out systems. 1 arid. 2 of the red cell amid regionus. I anud II of serunm, knee amid TMJ svntovial fltnids tire shownu. The results with serum, knee arid TMJ.
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
