS AND METHODS
2 . 1 Chemical
Reagents pur chased from S i gma Chemical Company , S t . Lou i s ( US A ) , wer e ; palm i t i c a c i d , stea r i c acid , ole i c a c i d , l i n o le i c ac i d , pa lmi toyl-CoA , oleoyl-CoA , l i noleoyl-CoA , ma lonyl-CoA , coen z yme A , L-carn i t i ne , DL � -glycerol
3 -phosphate , ATP , ADP , DTT , NAD+ , ant imyc i n D , albumi n ( Cohn Frac t i on V ) , EGTA .
DL- [ methyl-1 4 c ] carn i t ine hydrochlor ide and
L- [ u-1 4 c ] glycero l 3-phosphate ( ammonium s a lt ) wer e purchased from Amers ham Interna t i onal , Amer s ham , Bucks , U . K .
Other reagents were analyt ical grad e .
Butanol was was hed free of impu r i t ies wi th d i s t i l led water , dr ied with CaCl 2 overn i ght and re-di s t i l led twi c e , the i n i t i a l 5 0 ml and the f i n a l 1 0 0 ml from a volume o f 2 1 were d i s carded .
2 . 2 of
Solut i ons wer e pr epared us i ng dei on i s ed-d i s t i l led water . Micel lar solut i ons of saturated fatty acids wer e p r epared b y t i trat ing aqueous solut i on s of t h e pota s s i um
salts to between pH 7 to 8 with HCl . For mi cellar s olut ions of palmitate and stearate th i s operat i on was carr i ed out at 7 0 ° C to ensure the s o lub i l ity of these fatty ac ids .
Unsaturated fatty ac ids , present as the free ac i d , wer e t i t rated wi t h a n equ imo lar amount of KOH to form a m i c e l lar s olut i on of the potas s ium salt of the fatty ac i d . Oxygen was excluded at a l l s t ages by f lushing with N2 and s o l ut i ons wer e then s tored under ni trogen . The concentrat i ons o f the
fatty ac i d mi c e l lar s o lut i on s were determined by ad j u s t i ng the pH of the s o lution to 2 , us ing a p H meter , wi th HCl and t i trat ing the free fatty ac ids with NaOH us ing Ni le B lue A as an indi cator ( Chen , 1 9 6 7 ) .
Rad ioact ive substrates wer e prepared as follows ; 2 . 2 . 1
5 0 �C i of L- [ u- 1 4 c ] glycerol 3 -phosphat e , ammon i um s a lt ( 1 7 1 mCi/mmol ) was made up to a f i nal concentrat i on of 1 0 . 9 4 mM wi th unlabell ed DL a -g lycerol 3 -phosphat e .
2 . 2 . 2 [ 1 4 c ] carni t ine
50 � C i of D L [ methyl-1 4c ] carn i t ine hydrochl o r ide ( 5 1 . 4 mCi/mmo l ) was made up to a f i na l concentration of 4 . 3 2 mM with unlabelled L-car n i t i n e .
Both radi oactive substrates wer e stored i n 1 cm3 a l i quots at - 2 0 ° C unt i l requ i red .
Glas sware us ed in the preparat ion of mitochondr i a and mi tochondr i a l s o lution s was c leaned by soaking in alka l i then ac id-wa s h i ng before f i na l ly r i ns i ng with de i on i s ed water . No detergents wer e used for clean i ng g l a s swar e
as soc i ated wi th the i solat ion o f mi tochond r i a o r meas u r ement of mitochondr i a l activ ity .
2 . 3 of intact l iver
To is olate intact funct i onal mi tochond r i a from t i s sues the osmolar i ty of the suspending med i um mus t be such that the mitochondr i a retain the i r phys i olog ical shape and s i z e . The mi tochond r i a l membrane , l ike many other membranes , has elas t i c properties wh i c h a l lows i t to shr ink or expand to wi thin certain l imits depending on the osmo lar i ty of the medi um surround ing i t ( Wh i ttacker , 1 9 6 5 ) or the presence of agents that promote swe l l ing or contract ion ( Kutt i s et al . , 1 9 6 8 ) . Although t h i s swe l l i ng and contracting proce s s i s a n i n vivo response , the i solat i on o f mi tochondr i a i n a prolonged swol len or contracted s tate , due t o incor r e c t osmo la r i ty of t h e preparat i on med ium , h a s a marked e f fect on the metabo l i c act i v i ty of these organelles ( Vigna i s and
Vigna i s , 1 9 6 5 ) . I t i s therefore impor tant to ensure that , dur ing the i s olat i on of mi tochond r i a , the i r phys i olog i ca l shape and s i z e i s ma intained .
B es ides ma intaini ng the phys i olog i c a l s i z e and s hape of the mi tochondr i a , it is important that intact mi tochondr i a a r e relea s ed f rom the l iver cell . Th i s i s achieved b y us ing s hearing forces and , in order to carry out th i s proc e s s
succes sfully , mate r i a l that i s res i stant t o s hear ing mus t be removed ( Nedergaard and Cannon , 1 9 7 9 ) . One common met hod of s hear i ng intact cells is by a hand-operated , or motor
dr iven , homogen i ze r . The function of the homogen i z er is to s hear the cell membranes and release the intact cell
organel les into the homogen i z at i on medi um . The part i c le s i z e of the t i s sue to be homogen i z ed i n relat i on to the c learance of the homogen i z er is cr i t i c a l in achi ev ing succes sful s hear i ng . If the parti cles of t i s sue are too large , the pressure exer ted by the homogen i z er pestle may caus e the cells to bur s t and thereby damage the cell
organelles , r e sult ing in an i ncrease i n the percentage of broken mitochondr ia . On t he other hand , if the par t i c les are too sma l l in relat i on to the homogen i z er clearance , the lack of s hear ing dras t i cally reduces the yield of intact mi tochondr i a . In both i n s tances , the proportion of i ntact mi tochondr i a released i s lower ed . Part i cles in the range of 1 . 5 to 3 . 0 mm3 d i ameter were found to be suitable for an opt imal yi eld of mitochond r i a us ing a homogen i z er c learance of 0 . 2 5 mm .
Mos t publ i s hed methods of prepa r i ng l i ver mi tochondr ia sediment the mi tochond r i a at 9 , 0 0 0 - 1 5 , 0 0 0 g for 5-10 mi n , forming a compact pel let of broken and intact mi tochondr i a
( Johnson and Lardy , 1 9 6 7 ; Otto and Ontko , 1 9 7 8 ) . The broken , and les s dense , mi tochondria are then careful l y removed from t h e top of t h e pelle t and the rema i n i ng i ntact mi tochondr i a resuspended i n fresh pr eparat i on med i um . If the mitochondr i a l pel let is too compact , resuspens i on i s often incomp lete and a n increase i n broken mi tochondr i a i s observed after further centr i fugat ion .
The method des cr i bed her e fol lows the same procedure as des c r i bed by Johnson and Lardy ( 1 9 6 7 ) but uses a lower force of 4 , 5 0 0 g for 20 mins wh i ch leads to the format ion of a softer pel let . After centr i fugat i on , the broken mi to-
chond r i a form an opaque f luffy layer and this i s eas i ly removed with the supernatant by a gent le rocking of the
centr i fuge tube . The eas e of resuspen s i on of the relat ively soft mitochondr i a l pel let leads to the i s o lat i on of
mi tochondria with min imal damage .
2 . 3 . 1 Treatment of an imal s and l iver t i s sue to
the of rat and mitochondr i a
Femal e Sprague-Dawley rats we ighing between 1 5 0 - 2 0 0 g were kept at 2 5 ° C w i th da i ly cyc les of art i f i c i a l i l lumi nat ion between 6 . 0 0 a . m . and 6 . 0 0 p . m . and fed a d l i b i tum on a stock laboratory diet cons i s t ing of 2 1 . 6 1 % prote i n , 2 . 3 9 % fat , 5 . 1 7 % f ibre , 5 . 0 2 % as h , 0 . 7 9 % ca2 + , 0 . 5 % P , 3 . 4 6 % total n i trogen and 1 2 . 81 % mo i stur e .