CHAPTER II

l\lATERIALS AND METHODS

Induction of myocardi~ infarction:- Rat (Spragu-

Dawley strain 10~ gl - ~ody weight) were given normal diet*

for 3 months. After 3 months the animals were divid d

into 2 groups. (1) Normal Control group (2) Experimental

group. Il[yocar di al . infarction wa.s induced in the animal

of the experimental group by administration of isoproterenol

L-1-(3-4 dihydroxy phenyl-)2 isopropyl amino ethanol.

hydrocloride_7. (Sigma chemical Co-USA) as described by

Wexler and coworkers14 Isoproterenol in terile water

was inj cted to rats subcutaneously tl'lic 't'1ith a 24 hours

interval between each injection at a doze of 35 mg of

isoproterenol/100 gl body weight. Rat in the control group. .ree ived injection of physiological saline under similar

conditions. ' Rats surviving the second injection were

* Hi ndus t an lever rat feed.

:30

sacrificed at 1Y2 days after the first injection in both

groups. Th animals were stunn d by a blol'1 at the backand

of the neckLkilled by decapit ation. Blood was removed

from each animal, serum separated and stored frozen for

further anal ys i s . The heart , aorta, l i v er , kidney liT r

qui ckl y r emoved to ice cold containers. The number of

animals used for 'each experiment is mentioned in the

conce rned chapter . For histopathological examination,

h art from a represent at i v e number of animal. in control.

and experimental gr ou was removed and fixed in 10% buffered

neut r al formal i n;; .

Analyt i cal pro cadures

Estimation ,of Ser um CPK and GOT

Serum CPK (Creatine phosphokinas e E. C~2 .7 .3 .2) Wa

detarmined by the met hod of Huges 152 and GOT (Glutamic-

oxaloac tic transaminase Er. C,~.2 . 6.1 .1) by th procedure

described by Rei t man and Fr ankel . 153

The reaction mixture in the case of CPK 'cont a ined

0. 2 ml of 0.025 M Magnes i um acet~t , 0.25 ml 0.012 M

Creat ine phosphat e , 0.1 m1 of 0. 15 M Cysteine and 0. 1 ml

serum. The r eaction was started by adding 0.25 ml of

0.004 M ADE solution and incubated at 37°C for 30 minutes .

31

The creatine l i ber at ed was quantitatively measured by

colorimetric method . The serum CPK is expressed in terms

of mg creat i ne per minute per 100 mI serum. In the case

of GOT , the incubation medium cont ained 0.5 ml of the

substrat s (0.2 M DL-aspartic acid and 0.002 M ~-Keto

glutaric acid) and 0.1 ml serum and incubat ed for 60 minut s

at 37 0 c. The enzyme activity is expressed in t erms of mg

of yruvate per minute per 100 mJ. serum.

Extraction rold Es t i mat i on of lipids

The extraction of lipids from serum and tis su was

carried out according to the procedure of Folch et !!~54 .

The aorta, heart , kidney and liver vler rinsed in cold

physiological saline and freed of blood. The aorta wa

cleaned of adv ent i t ial f at . The t issues were pr es s d

between the folds of filter paper. The homogeniz d tissue

was extracted with chloroform: met hanol (2: 1 v/v). The

extraction was repeated 3 times. The combin d filtrat

was shaken with Y5th its volume of 0.7% potassium chlorid

and aqueous l ayer was di s car ded . The wash d lower l ayer

was made ~pto a known volume Wi t h chloroform and used for

various lipid estimation.

Total cholesterol was estimated by the method of

Abel l , 155 p osp hol ipids by t he met hod of Zi l v.er S .i t

32

and Dav i s156 and triglycerides by the met hod of Van Hand J.

nd Zi l ver smit 157 Trlith th modification that florisll

column wa.s used to r emove pho pholipids. Fr e fatty acid

were ~stimated by the method of ItBya158 •

Estimation of protein

Protein wa estimated in all enzyme extracts, after

trichloro a cet i c acid precipitation, by the method of

Lowry §! i:!.159

Preparat i on .of a cet ol).8 dr;y p, o.!.der of the tissues

The tis sues r emoved at autopsy were rinsed in

phys i ol ogi cal saline and freed of blood. The tissue

(heart and aprta) was homogeniz d, with cold acston at

OOe and l eft 'for 72 bra , at Ooc with change of ac ton

v ery 24 hours. It Was then ~iltered and extracted with

ethe r : acet one (3:1) at 37°0 for 1 hour, followed by

et her for 1 hour. The defatted tissue waS then dried

t o constant weight.

J.stimat i on of gl ycosaminogl ycan fraction in the tissu

The dry def at t ed tissue was subjected to digestion

With papai n (Y3 - Y4 the dr y weight of the t i s sue ) for48 hrs, a t 65°c in 0. 1 M phosphat e bu·fifer, p/H 6.5 containing

33

0.005 M ~TA and O. 005M cyst in hydrochloride according

to th .pr oc dure of Laurent. 160 Fresh pa ain Was added

very 16 hours. Th digest aft r centrifugat ion wa pass d

through a column of cellulose (microcrystalline, chromat o-

graphy grad E. l\i rck G, rmany) , Pr viously trash d t'l1th 1%

cetyl pyridinium chlorid ( OP O) solution. The diffexent

gl ycosaminoglycan (gg) fract ions ( 1) ' Hya J.uron i c acid (HA)

(2 ) H parin sUlphat (HS) (3 ) Ohondroitin-4-sulphate( Ch-4S)

(4) Chondroitin-6-sulphate (O~) (5) Dermatan sulphat (DS)

a nd (6 ) Hepar i n (H) was eluted a ccord ing to the procedure

of SVej ca r and Rob er t son . 161 Th e .individual gg fract ions

'tfere quanti a't ated by t he e s t ima t i on of uronic acid by the

modified carbazole r eaction of Bi tter and Mui r. 162

Es timat i on o!. enzym activities

For the est imat i on of 'gg degrading enzym s, the

t issue tTas homogenized, ill agueous 0. 1% Br i 'j - 35 solution

and t h e super natant dilut d l'1 i t h a n equal' vo~ume of

ap r opriat e doubl st r ength buffer. Th e activities of

!3- 'lu cur onidase ( EO 3 . 2. 1. 31) and f;5-N- a c et yl hexosaminida

( Ee 3 . 2 . 1. 30 ) wer e det e r ined by t h met 40d of Kawa i and

An 0163 using I?-ni t r o ,phenyl - f3-D glucuron i d in 0 . 1

acet'a.te b,uf f e r (p-JH4 . 5) and P- nitr op henyl - A-N-acetyJ.

34

glucosaminid 0 . 1 M citrate buffer (pH 4.5 ) respective1y

as substrates . The assay of arylsulphatase A (EO 3.1 .6 . 1)

was carried out us i ng 4-nitro catecho1 sulphate in 0. 1 M

acetate buffer (.pH 4.5) as substrate according to the

procedure of ROy. 164 The activity of cathepsin D

(EO 3 .4~ 23~ 5) was determined by' using 4% hemoglobin in

0.1 M acetat buffer (pH 4.5) as the subs'hrate and determin-

ing the amount of tyrosine liberat ad by the method of Fol in

and Ciocalte • 165 The acti~ity of Hyaluronidase was

d termined by using 0.5 m1 of the olution (3 mg of HA in

10 ml 0.1 Macetate bUffer) as substrate and 0.2 ml enzyme .

The hexosamines liberated were estimated by the m thod of

J. essig ~ !!l~ 66

Esti~:tion o;t the carbohydrate components of totalg;t.ycoproteinp

The dry defatted tissue was subjected to pa ain

digestion (crystalline papain, one third the dry weight of

th tissue) for 72 hours at 65°c in 0.2 M acetate buffer

(pH 7.0) containing 2 mg cysteine hydrochloride/m1. Fresh

papain lias added every 24 hours. he digest ~as then allowed

to cool t o room temperature and 4-5 volumes of ethanol wa

added at Ooc and kep·t at this tempers.ture for 24 ho rs .

35

The supernatent after centrifugation was eva, orated to, I

dryness in the cold in vacuum. The residue 1'1a S d issolved

in water and used for the analysis of carbohydrate

components . The procedure used i s similar to that described

by Wagh et a1 ,167 except that ethanol was used instead of-TeA to d'eprote'nise the digest, since T eA keeps in solut~on

the tissue gl ycogen and gg.

Total hexose was estimated by phenol - SUlphuri c

a cid met hod168 , fucose by the method of Di s ch and

Shettles16~ and sialic a cid by the thiobarbituric acid, '

met hod of War f en . 170

Prot ein-bound hexose was estimated in serum by the

me t hod of ~eimer and Mos hin, 1?1 Fuoose and sialic acid

i n serum wa.s est imat ed a ccord ing to the procedures mentioned

abov •

Det e rmination of gl ycohydrol ase actiVity--

Homogenat e ( 1: 3) of t he tis sue was prepared with

0 .1% BrijA?- 35 solution at 0°c. The supernatent obtained

by centrif ugation at 2000 x g for 10 minutes at OOc was

diluted suitabl y wit h appr op'r iat e buffer. The final

con oentrat i on of Br i j - 35 was l ess than 0.02% in every ca~e.

The enzyme activiti s were determi ned by the met hod of

36

Kawai and Anno, 163 using the following as sUbstrates: P-nitro

phenyl~-N-acetyl glucosamide i n 0 .1M citrate buffer (pH 4.5)

fo r l'-N- a cet yl glucosaminidase. (EO 3.2 .1 .30) , P-nitrophenyl

galactoside in 0.02M ,ci t r at e buffer (pH 3.7) for ,!.5-galactosidas

(EO 3.2. 1.23), P-nitrophenyl glucosid in 0.02M citrate buffer

(pH 5.5) forjB-glucosidase ( Ee 3.2.1 .21) and P-nitrophenyl

fucosid . in 0 . 0 214 citrate buffer (:pH 5.0) for ,8-fucosidase

( EO 3.4.4.23). The enzyme activit y in each case is expressed

in terms of micromoles of P- ni t r oph,enol liberat d per minute

per gram protein in .the case of tissue. \ihen serum was used

as the enzy~e source, activity in each case is expressed in

terms of micromoles of p-nitrophenol liberated per minute

per 100 ·ml serum.

~s~ imat ion of ~lucosamine Ehosphat e isomerase(glutamine forming)( EO 2 . 6 • 1, 16) in. t he heart

The h omogenr.aat s of t he t issue at' OOc in glucos -6-

phosphat e (0.012 1~1 , P'H 7.2) KCl (0. 154 I\'J:) , EDT A (O. 001Ivr)

was used. The react ion syst em contains 0~1 ml luco -6-

phosphat e sodium salt (O . 1M, pH '7 ~ 5 ) . 0.15 ml L- lutamin

(0.1 M) 0. 1 ml reduced glutathione (0 .1 M), 0.45 ml sodium

phosphate buffer pH '7., (0 . 1 r~) and 0.3 ml nzyme wai ncubated for 1 hr at 30°c. The reaction system wa

37

sto ad by 1 mJ.' of 0 ."4 N TCA. The hexosamine co nt nt of '

1 m1 supernatent was estimated by the met hod of FOge11. 172

In the control a l l the complete reaction ystem i s :f'ollov1ed ,

but glutamine is ab s ent . Glutamine is added just before

the additio· of TCA.

Sulphate metabolism

The concentration of ,PAPS ( 3' phospho ad noain~-5 1

phosphosulphate), and the activity of sulphate act ivating

syst em (which includes sulphate ad enyl transferas ( EO 2.7 . 74)

a nd adenyl s ulpha t e kinase ( EO 2.7 .1 .25) and aryl sulpho-

transferas (EO 2. 8.2.1) in t h e heart wer estimated by the

method of J ansen and Van kempen using methyl umbel l ifer n ~ 7 3 ,1 7 4

The method is described below.

PAP S present in a h ea,t inactivated extract of the heart

t issue Has e st imat ed by deter mini ng t he amount of m thyl

umb ell1ferone ' sUlphate ,( }IDS) form~d fro m thyl umbelliferone

.(MU) using aryl suI.- ho transfer~se p r esent i n no rmal. rat

liver extr act. The h eart tissue was extracted With isotonic

KOl (1 :5,) and the sup er nat ent is inactivated by placing in a

b oi l ing water bath for 60 seconds . Th e reaction system

cont ained the h a inactivated extra c·t (1.5 ml), 0..4 :[,1

38

Tris-HOl buffer pH 7.4 (1 .0 ml) containing 0.5 M EDTA, 1.5 m]

U, 0 .2 m [KH2P04( .pH adjusted - 0 7.4 with aN KOB) and th

supernatent from the homogenate of normal rat liver in

isotonic KOI as the sUlpho transferas (0.5 ml)s Two

controls wer e al so run, one using isotonic KOl ( 1. 5 ml) in

place of heat i na ct ivated extract and an ot her using distill d

wat er (0.5 ml) in lace of the norma.~ rat liver xtract.

I n cubat ion was carried out at 37 0 c for 30 '. The t Ubes wer

placed in boi l ing wat er bath for 60 s econds t o ar rest th

r ea ction. An al i quot of t he supernatent (2 ml ) was ass d

t hr ough a column of Do ex-50 (H+ for, 3 x 1 em) , and the

coLunm "Tashed wi t h wat er . The rID S present in t e eluate

and washing was hyd r olysed Wi t h 2 N HOl at 80 0 c for 30 minute

and est imatod by flur oscence intensity measurement a

described by VanKemp en and l anson174 ( lb - max , 324 nm and.

Em. rJIax . 484 nm) ,

2. §~lphat e apt i vating syst em

The au.l, hate a ct i·vat ing system pr s ent in t he s~per

nat ant (A) fr om a 20 I} homogenat e ( Iv) of t he t .issue f rom

th exper im ntal ani mal in i sotonic KCl s ol ut i on was used

t o f orm P S from ATP and inorganic sulphat e. The P- S

for med was es ti at ed a s described abov •

39

Th reaction system contained A (1. 5 ml) and 0.4 M tria-HO~,

pJH 7.4 (4 ml) containing 2 .2 mM Magnesium chloride, 50 mM

potassium sulphate and 4.4 roM ATP . The reaction was

top d after incubation for 1 hour at 37°c, by placing

the tub in boi~ing water bath for 60 seconds. A contro~

was a.Lso run u ing he t ine.ctivated A in place of the

enzyme. An al i quot of the supernatent (1 .5 ml) we.s

incubated l'1ith EDT A 250 mM (0.5 ml), M1J 1'.5 IIU~ (0 .5 ml ~

in Tr is-HOI ) buffer and su e~~atent .(0. 5 ml) from normal

rat liver as suJ..photransfer as (a mentioned ab ov e in thecase of est imat ion of PA1?S) f or :;0 minut s at 37 0 • After

ar rest ing t h e r~action by k~eping i n boi l ing water bath

for 60 seconds the I~S in the supernatent was es t i mat ed

a s be fo re .

3. f-xY"l sulphot ranaf'er a se actiVity

The sulphotra.nsferase in the t i ssue extract from

t he exper i mental ani mal was used "t o cat alys e transfer of

sulphat e from PAPS (generated from ATP and i norgani c suJ. hate

by using enzyme syst em p'res ent i n normal r at liver extract)

t o MU .

P.APS was fir s t generat ed, by i ncubating the au er-

nat ant from 1: 5 normal r at liver homogenate in isotonic

KOI (1.5 ml) wi t h tris-HOl buffer pH 7.4 (4 ml) containing

40

2. 2 mM ~g 012

50 roM K2S04, and 4.4 ml\[ Nr f or 1 hour at 37° c .

After arrest ing the r a ct i on by placing in boi l ing water

bath fo~ 60 seconds , an aliquot of t he supernat ant , (1.5 ml)

was i ncubat ed at 370 c , for 30· 1'l ith 0. 4 t r is HOI buf fe r

pH 7.4 (1 ml) contaL~ing 0. 5 mM EDTA, 1.5 roM . Up 0. 2 ~[

Xl!2P04 and the supernatent ( ) from 1:5 homogenate i n

isotoni c KC1 of t he t i ssue f rom experimental animal ( 0~5 ml).

The cont rol containing beat i na ct ivat ed A (0. 5 m1). The

r~s r esent in an aliquot of the supernat ent is es t imat ed

aft er arrest "ng the r eaction by placing in a bo iling water

bath for 60 a- conds , as described before.

Est imat i on of aldehyde dehydr?genase (EO 1 ~.1 . 3) and

alcohol dehydr ogenas ( EO 1.1.1.1)

5 gm of heart t issue was homogenized i n 20 ml of

s ucr os e med ium (0.25 M Sucr ose - 5 ~1 T. r i a-HOI 0.5 ~i EnTA,

pH 7.2) and the homogenate Was used as the enzyme medi um•

.I l dehyde dehydrogenase WBJS as aayed ape ot r-o ho tometrical ly

wi t h al dehyde as substrat e by measuring t he reduct i on of

AD+ at 340 roM , by t he method of Tot t mar gi a • 175 Th

assay mi xtur e cont ained 50 mlf 'od i um pyr r opho phat pH 8.8,

0 . 5 mM + + 0 . 1 ( t o i nhibit.AD or 2.5 mM NADP , roM pyr ozol

a l cohoJ. dehydr ogenas e ) 0 . 05 - 5 a cet al d ehy and

41

5 micromole rot none in m t hanol (t o inhibit mi t ochondr i al

~T.A.DH oxidase ) . The r ea.ct i on wa s star t ed by addition of t he

subst rate . A blank was run simult aneously wi t h omi s sion

of t he substrat . Un i t of ald ehyd e dehydrogena se is

mi cromoles of NADH formed / minute/mg of pr otei n . oOhOlI

d ehydrogena se 11J"aS a s aayed in principl e a s d e s cr ioed by

Butt ner 176 . The a ctivity was mea sured by f ol l owi ng the

oxidation of NAnll spect rophotometrically wi t h a cet aldehyde

as the substrate. The rea ct ion mixture contained 50 roM

K1I2P04 ' p~I 7.5, 0 . 15 DIM llADH and 8 ml'-1: acetaldehyde . The

reaction was st arted by addition' of acetaldehyde. A blank

was used with omis s ion of the subst rat e . Unit of al cohol

dehydrogenase is micromole of I AD+ formed /minute/gm prot ein.

Est i mat i on of u~PG- ehydrogena~ ( trrPJ- glucose-NADP-Oxido-

r edu ct a s e ) ( EO 1. 1. 1.22 ) •

The chil led heart was homogenised at OOc i n a warri .ng

blender with acet one , previously cooled at _10 °c. The

acetone drypowder ( 400 mg) lvas ext r act ed i

sulphat e was add d and t he prote i n pr c1 itate wa collect d

by c nt i6ugat i on at 15000 x g for 10' . T e pr . cipi tate was

di sol v d in O. 02 ~ a cetat buffer pH 5. 4· and dialy ad

against c t at buffer for 5 hours. The met hod described

by St rominge r ~ !i!177 lias used for determination of the

enzy a ct iv i t y i n t h dialy ad solution UDPG dehydrogenase

activity was determin d f rom the rat of DPH+ r eduction,

measur s e ctrophoto met r i cal l y at 340 mjU. The system

contained in addition t o enzyme 0. 1 micromole of UDPG and

0 .5 micromole of DPN~ i n a total volume of 0 . 5 ml 0. 1 M

glycine buffer pH 8. 7 . A blank without substrate w

i ncluded .