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Changes in apolipoprotein Band oxidized low‑density lipoprotein levels in gingival crevicular fluid after initial
preparation
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「 一 Keywords:
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ofP e r i o d o n t a l Research Dra
代O r i g i n a l A r t i c l e
n/a 一
I s h i z u k a , Motono
「i ;Showa U n i v e r s i t y School
ofD e n t i s t r y , Depa
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ofP e r i o d o n t o l o g y ; Showa U n i v e r s i t y
School of Pharmacy, Di v i s i o n
ofB i o
logical Chemistry, Department ofMolecu
lar B i o l o g y
kato, r
i n a ; Showa U n i v e r s i t y School
of Pharmacy, Di v i s i o n of B i o
logicalCh
巴m i s t r y ,Depa
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lecular Bi o l o g y
M o r i y a , Yumi; Showa U n i v e r s i t y School
ofD e n t i s t r y , Depa
代ment
ofP e r i o d
ontology; Showa Un i v e r s i t y School
ofPharmacy, D i v i s i o n o
fB i o l o g i c a l Chemistry, Department of Mo
le c u l a r B i o
logyNoguchi, Emiko; Showa U n i v e r s i t y School o f D e n t i s t r y , Department
ofPeriodonto
logyK o i d e , Yoko; Showa U n i v e r s i t y School of D e n t i s t r y , Department
ofP e r i o d
ontologyI n o u e , S h i n ; Showa U n i v e r s i t y School of D e n t i s t r y , Department
ofP e r i o p e r a t i v e M e d i c i n e , D i v i s i o n of Genera
lMedicine
I t a b e , H i r o y u k i ; Showa U n i v e r s i t y School
ofPharmacy, D i v i s i o n o
fB i o l o g i c a l Chemistry, Depa
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of阿olecularBi o l o g y
Yamamoto, Matsuo; Showa U n i v e r s i t y Schoo
l ofD e n t i s t r y , Department
ofPeriodontology
gingival crevicular
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xidizedl o w ‑ d e n s i t y l i p o p r o t e i n , a
polipoprotein B,i n i t i a l p
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Changes i n apolipoprotein B and oxidized l ow‑density l i p oprotein l e v e l s i n g i n g i v a l
c r e v i c u l a r f l u i d a f t e r i n i t i a l preparation
Motonori l s h i z u k a " ・
b'I ミ i n aKato¥ Yumi Moriya" ・
b'Em i k o N o g u c h i ' , Yoko K o i d e " , S i n
lnoue c , H i r o y u k i l t a b e 1
》，Matsuo Yamamoto"・*
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D e p a r t m e n t of P e r i o d o n t o l o g y , S h o wa U n i v e r s i t y S c h o o l of De n t i s t r y , 2
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D i v i s i o n o f B i o l o g i c a l C h em i s t r y , D e p a r t m e n t o f Molecu l a r B i o l o g y , Showa U n i v e r s i t y
Sc h oo l o f P h a r m a c y , I
・5 ‑ 8 H a t a n o d a i , S h i nagawa , Tokyo I 42
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c D e p a r t m e n t of Pe r i o p e r a t i v e M e d i c i n e , D i v i s i o n of G e n e r a l M e d i c i n e , S howa Un i v e r s i t y
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K e y w o r d s : g i n g i v a l c r e v i c u l a r f l u i d , ox i d i z e d l o w ‑ d e n s i t y l i p op r o t e i n , a p o l i p o p r o t e i n B , i n i t i a l
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A b s t r a c t
O 凶 e c t 1 v e :P e n o d o r
】t a ld i s e a s e i s a c h r o n i c i n f l a m m a t o r y d i s e a s e c a u s e d b y b a c t e r i a l i n 免 c t i o n t h a t c a n l e a d t o t o o t h l o s s G m g i v a l c r e v 1 c u l a r f l m d (GCF) c a n b e e a s i l y a n d n o n ‑ i n v a s i v e l y c o l l e c t e d We p r e v 1 0 u s l y d i s c o v e r e d t h e p r e s e n c e o f a p o l i p o p r o t e i n B ( a p o B ) , t h e main c o n s t i t u e n t o f l o w ‑ d e n s i t y h p o p r o t e i n , a n d o x i d i z e d l o w ‑ d e n s i t y l i p o p r o t e i n (oxLDL) i n GCF o f h e a l t h y s u 対 e c t s I n t h i s s t u d y , we m v e s t i g a t e d w h e t h e r p e n o d o n t 1 t 1 s a
伍e c t sapoB a n d oxLDL l e v e l s 1 1 1 GCF b y e x a m i n i n g t h e s e l e v e l s a f t e r i n i t i a l p
問p a r a t i o no f t e e t h ( s c a l i n g a n d r o o t p l a n n i i 】 呂 ， S R P ) .
M a t e r i a l s a n d m e t h o d s ・ The s t u d y p o p u l a t i o n c o m p r i s e d 1 1 p a t i e n t s w i t h c h r o n i c p e n o d o n t i t 1 s GCF s a m p l e s were c o l l e c t e d f r o m t h e h e a l t h y s i t e s a n d s i t e s w i t h p e r i o d o n t i t i s i n e a c h p a t i e n t a t b a s e l m e a n d a t 4 a n d 8 weeks a f t e r S R P . The volume a n d c o n c e n t r a t i o n o f p r o t e i n , a p o B , oxLDL a n d c y t o k m e s 1 1 1 GCF were m e a s u r e d .
R e s u l t s At t h e b a s e l m e , t h e volume a n d c o n c e n t r a t i o n o f p r o t e i n , apoB a n d oxLDL i n GCF w e r e h i g h e r a t s i t e s w i t h p e n o d o n t 1 t i s t h a n a t h e a l t h y s i t e s . ApoB a n d oxLDL c o n c e n t r a t i o n s i n GCF d e c r e a s e d a f t e r S R P . The volume a n d c o n c e n t r a t i o n o f p r o t e i n a n d oxLDL i n GCF c o r r e l a t e d w e l l w i t h p r o b i n g d e p t h .
C o n c l u s i o n : ApoB a n d oxLDL i n GCF a r e p o t e n t i 剖 l y u s e f u l m a r k e r s o f p e r i o d o n t a l 1 1 1 f l a m m a t 1 o n
J o u r n a l o f P e r i o d o n t a l Research
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Journal of Periodontal Research
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I n t r o d u c t i o n
P e r i o d o n t i t i s i s a c h r o n i c i n f l a m m a t o r y d i s e a s e c a u s e d by a n i n f e c t i o n from t h e b a c t e r i a i n
d e n t a l p l a q u
巴.I n t h e e a r l y s t a g e s , t h e i n f e c t i o n c a u s e s g i n g i v i t i s i n w h i c h t h e i n f l am m a t i o n i s
I i m i t e d t o s o f t t i s s u e s . Subseq u e n t l y , t h e d i s e a s e may p r o g r e s s t o p e r i o d o n t i t i s , i n w h i c h t h e
d e s t r u c t i o n of c o nn e c t i ve t i s s u e a nd a l veo l a r bone c a n l e a d t o t o o t h l o s s . Ging i va l c r e v i c u l a r
f l u i d (G CF) i s ex ud ed from t h e c r e v i c e between t h e t o o t h and t h e s u r r o u n d i n g g i n g i v a l t i s s u e .
GCF c o n t a i n s p l asma p r o t e i n s and components d e r i v e d
合om l oca l g i n g i va l c e l l s ( l ) and c a n b e
e a s i l y a n d n o n ‑ i n v a s i ve l y co l l e c t e d u s i n g d e n t a l p a p e r p o i n t s . Many s t u d i e s ha ve i n v e s t i g a t e d
t h e c o n s t i t
附1 t s of GCF; f o r examp l e ,
IL-I~.JL
・8 , a nd a s p a r t a t e a m i n o t r a n s f e r a s e i n GCF c a n
be u s e d t o d e t e r m i n e p
巴r i o d o n t a l i n f l a m m a t o r y s t a t u s ( 2
・4 ) .
Ox i d a t i v e s t r e s s , due t o an i n c r e a s e i n dang e r o u s p r o ‑ o x i d a n t s and / o r a d e c r e a s e i n p r o t e c t i ve
a n t i o x i d a n t s , i s i n vo l ved i n a va r i e t y of p a t h o l o g i c c o n d i t i o n s , s uch a s t h e i n f l ammatory
r e s p o n s e and t i s s u e damage. R e a c t i v e oxyge n s p e c i e s (ROS) and p r o d u c t s of c e l l u l a r a e r o b i c
metabo l i sm p l a y a c r u c i a l r o l e i n t h e a n t i b a c t e r i a l d e f e n s e . Howev e r , t h e ove r p r o d u c t i o n of ROS
c a u s e s t h e p e r o x i d a t i o n of membrane
1i p i d s , d e s t r u c t i o n of p r o t e i n s 、 and e x c e s s i ve p r o d u c t i o n o f
i n f l a m m a t o r y c y t o k i n e s . I n a d d i t i on t o i n f l a m m a t i o n and m i c r o b i a l i n f e c t i on , o x i d a t i v e damage
c a n t r i g g e r c a r d i o v a s c u l a r d i s e a s e (CVD), d i a b e t e s mel l i t u s (OM) , and p e r i o d o n t a l d i s e a s e s .
P r e v i o u s s t u d i e s r e p o r τ e d t h a t an i n c r e a s e i n p l asma l e v e l s of o x i d i z e d l ow‑de n s i t y l i p o p r o t e i n
Journal of Periodontal Research
. .
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，
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(oxLDL) i s a m a r k e r o f CVD ( 5 , 6 ） 剖 1 d t h a t h y p e r g l y c e m i a i n c r e a s e s u r i n a r y 8 ‑ h y d r o x y d e o x y g u a n o s i n e l e v e l s m p a t i e n t s w i t h t y p e 2 D M ( 7 ) S e v e r a l r e p o r t s h a v e shown a n a s s o c i a t i o n b e t w e e n p e r i o d o n t a l d i s e a s e a n d o x i d a t i v e s t r e s s Po1phyromonas g i n g i v a l i s , a m 旬。 rp a t h o g e n m p e r i o d o n t i ! J s , i n d u c e s ROS a n d r e g u l a t e s t h e p r o d u c t i o n of!L
・6a n d
Iレ I~i n g i n g i v a l e p i t h e l i a l c e l l s ( 8 ) . A d d i t i o n a l l y , we showed t h a t oxLDL i n d u c e s t h e p r o d u c t i o n o f p r o
司i n f l a m m a t o r ym e d i a t o r s , s u c h a s I レ 8 ,I L ‑
I~.a n d P G E , , 1 1 1 human g m g 1 v a l e p i t h e l i a l c e l l s ( 9 ) .
OxLDL i s c o n s i d e r e d t o p l a y a p i v o t a l r o l e m a t h e r o g e n e s 1 s LDL 1 s composed of a l a r g e number o f I i p i d m o l e c u l e s w i t h a s i n g l e m o l e c u l e o f a p o h p o p r o t e m B ( a p o B ) P r e v i o u s r e p o
巾h a v e shown a p o s s i b l e a s s o c i a t i o n b e t w e e n p l a s m a l i p o p r o t e i n m e t a b o l i s m a n d p e r i o d o n t a l d i s e a s e s LDL‑C l e v e l s a r e s i g n i f i c a n t l y h i g h e r i n p a t i e n t s w i t h p e r i o d o n t a l d i s e a s e s t h a n t h o s e m c o n t r o l s u 吋 e c t s( I O ) . When p e r i o d o n t a l t r e a t m e n t i s g i v e n t o p a t i e n t s w i t h c o r o n a r y h e a r t d J S e a s e , s e r u m oxLDL l e v e l s d e c r e a s e s i g n i f i c a n t l y ( I I ) . We p r e v i o u s l y f o u n d t h e p r e s e n c e o f apoB a n d oxLDL i n GCF c o l l e c t e d f r o m h e a l t h y p e n o d o n t r n m usmg a s a n d w i c h ELISA method ( 1 2 ) . H e r e , we 1 1 1 v e s t 1 g a t e d w h e t h e r m f l a m a t 1 o n a n d / o r d i s e a s e 1 1 1 l o c a l p e n o d o n t a l t 1
田u e sa f f e c t apoB a n d oxLDL l e v e l s i n GCF.
M a t e r i a i s and methods
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Pa t i e n t s a n d s t u dy d e s i g n
T h e p a t i e n t s e n r o l l e d i n t h i s s t u dy were f u l l y i n f o r me d a b o u t t h e s t u dy p r o t o c o l , a n d w r i t t e n
i n f o rm e d c o n s e n t was ob t a i n ed i n acco r d a n ce w i t h t h e D e c l a r a t i o n o f H e l s i nk i . The
ex p e r i m e n t a l p r o t oco l f o r t h i s s t u dy was a pp r o v e d b y t h e e t h i c s co mmi t t e e o f S h owa U n i v e r s i t y ,
Tokyo, J a p a n (No.20 1 4 ‑ 0 0 6 ) . E l e v e n pa t i e n t s w i t h c h r o n i c p e r i o d o n t i t i s a c c o r d i n g t o t h e c u r r e n t
c l a s s i f i c a t i o n of t h e A m e r i c a n Academy o f P e r i odonto l ogy ( 1 3 ) were r e c r u i t ed f r o m t h e
Depa r t m e n t of Pe r i odo n t o l ogy o f t h e De n t a l H o s p i t a l , S h owa U n i ve r s i t y , f r o m J u l y 2 014 t o J u l y
20 1 5 . T h e exc l u s i o n c r i t e r i a i n c l u d e d : t r e a tm e n t f o r p e r i odont i t i s i n t h e p r e c e d i n g 3 m o n t h s , u s e
of a n t i b i o t i c s a n d a n t i ‑ i n f l a m m a t o r y d r u g s w i t h i n t h e p r ece d i n g 3 mon t h s , know n s y s t e mi c
d i s e a s e ( e . g , . D M , o s t e o p o r o s i s , t um o r , a u t o ‑ immune d i s e a s e s , c e r e b r a l i n f a r c t i o n , myocardia l
i n f a r c t i o n ) , p r e g n a n cy o r l a c t a t i on i n fema l e s , a nd c u r r e n t c i ga r e t t e s mok i n g .
C l i n i c a l exa min a t i o n a nd GC F s a m p l i n g we r e pe r f o rm e d by t h e s a m e exa min e r t h r o u g h o u t t h e
s t u d y a t b a s e l i ne a n d a t 4 a n d 8 wee k s a f t e r i n i t i a l p r e p a r a t i o n ( s c a l i n g a nd r o o t p l a nn i n g , SRP),
w h e r e a s , b l ood s a m p l i n g was p e r f o rm ed a t b a s e l i n e o n l y ( F i g . 1 ) .
C l i n i c a l m e a s ure me n t s an d p e r i odo n t a l t h e r a p y
To a s c e r t a i n p a t i e n t s ' p e r i odo n t a l s t a t u s , t h e p r o b i n g pocke t d e p t h (PPD) a n d b l e e d i ng on
p r
叫ヲi n g(BOP) of t h e f u l l mo u t h we r e r e c o r ded i n e a c h pa t i e n t . T h e n , we s e l e c t e d s i t e s wi t h
p e r i o d o n t i t i s (PPD と 4mm) and h e a l t h y s i t e s [PPD三3m m a n d BOP （ 一 ） ］ 合omamong t h e
Journal o f Periodontal Research
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n a t u r a l m a x i l l a r y a n t e r i o r t e e t h o r p r e m o l a r s 1 1 1 e a c h p a t i e n t . C l m i c a l m e a s u r e m e n t s p e r お rmed a t t h e sam 判 i n gs i t e s i n c l u d e d PPD, g i n g i v a l i n d e x ( G I } , a n d p l a q u e i n d e x ( P l l ) . A l l p a t i e n t s r e c e i v e d 1 1 1 1 t i a l p e r i o d o n t a l t h e r a p y , mcludmg SRP and o r a l h y g i e n e e d u c a t i o n .
G i n g i v a l c r e v i c u l a r f l u i d sampling
GCF s a m p l e s were c o l l e c t e d
骨oma s i t e w i t h p e r i o d o n t i t i s a n d a l s o a h e a l t h y s i t e i n e a c h p a t i e n t . The GCF s a m p l i n g method u s e d was d e s c r i b e d p r e v i o u s l y ( 1 2 , 1 4 ) . B r i e f l y , t h e s i t e of GCF c o l l e c t i o n was i s o l a t e d w i t h c o t t o n wool r o l l s , a n d s a h v a was g e n t l y removed f t o m t h e a d j a c e n t t o o t h s u r f a c e a n d g i n g 1 v a u s i n g a n a i r b l o w . Two s t a n d a r d p a p e r p o i n t s (DENTSPLY l n t e r n a t 1 0 n a l I n c , Y o r k , PA, USA) were g e n t l y i n s e r t e d i n t h e g i n g i v a l c r e v i c e f o r I min a n d t h e n t r
剖lS自e r r e dmto I 00 μL of i c e ‑ c o l d s t e r i l i z e d p h o s p h a t e ‑ b u f f e r e d s a l i n e (PBS) c o n t a i m n g p r o t e a s e m l u b 1 t o r c o c k t a i l ( S i g m a ‑ A l d r i c h . P27 l 4 } . A 抗 e r5 m i n , t h e same p r o c e d u r e was r e p e a t e d w i t h new p a p e r p o i n t s . A l l s a m p l e s w i t h b l e e d i n g we
日e x c l u d e d .The volume ofGCF e x u d e d a t t h e s i t e was a n a l y z e d by one p a p e r p o i n t g e n t l y i n s e r t e d i n t h e g i n g 1 v a l c r e v i c e 自 由 1 0 s a n d m e a s u r e d u s i n g a p r e ‑ c a l i b r a t e d P e r i o t r o n
⑧8 0 0 0 ( P r o F l o w I n c , New Y o r k , NY, USA) P r o t e m c o n c e n t r a t i o n s w e r e d e t e r m i n e d u s i n g M i c r o BCA P r o t e i n A s s a y R e a g e n t (Thermo F i s h e r S c i e n t i f i c I n c , R o c k f o r d , I L , USA) w i t h b o v i n e s e r u m Albumin (BSA) a s t h e s t a n d a r d . GCF s a m p l e s were s t o r e d a t 4°C u n t i l t h e a n a l y s i s ofapoB a n d oxLDL, a n d t h e n a t ‑80°C u n t i l t h e a n a l y s i s of c y t o k i n e s .
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Blood sampling
Venous b l o o d ( 1 4 m ） し was drawn from t h e b r a c h i a ( v e i n of p a t i e n t s u s i n g a co l l e c t i n g t u b e .
Measurements of t h e f o l i o
仙i n gp a r a m e t e r s were p e r f o r m e d a t BML, I n c . ( T o k y o , J a p a n ) :
g l y c a t e d hemoglobin ; t r i g l y c e r i d e ; LD しC ;HDL‑chole s t e r o l ; t o t a l cho l e s t e r o l ; L i p o p r o t e i n ( a ) ;
h i g h ‑ s e n s i t i v i t y C ‑ r e a c t i v e p r o t e i n ; a n d ma l o n d i a l d e h y d e ‑ m o d i f i e d LDL (MDA‑LDL). P a r t of
t h e b l o o d s a m p le ( 5 mL) was c e n t r i f u g e d a t 1 5 , 0 0 0
×g f o r I 0 min t o s e p a r a t e t h e p l a s m a .
C hy l omicron (CM); v e r y l ow‑de n s i t y l i p o p r o t e i n (VLDL) and LDL f r a c t i o n s were s e p a r a t e d by
s e q u e n t i a l u l t r a c e n t r i f u g a t i o n , a s d e s c r i b e d p r e v i o u s l y ( 1 4 , 1 5 ) . Then , t h e apoB and oxLDL
c o n c e n t r a t i o n s of e a c h l i p o p r o t e i n f r a c t i o n we r e u s e d t o c a l c u l a t e t h e i r t o t a l co n c e n t r a t i o n i n
p l a s m a . The CM, VLDL, a nd LDL s a mple s were s t o r e d a t 4 ° C u n t i l a n a l y s i s . No e f f o r t s we r e
made t o modify t h e d i e t s of t h e p a t i e n t s p r i o r t o b l o o d co l l e c t i o n .
Measurements of a p o l i p o p r o t e i n 8 and o x i d i z e d LDL c o n c e n t r a t i o n s
The co n c e n t r a t i o n s of apoB i n GCF s a mpl e s were measured by t h e s a ndwich ELISA p r o t o c o l
w i t h s l i g h t m o d i f i c a t i o n s ( 1 4 ) . B r i e f l y , 96
・wel l m i c r o t i t e r p l a t e s ( 3 3 6 9 ; C o s t a r
⑧；Co r n i n g I n c . )
c o a t e d w i t h I 00 μL o f an a n t i ‑ h u m a n a poB monoc l o n a l a n t i b o d y s o l u t i o n (H454640M;
M e r i d i a n L i f e Sc i e nc e I n c . , Saco , ME, USA; I 0
~tg/mLi n PBS) f o 1
・2 h a t 37 ° C we r e b l ocked
w i t h 20
・m MT r i s ‑ b u f f e r e d s a l i n e (TBS) con t a i n i n g I % BSA a t room t e m p e r a t u r e f o r 2 h .
Samp l e s of I 00
~tLwe r e i n c u b a t e d i n t h e we l l s f o r 1 . 5 h a t 37 ° C , t h e n washed f o u r t i m e s wi t h
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340 μL o f TBS c o n t a i n i n g 0 . 1 % Tween‑20 ( T B S ‑ T ) . G o a t a n t i ‑ h u m a n apoB p o l y c l o n a l a n t i b o d y ( P 0 4 1 1 4 ; 1 : 2 0 0 0 d i l u t i o n i n PBS; A c r i s A n t i b o d i e s G r n b H , H e r f o r d , Germany) was a d d e d t o t h e w e l l s a n d i n c u b a t e d f o r I h a t 3 7 ° C . A f t e r w a s h i n g 五 o u rt i m e s w i t h 340 μL o f TBS‑T, a l k a l i n e p h o s p h a t a s e ‑ c o 町 u g a t e dd o n k e y a n t i ‑ g o a t l r n r n u r
】o g l o b u l mG ( l g G ) p o l y c l o n a l a n t i b o d y ( I 1 5 0 0 d i l u t e d
111TBS contammg I% skimmed m i l k ; J a c k s o n l r n m u n o R e s e a r c h L a b o r a t o n e s , I n c , West G r o v e , PA, USA) was a d d e d t o t h e w e l l s a n d i n c u b a t e d f o r I hat 3 7 ° C . The p l a t e s w e r e washed
自o u rt i m e s w i t h 3 4 0 μL ofTBS‑T a n d o n c e w i t h T r i s ‑ H C I , pH 8 . 8 . The p l a t e s w e r e m c u b a t e d w i t h I 0 0 μL p e r w e l l o f p ‑ n i t r o p h e n y l p h o s p h a t e 抗 o rI h a t 3 7 ° C . The a b s o r b a n c e s m e a s u r e d a t 4 0 5 a n d 490 nm w e r e r e c o r d e d u s i n g a r n i c r o p l a t e r e a d e r ( M o d e l 6 8 0 ; B 1 0 ‑ R a d L a b o r a t o n e s , I n c . , H e r c u l e s , CA, USA)
The c o n c e n t r a t i o n s o f oxLDL m t h e GCF s a m p l e s w e r e m e a s u r e d u s i n g a s a n d w i c h ELISA p r o t o c o l ( 1 4 ‑ ‑ 1 6 ) t h a t was m o d i f i e d s l i g h t l y t o i m p r o v e
出s e n s i t i v i t y . B r i e f l y , 9 6
・w e l l m 1 c r o t 1 t e r p l a t e s ( 3 9 2 2 , C o s t a r R ; C o r n i n g I n c . ) c o a t e d w i t h 1 0 0 μL o f a n t i ‑ o x i d i z e d p h o s p h o l i p i d m o n o c l o n a l a n t i b o d y DLH3 s o l u t i o n ( 7 . 5 ド ダ mLi n PBS) o v e r n i g h t a t 4°C w e r e b l o c k e d w i t h 3 4 0 μL o f B l o c k i n g B u f f e r ( T h e n n o F i s h e r S c i e n t i 五 c )a t room t e m p e
問t u
問f o r1 5 min S a m p l e s o f I 00 ド Lw e r e i n c u b a t e d m t h e w e l l s f o r 2 h a t 4 。 C ,t h e n washed f o u r t i m e s w i t h
340 μL o f TBS c o n t a i n i n g 0 . 1 % Tween‑20 ( T B S ‑ T ) . G o a t
田1 t i ‑ h u m a napoB p o l y c l o n a l a n t i b o d y (P04 I I 4 ; I : 2 5 0 0 d i l u t i o n i n PBS; A c r i s A n t i b o d i e s GmbH) was a d d e d t o t h e w e l l s
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a nd i n c u b a t e d f o r I h a t 3 7 ° C . A f t e r was h i n g f o u r t im e s w i t h 34 0 μL of TB S
・T
、ah o r s e r a d i s h
p e r o x i d a s e ‑ c o n j u g a t e d donkey a n t i ‑ g o a t l gG ( 1 . 1 2 , 5 0 0 d i l u t e d i n TBS c o n t a i n i n g I % s k imm e d
mi l k ; J a c k s o n lmmun oResearc h L a b o r a t o r i e s , I n c . ) was a dd e d t o t h e we l l s and i n c u b a t e d f o r I h
a t 37 ° C . The p l a t e s we r e was h e d f o u r t i m e s w i t h TBS ‑ T a nd o n c e w i t h 1 0 0 μL of 50
・mM
T r i s ‑H C I , p H 8 . 8 . The p l a t e s we r e i n c ub a t e d w i t h I 00 μL p e r w e l l of ELISA Femto Ma x imum
S e n s i t i v i t y S ub s t r a t e (T h e r m o F i s h e r S c i e n t i f i c ) w i t h s h a k i n g f o r I min . C h e milumin e s c e n c e
was m e a s u r e d u s i n g a s p e c t r a l s c a n n i n g multim o d e r e a d e r (Varioskan™ F l a s h ; Th e rm o F i s h e r
Sc i e n t i f i c ) . Co pp e r ‑ i n d u c e d o x i d i z e d hum a n LDL was u s e d a s a s t a nd a r d .
Measurements of cy t o k i n e co ncentr a t i o n s
Th e c o n c e n t r a t i o n s o f c y t o k i n e s i n GCF s a mpl e s we r e m e a s u r e d by f l o w c y t on
】e t r yu s i ng a
Hum a n I n f l a mmator y Cytok i n e s K i t (BO B i o s c i e n c e s , F r a n k l i n Lak e s , N J , USA) t h a t a l l owe d
d i s c r i m i n a t i o n o f t h e f o l l ow i n g c y t o k i n e s : I L ‑
I~，I L
・6 , I L
・8 , I L ‑I 0 , I L ‑ I 2 p 70 , an d tum o r
n e c r o s i s f a c t o r (TNF). Sa mp l e p r o c e s s i ng a nd d a t a a n a l y s i s we r e p e r f o r m e d a c c o r d i n g t o t h e
manufa c t u r e r s i n s t r u c t i o n s . B r i e f l y , GCF s a mple s we r e i n c u b a t e d w i t h t h e s i x cy t ok i n e c a p t u r e
b e a d s a nd ph y c o e r y t h r i n ‑ co n j u ga t e d d e t e c t i o n a n t i b od i e s a t r oo m t e mp e r a t u r e f o r 3 h an d were
t h e n was hed . Cyto k i n e l e v e l s we r e a s s e s s e d u s i n g a FACSVe r s e T M F l ow Cy t o met e r (BO
B i o s c i e n c e s ) . T h e d a t a a c qu i r e d we r e a n a i yze d u s i n g FCAP A r r a y Softw a r e (BO Bio s c i e n c e s ) .
S t a t i s t i c a l a n a l y s i s
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R e s u l t s a r e p r
田e n t e d
田t h em e d i a n w i t h t h e t o p 2 5 t h a n d b o t t o m 2 5 t h p e r c e n t i l e s 五 （ r s ta n d t h i r d q u a r t i l e s ) r a t h e r t h
叩t h emean a n d s t a n d a r d d e v i a t i o n . The c o m p a r i s o n b e t w e e n s i t e s w i t h p e r i o d o n t 1 t 1 s a n d h e a l t h y s i t e s a t b a s e l i n e was p e r f o r m e d u s i n g t h e W i l c o x o n s i g n e d ‑ r a n k t e s t V a l u e s o f p
く00 5 w e r e c o n s i d e r e d s 1 g m
自c a n tI n t r a g r o u p c o m p a r i s o n s w e r e a s s e s s e d u s i n g t h e Bon 日 e r r o m ・ c o r r e c t e dW i l c o x o n s i g n e d ‑ r a n k t e s t t o a n a l y z e s i g n i f i c a n t d i
俗r e n c e sb e f o r e a n d a
自e rt r e a t m e n t V a l u e s o f p < 0 . 0 5 / 3 = 0 . 0 1 6 7 w e r e c o n s i d e r e d s i g n i f i c a n t . The c o r r e l a t i o n was e v a l u a t e d usmg Spearmans c o r r e l a t i o n t e s t V a l u e s o f p < 0 . 0 5 w e r e c o n s i d e r e d s i g n i f i c a n t . F o r a l l s t a t 1 s t 1 c a l a n a l y s e s , we u s e d JMPR P r o 1 1 (SAS I n s t i t u t e I n c . , C a r y , NC, USA).
R e s u l t s
Study p o p u i a t i o n and c l i n i c a I p a r a m e t e r s
The b a s i c c h a r a c t e n s t r c s o f p a t i e n t s w i t h chromc p e n o d o n t i t i s e x a m i n e d i n t h i s s t u d y a r e p r e s e n t e d 1 1 1 T a b l e 1 The p
剖1 e n
臼a
呂田r a n g e d 世 om5 7 . 5 t o 6 7 y e a r s , w i t h a m e d i a n v a l u e o f 6 1 y e a r s A l l p a t r e n t s h a d a t l e a s t two s i t e s o f p e r i o d o n t i t i s a n d BOP. P a t i e n t s b i o c h e m i c a l p a r a m e t e r s w e r e , o n a v e r a g e , a l l wrthm t h e n o r m a l r a n g e s .
GCF s a m p l e s were c o l l e c t e d f r o m a s i t e w i t h p e r i o d o n t i t i s a n d a h e a l t h y s i t e i n e a c h p a t i e n t The c l i 1 1 1 c a l p a r a m e t e r s o f t h e GCF c o l l e c t i o n s i t e s a r e shown i n T a b l e 2 . At b a s e l m e , PPD, G I , a n d P l l w e r e s r g m 日 c a n t l yh i g h e r a t s i t e s w i t h p e n o d o n t i t i s t h a n a t h e a l t h y s i t e s . A f t e r
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pe r i o d o n t a l t h e r a p y , PPD v a l u e s a t 4 and 8 weeks a f t e r SRP d e c r e a s e d s i g n i f i c a n t l y from t h o s e
a t b a s e l i n e a t s i t e s ofpe r i o d o n t i t i s . GI was d e c r e a s ed a t 8 weeks
合om t h a t a t b a s e l i n e a t s i t e s of
p e r i o d o n t i t i s
目P I Iwas a l s o r e d u c e d
什om 1 t o 0 a
仇e ri n i t i a l p e r i o d o n t a l t h e r a p y a t s i t e s of
p e r i o d o n t i t i s ; howev e r , t h e v a l u e s d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e .
The c h a r a c t e r i s t i c s of g i n g i v a l c r e v i c u l a r f l u i d samples a t b a s e l i n e
The vo l ume of GCF exuded a t t h e s i t e and co n c e n t r a t i o n s of p r o t e i n , apoB , and oxLD L i n
GCF s a mpl e s were d e t e r m i n ed a t b a s e l i n e . The GCF vo l ume mea s u r e d u s i n g t h e P e r i o t r o n
⑧8000 was s i g n i f i c a n t l y h i g h e r a t s i t e s of pe r i o d o n t i t i s t h a n a t h e a l t h y s i t e s ( p
く0 . 0 I ; F i g . 2 a ) .
GCF was co l l e c t e d u s i n g f o u r pap e r po i n t s a t a s i t e , a n d e a c h s ample was r ecove r e d i n I 00 pL
of PBS . Becau s e t h e a c t u a l vo lum e of e a c h GCF s a mpl e c o u l d n o t be m e a s u r e d , t h e
conce n t r a t i o n s of p r o t e i n , apoB , and oxLDL i n t h e GCF i n PBS were d e t e r m i n e d . ApoB and
oxLDL i n t h e GCF s a mple s were measur e d by s a nd wi c h ELISA u s i n g a n t i ‑ a p o B and DLH3
a n t i b o d i e s . A l l t h r e e paramet e r s were s i g n i f i c a n t l y h i g h e r a t s i t e s of p e r i o d o n t i t i s t h a n a t h e a l t h y
s i t e s ( F i g . 2 b ‑ d ) .
Changes i n g i n g i v a l c r e v i c u l a r f l u i d samples a f t e r i n i t i a l preparat i o n
We a n a l yzed G CF s amp l e s a t b a s e l i n e and a t 4 and 8 weeks a f t e r i n i t i a l p r e p a r a t i o n ( S R P ) .
A
仇e r t h e i n i t i a l p r e p a r a t i o n , t h e GCF vo lum e , t h e p r o t e i n c o n c e n t r a t i o n and t h e apoB
c o n c e n t r a t i o n i n t h e GCF s amp l e s s i g n i f i c a n t l y d e c r e a s e d a t s i t e s of p e r i o d o n t i t i s a t 4 and 8
Journal of Periodontal Research
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weeks
骨omt h o s e a t b a s e l i n e ( p < 0
05β~0 . 0 1 6 7 a n d p < 0
OJβ~0 0 0 3 3 , F i g 2 a ‑ < : ) . I n a d d 1 t w n , t h e oxLDL c o n c e n t r a t i o n i n GCF s i g n i f i c a n t l y d e c r e a s e d a t 4 weeks a t s i t e s o f p e r i o d o n t i t 1 s ( p < 0 0 5 1 3 = 0 0 I 6 7 , F i g 2 d ) T h i s d e c r e a s e m oxLDL l e v e l s b e t w e e n b a s e I me a n d 8 weeks d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e : a l t h o u g h t h e m e d i a n v a l u e s w e r e w e l l s e p a r a t e d ( 1 7 c o m p a r e d w i t h 0 ) , t h e d i s t r i b u t i o n o f t h e i n d i v i d u a l v a l u e s v a r i e d s o m e w h a t . GCF s a m p l e s f r o m h e a l t h y s i t e s d i d n o t s i g n i f i c a n t l y c h a n g e a f t e r m 1 t i a l p r e p a r a t i o n
C y t o k i n e s ( i n t e r l e u k i n ‑ I f } and 8 ) i n g i n g i v a l c r e v i c u l a r f l u i d s a m p l e s
We m e a s u r e d t h e c o n c e n t r a t i o n s o f I L ・ I [ } ,I L ‑ 6 , I L ‑ 8 , I L ‑ I 0 , I L ‑ 1 2 p 7 0 , a n d TNF i n GCF s a m p l e s usmg f l o w c y t o m e t r y The I レ I f }a n d l レ 8c o n c e n t r a t i o n s m t h e GCF s a m p l e s a r e shown
111T a b l e 3 . At b a s e l i n e , t h e c o n c e n t r a t i o n o f
Iレ I~ 111t h e GCF s a m p l e s was s i g n i f i c a n t l y h i g h e r a t s i t e s o f p e r i o d o n t 1 t i s t h a n a t h e a l t h y s i t e s ( p
く0 . 0 5 ) .No s i g n i f i c a n t d i f f e r e n c e was e v i d e n t m t h e c o n c e n t r a t i o n o f I L ‑ 8 i n GCF b e t w e e n s i t e s w i t h p e r i o d o n t i t i s a n d h e a l t h y s i t e s . We w e r e u n a b l e t o d e t e c t I L ‑ 6 , I L ‑ 1 0 , I L ‑ 1 2 p 7 0 , a n d TNF m GCF t h e c o n c e n t r a t i o n s o f t h e s e c y t o k i n e s w e r e h k e l y b e l o w t h e l i m i t s o f d e t e c t i o n ( d a t a n o t s h o w n ) . A l t h o u g h t h e m e d i a n I L ‑I l f c o n c e n t r a t i o n t e n d e d t o d e c r e a s e a f t e r S R P , c h a n g e s i n t h e I L ‑ I l f a n d I L ‑ 8 c o n c e n t r a t i o n s
of t h e GCF s a m p l e s d i d n o t r e
鴻c hs t a t i s t i c a l s i g n i f i c a n c e C o n v e r s e l y , t h e c h a n g e s i n c y t o k i n e c o n c e n t r a t i o n s r e l a l ! v e t o t h a t e v i d e n t a t h e a l t h y s i t e s a f t e r i n i t i a l p r e p a r a t i o n w e r e s i g n i f i c a n t . C o r r e l a t i o n b e t w e e n g i n g i v a l c
問v i c u l a rf l u i d and p r o b i n g p o c k e t d e p t h a t b a s e l i n e
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T a b l e 4 s ho ws t h e c o r r e l a t i o n b e t wee n G C F param e t e r s and PPD o f t h e s i t e s o f GCF c o l l e c t i o n
a t b a s e l i n e . Th e vo lum e o f a nd co nc e n t r a t i o n s o f p r o t e i n and ox LDL i n GC F s a mpl e s p o s i t i ve l y
c o r r e l a t e d w i t h PPD . A l t h o u g h t h e a p o B and I L
・l 0 co n c e n t r a t i o n s i n G CF s h ow ed a t e n d e n cy
tow a r d a p o s i t i ve co r r e l a t i o n w
仙PPD , t h ey d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c anc e ( p = 0 . 0 5 9 and
p =
0 . 08 9 , r e s p e c t i ve l y ) . I L
・8 co n ce n t r a t i o n s i n G C F s ampl e s d i d n o t d i s p l a y a c o r r e l a t i o n w i t h
PPD .
C omparison of t h e r e l a t i v e r a t i o o f o x i d i z e d l o w ‑ d e n s i t y l i p o p r o t e i n : a p o l i p o p r o t e i n B i n
t h e plasma and g i n g i v a l c r e v i c u l a r f l u i d
We co mpar e d t h e r e l a t i ve ox LDL : apoB r a t i o s i n GC F a nd p l a s ma s ampl e s a t b a s e l i n e ( T a b l e
5 ) . T h e ox LD L : ap o B r a t i o r e p r e s e n t s t h
巴r e l a t i ve s u s c e p t i b i l i t y of LDL t o ox i d a t i ve
m o d i f i c a t i o n . Th e o x LDL : a p o B r a t i o i n GC F was d i s t r i b u t e d i n a w i d e r a n g e , a s s h ow n b y t h e
t o p a nd b o t t o m qu a r t i l e s , w h e r e a s i n p l a s m a , t h e ox LDL: a p o B r a t i o r ema i ned n e a r l y c o n s t a n t ,
r a n g n g
合o m 0 . 1 3 t o 0 . 2 9 n g / ド g .Th e me d i a n ox LDL:ap o B r a t i o s i n p l a s m a and GC F f r o m
h e a l t h y s i t e s a n d s i t e s w i t h p e r i o d o n t i t i s we r e 0 . 1 7 , 6 . 2 , a nd 34 n g / μ g , r e s p e c t i ve l y . Th e
d i f f e r e n c e i n ox L DL:a p o B r a t i o s b e t w e e n GCF f r o m s i t e s w i t h p e r i o d o n t i t i s and p l a s m a w a s
s i g n i f i c a n t . T h e oxLDL:apoB r a t i o i n GCF f r o m h e a l t h y s i t e s was ab o u t 36
・f o l dh i g h e r t h a n t h a t
of p l a s ma ; h oweve r , i t d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . Th e s e r e s u l t s s u g g e s t t h a t ox LDL i s
e n r i c h e d i n GCF s a mpl e s .
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GCF 1 s a u s e f u l p h y s 1 0 l o g 1 c f l u i d t h a t c a n b e c o l l e c t e d 1 1 0 1 1 ‑ i n v a s i v e l y . The p r e s e n c e o f apoB a n d oxLDL m GCF d e r i v e d 世 omh e a l t h y p e r i o d o n t i u m was d e m o n s t r a t e d i n o u r p r e v i o u s s t u d y ( ! 2 ) The a s s o c i a t i o n b e t w e e n c y t o k i n e s , s u c h a s I L ‑ I J 3 a n d I L ‑ 8 , a n d p e r i o d o n t i t i s h a s b e e n e s t a b l i s h e d ( 2 , 3 ) . I n t h e p r e s e n t s t u d y , we c o l l e c t e d GCF
骨oms i t e s w i t h p e r i o d o n t i t i s a n d h e a l t h y s i t e s i n e a c h p a t i e n t t o i n v e s t i g a t e w h e t h e r l o c a l i n f l a m m a t i o n a n d / o r d i s e a s e i n p e r i o d o n t a l t i s s u e s a f f e c t t h e apoB a n d oxLDL l e v e l s
111GCF We 五 o u n dt h a t t h e apoB a n d oxLDL c o n c e n t r a t i o n s w e r e h i g h e r m GCF d e n v e d f r o m s i t e s w i t h p e n o d o n t a l d i s e a s e t h a n i n GCF o b t a m e d
合omh e a l t h y s i t e s . I n a d d i t i o n , a s t h e p e r i o d o n t i u m h e a l e d f o l l o w m g c l i m c a l t r e a t m e n t , apoB a n d oxLDL c o n c e n t r a t w n s i n GCF d e c r e a s e d Our r e s u l t s s u g g e s t t h a t apoB
a n d oxLDL l e v e l s
111GCF r e f l e c t t h e d i s e a s e c o n d i t i o n o f t h e p e r i o d o n t i u m .
We m e a s u r e d oxLDL c o n c e n t r a t i o n s
111GCF s a m p l e s b y s a n d w i c h ELISA u s i n g t h e a n t i ・ o x i d i z e d p h o s p h o l i p i d m o n o c l o n a l a n t i b o d y DLH3 ( I 2 , 1 4 ) . S a n d w i c h ELISA h a s p r e v w u s l y b e e n u s e d t o d e t e r m i n e t h e oxLDL l e v e l s i n p l a s m a f ト omp a t i e n t s w i t h CVD a n d p a t i e n t s w i t h r e n a l f a i l u r e ( 5 , 6 , 1 5
・1 7 ) .I n t h e
戸田町】ts t u d y , we m t r o d u c e d a c h e m i l u m m e s c e n t d e t e c t o r t o i m p r o v e t h e s e n S 1 t 1 v 1 t y o f t h e m e t h o d , s o t h a t s m a l l v o l u m e s o f s a m p l e s l i k e GCF c a n b e s t a b l y m e a s u r e d
The c h m c a l p a r a m e t e r s o f p e r i o d o n t a l d i s e a s e d e c r e a s e d a f t e r S R P , c o n f i n m n g t h a t t h e p e r
旧d o n t a lc o n d i t i o n i m p r o v e d . The c o n c e n t r a t i o n s o f p r o t e m , a p o B , a n d oxLDL
111GCF, a s
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w e l l a s i t s vo l u me , d e c r e a s e d a t 4 a nd 8 wee k s a f t e r S RP . A t 8 wee k s , t h e f o u r m e a s u r e s b e c a m e e q u i v a l e n t t o t h o s e of h e a l t h y GCF a t b a s e l i n e . P r e v i o u s s t u d i e s r e p o r t ed t h a t t h e vo lum e a nd c o n c e n t r a t i o n s of p r o t e i n s i n GCF i n c r e a s e d co n co mi t a n t l y w i t h t h e de s t r u c t i o n of t h e pe r i odo n t ium ( 2 , 1 8 , 1 9 ) . F u r t h e r m o r e , s e v e r a l s t u d i e s s h owe d t h a t l eve l s o f MDA ‑L D L a n d 8
・i s o p r o s t a n e i n GCF we r e h i g h e r i n p a t i e n t s w i t h p
巴r i o d o n t i t i s t h a n i n co n t r o l s u b j e c t s ( 2 0
・2 2 ) . O u r r e s u l t s de m o n s t r a t e t h a t t h e vo lum e a nd co n s t i t u e n t s of GCF a r e a f f e c t e d by l oca l t i s s u e c o n d i t i o n s , b u t r e v e r t t o n o rm a l f o l l ow i ng t r e a t m e n t .
T h e co n c e n t r a t i o n s o f p r o t e i n , a p oB, a n d oxLDL i n GCF b e f o r e t r e a tm e n t we r e 4 ・ ， 1 2 ・ ， a n d
1 7
・f o l dh i g h e r , r e s p e c t i v
巴l y ,a t s i t e s of p e r i o d o n t i t i s t h a n a t h e a l t h y s i t e s . Beca u s e t h e GCF
vo lum e was i n c r e a s e d by o n l y 1 . 3 ‑ f o l d a t s i t e s of pe r i o d o n t i t i s r e l a t i v e t o t h a t a t h e a l t h y s i t e s a t
b a s e l i n e , i t i s l i k e l y t h a t p l asma p r o t e i n s a n d l i p o p r o t e i n s p e r m e a t e GCF a t d i s e a s e d s i t e s . T h e
i n c r e a s e s i n t h e co n c e n t r a t i ons of a p oB and oxLD L we r e l a r ge r t h a n t ho s e i n p r o t e i n
co n c e n t r a t i on , s u g g e s t i n g t h a t t h e i n c r e a s e s i n apoB a n d oxLDL l eve l s i n GCF a r e n o t s im p l y
d u e t o i n c r e a s e d ex u d a t i o n o f GCF
、：r a t h e r , l i p o p r o t e i n s m a y s e l e c t i ve l y t r a n s f e r i n t o GCF. S h a h
e l a l . ( 2 3 ) r e p o r t e d t h a t t h e ox L DL i n GCF o b t a i n ed f r o m p a t i e n t s w i t h p e r i odo n t a l d i s e a s e was
1 . 8
・f o l dh i g h e r t h a n i n t h a t o b t a i n ed from h e a l t h y s u b j e c t s . I n o u r s t u dy , GCF and b l oo d were
co l l e c t ed f r om t h e same p a t i e n t s ; t hu s , t h e d i
仔e r e n c ei n GCF between s i t e s of p e r i o d o n t a l
d i s e a s e a n d h e a l t hy s i t e s i n t h e same p a t i e n t s were d i r e c t l y co mp a r a b l e . T h e d i 斤 e r e n c e s i n apoB
and oxLDL conce n t r a t i o n s b etween GCF d e r i v e d f r om s i t e s w i t h p e r i o d o n t i t i s a n d t h a t co l l e c t ed
f r om h e a l t h y s i t e s d o e s n o t r e l a t e t o t h e co n c e n t r a t i ons of l i p o p r o t e i n s i n p l a s ma , b u t
Journal of Periodontal Research
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p e 1 m e a b i h t y c h a n g e s a n d o x i d a t i v e s t r e s s c o n d i t i o n s i n p e r i o d o n t a l t i s s u e s .
ApoB, a m a c r o m o l e c u l e w i t h a m o l e c u l a r w e i g h t e x c e e d i n g 5 0 0 k D , 1 s t h e m 句 o rc o n s t i t u e n t o f LDL. I t i s t h o u g h t t h a t t h e apoB i n GCF i s o f p l a s m a o r i g i n ( I ) , b u t t h e mechanism o f apoB e x u d a t i o n r e m a i n s unknown. P r e v i o u s l y , we showed t h a t t h e c o n c e n t r a t i o n s ofapoB a n d oxLDL w e r e s 1 g 1 1 1 百 c a n t l yh i g h e r 1 1 1 GCF
仕omp a t i e n t s w i t h D M t h a n i n t h a t f r o m h e a l t h y s u b j e c t s , r a 1 s m g t h e p o s s 1 b i h t y t h a t t h e p e r m e a b i l i t y o f t h e g i n g 1 v a l m i c r o c i r c u l a t i o n c h a n g e s u n d e 1 h y p e r g l y c e m 1 c s t a t u s ( 1 4 ) T r a n s c y t o s i s o f LDL w i t h i n t h e e n d o t h e l i a l l a y e r was d e m o n s t r a t e d a s a p o s s i b l e s e l e c t i v e t r a n s p o r t p a t h w a y f o r LDL ( 2 4 ) . T h i s s t u d y shows t h a t t h e s e l e c t i v e t r a n s p o r t o f h p o p r o t e i n s 1 s e n h a n c e d n o t o n l y b y D M s t a t u s , b u t a l s o b y o t h e r t i s s u e c o n d i t i o n s , i n c l u d i n g i n f l a m m a t i o n o f t h e p e r i o d o n t m m
The r a t i o s o f oxLDL apoB c o n c e n t r a t i o n s 1 1 1 GCF f r o m s i t e s w i t h p e r i o d o n t i t i s a n d h e a l t h y s i t e s were h i g h e r t h a n t h a t o f p l a s m a T l u s i s c o n s i s t e n t w i t h o u r p r e v i o u s s t u d y , which showed t h a t t h e oxLDL:apoB r a t i o i n GCF f r o m h e a l t h y
su~リectswas I 7 ‑ f o l d h i g h e r t h a n t h a t i n p l a s m a ( 1 2 ) . OxLDL i n GCF i s l i k e l y t o o r i g i n a t e e i t h e r f r o m LDL e x u d e d b y o x i d a t i v e m o d i f i c a t i o n a t t h e s i t e , o r f r o m oxLDL p r e s e n t 1 1 1 c i r c u l a t i n g p l a s m a . The mechanism o f t h e o x i d a t i v e m o d i f i c a t i o n ofLDL i n p e r i o d o n t a l t i s s u e 1 s u n c l e a r ; h o w e v e r , immune c e l l s s u c h a s n e u t r o p h i l s a r e t h o u g h t t o b e m v o l v e d . N e u t r o p h i l s c r o s s t h e j u n c t i o n a l e p i t h e l i u m
骨omc a p i l l a n e s t o p e 1 i o d o n t a l p o c k e
回Thep r e s e n c e o f n e u t r o p h i l s i n t h e p o c k e t s c a n b e v e r i f i e d b y t h e p r e s e n c e
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of n e u t r o p h i l ‑ r e l a t e d s ub s t a n c e s , i n c l ud i n g s e c r e t o r y l e uko c y t e p r o t e a s e i n h i b i t o r an d hum a n
n e u t r o p h i l p e p t i d e s 1 ‑ 3 , i n t h e jun c t i o n a l e p i t h e l i u m a nd GCF ( 2 5 , 2 6 ) . Ne u t r o ph i l s r e l e a s e
m ye l o p e r o x i da s e (MPO) a nd s up e r o x i d e a n i o n s (0 2 ・ ・ ) , t hen MPO produ c e s h y p o c h l o r o u s a c i d
u s i n g h y drog e n p e r o x i de ( H 2 0 2 ) co n ve r t e d from t h e s u p e r o x i d e a n i o n ( 2 7 ) . ROS , s up e r o x i d e
a n i o n s , h y d r o ge n p e r o x i d e , and h y p oc h l o r o u s a c i d f u n c t i o n a s a n t i b a c t e r i a l a ge n t s a n d ox i d a t i ve
m od i f i e r s of LDL t o ge n e r a t e oxLDL. T h e oxL DL: apoB r a t i o i n GCF t a k e n from s i t e s o f
p e r i o d o n t i t i s was m o r e t h a n 5
・f o l dg r e a t e r t h a n t h a t o b t a i n e d from h e a l t h y s i t e s , a l t h o ug h i t d i d
n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e a t b a s e l i n e . T h e p a t i e n t s e n r o l l ed i n t h i s s t u d y h a d m i l d t o
m o d e r a t e p e r i odo n t a l d i s e a s e s ; t h e r e f o r e , ox i d a t i ve s t r e s s a t t h e i n f l a m e d p e r i o d o n t a l t i s s ue s
mi g h t h ave b ee n m ode r a t e .
A n o t h e r p o s s i b i l i t y i s t h a t t h e ex u d a t i o n of ox LDL
合o m p l a s ma i n c r e a s e s . L i ke LDL , ox LDL
m ay b e t r a n s c y t o s e d wi t h i n e nd o t h e l i a l c e l l s , a nd o u r d a t a t oge t h e r wi t h p r e v i o u s o b s e r v a t i o n s
( 1 2 , 1 4 ) s u g g e s t t h a t oxLDL ma y b e a mor e f a v o r a b l e s ub s t r a t e t h a n LDL i n t h i s p e r m e a t i o n
p r o c e s s . Ho weve r , t h e d e t a i l s r e m a i n unknown , a n d f u r t h e r s t u d y i s ne c e s s a r y .
I n f l a m m a t o r y c y t o k i n e s produc e d by p e r i o do n t a l t i s s u e c o n t r i b u t e t o p e r i o d o n t a l d i s e a s e
p r o g r e s s i o n . P r e v i o t
』ss t u d i e s h ave r epo 1 ted t h a t l eve l s of I L ‑ 1
~a n d I L
・8i n GCF d e c r e a s e a f t e r
p e r i odo n t a l t r e a tm e n t ( 2 , 3 ) . Cy t o k i n e s a r e c o n s i d e r e d m a r k e r s t h a t r e f l e c t t h e s t a t e o f
p e r
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s i t e s o f p e n o d o n t 1 t i s t h a n a t h e a l t h y s i t e s a t b a s e l i n e . The t e n d e n c y o f I L ‑I p t o d e c r e a s e a 白 e r SRP i s c o n s i s t e n t w i t h p a s t r e p o r t s ( 2 , 3 ) The o t h e r c y t o k m e s exammed m t h i s s t u d y w e r e n o t d e t e c t e d
111GCF P a p e r p o m t s were u s e d t o c o l l e c t GCF b e c a u s e t h e y a r e s u i t a b l e f o r LDL r
田o v e r y ;h o w e v e r , GCF c o l l e c t e d b y p a p e r pomts was r e p o r t e d t o b e l e s s s e n s 1 l i v e t o c y t o k m e d e t e c t i o n t h a n t h a t c o l l e c t e d b y p a p e r s t r i p s ( 2 8 ) .
P e r i o d o n t a l d i s e a s e i s c a u s e d b y b a c t e n a l m f e c t i o n , a n d o x i d a t i v e s t r e s s 1 s a s s o c i a t e d w i t h i t s p r o g r e s s i o n ( 2 9 ) A l t h o u g h I L ・ I pa n d I L
圃8d i d n o t h a v e a p o s i t i v e c o r r e l a t i o n w i t h PPD, t h e oxLDL c o n c e n t r a t i o n i n GCF showed a s t r o n g c o r r e l a t i o n w i t h PPD a t b a s e l m e , s u g g e s t i n g t h a t 1 t r e f l e c t s t h e s t a t u s o f p e r i o d o n t a l mtlammatwn a n d t i s s u e c o n d 1 l i o n Our f i n d m g s s u g g e s t t h a t 1 t i s i m p o r t a n t t o e v a l u a t e t h e c l i n i c a l c o n d i t i o n o f p e r i o d o n t a l d i s e a s e u s i n g i n d e x e s a s s o c i a t e d w i t h o x i d a t i o n ： 日 o rt h i s , apoB a n d oxLDL i n GCF 1 e p r e s e n t u s e f u l m a r k e r s .
Acknowiedgments
T h i s work was s u p p o r t e d b y JSPS KAKENHI ( G r a n t Number 2 6 6 7 0 8 9 6 ) a n d g r a n t s f o r t h e p r o m o t
旧no f t h e a d v a n c e m e n t o f e d u c a t i o n a n d r e s e a r c h i n g r a d u a t e s c h o o l s i n s u b s i d e s f o r t h e o r d i n a r y e x p e n s e s o f p r i v a t e s c h o o l s 品 。 mt h e M m i s t r y o f E d u c a t i o n , C u l t u r e , S p o r t s , S c i e n c e s , a n d T e c h n o l o g y o f J a p a n The a u t h o r s r e p o r t n o c o n f l i c t s o f m t e r e s t r e l a t e d t o t h i s s t u d y .
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