esterase isozymes from rabbit synovial fluids normal ... - SAGE Journals

2 downloads 0 Views 1MB Size Report
J Clint Invest. 37:57,. 1958. 17. Harris. E, I)ibouma ... Black. AB, Zimmerman. HJ: Enuzyme activity in synovial fluids. J Lab. Clin. Med. 62:175,. 1963. 36. White.

THE

JOURNAL ©

OF

IIusTOCHEMusTRY

AND

ESTERASE

of

FROM

and

Oral

amid

starch

gel

fractture

of

esterase the

red

that

cell.

that

produces

or

maintains

esterase

rates

comparable the fluid

mennhrane in

huave i)eeum used

to study

the

among

gemuetic

enuzymes

(8,

29),

origin arid deve!opuuienmt to examine phylogenetic

vertebrates

(18)

as to of re-

apl)licationis

antd to explore

tropitotonutetric

of electrophoretic,

autd

huistochenmical

esterase eumzyutmo!ogy. Jut the currenut project,

as

l)roblenut coutcerned witlu joint (TMJ) dysfurtctiotts, exautiiutinig fluids

tite

froun

antalvses

part

of att

nuontspecific

esterases

subcouudylar

Murnuanue

iutvestigatiomt decided

of the

to

area.

antd

Doku

false

compare

joiuit

it to

ottgoirtg

the

the

TMJ

becomes

tenuuporal

or

zygontatic

itt

part

tiomual

mutaterials

tissue

response

red of

similar findings

membramue

normal

temporo-

itt

fused bonte

l)y American

produces

(i.e.,

to

a

Silastic

mmtterposi-

as

produce

miutimal

a utumique amid plasuuua

couuubmnmationt of trautsudate (16,

production of hyaluroumate protein stituettt of nmormttal syntovial fluid some joint diseases tlte conintective

a con14). Itt mm(lose

by

lubrication

arm

articular tiout

to

the

(35), (3, 13, tissue

nnentbramte

as

have

proper

been

well as showtt

in increased mucopolysaccharide A “weeping bearing” concept suggests

cartilage of svutovial

thuat

a

porous

the to l)roof

defornmable

may conttribute to the COntl)Osifluid (16). mu disease states tue

uttenutbrauteloses its selective l)ern1eabilit’ amid the fluid

to

contcemmtrates

as

of

(15).

defects, correctiont (1 2). This proCatmcer

Thtis

imisertiont

comunueceutzynte out the

(antkv!osed) a result

fracture

arid

36). The syntovial menutbraute, a specialized tive tissue, luas oxidative amid hydrolytic activity (5) which nutaumifests its actiomu

(7).

seruutt

foumud

fluid is exudate

particil)ate ductiont

thterefore

joirut

reuntionu.

autci

nutenibrane

cells,

of a surgical

fixed

synmthuetic alloplastcs have beent evalutated

svutovial

work

to tite

(12).

cell

syntovia!

traunuta, iutfectiott or contgeutital can be attainmed by toutdylotouutv Stnpported Grautt 1N23M.

to prevenut

al)!)roxintlatioui

We

blood

of a rrmaterial

attd a false a sheet of seguneutts

suggested

fluid.

red

itt

amid other syniovial fluids. Wheut

the

the

time (reationu

distal

membrante,

Previous (12)

inivolves

at souiue point

svnuovia!

of

founid

syutovial

temporonumandibular joinut,knee joiutt joiuut which was produced by placirug Silastic between two nuobile fractured Habbi,

the

a of

those

had

but

to

Syumovial

spec-

teutuporouuuaumdibuhar we were iuiterested

hormiogentized

of tIme rabbit

2 of from

microscopic

novo

activity

of

regions

fluids

light

pro-

segments 1 and

joint

false joimmt. The arid Supranumid)

diseased ntauitmahiatt ceuutral (2, 28). These are just a few of

systent

mttarker

false

by

fluid

major

esterases

de

Ce(lure

(outtrol

niorutual autd

utervous

joint

two

systems

reinforced

esterase

compared

between

fluid

differentiate

similar

false

had to

and

joint

were

a

Silastic

body

joint joint

rabbits

studied

differentiated false

a fluid

of titanuunaliant

latiomushmips

thue

msozynies

markers

time eutubryologic cells (4, 26, 27),

the

of

a synovial

temporomandibular

from

of

fluids

and

joint.

Noutspecific

uttechantisnis

All

temporomandibtular

does

mandibular

at

and

obtained

of Medicine

1971

White

a sheet

area.

Schools

02111

30,

knee

Zealand

isozynies

FLUII)S

University usetts

mum Auigu.st fromn

New

interposing

tests

Knee,

only

the

migrated

Analysis

not

biochemical

by

of

subcondylar

inhibition

zymograms.

the

left

However,

cells

to

animals

thte

activity

cell.

red

1972

ISA.

SCIT 1FF

Tufts

Massach

punbhicat

isozymes

and

electrophoresis

mu three

duced

trace lunug

l)utyrylesterase

serum

fluid,

(L.E.),

Boston,

in

JOINTSt

ROBERT

AN!)

Surgery

for

SYNOVIAL

ARTIFICIAL

Medicine,

Received Acetyl-

RABBIT

ESTABROOKS

(ILS.)

_4natoin

ANI)

Dental

snoviah

Printed

ISOZYMES

LEWIS

2mn-2u9,

3, pp.

Society,

XOR1’sIAL

Departments

20. No.

Vol.

CYTocnuEunts’rntv Inc.

iuistocnemican

1972 by The

Copyrighr

has

ant iutcrease

autd a chtantge

‘flue svntovial

fluid

source of umutrieutt supply tissutes. ‘fhue

Society

rtontspecific 211

sauuup!es acety!-

unmder antd

of plasuuma

itt hyalurontate

serves for study

1)roteiut proteint

as a lubricamtt the were

butyrylesterase

arid

avascular

a

local

anmalyzed activity

for

212 by

ESTABROOKS

starch

choseut thie

gel

electrophtoresis.

because

rabbit

the

have

This

Peri!)heral

beent

method

blood

classified

AND

was

esterases

of

Itistochemicalk-

(26) anid their genetic control has been determined (29) . Jut addition, relative isozynte activity was amualyzed activationt identification

by densitontetry. ageutts and

were

classification

MATERIALS

Surgery tnumderweuut

Various

inthibitiomi

ennp!oyed

AND

to of the

aid

and in

the

esterases.

METHODS

: Four New Zealand White rabbits low condylotomies with the positioninmg of Si!astic as described by Habbi etal. (12). Out the day of surgery each rabbit was weighed and givenm 20 mg/kg of body weight of intramuscular chloropromaz i tie hydrochloride (Thorazine). Fi fteenu miumutes later 20 mg/kg inmtnaveuuous pentobanbitah were inujected. The left condylar region was exposed via a submandibular inucisiont with reflect ion of the overlyirug muscle anud periosteum. A surgical fracture was created 1 cm below the condyle with a 557 buir ott a denuta! drill. With a no. 1 round bunr a hole was drilled in the distal segmentt. A sheet of Silastic (12), 2 x 2 x 0.1 cm thick, was adapted to the distal segment and secured mt place with 00 chromic gut suture, which was passed through the hole in the distal segment anud the Silastic in a mattress fashiont. The wountd was closed in layers amid each rabbit was given 300,000 units of itutramuscuiIan Bicilhinu. After ii monuths the rabbits were bled by cardiac puncture antd 50 ml whole blood were removed. One-half was treated for routine red cell !ysate preparatiout (29), arid the other half for serum collectiont. The rabbits were thert sacrificed by ether inhalation amid perfused with 1 liter nuormal sa!iuie to reduce the likelihood of blood contamituation during dissection. Sample preparation: All fluids were obtained withint ant hour of death. Any syniovial flunid that clotted or appeared to be contamintated by blood was discarded. The posterior kmuee joint provided kutee syntovial flunid. A skin incision was made along the anterior aspect of the leg. The patella was exposed and the leg joint was held mu a flexed position while the patella was cut transversely. This exposed the joint, cavity. Small beads of straw colored viscous fluid were aspirated with a tuberculin syringe through a 27-gauge needle. A total of 0.15 ml of fluid could be aspirated from the two knee joints. In most cases, an equal amount of normal saline was used to flush the syringe. The fluid was then diluted to make a 4:1 ratio of saline to synovial fluid. The TMJ was exposed with a preaunicunlar incisionu. The mandible was mobilized to identify the capsule which was incised. The mantdible was

SCHIFF

thent forced into a retnunded position so as to squeeze the fluid to the sunpenior surface. A lambda synintge was tised to aspirate the fluid. The bevel had to be held next to the articular disc arid the surface of the svnuovia! membranue. Sixty microliters of fluid were aspirated from both joinuts. The syringe was flushed with 120 Ml of nuormal salinue arid the aspirate was diltuted with arm additionta! 120 Ml of saline. In additionu to the above flunid, which was co!lected front both the experintenuta! arid coumtrol antimals, fluuid was obtainued from the implanutationi articulationu site of the operated rabbits. Following removal of the above menitioumed flunids, the left conudyle, ramus anmd body of the mautdible were dissected otnt. Unider a (!issectinug nuicroscope the overlyiuug muscle arid cormntective tissune were meticulounsly removed . The pseuudoarthrot ic j omit had a fibrouts capsule-like hinuinug which was easily discernible. The iuuutersurface had nu glisteninug membraute which also covered the Silastic implant. The flutid was aspirated front the glistening surface. Thirty to sixty microhiters of fluid were obtained and the syringe was flunshed with art equal quantity of sahinte arid fuirther diluted to a 4:1 ratio. A sample of synuovial mentbranue was removed front several conutrol animals anud homogemtized with 1 ml of saliume. Followinug ceuttrifugat ion the superumatanut was retrieved antd stored. Samples were either tised ott the day of preparation or stored at -50#{176}C. Ehectrophoresis: ilorizonutal starch gel electrophoresis was performed on all samples by the methods of Schiff et al. (27), Stormont arid Suzuki (32) anud Schiff amid Stormonit (29). The latter procedure gave the best resoluutiouu arid was used nount ituehy. The electrode tray bunifer (pH 8.1) counsisted of 2.4 g !ithiiuum hydroxide, 23.6 g boric acid arid 2000 ml distilled water. The gel btnffer (pH 7.3) conusisted of 40 ml electrode tray buffer, 140 ml distilled H20 and 70 ml Tnis-citrate buffer (pH 6.6). The Tris buffer was composed of 17.32 g Tnis (Sigma 7-9), 9.68 g citric acid (momuohydnated) antd 2000 ml distilled water. Each gel contaimted 37-42 g starch (Couunauught Medical Research Laboratories). The amountt depended oru the lot used. The gels were prepared autd molded mm frames as described by Kristjansson (19) and Gordon (10). Each gel was i.nsed within 24 hr of preparatiomi. A cut was made 5 cm from the cathoda! enud, perpenudicular to the long axis. Paper wicks, 6 X 10mm thick, were satuirated with the samples, blotted antd imuserted into the cuut. Voltage was applied at 150 v for 30 mini. The wicks were then removed. A glass slab with ice packs was placed on the

uipper

surface

of the

gel.

The

voltage

was

ESTERASE

ISOZYMES

mN

SYNOVIAL

213

FLUID

at 350 v mutt il the borate houmnidary ni1) cm front the origint. The timtte (if the runum betweeni 3 anmd 43/a hr. the comphetionm of the run, the gels were

coumi iumucd

grntted varie(1 At sliced

huorizonntally.

solut

Eachu

slice

mum vomit ainmi umg 2 ml of

itt acetonie,

salt

100 nttg

Ittt(l

ported,

98

ml

blrue

fast

(list

illed

bunifer

tm

was

imnuersed

itt

Tt1 \\‘

a

stnbst rat e solunt iont BB salt or fast blune Hit

l1O.

foutnid

As

to

he

previouisly umeces.sary

rein the

ioti nueditiun (26) . Rouut iutel the top slice placed itt a-nmapht huyl mucetate as substrate anid t lme hot tont itt a-nmmiphtthyl buntyrate. After 13’-2 hr at 37#{176}C the gels were rinised mt distilled 1I() aumd fixed itt 50 ntet haumo! overutighut Inhibition audi activation tests: For punrposes of i(leutt ificat mont anud classificat motn, act ivntt i(iti arid iumhibitionm tests were perfornted (26). Folhowintg electrophoresis a luorizonital gel slice was inrmnutersed itt 100 ntul of preheated (37#{176}C) test reagenmt fur 30 nuint. Two nuilhihiters of sunbst rate

2 t2

S(ulult mum in

C.-

itiCtil)ltt

\%.I1.s

acetoume

of

a-nnaphtiuyl

acetate

or

(b)

eserinme

disodiuum sunlfate

(l0’,

(El)TA) 10,

10

(10’’ M)

; (e)

1f’

a-

niaphit hyl buntyrate aumd 100 mg fast blue BB salt were t hteni added to each tray. Gels were inicuihated for ant add it ioumal 13 hr at 37#{176}C anud I huenm examititued. TIte gels were fixed as ntenmtioumed above. The act ivat ors atud inmhibit ors were : (a) et hvlenuedi auutiutetetraacetate,

7.,,

-

ill); diiso-

(l)FP) (l0 to l0 111). Jut sonic experintteumts these agenmts were dissolved in (list illcd water. Jut other cases I)FP anal eserine were diluted in 10 M EI)TA, whuich huas heent shuowni to activate ntost red cell esterases wit hount iumluihit i nig the remumaitmder (26). The I )FP (dissolved propylene

sohiitiott

-

e

pr(ipV!fltiOrOphOsphtlit

itt

/E’J

glycol)

nut 102

was

mnaiuitaiuued

H

--

____ _______ ____

-ci

mm a stock

J

Relative isozynte activity of (leterminted with a Joyce Loch! Churonutoscaut denusit omet er. Ant optical reflect aumce systemut witit a conutplenteumtary filter of 610 mM waveleumgt It w unsed to scant t he zvmogranuu Itroungh a 1 nun x 2 mun aperture. A 101) wedge rind a B cant wit It a specimenu record ratio of 3:1 provideul a sun it able ratige of expaumsioum. Areas unuttler thue (unrves were auntomaticahly iumtegrated. The racintgs were therm scaled dowum for puuhhicat mum to represenit thtree-founrths of the zvmogrant heumgthu. l)ensitometry:

the samples

was

HESUL’I’S

The

Mectropluoresis:

anid

1)1:11ehet

nmaphut hyl strates

acetate

(26, is

esterase

a

isozymes

which

flunids

amid

to

1

rabbit

of

regions

ivit

a-nuapltt

Figuire

diagrant

responding

joinit

anid

29).

zoumes

act

idenmticntl

were

scribed

26)

esterase

will

esterase seruim. of

of rabbit reul cell (iht ainted wit It aIuy! huu yrat C as stth) those previotishy dev

(tmukenm

from

-

referenice

Hb-orig

-----

I

II

Fro. 1. A diagram of the zommes of esterase activity obtained with a-uiaphthyl acetate (I) aund a-nmaphthy! buntyrate (II) as stnbstrnttes. Those zonues that were invariably present with any onme substrate are designuated by lower case letters amid are shown as solid bars. The zontes whichu exhibited phemiotypic vaniatiotu are cross-hatched amid are shown by systems designiated 1, 2, 3. Only the heterozygous phenuotypes are shown mm the diagram. The symbol lib indicates hemoglobimu. Taken directly front Schiff (26).

red cell arid platelet aid

itt describing

zontes

derived

Figunre

cor-

front

of

fltnids

synmovial

2 shows obtainued

flunids

thue butyrylesterase zyntogrants from tue TMJ (mu) nunud kumee (h) compared to serunm (c, d, e, f) arid

214

ESTABROOKS

-n iii abc FIG.

2. A

compares

ef

butyryhestenase synovial

activity

zymogram flunid

(a),

which

knee

The

symuovial

fluid (b), serum dilunted (1:10) with saline (c), serum di!unted 1:5 (d), senunm diluted 1:2 (e), tnutdiluted serum (f) amid red cells (g). Those areas of joint flunid amid serum that migrate at the same rate as systems 1 and 2 of the red cell are designiated as regions I and II, respectively. red cells (g). The joint fluids arid serunm coumsistently demonistrated multiple bamtdimtg patterns which migrated at rates similar to the red cell zonies previously designated as systents 1 arid 2 (Fig. 1). Acetylanud buutyrylesterase zyntograms were

virtually

idenutical

for

synuovial

fluids.

Jut

order to mainmtaium a conutinmunity of nmomenmclatuure but uuot to imply that the esterases are the same mm the red cells as inn the serum or symuovial flunids, the most cathodal zones are designated as regioum I anud the more anuodal as region Ii for joint flunids anmd serutnn. The quest ionm of diluntinug the syntovial flunid durinmg

its

acquisitioum

aumd

thereby

losinmg

count acted. comparinmg

a sheet

joinit

of Silastic

limited activity

to of

the

areas

region

of

I

was

regions ntuore

I arid

II.

pryniounuced

hart anuy of the sytiovia! fluids or serum (Fig. 4). As menut ionued above all joinit flunids showed two major conicenitrat iotis of acetylanmd huntyryhesteruse activity which have i)eenm designated its regions I anud II. however, there was nnore rapid iiydrolysis of the butyrate subst rate. Each region had one or more zoutes with as niaumv as three zones in atty otte regionu. The inutensity of t lie at aiutinmg varied front animal to anuimal. For example, the cathoda! zone of region I was nutore iumteutse t hart the anuodal zoume mm the aumintal of Figunre 3a while the reverse was trune itt the sample of Figuire 2a. however, the nuajority of animals did riot show sunclu art oI)viouns differeutce. Some of I hte zoutes (if regiont I dentonust rated apparent genuet ic variat ion which appeared to corresponid to the hteuuotypes fotinud itt serutnt (11). These were autahogouis to the zonues produuced However,

by

the

because

Es-l

!ocuus the

of

of

limited

red

cells

(11,

29).

of

anti-

utuimber

esterase

zonmes was examiumed by companinug uuutdiluted with diluited samples. All isozymes presentt in the tintdilutted fluid were fouutd itt the dihunted nr,aterial. The technical difficulty of aspirating the miumute quantity of synuovial fluid forced us to aspirate directly along the synuovia! membranue, especially mu the TMJ amid false joinit. Jut the last fotur auuimahs that were exantinmed, regiort I esterase activity appeared to be related to the technuique of (ibtaiuminig fituids. Whenm beads of knee syniovial flunid were aspirated without couttactinug the syntovial membrane, regioum I activity was minimal. Wheum the fluid was obtainted by aspirating along the membraute, there was marked imicrease in region I ac tivity. Because of the minuimal quanmtity of TMJ fluid the synuovial membrane surface was milways Figuure 3 is a zymogrant front a rabbit that had

SCHIFF’

posed betweenm the stirgically fractutred boric ends of the left stibcouudylar area. TMJ (a, c) anud false joittt (b) flunids were diluited 4: 1 witiu utorrnal saline whereas kuuee svnuovial fluid (d) was niot (liluited. Regioum I esterase activity front tite knuee (Fig. 3d) is tmot obviouus in the photograph buit a very weak zone counld be seen arid demoumstrated by denusitometry (Fig. 4a). This ntiuuinutal activity was always less than seruuni or TMJ fluid. Jsozymes in fluid front the left TMJ (c), above the couudylotomy, were contpanable to the coumtralateral TMJ (a) except for reduced activity. however, mt onue case, ani additionual zonue was foutumd that ntigrated ntore rapidly toward the aumode t han univ other zonue of either red cells or serunm. This rentains uumexplainued. The false joinit (b) also had esterase

0. d

TMJ

AND

fluids muter-

+

IS

a

I

C

d

FnG. 3. A zymograun which contpares the buntyry!estermtse activity of joinut flunids front a rabbit with a false condylar joinut. Right TMJ (a), false joinut (b), heft TMJ (c) amid knuee (d) svntovial fluuids. The false joinut was constructed out the left side below the umatunnally occurring TMJ.

ESTERASE

ISOZYMES

IN

SYNOVIAL

215

FLUID

11 (I

(1 It I’

jt

#{149}-92%

I Ii

I

I

It it I

I

I

I

J1b ;

I’

11

ii

,‘

It

.\

I

-/ll

Frc.

4. 1)enusitometer (b),

the zymogram. regiomi to the muds

(c) arid numbers

The wluohe.

irm this

stundy,

the

regioum

vial flunid canunmot at this Es-i locuis. Regiotu II appeared fluids.

The

activity

zyntogranut bunt

while the

systcuuu sante

2 of

rate,

the

flunids,

anmalyzed

three

by

showed

less

withint

of

±5%

of each

nuals

thuat

did

scent

in TMJ

samples

was

surface. ±10%

of

ntiainmitug

fltnids

(Fig.

aspirated

showed

False

joiumt II

wit hint

±10

suuovra!

titan

flumid I arid for

ntentmbraute

all

mm Figure

to

uinuab!e

knee

the

4d) areas

three

this

suggests

that

differences

of

scale

is ott the ordiuuate

the

itt region

anti-

finmdinngs two

membrane

reagenmt

Es-2

The

re-

itt regiont

arid

the

fitnid.

is

out at

the

activity regiomu The

itt

ovial

fitnids

fluuid

esterases

knmee

acetate

proumounnmced

of

as

buntyrate,

esterases

titouugh

the

to

eserinue

itt

itt

the

the

itt

1)FP

ium

limited

5.

were

where most

amid

Figure

amid

used

serum

anud

but-

TMJ

The

with

the

change.

acetyl-

kutee

untuaffected

synm-

synmovial a-uiaphthyl

however, mm the presenice II were both activated.

of

I anud showed

enzymes

arid the

cell,

fluuids.

or

ranuge

ott

red

sunbstrate.

Serunntu

test

Es-i

of the

fituids

eserinue shownm

regioums

synmovia!

Becaunse

showed

the

to

eseninue

molarity

were

activity.

I activity

mm the

synmovial rountinuely

with

syntovia!

are

of the

responuse was

tests.

mott

of

were

or unit

the regioum I anmd II TMJ

of

esterases

resunlts

less

=

inuteumsity

mo!arity

anud

mediuunt

inmhibit

The

(150%

the

30-

=

activatiomm figuire

EI)TA

of sample,

tyrylesterases

2

marked amid the

(26),

aum(I Es-2

unit

inttenusity,

0

similarity

oumly

were

ant arbitrary

of umormal,

knee

preinmcuubationm

anmahyzed

results

activated

M

the

The out

abscissa.

10

of

of eserzones

10-209

Jut each

itt sertunt,

quiaumtity

At

=

5

Figures

effects esterase

niorma!

=

=

4

isozymes

Es-i

restult.

1

nmormal).

suibsequienut

withuium

4c.

tested. had

I

umornial,

El)TA

mm tltese

region I act ivit v (Fig. 4b), alt bough the fluid frouuu t hue sante joinmt showed minuimal This

4-5

ntore

Becauu.se

ntore

of each

anmimals

htontogenuate

thaum

two

syniovia!

had

four

were

The

fell

3

rtuled

the ott the

graded

26) where

(9,

esterases

activity

to exp!aium

(Fig.

visuiallyanmd

extibe

tests:

ehectrophoresis.

by

caumumot

activation

iume aumd DFP,

4).

the

an(l

separated

tracimugs

fluuid fluids

mniumimal

synuovial

however,

cartilage

rate graphically respectively,

6 dentonust

had the

front

TIJ

scent

similar

are

region

15

homogemuized

acqtiisitionu.

porouts

of

tinder

(Fig.

approached Thue

(a),

sample

40

J{egionu

4a).

directly

pattern

I anmd appeared

prescntt

II

(Fig. 4c).

of

samuiple

anmd

to the

compared

knee

fluids

samples

other

coniform

the

flunids areas

regionu

flunid

riot

Fourteeum

anid

due

lnthibitiont atid

There

TMJ

inmtegrated.

thanu

of knee

from (16).

scale

amid the

15 kumee

be

dates ount

synmovia!

II had ntore fluuids.

joinmt

mechianuically activity

Thirteeum

a!!

mnentbrauie

deumsitomutetry

were

inn

separate

false

activity

nutay

the

to

variability.

synmovial

ctirves

esterase

of synmo-

region

syumovial

Fifteeum

htonutogeutated

the

observed in this region red cells, which migrates at

showed

1)enusitotnetry: synmoviah

siuiti!ar the

variationu

show

be attribuuted

of sertnuti

d

joint (d) synmovial fluids. Tracings are three-fourths of the length of to the curves of regionus I amid II represenut the percentage area of that

J esterases

time

rcsenubled

umo genuetic

was

that

false nuext

I

C

b

recordinugs

TMJ

1

,1II

1\

#{149}J,

a

memimbranue

It.

a

conuphex

in regionis same

nianuter

J arid as

reaction, II

al-

respouuded

syniovial

fluid

216

ESTABROOKS

0-#{149}---

AND

0-

I

SCHIFF

‘cSIEA

I

I

I

I

2

iO_8

Molarity FIG. 5. The effects of esenine out systems 1 arid 2 of the amid TMJ svntovial fltnids tire shownu. The results with serum,

lB antd JIB with acetate.

represenut

the

butyrylesterases

of the

red

cell

knee

respective

amid regionus

arid

TMJ

regionms

I anud II

of serunm, knee flunids were idenutical. JIA show the activity

synovial IA arid

whtile

4. 3. U)

,0 ,

C

I.

_0”

> #{163} 0.

r

I

I

C C

‘-4

a) > 0 0)

-

i-

1-

‘1-

-5

10-6

T

I

,Q-7

I

10-8

IO

Motarity The effects II of senuim, knee

FIG.

and

6.

etizymes. described Figuure ases

of

of diisopnopylfluorophosphate and TMJ synovial flunid.

The red cell react (26). 6 shows the effects systems

1 amid

2 of

ionm was of red

1)FP cells

as

previotushy

out the anud

(DFP)

arid

II

ned

cell

of serum

region I esterases inmcunbated itt i0

ester-

regionts

on

I

regionm

II

esterases

systems

atid

1 arid

synmovial to

were i0 showed

fluids.

2 arid

regions

System

inmhibited 1)FP. Systent

totally M

total

inihibitionu

I

1 anid whent 2 arid at

ESTERASE

ISOZYMES

i04H l)FP 10-v A! the the cotmtrol.

isozyme activity was comparable This was true with either acetate

butyrate

as

sunbstrate

ported

resunhts

but

as

the

conucenmtrationi

anud

of red

cell

IN

approached

coumfirms

related

to or

previotushy

esterase

inhibit

re-

ion

(26).

which

(‘onidylotouuuy,

iii

produces

a pseudoarthro-

(12)

successfully

doarthtrosis tissue

!)roduced

66

itt

that

showed

rabbit

Silastic

reactionu.

fumuctionual

conidylar

Additional

give

studies

1w

syntoviah

(6)

a ntienitbrante muienuubrane

have

that

joint

fereuutiated

niotiont

devehopittg

cartilage

nutodels

to special Skuble,

Doku (23) fibrous cartilage dyhectomy tiont (23) all

branie

contdeuuse

characteristics

thue forutiationi sites after

of

the

syntovial

connective

fronut

service

anud

favorable

are

ott a clontal predestined

reunaint

stability for

of

tive tissue cells to fornim syniovia! also feasible since mi joirut diseases chiaride couttenut is intcreased fluid

amid

the

beneath

the

syntoviah

cells

coutnuective syntovial

htave

beent

of cells

arid

situated

fibrous

type

believed syntovia!

to reutuove

!)ronutintenut

cavity

A

cells as

directly

well

as

lanutinta

tissue

cells

are

(21).

two

Itight

of

the

type

A

anud

thuerefore

the

reagents

this

is I

used

for

the ester-

of

iui

have

I isozymes.

This

of vhuat

synuovial

close A

These

is

the

)hagocytic of lysosomes

may

comttaint being

about

based

to

are

is currenutly

is krtowum

fluids

h)roximity

cells

quauttities

large

(22).

are found be presenut

th#{236}ey may

Type

surface

the

out

(13, 16, 31). The fluids are clear, viscous with it 1)11 of 7.3-7.4.

the

investi-

compositiout

hturnant

studies

pale

yellow, and They have a

of about 1.010, a I #{182} proteimu auud ant albuntint to globuhimu ratio anud

lipids

are

absent.

Non-

with plasmtia tranusudates aside front anud how cell count (36). Plasnta l)rotemmts

coutsistenut viscosity

are

founud

synovial

itt

electrophioretic cemutrationts

ever, these proteins those of plasma high

untits

the is a is

certaini

joint

fectious arid two

of

to

appears is

diseases

arthritis),

acid l)htospluatase of the enzymes

to

fluid nuot lactic

the

presence

of

of

a!-

arid glucuronate be bound to

called

found (e.g.,

How-

i(leuttica!with studies and

consisting

glucosamine

synovial

cont-

plasma.

(16).

due

a hiohysacchtaride

in

are

is

different

reduced

be

to

Prol)erties

Hyaluronuate

have

and to the

imutmunologic

viscosity

htyaluronuate, (13).

but

appear itt

sedintenitationt

‘rite

fluid

distributions wheut compared

whuich

arid

from

but

I esterases

regiomi cells,

surface.

proteinu

cell

fluid

idenutical,

to the

conicenitrationt

mutenibranie

proteint

uimore and

be

itt the degrees of activity However, both region

Because mtiembrane

The

phagocytic

electron

syntovial

to

sintuilarly

of adipose

breakdowmi products front (22). The Golgi apparatus

organielle

speculative. in synuovial

ternatinug

itt a loose

3H-ghu-

and

of regiout I nay be due to the difficulty itt acquirimug adequate fluid, although this is quite

thieir itt

by

antibody

(3),

ases

tlte

to Particil)ate

is characterized

coutntective

superficial

tissue shuownt

by a conimuec-

niembrante is mitucol)olysaCthe syntovial

mt

nuienibranue

its inicrease (7). Syntovial ntieuuibranie types

activated

Metap!asia

beemu

proteiuu niitrogentous substauuces are less common than itt plasuuia amid the electrolyte conipositioum is comusistenut with serumii. These finudimmgs are all

thteory (16) a specialized

unttil

dormant

eumvironinmentt.

by

cells

expected

of 4: 1. Fibrintogeti

nioruttal joint to the false joint is unlikely because of its locationi. However, the de novo utiembrante citnt be explainted itt whicht cells

syruovial

TMJ

specific gravity contcenttratioui

the

(1). has

amid imihibitionu amid are ProbablY differemice itt the inmtemtsity of the

gated. Mucht

tissue.

uuienibrauue

have

react

region

utteni-

with

and

imuttuniofluorescemuce tissue culture (16)

autd

The

ruear

of conu-

grant-

conistituents

is synthesized

riot the case, especially of regiont I esterases.

at

inivestigaruientbranue

of syuuovial

frouuu

extenisioru

unidif-

entds of amid differ-

tissue. (30) auu(l Murnuamue

att(l conidylotomy. Further revealed that a ghisteumintg

differentiated

Direct

for

that the

at

have observed mu the surgical

of the

Sokoloff

h)rimitive joinit capsule. Uteri comitintues amid leads

fornns of contutective Chtoukas anid Toto

amid

to

umecessary

amid

cells

to fornt thue differenitiationt

arid

knuee

sanne.

The

associated

(25). may

activationu

segntentts This was

antalogous

is

developntienut

uuieseutchtynu’ia!

euttiate Cellular

was

mu

(13).

also

fluid

tlte

itt

and

are

syntovial

dentonistrated

esterases

demonstrated

. h)rachmant

(23)

shtowni

emiubryologic

with

which

antd

ntiutintal

(21)

l)rotemmt

One

pseu-

areas

amid Supranitid

thuat at the eumd of 26 weeks the bonty had renutodeled arid fornied ant arthrosis. liuted

of

Hyaluronate

the

l)roductiomi

B cells

miticroscopy

treatuitenut of choice to restore furictioni anikylosed teutil)orouttanidibular jointt. Habbi

et a!.

iuya!uromuate

type

production

is the

the

to

ular,

217

FLUID

fixationu cosantine

DISCUSSION

sis,

SYNOVIAL

hiyaluronate

jut serum rheumatoid

dehydrogenuase-5

isozynite activity thtat are elevated

a

(3). amid

Jut mu(33)

(34) are (35). The

218

ESTABROOKS

ability

of

the

synioviah

niteutibrane

to

AND

whuichi

uttainutaini

its selective l)enutieabihitY is destroyed amid hyalurontate, synthesized front type A cells (22), is (lecreased

(13).

I)FP

is

binds

to

active

sites

a

sl)ecific

esterase

anud

htistidinue

seritue (26,

31,

of regionu

I at

activity

inudicated

isozymites

of

region separate has

II.

(26,

EDTA

sitowed

mu rabbit

activation

of as This

for

systems

where there was esterases without

of

cells

region

synuovial

I

fluid

results

cells

have

as

marty

heterozygous pheniotype zymes, respectively (26, study were riot able to three

bands

itt

exauttinted.

regiont

However,

as

kutee

differed

imihibitiont of mu the Es-i

for

Es-i

the

presently

atud

Es-2

the

dernonustrate

niore

I or II of the

syniovial iutteuisity

fluids anid

differences

iii

itt

utumben of zones mt region I were observed but insufficieuit ntunttber of antinuials ntade it inttpossible to demonistrate of

geutetic It has

confidence.

that

the

uvstent

2 esterases

rived front platelets isozynties of region fluid,

although

teristics

niot

as

appear

enuzymes

vaniatiomt previously

II

in

havinig

red

serunt

similar

2 with to show genetic

system

of the

cell

rabbit

However, the

ant

with arty degree beemi reported of

(29).

fornmable

fact

were

that

two

isozyuiiic

role

thtis

prol)ortiolt

tologic in the

The

TMJ I amid

syruovial

fluid

fluid II.

front

with There

tlte

respect was

false

joint

to the nitore

region

presenuce activity

was

space

of

of

kutee

thte for

of

the

‘flue

‘FMJ is

at

time

in

conutaitis

utuigrate

is

htya!iuue

(hifferenuces

fluid

wbuicht

(he-

(13).

at

least

the

same

1 arid 2 of

systeutus

the

which

this

at

time

It

m probah)le

syntovia!

fluid.

synmovial

nuternbrauue

throughu

active

This

is to

does

the

secretionus

nmembranue

produces

to

fluid

syniovial

de

novo

tissue but,

or nuuainitaints of the

selec-

or

comufirmuis

study

comintective

uuiemtibramie

that

nutainutaitu

the

his-

utuenibranie (23). Not

differemttiate mu additiout, a fluid

siniihtr

mr

tentiporoutiautdibu

ut

this joint.

ACKNOWLEDGM

We Dr.

wish H.

to thauik Dr.

C.

Doku

for

their

also wish assistance

of

the

suggestioums

ENT5 Thomas

Murniante

1)epartnrmenut

anid

to thanuk Dr. mu l)reparationt

guidanuce.

amid of

Oral

Thte

W. I)uante Belt of the photo-

REFERENCES 1. Barlnuuud P, Novikoff AB, Hamerntaum 1): Electrout microscopy of the hunmanu synuovial membnane. J Cell Biol 14:207, 1962 2. Banronu K, Bennsohut J, Hess A: Abntorma!ittes

.

of a regioni regiont

jo)inut

that

porous

graphis.

nuorma!

mt

account

the

Es-2

II zontes

resembles

also

TMJ. of jo)iuit

suggests

from sunrface

syntovial

of the

imihibitioni

ntentbrante

the

of thie

finudiutgs of a funuctionial symiovial false joint of the rabbit rauiuus

authors for his

I asufficienut

that

h)eruiteabilit’.

synuovial

region II esterases are extrato couuipare thteutu to l)latelet

to determinue whether genetic polymorphismti.

atud

found front

contcel)t

which

the

regionms

the

the

Surgery

presenut showed

(i.e.,

seenui to be

jecturah. cellular, isozymes.

proposed

proportions to

with eseniute while the serum amid joinut fluids had increased activity. Whether or riot the isozymes of regiout II are the sante as systent 2 is still coutSinuce the it is difficult

was

TMJ

varyimig untique

omily

partial

joint

the

more tltaut present iii

symiovial

did

‘FMJ

red cell. Eacht of these regioums conutaints one zonte of activity. The enizyntues are

amid

charac-

the

by the

knee

to

that

occurs

rate as the esterases

tive

de-

the

bearinug

articular

may

arid

as judged

closely

into

thue

cartilage

esterase

inihibitiont

been fluid

cartilage

the

DFP amid EI)TA, vaniationu. The

showed

of

two fluids. Titus, rabbit

in

Separations

has

false for

nutore activity the esterase l)atternu

more

a trarusfer

mmknee

sinutilar

a vee)iuig

fibrocartilage

enthis than

29).

resenmbled

lubrication

the

at

accounut

wluent

II

2 of the

bands

However,

Additionually,

fluid of a

l)roduce(l process. The

mtuernbrante

regiomu I uiot idenutical

membranie), I, so that

1 arid

six

of are

alonug the in regioni

was

in synuovial developuuiemmt

activity.

.1!

understood. Red

activities fluids

techutuique

(35) huave shuownu

nmay therefore

mu a niarunier

fiutdimig

is not

synuovial

esterase

The synovial

of the

et al.

activity Thte

aspirated

amid

were independent to account for

West

i ut a surgically of a pathologic

rabbit

zymogranuis.

(26,

but

partial citauige

enizymes. These latter results of substrate. The nuiechantismut differenutia!

red

mi the

increased

the

becaunse

fluid.

muew developing

of those

substrate

acetate.

observed

cell, Es-2

front

suuspect

the

nierubranue is a repair

joimit

sites

differemtt

butyrate

with

that

active

be

inucreased enzynuatic duriumg (hisease states.

miormal

regiont is thus probably under comitrol. A similar situation

witht

umichanuged

inihibitionu

II had

uttay

acquiring

iii

syntovial fracture

their

sulfate, a reversible cholintesterase 31, 36), in the presenuce of 10

inhibitor

red the

the

dentonistrated

esterases

which at

complete region

I are

regionu

29) . Eseninue

front

The

while

that

Each genietic

beeni

36).

lI

10

imihibitor residues

SCHIFF

I,

ESTERASE

itt

brainu

esterases

itt

ISOZYMES

multiple

sclerosis.

Proc

Soc Exp Biol ‘Ied 113:521, 1963 3. Blttun S, Januis H, hlamermamu D Cellurlar origiuu of hyahunronateprotein in human syniovial membrane. Science 150:353, 1965 4. Bnunnustetter MA, Hardie JA, Schiff H, Lewis

:

JP,

Cross

CE:

nuacrophages: Es-2 marker 1064, 1971 5. Cohen AS:

Origin

sturdies of

mice.

Lactic

of

punlmoumary

of stem Arch

6. I)rachmanu

7.

Med

Sokoloff

127:

(L!)H) of synuovial states,

arid fluuid with a

isozymes.

L: The

Ar-

role

of move-

embryonuic joint developmeuut. Biol 14:401, 1966 Ghadiahly FN, Roy A: Histochemistry oviurm itt experimeuttal haemarthrosis rabbit. Autut Ithetum I)is 26:117, 1967

I)ev

merit

1)B,

unsinug the

Intern

dehydrogenuase

trammsamiuuase (GOT) activity and seruim in rheumatic disease note out synovial flunid LI)H thritis itheurm 7:490, 1964

alveolar

cells

in

of synuitt the

Giblett ER: Genetic Markers in Human Blood. Bhackwelh Scientific Publications, Oxford, 1969 9. (1omoni (1: Histochemistry of huimant esterases. J hhistochem Cytochem 3:469, 1955 10. Gordon AH: Elect rophoresis of Proteinus mi S.

Pohyacrylamide

Elsevier 1969,

Publishing

13.

14.

15. 16.

17.

18.

Starch

Gels.

Company,

New

York,

AA, Sartore mu red-ce!!

G, Stonmonmt estenases

C: Genetic of rabbits.

Geuuetics 52:1545, 1965 Habbi I, Muirnanme TW, l)okui HC: Silastic arid Sunpramid mu anthrophasty of temponomanudibular joinut itt the rabbit. J Oral Stung 28:267,

1970 Hamenman I), Hosenubert L, Schubert M: Diarthrodial joinuts revisited. J Bone Joint Sting 52:725, 1970 Hamenman I), Sanidsoum J: Isolatioum of hyalunrontate from syruovial flunid by zone elect rophoresis. Natuire (Lonmd) 188:1194, 1960 Hamermanu D, Sandsoum J: Untustnal properties of hyalunrouuateproteiuu isolated from pathological synmovial flunids. J Cliut Invest 42:1882, 1963 Hamerman D, Schtnster H: Hyalunronuate mt nuormal htumant synuovial fluuid. J Clint Invest 37:57, 1958 Harris E, I)ibouma I), Krane SM: Collagenmases itt htumaum synovial flunid. J Clint Invest 48 :2104, 1969 Holmes H, Masters C, Weh)h) E: A comparative sttndy of vertebrate esterase ntuu!tiplicity. Conip Biochem Physiol 26:837 1968

SYNOVIAL

219

FLUID

19. Kristjantssonu FK : Genetic control of two blood serum proteimus in swinue. Cant J Genet Cytol 2:295, 1960 20. Linn FC, Itadinu EL: Lubrication of animal joints. III. The effect of centaini chemical alterationu of the cartilage and huubnicant. Arthritis Rheuim 11:674, 1968 21. Munrmuaute TW: Uptake of thonionu dioxide by synuovial membranue. Anmat Rec 160:398, 1968 22. Muurnamte TW: Effect of sclenosannts on the syntovial lining tissures of the rat squamosontanudibunlar joinmt.J Oral Sung 28:96, 1970 23. Munrmtane TW, l)okui HC: De novo formation of a syntovial membrane from fibrous connuective tissue. Jnt Assoc I)ent Res Abstr 151, 1971 24. O’Hare KH, Newmamu JK, Vatter AE, Reiss OK: Esterases in developing an II. Ant electrophoretic analysis.

25. 26. 27.

Amenicanu

p 43-127

11. Grtnnder variatiomus 12.

tumid

IN

28. 29. 30. 31. 32. 33.

Cytochem 19:116, 1971 Roy 5, Chadially FN: Synthesis of hyalunontic acid by synuovia! cells. J Path Bact 93:555, 1967 Schiff It: The biochemical genetics of rabbit erythrocyte esterases histochemical chassificationu. Schiff

J Histochem It, Brunustetten

Isozymes

alteratiomu J Clint

35. 36.

Cytochem MA,

18:709, Htnnuter

1970 IlL, Cross

CE: Electrophoretic separationt of estenases of pulmontary alveolar cells. J Histochem Cytochem 18:167, 1970 Schiff R, Jacobson 5: Genetic conutro! of esterase isozymes from rabbit brain. Genetics (14:S56, 1970 Schiff H, Stormonut C: The biochemical genuetics of rabbit erythrocyte esterases: two nuew esterase loci. Biochem Genet 4:11, 1970 Skunble 1), Choukas N, Toto P: Crantiomanudibunlan bone chanuges mt rhesus monkeys induced by condylectomy. J Oral Sung 28:273, 1970 Smith I, Ed: Chromatographic and Electrophoretic Techniques: Vol II. Electrophonesis. Interscience Publishers, New York, 1968 Stormonut C, Suuztnki Y: Atropinesterase of rabbit seruim: localization of the enzyme activity in isozymes. Science 167 :200, 1970 Vesell ES, Ostenlammd KC, Beam AG, Kinke! HB:

34.

adunlt rat lung. J Histochem

of

lactic

in arthritic

Inuvest

41:2012,

dehydrogena.se:

synovial

fluid

their

and

sera.

1962

Veys EM, Wieme RJ: Lactate dehydrogenase mu synoviah fluid. Aunt Itheum Dis 27:569, 1968 West M, Pashe RM, Black AB, Zimmerman HJ: Enuzyme activity in synovial fluids. J Lab Clin Med 62:175, 1963 White A. Handler P, Smith E: Principles of Biochemistry. Ed 3. McGraw Hill Book Cornpanty, New York, 1964, p 703-705

Suggest Documents