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Pyridoxal 5′-phosphate deficiency in uremic undialyzed, hemodialyzed, and non-uremic kidney transplant patients
h,~
etg
127 ( 1983 ) 2 0 5 - ~ 15
205
Etsevier Bio edical Press CCA
2364
P.v"idoxal 5" phate deficiency ure undiaiyzed, he .nod alyzed, and non-u remic kid ney transplant pa tien ts r
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c s ~', M . B. Lacour a . , C. Parry ", T. DrOeke b, M . u a m " , H . K r"i and D. D u r a n d " "
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I n lliis s t u d y , w e h a v e i n v e s t i g a t e d p ~ a s n l a , pyridox;Lt 5 ' - p h o s l s b a t e ( P L P ) c o n in u n d i a l y d mad d i a l y z e d u r e m i c p a t i e n t s a n d i n k i ~ n e v t r a n s p l a n t st b j e c t s , u s i n g a n e n z y a t i c tccl~! i q u e witii i h e r m a | d e p ~letn~ t i ~ to | i b e r a t e [3Lt~ i~ti f r o m p l a s m a p r o t e i ns. T h e, ..s p......e, c"i f i c i t y o f tl~e r e a c t i o n i n d i c a t n o i n t e,r f ~e r ,e n c e 4 p y r i d o x a i a n d o n l y 3 .% i n t e r f e r e n c e w i t h p y r i d O X i l. ....1' ll e .p. .h. o s p h a t e . I n 17 h e ~ o d ia | y z e d p a t ic a Is, a d e fi c i e n c y o f a b o u ~ 50 % .of a s m a P i....1-, c o n c e n t rat i o n is f o u n d as c o m p a r e d to 25 l~ea!~lay s u b j Is (22,2_*.:2.47 vs. 4 8 + _ 3 . 0 0 . . S. E .M .) . . In . . . ,,,e,l . u n d i a l ) 7 + .... ed n m o l ... | :I, a s ni .... ' ° ........... n_+ u ev m t ...... c w~th e ..... ' s ............ • o n c e~.n | .r a. .i o.n . is. a.! s o . d ' .....er -a.s...e...d..... (....... 2 9 . 3 N~ i . 7 4 n I-I i renal !u:re, ! h e p i n s a P L P c............. T h e absence: o f P L P in p l a s m a ult ntfil i r a t e s d e m o n s | r a t e s tila t n o loks o f P L P ~.~'-ccu d u e tO h m o d l a l y s l s . Tile daily oral supplementation itil 2 5 0 7 5 0 m g p y r i d o x o l induces a y s i:o l o g i ~c .... a l i n c r e a s e in p l a s m a P L P CO .... 1"S t l p r,a p h........... ccfllratiOll i n | ~e" ....o d i a l y.....~° e. d as we II as G ~
in
u nd iaiyzed
pa tien
t s.
t n.... a PLP conccntraIn i 1 6 n o n - u m i c k i d n e y t r a n s p | a n t s t ib.... j e' c~ t. s ., . t.h e. . m. e. a n p l , , s....... l i o n w a s 33,8 5 : 3 . 5 0 n m o l . | - , i . In o f t.h'..... ese~ p a t i e n t s , a deficit (helo 20 n O [ * i ..... ) a s o b rved_. :lion in n c l u s i o n , u r e m i c p a t i e n t s h v" a d e f i c i e n t B 6 .......i ....." | t s c o t o p... y r i d.o x o | c. o . : p . iotogica! p l a PLP content :tion tl ........¢'ssi::la , ' s o suppie entation ith lower doses that ihose i d e i y u S: e~d a | p r e s e n t . In k i d n e y Iranspiant a s i t n i l a r p l a s m a P L P d e f i c i e n c y is o b s e r v e d in t i absence of chronic a ' ...'. failu .
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Vitamin B6 actualty cormspoJlds to thr active ~ e t a b o l i t e s , i.e: pyridoxol. pyridoxai and pyridoxamine, which play a role in man3 metabolic functions after ]~aving undergone p h o s p h o w l a t i o n . In tissues~ tl!e major vita in B6 c o m p o u n d s appear to be pyridoxa[ phosphate (PLP) and p2~ tdoxamine phospllate ( P M P ) ill. In pl as tn a, py ri d a I (P L) a n d P L P reptesent tln e ~ ai i1 for s of vi t a in B 6. T h e ~ for plasma PLP reflects accurately the vita in B6 S t a t U s . The deter i n a t i o n of p l a s m a PLP is of particular interest because hu an b o d y sto s of vitamin B6 are ~ all They can be quickly depleted. . . Moreover, . . sonde. drugs antagon,;ze the m e t a b o l i s m of vitamin B6 to i~s active metabolites [2]. The study of clinical conditions potentially complicated by a mild vitamin B6 deficiency ~ q t t i ~ s a sensiti and reliable assay system for the precise ' ca~u c sn rt e- ' " of P L P concen t tion i n p~asma or tissues. SCV .... e I 17uo met tic [ 3.5 ~ 7] spect pllo, to~etric [8,9 i, and microbiological m e t h o d s [10-13] a presently available far the assay of plasma PLP, bui ihey are not sufficiently sensitive a n d / o r specific. E ym ic assavs~ [4,14~20~ a,e'r- a o n g the o s t satisf'actory tnethods, .... associating :peclficity and sensilivity in the estimation of P L P in biological a t e r i a l s ~thout ils prior isolalion or purification. Enzy aiie metl~ods are all based on tl~e abilily of P L P to restore the aclivity of an a p o e n z y ~ e to which P L P is the coenzy e T h e enzymatic p c e d u used in this s t u d y has beeo derived f r o m the techniq~ae described by C h a b n c r a n d Livingston [4]. It is based on the rate of t~CO2 ~ r ation f r o ~ L-[ ~'~C]tyrosine u n d e r the influence of PLP-sti vlated ty sine apodecarboxy~ last. The p u r p o s e of tl~e s t u d y as to d e t e r m i n e ~he serun~ P L P ncent lions (1) in ure ic patients treated or not by hemodialysis and s u p p l e m e n t e d with p y r i d o x i n c or not, and ( 2 ) i n kidney tra piant patienls ith normal or near normal renal f u nC' "r o O n . wl~o all ceived i m m u n o s u p p ~ s s i v e tl~e py. "
Patie:nts and
ethods
tien~ Tl~rcc g ups of palients were studied" hcaIlhy subje-cl~, ur,i c e patients, and kidney t nsplant patients. V e n e p u n c t u r e was d o n e on twenty-five hea|thy subj ts in tl~e morning. The blood was collected into heparinized vials (10 I U . 1 - l ) . These control subjects received n o e d i c a l t at ent, and they had no llistorY of he ~ic or e n d o c r i n e d : isease. ' - ..... W o m e n ho were laking a 1 contraceptive drugs ere- discarded fro th e s t u d y. u s e h av¢ s t u d i ed ! 2 wo ~nc n, w it tl a nl e a n age o f 36 yea ~ (rang e, 20 - 54 years), and 13 men with a men J1 age of 28 year~ (range, 20 ~ 38 y,e a1. . . . )j Thirty-six patients itl~ chron;c renal [nsuffici~ cy ere studied. T e a t y - n i n e were be odialyzed for a can of 3 6 ont:ns~ their erage age as 42 y e a ~ (range, ~ 2 - 7 0 y e a ~ ) , Se n u ~ i e patienls w rc s~udied a! the ter ina! stage of f e n af l: insufficiency j u s t prior to tl~e beginning of hemodialysis (c .... ~ a ! inine cleara e < 5 ~ 1 / in). T h e a rage, age w'as .... 38 years (range,. 25~51 y e a ~ ) .
207
T h e third g r o u p consisted of 116 kidn,.ey t r a n s p l a n t n o n - u r e ic palients, ith a e a n age of 33 years {range, 16~58 years). T h e p l a s m a P L P d e t e r inaIion was : e ti ....... al tra nsp Ia n ~a*l l O :l l . . . . .... p c r f o r m ed a ~ V, ar~:: a bl e i n ter val s afle r r: e:z~
Meth T h e e n z y atic t - .......: ............. d e s c r i b e d here. w h i c h h been derived from the o r k o f S u n d a r 'c-s a n a n d C o u r s i n [ia] a ~ d was m o d i f i e d b y C h a b n e r a n d L i v i n g s t o n [4], uiili s the following reaction"
-~ C] yrosine ...
Iy'rosl":
a
rbox ylase
y r a m i t ~e~ ........
P L P dcpendent
CO~ "
S o d i u a c e t a t e buffer, p H 5 5. 0.1 n~ol . i° ~ in distil ed w a t e r (',Bake-r. D,vcc a r e ",r, T h e N e t h e r Ia n d s). ~ridoxai s o l u t i o n (Calbi h e m . La Jolla. C A 92037, U 37.7 y moi isi d i s l i | | e d a t e r .... T h i s s o l u t i o n was atiq u o ! c~d i n t o |i | u es ( o f 500 ~1 e a c h ) a n d s t o :at ~ 2 0 ° C until u .... T h u s , o n e t i a ~ e d for e a c h to p r e p a r e a fresh w o r k i n g P L P of 377 a n d 75.4 n o 1 . ! - d i l u t e d i n , c e t a t e , ~" b u l f e r a t p H 5,5. L-Tyrosine solu!ion (BDI--1 C h e m i c a l s Lid, Poole, U K ) , 5 o I . 1.... ~ in acet~ttc b u f fer a t p H 5.5. L-[ i i4 C ] T y r o s i n e s o i u t i o n , p r e p a r e d b y a. d..d...~l t i o n o f e - [ ! - !a C ] t y r i n e ( N E C ....170, s p ific aclivily 5 !....9 m C i o ~ m o l - ~, N E N , B o s l o n M A 02 ! 18, U S A ) ~o an L-ty s~nC s o k i i i o n : o o b t a i n a fi a~ s p ~fic activity be~ n 4 al~d 8 / l C i - : .... $: A Tyro~Inc ('F-4629, I . 1 U . g ...... i I U p r o .... d u c i n g !/~ o i / I CO~ fro: L-ty sine p,..,.re' rain at p H 5.5 al 37°C,° S i g m a (2"he l!lc,~l: " C o , St Louis, M O 63178, U S A ) c o n t a i n i n g 2.5 g - 1 ~ in dislii.ed wa~er. E D T A so Iu t io n, 2 0 g * I -~ (M e r "c k , D a r n~s ta' d t, F R (3). HCI 5 o1.i-~. H y a l i n e h y d r o x i d e , 1 0 x P a c k a r d ( K h ....L~gh| C o l n b r o o k , Bucks, UK). .... H e p a r i n soluti 1% (Vi r u m , S e d .
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PLP w e r e c e n tri fuged a ' 1 4 ~C
[or 10 rain a l 5000 ~ g., I~ was n e c e s s a r y o c , n e ~ri f,u ge- i ~ e d i a t e l y a, f ler bl d s a m p. .l .i .n g in o r d e r to limit p o s s i b l e h y d r o l y s i s of P L P by a l k a l i n e .....p.h. .o. s p h a t a s e aclivify c" on t a ined i n eryt , . . .hr . ocy tes or leukocy ies .... E sa p l e ,,as " tested in !riplicale a n d ' c o ....... n p~ a d ilh a ....s t a n d a r d P E P rye perfo ith a p o o l o f se o b a t n e d f m h e a l l h y s u b j is. l n i o 5 - • Iub , | o 800 ~i o f ch plas a 3 2 / a l o f E D T A l u | i o n were: a d d e d a n d to 1000 using ........... " .....e b u r a pl-i . . . . . .5,5, . . . . . .At . . . . .ibis . . stage, the s u p p l e m e n l a t i o n o f p t a s ~ a with P L P was p e r f o r ed by ~ p l a c i n g the a b o v e I68/~1 o f acetate' •'' b u f f e r by s o l u t i o n s c o n t a i n i n g i n c r e a s i n g a m o u n t s of PLP. A f t e r a g i t a t i n g for | 0 s in Blood
s a n~ p ! e s
20g
a Vort ~ i x e r , the: t u b e s e p i a c e d in t l l e d a r k , a t r o o te peraturc, for 30 in. pt|. o f a t a r e "l'hcn 2 5 0 / a l ~ e a c h t u b e ,~e . . . . . . .t .a k e n :in . .~ t o . w h i c. h 95(i . b u f f e n p H 5.5 .... w e added. Afler |0 s of agitation by the Vortex ixer. they ere
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m a i n t a i n e d in c o n s t a n t o t i o n f o r 10 r a i n a t 8 0 ° C in a s h a k i n g w a t e r b a t h , A f t e r 5 n:~in t n ' f u g......a t i o n l ....5 0 0 0 × g , 6 0 0 # i o f e a c h s..u. .... p e•r n a t.............. a n t w e~r.....,e ....s a m p l e d a n d I0t3 of a zyme in we added Then 0a.....i t e,r 3 0 s o f a g i t a t i o n the Vortex, the vials ~ we...... . . . . . . i n t h e d"ar."k a~ o m re t e~n.......... J p e r a• t u ~ r 3 0 ~ i n t o al}o P i . P ..... r e c o ~..... b ~....n:...... a ti ..... on i t h a p o e n z y .n ~ e . T t l e n t ~[........ c, | u b e s w e r e t r a n s f e r r e d t o 4 ° C a n d k e p t at t h a t te p e r a t u r e f o r 5 i n b e f o r e 1000 p l o f ! . - [ l - ! a C ] t.:~ ' rosa ..... : n e s o lu t i o n w e r e a d d e d . I e d . i a ! e l y.,. a f ..... t e . ,........... a~ d s. .t h. e y. . w.e r. e c'io....... s c "d wi tb a stopper . . . . t - : ......' a sheet of paper (4×2 c )folded ~eogtbwi i n t o t o a n d i p. . . . a l e d w i t h 8 0 g ! o f h y a m i n e h y d r o x i d e : s o l u t i o n a~,,'s {|i u s t r a t e d in F i g i. asperfor edforexactlyl5 mi,lat37°C ith c o n,,,s ,t a]x t a g i t a ti o n in a I n c u b a t i o n w', ..... s h a k i ~ g w a l e t b a t i~ e n , u~,,,,ing s: a ~ r i n g e , ? m I I-ICI w e r e i n j e e r e d t h r o u g h i he s t o p. .p. e. .r. . .t o s t o p t h e enz,,., ..... : y ....... a ric, r e a c i i o n . F o r t h e n e x t 2 h t h e and capture of iaeo ,o,,. hydroxide was performed at 20-22°C. .... t h e s h e e t s o f pap., r r e c u i o f f a n d i t r o d u c e d i n t o v i a l s c o n i a i : n i n g ] 13 m l o f s c i n ~alion licluid .... T}le* t a d :i.... o a"...... c l i:v l: t ....... y W a S d 'e r ....~"! ...... n e d i n a, ! i q u i d s c i n ! l i a t i o n s............ .......~.... ( S L 30. p e*c t........ r o n ~e~er l n t e r ~ c c h n i q u ~ Plaisir~ F-rat~ " ) , Results
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]twubatioJ~ tinge at 3 7 ~C. F i g .... 2 s h o w s t, b a..... ........ t ,,he action velocity was constant b e t w e e n a a n d 30 r a i n a n d t h a t a f t e r 3 0 r a i n t h e e n d - p o i n t o f enzymatic., r e a c t i o n w,,,:as hie ......A l l m e a s u ~ m e n ~ s w e r e p e r e d w i t h oil i n c u b a t i o n ti e o f 15 r a i n . Ap tl2e i
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Fig .....2, E f ~ c t o f i n c u b a t i o n tinle at 37~C the M e t h s |i ....
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T h e e n z y m a t i c a c t i v i t y ~ncrea d a~ t e a s e ~f t y r o s i n e c o J l c e n t r a t i o n in t h e a s s a y m e d i u bet e e n 0 . 7 3 5 a n d 1.471 t o o l - 1 " .... A b o v c 1.47! ~ o i . l - i , l h e e n z y m a t i c ac -t i v i t y w a s ~ a.~: t n t a i n e d c o n s t a n t a n d we h..... a .VC :,Se !~e c l e d a t y r o s i n e c o n c e n t r a t i o n o f 2,941 ~ m o i . i " ~ to a v o i d su b s t r z t e c o n c c n t ~ t i o n b e i n g a Iim i ring f a c t o r in t he z y m a ~ i c r e a c t ion u s e d ... .... ~
.
.
.
.
in"luence# ....... of E D . The fiuence of EDTA as s t u d i e d ~ r c o n c e n t r a t i o n s " ran,ingg b e t ~ e.... e n , 0 a n d 1.6 n g . ~1 ..... ~ p l a s m a ° W e h a v e o b s e r v e d a a x i m a l sti~nula. t i o n of e n z y atic a c t i v i t y ( 3 " / % ) a t 0.8 n g m l " i E D T A as c o p a r e d to the a c t i v i t y .. "~ ....: t .....)'' de m i n e d in a.......b....... s e-n....... t e of T A .... I n f l ~ n c e of l ~ q ~~lity it~ t h e pture CO,, W h a t m a n p a p e r i n ~ r t e d i n t o ~ial s t o p p e r s w a s ~ p r e g n a l e d w i t h 40, 80, i20 o r 2 0 0 / a i o f h y a m i n e h .y d rox . . ide. so. lu .ti on, . .U si. n g. a hi g h P L P c o n c e n ira ~io n o f ~'50 n o l . I - J, n o d i f:l'ere 1iec in r a d i o t i v i t y s u r e d was obser between ca p t u ~ . T h u s ~h e 8 0 t~ l o f h y a m i n e b y d r o x i d e wla icla w e re u s d i n o u r st u ~1y w e s u f f i c i e n t to k e e p all t h e f o r t d. ]n, fhte~Ice o f hepari!~, By a* ....n a' l y....z i n g s e r u m in the a b s e n c e o r in p r e s e n c e of l 0 | U. l - 3 h e p a r i n , w e o b s e r v e d th h e p a r i n n d u c e s a n ir e a s e o f i 9 . 5 % in t h e efficiency of zymatic reaction, a is t h e a n wily bl samples col !eeted i n t o vials c o n t a i n i n g h e p a r i n to o b t a i n a final c o n n t r a l i o n o f i 0 I U - 1....
teinizati ,t Jono, t h e fi ....
pro We have studied p u s i n g p e r c h l o r i c a c i d ( H C i O 4 a n d t,h e
u r e s f o r d e p leini , , n d u s i n g t 'h, ....................a-......... guta-
2:I 0
t,ion W h using the a c i d p r o c e d u r e , the excess:, - ' .... s o f t t C I O 4 will be n e u t r a l i d t o obtai n a supcrnatan t with a pH nea r 5 T h e a d d ilion o f ....... s ' p o t a s s i u m h y d r o g...e n ca r b o n a t e is a~socla led wi:th t h e a t i o n o f i n s o l u b ! e KCIO.~ 0 w h i c h is eli n............ atedi b y C¢~ [..... l h e s u p e a i a I, Wh u s i n g this t e c h n i q u e is t o o r ifugation: . . . . . ...... .t t .o ~. e .v e ~ a l k a l i n e (pI-I 8.0), prohibits PLP use h e- b u f ~:~ " ..... o f the i n c u b a t i o n m e d i u :is F u r t h e r o ~ , this p r o c e d u h u l d be a s s o c i a t e d w i t h loss. T I u s e induces: an i m p o r t a n t g a s e o u s re.~.ase, whic ~' o f c a ici utn c a r b o t~a t e s u p p I ie s a s u p e r n a t a n t w i t Ix p H 6.2, w h ici~ a i Io s en zy n ~a t i c : ~ N e .............. d e t e....~ m~nation. T h i s p c e d u r e d o e s :not i n d u c e i m p o r t a n t gae, s lease. W h c n u s i n g | h e r m a | d e p r o t e i i z a t i o n at 80 o C f o r 10 ino ttie s............. u p e r 1~"a t a n Is ~r a n d t h e p H was: after ..... I .... ~ ..... . . . . . . . . . T h e c o pari a b e l n the t o p ceriums using o r t ! C l O , w i t h n e u t l i z a t i o n o f the: s u p e r n a ~ a ~ t b y c a l c i u m c a r b o n a t e , s h o w s a higher. . . . e. f f. i c. i e. n c y with t h e r m a l d e p r o t e i n i z a l i o n (Fig. 3). i n a d d i t i o n , t h e ,::atteri' .......... p r e d tire is easily a p p l i e d t o a la series of vials. .
Cha
of the m~kod
t
p l a ~ a PLP
.
.
.
nination
F:~g0 4 s h o ....w ...... ~ a linear a~ed b y U V light p s o r to b e t w e e n 0 a n d 140 n m o ! . i ~ P L P
to a . . . . the
~sooJ 800 g
0 1020
40
6~
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PLP ¢0ncenlta!ioa |am01 I"~t F i g :3. . .|.n. u c n c e o f tlie d e p r o i c i n i {ion p r e d u ~ o n t}~e P L P recover, ..... from dif~ren p|as PLP c ~ ....~. . t~: ........ s. e al 8 ~ .p.r. o. .~. n i ~ t i (~ 8 0 ~ C ~ r 10 rain u ~ i ..... . . . . . . . cl .. ....... w i t h ~ "~ate bL~ r p l t 5,5 I n . . ~ I .~ i c. .acid . d~~eini p g O X ~he a ~ d c s s e~ w a s te d cak~u m F i g ; 4 ~ S~and~rd c u r v e ~ r
plas
PLP deiermina~ion,
e incubatiot~
i x t u r e is g i v e n i n the
Metkods
21 t BLE I S l"ECI FI C 1| Y O F T H E E N Z Y M A ~ I C R EA {- I O N Subsiance 80
n
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as +
Pyridoxit~e Pyridoxa~inc Pyridoxan+ineophasphate 4- Py ri d ox +c aci d Pyridoxal
PLP
~t
r~i~iO~ expressed (n,no|,l
I nte r fe re n c e
...... + )
.............................................................................................................
+
4
16 2.4 0.4 0
2 3 0.6 0
+o+. , .... T h e r o v e r y ,+ ' .......v " o f P L P . R+.~++,++............ d e t e r m i n e d .at t,w d i f f e n t |,e..++..els n p l a s , n a s p i n ~ n s w,+........... +,e s u p p l e e n t e d w i t h a l h e o tica .....c o n t e n t t i o n o f 37.7 n o l * | .....) P L P ,o M e a n t v h s 95 ....l e 2.41% (r S E M ) . plas :spcc)- -+ mens were supplemented with a o f 75.4 n ~ o l . I PLP. Under c o n d i t i o n s ~ e a n r e c o v e r y w a s 89.9 _+ 3.30% ... Sp~.~+c./t);
We
have
measured
the
radioactivity
produced
by
80 n m o + | - i -+
OYri+:doxa i+n e p h o s p h a t e , p y r i d o x a m i n e , p y r i d o x i n e , p..... y ' ri d o........ x a.....! a n d 4 - p y r i d o x i c ,'icid b y... . .c.o . p a r's oi n witti t h e r a d i o a c t i v i t y p r o d u c e d by a P L P c o n c e n t r a t i o n o f 80 n ~ o l . 1- ) ( ble I). T h e r e s u l t s s h o that only pyridoxine, p y r i d o × a m i and pyridoxa~ine a n d 3%. )
~
...... ,
p h o s p h a t e .¥ie!d an i n t e r f e r e n c e r e a c t i o n
,
h"llc 1 w+.... as r e s p e c t i v e l y 5, 2
pool of rum a l i q u o t e d in ° a n a t y z e d in i 6 d i f f e s i o v e r a 2 ....~ o n t h ' s p e r i o d ......F o r a . . . . t h e y .........: l+on fficie++t w ~0.97% . 38.98n ol . .I+ ...... A
p~es which e a n c o n c e , trat+ol~ o f
)
"+..... ~+~,+++e+ ....t tOtL E i g h t s+e e a n a l y d t h e d a y+ oP +san.+,pling a++nd 15 d a y s ....~ft+er at ~ 2 0 + C , T h e m n P L P c o n c e n t .... tion ~ a s 47°57 6.21 a n d 4 6 . 9 2 _+ 6 . 9 4 n n ~ o l - I ~1, r e s p e c l i The ean difference for paired ~0,65.4-_ 0+93 n m o i , i ......) w h i c h a s n o t signifi n t l y d i l u e n t from zero.
13io
¢~! dot
)inations in umn~ic and kid~wy
nsplanl patients
Mean plasma PLP concentration as 48,8 __. 3,00 ( S E M ) n ~ ~ t . i - ) in 25 h e a l t h y i ng n o ora, . 1++s u p p l e ' a l l , / s i g n i f i c a n t f....i "i f f e r e n c e e x i s t e d s u b j e c t s tak+ e n t a t i o n , A sta t'istic bet n m e a n p l a s m a P L P c o n c e n t r a t i o n s d e t e r m i n e d in m e n ( n 13) a n d in en (n 12): 56.7 5 : 4 . 3 0 v e us 40.2 ± 2,52 n m o l . l+ i p < (1.005 ( F i g . 5). ++. u ~ ~ c p a i e n t s r e a t e d b y i n t e r m i t ten t h e m o d i a ! y s i s h a d a s i g n i f i c a n t ( F < 0 , 0 0 7 ) de+ r e a s e in ~ e a n p l a s a P L P as copared to c o n t r o l v a l u e s ( 2 2 . 2 ..... 2 . 4 7 n ~ o i , ! ..... , n 17) .... I n t ';|+e ....'p a t i e n t s n o s t a t i s l i c a I l y signi fic a n t d i f f e r e n c e w a s o b rved b e t n the sex 23.6 :t: 3,74 f o r e n .+and f o r ni w e e n ( F i g . 5) ! by 250 to 750 g ....
+ e
212:
pyridoxo~ per d a y i n d u c e d on increase in tnean p l a s m a P L P c o n c e n t r a t i o n , w h i c h as higher than t o b s e r v e d in h e a l t h y s u b j e c t s .... A f t e r 30 d a y s of s u p p l e m e n t a t i o n , c e a l r "a t :t o n o f 8 9 . 5 0 ~ | qo.59 12 tents(5~c-n~nd 7 ]iadap|as a P L P c....o n................ _
m
I
w
ff
0
~
25"
488~30
~930¢~
2~2~247
f:ig 5 PMs~a PLP t~ ntraiions in h e a l l h y niro|s (!eftL in non dialy d u mic ~t~ien|s with e - ~ t a. . ic. . ~ ~ i l.u ~ .~CRF,. m M . d ~ . ~ ) .....d in ~ m ~ c ~ t ~ n i s trc~tc° ....~ (t'tD, righi). T h e horizont~i corresponds to m ii plasma PLP concentration in e,,:h group and tlie hatched ar¢~= to .& SEM. Student's : test indic:ires p ¢ O.OOl ~ r ht:atthy controls vs° CP, F or vs. ! ! t ) lien ts. O a n d o d e n o!e ~ m a i e d tnale su bj eels. respec ti ty,
n O[ . ~ , W e |l......... I" ..... a v e" a | s o PLP in o b t a i n e d 'a,t ~he b e g i n n i n g or at the e n d o f several h e m o d i a l y s i s s e s s i o n s . P L P w a s | ndcte;ctable :.... ~ .... V cry t rong bi ndi ng to pi 'i',s ~ a p leins, in t h e s e s a m p l e s i :- i u.......r-c .........:lc... p a t i e n t s w i t h c h r o n i c renal failure ai ....I ~ e se n n o n - d i a r y ........
4i
...........~a
at . o
vaiues
i i
0
10
i
30
40
50
SO
PEP coneenlrati0n
~
eO
~
~0
01
Fig. 6~ Distribation of plasma PLP ncentrat .... ....... i ons in kidney t~anspl range if normal valu ~ u n d in 25 hCaithy coat Is.
t patients. The arrows indicate tt~e
213
hod d e p l e t i o n of p! m a P L P : 29.3 1.74 n m o l . 1- I ( p < 0 . 0 0 1 vs n t ~ l s ) . Ora! supple in the n patients with 300 d a y pyridoxol for 15 day:,.,, i uced a striking increase o f p i a P L P concet~trations to 90 _+ 129 n oi ....1 ....i. . in kidney. t r a. n s p l. a n t . patients, mean plas a P L P " .... n .... . . co; cent tion was d e c r e a s e d by c o - - p a r [ s o n i t h n o r ~ al su bj ec ts (33.81 __+3.54 n in o l . l - ~, n i 16). T h e d istr ib u tio n o f all , a l u e s is shown in Fig 6. Ttl~s Fig. de o n s l r a t that 97 subj ts had a concert tion below 40 n o l . 1 i 75 of which h a d a deficiency (belo 20 nmol - I" ). a n d p e doxicaily f o u r subj ~s h a d a high pl~ls~a P L P ............. .... con ~1 a tion: (89, 116, 238 a n d 2 5 1 n m o l . i ...... ). .
.
.
.
.
.
.
.
~
w
S
P
Oi'scussion he e n z y m a t i c m e t h o d used in the p r e s e n t s i u d y sho s su rficient sensitivity. .lnation ....... * s".p e e t f ....1 0 a n d r e p ucibi~ity for the di e e r of pia~ma P L P n ntrationo .... in lion is always u probie b e "c a u s e of the ex~ste e o f for s of v i t a m i n in blood. Microbiological a~says yield values with ide v a r i a t i o n s and scatter b e c a u s e oi' the lack of specificity of lnicr rganis s for vitamin B6 co p o u n d s [i31° Using c h e ~ i ca m e t hods, wide va ri a t ion s res u ] t fro in i t~terreren ce s a n d i n s t a bili ty o f c o m p o u nds for ed b2~' ~he t h e i I r,eactlo)l [%21 }0 O n tl~e "........... nt ...... rary..... !he e v m a t i c tcchJ~iques a r e specific for P L P . Pias a P L P d e t e r m i n e d in o u r s l u d y for h ItbV subjects arc co p a r a b l e to those o b t a i n e d by C h a b n e r a n d Livingston [4}, b u t t h e y a r e io r than ihose o b t a i n e d by Hines a n d Love [18] w b o used apophosphoryla~,e ....... .s as a p o e n z y m e . The ai Jx p r o b i e ~ wi til P L P mea sure ent i s i t s b i n d ing t o p Ia s ~ a pro t e i n s. P L P a n d PL f o r ~ Schiff ba s ith free an~ino g ups of p r o t e i n ....In t h e e pr. s................. e n ' of protein-pr ~ a g e n t s , all the Schiff bases d o n o t b ak a n d P L P m ains b o u n d to pr ipitated proteins, at [east in par{ [71. . . . us P L P e x t r a c t i o n f ~ ~ p r o t e i n s is i n t o piete, a n d this p b l e m could ex p l a l n the di fferences in p l a s m a P L P c o n c e n t r a t i o n s r e p o r t e d in the litera!ure. P e e h l o r i ¢ acid and trichto acetic acid ° h a v e been p o s e d for d c p r o t e l' n i'z a I ion. However, the e x. . . . . . ss• use of these acids nece~itat ihe|at eli i tion of the acid exc s b y p r ipi i i o n ( K C I O 4 ) o r b y e x t el[on with e t h e r for t r ic h l o ............. ...... acetJcacido Neutraii ln~.ufficmnt . for trichioroacetic acid. which inhibils the enzy atic reaclio u d for assay. F u r t h e r ore the recovery of P L P f r o ~ trichioroacetic e x t r a c t s o f hemolysates is i n c o m p l e t e [ 7 ]. U sin g t h e ~ et hod d escri bed h e ~ d eprotei ni za, ion b y ode ra tc hea t coagu i a t io n ~: al oW. s good recoveries of P L P fro p l a s m a s a m p l e s to be ob [ ' ........ ned. P vious i n v e s t i g a t i o n s indi te the possibility of P L P deficiency n h e m e d i a ! y s i s ina~ion of p l a s m a P L P n ntrations d e m o n s t r a t e s clearly an i p o r t a n t d e c r e a s e c o ~ p a r e d ith h e a l t h y subjects. O u r results are in a g u e e a t ]th those of S t o n e et al [23]. e d ~ a s e o b s e r v e d in l~emodi.a l y s i s p a t i e n t s d s not a p p e a r to be due to a loss o f P L P into the dialysis ft uid, since P L P is st ngly bou nd protei as [26,27]~ a n d e h a v e been u n a b l e to sure a n y P L P in p l a s m a ultrafi ttrates ......F u r r i e r ore, a defie:it of similar i rend-stag,, : ha| failure n o t yet t a t e d tan is also o edin u~micpati ts by hc~ ialysiS.
++4
++
Wlien t din 8 he odialysis p a t i e n t s ith p y r i d o x ~ t h e i r p | a s a P L P ncent~tions increase a c c o r d i n g to the vita in B6 ~ e t a b o l i s p r e v i o u s l y suggested [+++,+8}+in hich pyHdoxol is c o n v e r t e d to pyr[doxoi p h o s p h a e a n d p y r i d o × a l a n d m e t a b o l i z e d to ~s a d e f e c t in P L P s y n t h e s i s f r o m p y r i d o x o | c a n be d i s c a r d e d as ~n ex p!an a i ion for P L P defici e ~ y in h e r o d ialys~s p a t leo ts. T h e d e ri ci e ncy ~ u st b e d u e to an i n c ~ a s e in et~bo!~c c l e a r a n c e o f P L P in ~ c h as d e o n s t ted by Spa n n u t h et a I [ 22} + F u rt h e rm o re + o u r re s u I t s w it!~ o ra i su pp! e e n t ~ t i o n t t h c r z p e u i i c doses of +pyridoxol+.++ widely used in h e ~ o d i a l y s i s p a t i e n t s , i n d i c a t e a'n ~ o f p l a s m a P L P concert l r a t i o n compared w: .....~th physiologic~l over o ad ..... . . n.c e n .l r ~ l i o n s . d recently by at c h is excessive vit~ t36 has b n ;++tr s K o p p | e et i [29) In k i d n e y t r a n s p l a n t p a t i e n t s ith n o r m a l or n e a r n o r ~ a t renal f u n c t i o n , ++e° a s o obscrv'ed a m n r k e d d e f i c i e n c y in p l a s m a P L P nc'entr+ations. A ~ ~ o r deficit ( b e l o w 20t~ o l + l ~ ) i s p r e s e t i n 6 5 % o f t h e p a t i e n l s s t died. ..... + t + lh¢+~i+ T e pa+|++ents +a++ +dy have a deficit o:f p l a s m a P L P + n+ ntrat++on whe+++l +++++++v+ re / +tI+cir kid+cy g+ ft .... Afte+r+ tamp i a n t a t i o a they mainta+n th+e de+tic+rat :unde+r i m u o o s u p p r e s s i v e t h e r a p y ( g c n e r a | | y zzath+ fine a n d steroids)+ It is p o s s i b l e that these d r u g s have an i n h i b i t o r y action o n pyridoxa! p h o s p h a t e kinnse in the liver+ n o :+.uch ct'fect l~as b e e n ~ p o r t e d in the l o dat+e T h i s p | a s a P L P c o u | d p. .e.r.h. a p s t h e ....very +~p t~ s t i m u | a t i o n o f alan~ne aminot activity i n d u c e d by P|+P s u p p l e e n t a t i o n i vii using pl a from r+,+n~lc t r a n s p l a n t p a t i e n t s {301 H o w e r. the c h r o n i c d e f i c i e n c y in tissue P L P +, oct+tration i n d u c e s a dr;crease e .... ' is gel+e r a fly c~n in a p. o t .r a n .s a . i n' a........ s y n t h e s i s, w h i c h . . . . . p a t i e n t s in th e• abscrl of c y+....... t o l y....t i c ~i r d i s e a s e [ 23, 31,32]..... o b s e r v e d in l~en+odiatysis T h u s .... in ~1 d e i e r m i f i n n of the sti u l a t i o n inde× o1" t ~ n s a ~ n se tivi ty b y P LPd esnoiallowt e ofthePLP In k i d n e y. . .t.r a n s p l a n t p a t i e n t s the p ! a s ~ a I'LP deficit c o u l d indic a,t....e a tissue P L P deficit. H o vcr. the lat te+r +c o u l d c o n s t i t u t e an a d v a n t a g e for s t e r o i d ~ i ~ n on target .... c"o ]S, since P L P h a s been r e p o r t e d to d e c r e a s e the f o r m a t i o n o f c o plexes betw++e C....~ sic id a n d c y t o s o l i c p t o r a n d their t r a n s f e r t o the nucl r ste icl ~ rcee p i o r 133].
R¢+~ nees ! [..... n l BL Bain JA ....WilIiams i{ L, q'he dist ribu lion of vitami n ~6 in t h e t s " of two i n b r ~ strat ins of +~ice ~ple|e and ~i~amin ~ef~ient s .....J B i ~ C h e m t962: 237: 1989~ i ~ I. 2 Sau HE. JE. ~:ke+ l, : N Jr. as en, o¢ t * nutri~ionnl status o f vitami+i B6 in the hu an ....A J Clin N v t r I972 25: 629+b42. 3 C h a u h a n MS, Dakshinamurti K+ N u o m e ! r i ¢ assay of pyrido~ai and pyridoxa| 5'.ph phate. Anal Bi tern 1979: ~ ; 426-a32, 4 Chabner B+ i v i n ton |)+ A sinlp}e c h e m i c assay for pyridoxa| O~1o~Pi+ate+ Anal Bi hem ~970: 34: 413 ....423. 5 C h a u h a n MS+ K F|uo a y of B6 vi~ame~ in ~o!ogica! |e~Mo Clan O i i m Acla 198 | M : 159+ 167. 6 qStmtJr0 Z+ T~kanash~ S: F l u o 6 m e t r i c determination of pyridoxal and pyridoxal 5 ¢- p hO ~sph~le in b~ologi 1 m~tetiAis by the reaction with ~un+de+ . . . . i + In: + M cColmtck : , DB, Wright LD, cds+ M e t h ~ s in eazym~)t~y+ i. f 8A, New York: A demie P s+ 1970:471 ~475+ 7 St+~ + SK+ Beutier | A new fluori trig me:ih e determination o f pvrido×al 5-+ h o ~ h e + Bi hi Acta : ~5-77~
8 Hender.~on HM. e conversio41 of ~ ~ d o x i n e pb.osph~e ~nm pyr oxa| ph,~:,F le in ~erto~ia B i I iem 3 i% 5; 9 5: 7 7 5 - 779. 9 Whi(e RS, Deln ey WB, Purification ~nd p ~ c r t , ~ s of vito in i hemistry t 970: 9: 4057-4064. 0 Ander~,on .... B B, art MB F u l - 3 o ~ The measurement of rum pyrido×a| by a .................. ic biliously u~i ! ~ ¢ t ~ h aci| !u s i ....2 C li ~ Pa t h o i 1970,: 2 3 ~ ......~ ... I~ ME. {of h | ~be vitamin B6 m~k° ~ic~b/o~ I $ Da~ R 26: -214~ ~2 Rab~owi~z 3C. M ~ a d y NI. S~i~ EE ...... ; ro p u t e r m / ~ ' ......~ ef p v d d o ~ ~ h L ~ s~o J B ~ ~ ; ~75: ~ - i ~ . | 3 Ark:in L Sci~!!z AS. w,[ a~ns WL. F ~ v CN. Yeasl ~ i c r o b i o l ~ / c a ! e t h o d s l~r deter ~ a l i o n of viVa.ins Pyrido×ine ...A~al . {rid Eng C h e m ~943; 15; i41-144. 14 Sundare.,~n PR. C o u s i n 1)11. Mi" ........ Of pyr~doxal phosphate u~,~ngt..~y s~a~ si si~ic.i. i~ C a n d ~ y r o s i n c ap~ afboxytase. In' McCormick DB, Wrigh! LD, eds. Methods in e n z y m o i o ~ , Vol. 18A. New York: ademic Press. 1 9 ~ : 509-512, 15 t|~,m~li A. E~ ~;~tic de|er inafion of pyrido×al ph ph~,~e in p: mz by degarhoxy|z~ion ~f I | y sine ....14C(U) ~nd a co par/son wi~h lhe !ryptophzn load lest0 Sc;~nd J (?iin [,~b invest 1967 20: !
....
!6 li~ske|| BE. ~ e ~ l EE. A~ a~~pt a ~y for pyridoxal p~ p t~ Anal Hi he i : 45: 5 6 ~ . 17 Okndz K+ FIU+i S+ w++dz M+ M ~ + y+ ef py+~d al phosphate ~++ng t~ptophan+laC+ ++ + it+ ++ topha+ e .... in: M c r ick DB .... Wrigh| LD+ ~s+ Meth s e n ~ m o i o g y . Vo| .... 18A. Ne York' < A demic Press, t 9 7 0 : 5 0 5 :0,,, I~ ilines JD, Lo DS. of serum z~d b| pyridoxa] p:hospi~le n n~r~iio~s with purified rabbi| skelelal m u s i c apopi~osphorylase b. J Lab C!in Med 1969; 7 3 : 3 4 3 ....3 19 Pierce Hi, CampBell C, Moore ~ Hillman RS. Modifications i~ the p u r i f i ~ r~bbit skeletal muscle a p o p h o s p o ~ i ~ s e b. Assay of serum phosphate levels. Biochem Med 1974; t0: 360~367. 20 A l l e n ~ a r k S, H i e | | E, [.a n.Cohn U. New mclhod f~r qua~t}~a~ive :~a~y~s of p y r i d o x ~ , l5-~p h o s phate in biological malefia!. J C h r o n ~ l o S r t978; i~6::485 ....489. 2i SF, $hane B. ~ l i ztioa o~ v i ( a ~ n B6 c o ~ unds in h n : ~ biood ~ad ur~neo C I ~ Ch/m A c ~ ~ 8 ; 21: 7 1 - ~ ° 22 C[ ......Jr. Wi,,,a oek LG0. W.a g . r C. . S~ . . WJ. . .| ~. a pta a ~ ............. f a r c e ~f pyrid~xa| 5".p ~ha~e ~n r i t z in u ie m $ C l ~ Meal i ~ ~: 2-7. 23 Stone Warn k LG, W~g r C, B6 d~icie y in n m~a......A ~ J Cli~ N u l r 19t5; 28: 24 Kleiner M J, T~ie SS, Sulii n IF, C h a ~ i j. Vitamin B6 d e f i c i e n ~ in ~ a ~ , | e ~ z n c c din|ysis patients: me|abo|ic e{ Is of repletion, A m J C!i~ Nu|F 1980: 33" 1612-1619. 25 Dobbclsiein H, K;S cr W ~ Mempel W, Gr s ~ W i ! d e H. Edcl HH. Vi|zmin B6 deficiency in ure ia and iis i,nplic ions ~ r the depression of immunc respon s. Kidney [nter~ 1974; 5' 233-239. 2.6 Lumeng L, Brashear RE, Li TK. Pyrido~ai 5'.ph phate in plasma; s o u s e .... protein-binding and ceilu~a~ | nsport J Lab Clia Med i974; 84: 3 ~ - 3 ~ 3 . 27 A ~ d e r s B N e ~ m a r k PAo Ra fins M, (3teen R P s z binding of v i t ~ i ~ B6 compounds. Na~u ( ~) i :2~: ° 28 S n ~ l H~skd] ~Eo T ~ tabo|~ . . . . of vi|~m~n 1t6 pr B~. 197| ° 2 ! ....7! 29 KoppIe jl'). u ~ KC, n k r a n ~ M.t' ~ a~o ily r ~ i ~ l ~ a ~ ~ r p idox/ae s ~ ~ l e m e n l s in ch nic i fa/t~tre0 K ~ n e y i 98!; 694 .....7 ~ .... 30 Laconr ~, V a ~ u l t A , Deg F, Brunezu M. K ~ i s Baitly M. de in vitro de p h osphale calzly~ique d ~ ~ ses dans une . . . . . . . • .. e pyridoxal dans la d~ter i n a t i de puiation de t nsplanl~s r6naux. Clan Chim ,&eta 1982; 120: | - ! 2 .... 3t Wa k L G , "St one .... , W J. Wagner . . C. . D ~.r ~ ~. d a ~ a r ! a ! e a m i n o t r a n s ~ f a s e (SOOT) livity } n rum of u ~ m i e patients. Cli n Chem 1974; 2 0 : [ 2 i 3-1216. 32 Wolf P L Wii[iams D , plon N, utsoa AS. Low aspartate lrans~m!n~se activity in serum of paiients u n d e ~ o i n g c h r o n ~ hemodial~'s~s. Ciin Che 1972; ~8: 567-568, L i l w a ~ G. Modn!ator a~d lhe glue or~icoid recep| . Trends B ocher, i 979; 2 i 2 ~ .
etg
127 ( 1983 ) 2 0 5 - ~ 15
205
Etsevier Bio edical Press CCA
2364
P.v"idoxal 5" phate deficiency ure undiaiyzed, he .nod alyzed, and non-u remic kid ney transplant pa tien ts r
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Is|. 1982)
SI| lll~at~."
I n lliis s t u d y , w e h a v e i n v e s t i g a t e d p ~ a s n l a , pyridox;Lt 5 ' - p h o s l s b a t e ( P L P ) c o n in u n d i a l y d mad d i a l y z e d u r e m i c p a t i e n t s a n d i n k i ~ n e v t r a n s p l a n t st b j e c t s , u s i n g a n e n z y a t i c tccl~! i q u e witii i h e r m a | d e p ~letn~ t i ~ to | i b e r a t e [3Lt~ i~ti f r o m p l a s m a p r o t e i ns. T h e, ..s p......e, c"i f i c i t y o f tl~e r e a c t i o n i n d i c a t n o i n t e,r f ~e r ,e n c e 4 p y r i d o x a i a n d o n l y 3 .% i n t e r f e r e n c e w i t h p y r i d O X i l. ....1' ll e .p. .h. o s p h a t e . I n 17 h e ~ o d ia | y z e d p a t ic a Is, a d e fi c i e n c y o f a b o u ~ 50 % .of a s m a P i....1-, c o n c e n t rat i o n is f o u n d as c o m p a r e d to 25 l~ea!~lay s u b j Is (22,2_*.:2.47 vs. 4 8 + _ 3 . 0 0 . . S. E .M .) . . In . . . ,,,e,l . u n d i a l ) 7 + .... ed n m o l ... | :I, a s ni .... ' ° ........... n_+ u ev m t ...... c w~th e ..... ' s ............ • o n c e~.n | .r a. .i o.n . is. a.! s o . d ' .....er -a.s...e...d..... (....... 2 9 . 3 N~ i . 7 4 n I-I i renal !u:re, ! h e p i n s a P L P c............. T h e absence: o f P L P in p l a s m a ult ntfil i r a t e s d e m o n s | r a t e s tila t n o loks o f P L P ~.~'-ccu d u e tO h m o d l a l y s l s . Tile daily oral supplementation itil 2 5 0 7 5 0 m g p y r i d o x o l induces a y s i:o l o g i ~c .... a l i n c r e a s e in p l a s m a P L P CO .... 1"S t l p r,a p h........... ccfllratiOll i n | ~e" ....o d i a l y.....~° e. d as we II as G ~
in
u nd iaiyzed
pa tien
t s.
t n.... a PLP conccntraIn i 1 6 n o n - u m i c k i d n e y t r a n s p | a n t s t ib.... j e' c~ t. s ., . t.h e. . m. e. a n p l , , s....... l i o n w a s 33,8 5 : 3 . 5 0 n m o l . | - , i . In o f t.h'..... ese~ p a t i e n t s , a deficit (helo 20 n O [ * i ..... ) a s o b rved_. :lion in n c l u s i o n , u r e m i c p a t i e n t s h v" a d e f i c i e n t B 6 .......i ....." | t s c o t o p... y r i d.o x o | c. o . : p . iotogica! p l a PLP content :tion tl ........¢'ssi::la , ' s o suppie entation ith lower doses that ihose i d e i y u S: e~d a | p r e s e n t . In k i d n e y Iranspiant a s i t n i l a r p l a s m a P L P d e f i c i e n c y is o b s e r v e d in t i absence of chronic a ' ...'. failu .
* Address
~r
correspond ce: B La ur, Lab P a r i s ~. . . . . e x 15. Fa n........ c e . : ' •r
-
~
1 83
azoi~de
vler ~io
Biochi
icat
P
ie A , H ~ p i I
s
Necker,
149, r u e d e
206
In![
ue t *
Vitamin B6 actualty cormspoJlds to thr active ~ e t a b o l i t e s , i.e: pyridoxol. pyridoxai and pyridoxamine, which play a role in man3 metabolic functions after ]~aving undergone p h o s p h o w l a t i o n . In tissues~ tl!e major vita in B6 c o m p o u n d s appear to be pyridoxa[ phosphate (PLP) and p2~ tdoxamine phospllate ( P M P ) ill. In pl as tn a, py ri d a I (P L) a n d P L P reptesent tln e ~ ai i1 for s of vi t a in B 6. T h e ~ for plasma PLP reflects accurately the vita in B6 S t a t U s . The deter i n a t i o n of p l a s m a PLP is of particular interest because hu an b o d y sto s of vitamin B6 are ~ all They can be quickly depleted. . . Moreover, . . sonde. drugs antagon,;ze the m e t a b o l i s m of vitamin B6 to i~s active metabolites [2]. The study of clinical conditions potentially complicated by a mild vitamin B6 deficiency ~ q t t i ~ s a sensiti and reliable assay system for the precise ' ca~u c sn rt e- ' " of P L P concen t tion i n p~asma or tissues. SCV .... e I 17uo met tic [ 3.5 ~ 7] spect pllo, to~etric [8,9 i, and microbiological m e t h o d s [10-13] a presently available far the assay of plasma PLP, bui ihey are not sufficiently sensitive a n d / o r specific. E ym ic assavs~ [4,14~20~ a,e'r- a o n g the o s t satisf'actory tnethods, .... associating :peclficity and sensilivity in the estimation of P L P in biological a t e r i a l s ~thout ils prior isolalion or purification. Enzy aiie metl~ods are all based on tl~e abilily of P L P to restore the aclivity of an a p o e n z y ~ e to which P L P is the coenzy e T h e enzymatic p c e d u used in this s t u d y has beeo derived f r o m the techniq~ae described by C h a b n c r a n d Livingston [4]. It is based on the rate of t~CO2 ~ r ation f r o ~ L-[ ~'~C]tyrosine u n d e r the influence of PLP-sti vlated ty sine apodecarboxy~ last. The p u r p o s e of tl~e s t u d y as to d e t e r m i n e ~he serun~ P L P ncent lions (1) in ure ic patients treated or not by hemodialysis and s u p p l e m e n t e d with p y r i d o x i n c or not, and ( 2 ) i n kidney tra piant patienls ith normal or near normal renal f u nC' "r o O n . wl~o all ceived i m m u n o s u p p ~ s s i v e tl~e py. "
Patie:nts and
ethods
tien~ Tl~rcc g ups of palients were studied" hcaIlhy subje-cl~, ur,i c e patients, and kidney t nsplant patients. V e n e p u n c t u r e was d o n e on twenty-five hea|thy subj ts in tl~e morning. The blood was collected into heparinized vials (10 I U . 1 - l ) . These control subjects received n o e d i c a l t at ent, and they had no llistorY of he ~ic or e n d o c r i n e d : isease. ' - ..... W o m e n ho were laking a 1 contraceptive drugs ere- discarded fro th e s t u d y. u s e h av¢ s t u d i ed ! 2 wo ~nc n, w it tl a nl e a n age o f 36 yea ~ (rang e, 20 - 54 years), and 13 men with a men J1 age of 28 year~ (range, 20 ~ 38 y,e a1. . . . )j Thirty-six patients itl~ chron;c renal [nsuffici~ cy ere studied. T e a t y - n i n e were be odialyzed for a can of 3 6 ont:ns~ their erage age as 42 y e a ~ (range, ~ 2 - 7 0 y e a ~ ) , Se n u ~ i e patienls w rc s~udied a! the ter ina! stage of f e n af l: insufficiency j u s t prior to tl~e beginning of hemodialysis (c .... ~ a ! inine cleara e < 5 ~ 1 / in). T h e a rage, age w'as .... 38 years (range,. 25~51 y e a ~ ) .
207
T h e third g r o u p consisted of 116 kidn,.ey t r a n s p l a n t n o n - u r e ic palients, ith a e a n age of 33 years {range, 16~58 years). T h e p l a s m a P L P d e t e r inaIion was : e ti ....... al tra nsp Ia n ~a*l l O :l l . . . . .... p c r f o r m ed a ~ V, ar~:: a bl e i n ter val s afle r r: e:z~
Meth T h e e n z y atic t - .......: ............. d e s c r i b e d here. w h i c h h been derived from the o r k o f S u n d a r 'c-s a n a n d C o u r s i n [ia] a ~ d was m o d i f i e d b y C h a b n e r a n d L i v i n g s t o n [4], uiili s the following reaction"
-~ C] yrosine ...
Iy'rosl":
a
rbox ylase
y r a m i t ~e~ ........
P L P dcpendent
CO~ "
S o d i u a c e t a t e buffer, p H 5 5. 0.1 n~ol . i° ~ in distil ed w a t e r (',Bake-r. D,vcc a r e ",r, T h e N e t h e r Ia n d s). ~ridoxai s o l u t i o n (Calbi h e m . La Jolla. C A 92037, U 37.7 y moi isi d i s l i | | e d a t e r .... T h i s s o l u t i o n was atiq u o ! c~d i n t o |i | u es ( o f 500 ~1 e a c h ) a n d s t o :at ~ 2 0 ° C until u .... T h u s , o n e t i a ~ e d for e a c h to p r e p a r e a fresh w o r k i n g P L P of 377 a n d 75.4 n o 1 . ! - d i l u t e d i n , c e t a t e , ~" b u l f e r a t p H 5,5. L-Tyrosine solu!ion (BDI--1 C h e m i c a l s Lid, Poole, U K ) , 5 o I . 1.... ~ in acet~ttc b u f fer a t p H 5.5. L-[ i i4 C ] T y r o s i n e s o i u t i o n , p r e p a r e d b y a. d..d...~l t i o n o f e - [ ! - !a C ] t y r i n e ( N E C ....170, s p ific aclivily 5 !....9 m C i o ~ m o l - ~, N E N , B o s l o n M A 02 ! 18, U S A ) ~o an L-ty s~nC s o k i i i o n : o o b t a i n a fi a~ s p ~fic activity be~ n 4 al~d 8 / l C i - : .... $: A Tyro~Inc ('F-4629, I . 1 U . g ...... i I U p r o .... d u c i n g !/~ o i / I CO~ fro: L-ty sine p,..,.re' rain at p H 5.5 al 37°C,° S i g m a (2"he l!lc,~l: " C o , St Louis, M O 63178, U S A ) c o n t a i n i n g 2.5 g - 1 ~ in dislii.ed wa~er. E D T A so Iu t io n, 2 0 g * I -~ (M e r "c k , D a r n~s ta' d t, F R (3). HCI 5 o1.i-~. H y a l i n e h y d r o x i d e , 1 0 x P a c k a r d ( K h ....L~gh| C o l n b r o o k , Bucks, UK). .... H e p a r i n soluti 1% (Vi r u m , S e d .
....
t
....
i
.
0 i .............. ~ •
,
.
~
.
¸i
.
PLP w e r e c e n tri fuged a ' 1 4 ~C
[or 10 rain a l 5000 ~ g., I~ was n e c e s s a r y o c , n e ~ri f,u ge- i ~ e d i a t e l y a, f ler bl d s a m p. .l .i .n g in o r d e r to limit p o s s i b l e h y d r o l y s i s of P L P by a l k a l i n e .....p.h. .o. s p h a t a s e aclivify c" on t a ined i n eryt , . . .hr . ocy tes or leukocy ies .... E sa p l e ,,as " tested in !riplicale a n d ' c o ....... n p~ a d ilh a ....s t a n d a r d P E P rye perfo ith a p o o l o f se o b a t n e d f m h e a l l h y s u b j is. l n i o 5 - • Iub , | o 800 ~i o f ch plas a 3 2 / a l o f E D T A l u | i o n were: a d d e d a n d to 1000 using ........... " .....e b u r a pl-i . . . . . .5,5, . . . . . .At . . . . .ibis . . stage, the s u p p l e m e n l a t i o n o f p t a s ~ a with P L P was p e r f o r ed by ~ p l a c i n g the a b o v e I68/~1 o f acetate' •'' b u f f e r by s o l u t i o n s c o n t a i n i n g i n c r e a s i n g a m o u n t s of PLP. A f t e r a g i t a t i n g for | 0 s in Blood
s a n~ p ! e s
20g
a Vort ~ i x e r , the: t u b e s e p i a c e d in t l l e d a r k , a t r o o te peraturc, for 30 in. pt|. o f a t a r e "l'hcn 2 5 0 / a l ~ e a c h t u b e ,~e . . . . . . .t .a k e n :in . .~ t o . w h i c. h 95(i . b u f f e n p H 5.5 .... w e added. Afler |0 s of agitation by the Vortex ixer. they ere
Whatman :paper :l~Vam~ iwam
....ASSay m e g i u m F i g ... | . R @ imp
e n l a i. } O | i
gna led
t h e i.
i ih h y a m i n e
u b a t i o n ~.: a m ~ r .
~e:
Whz:tman
sh
............. t w a s :m ri ed
into.,
st o p. . . . ........ .r and
hydroxide,
m a i n t a i n e d in c o n s t a n t o t i o n f o r 10 r a i n a t 8 0 ° C in a s h a k i n g w a t e r b a t h , A f t e r 5 n:~in t n ' f u g......a t i o n l ....5 0 0 0 × g , 6 0 0 # i o f e a c h s..u. .... p e•r n a t.............. a n t w e~r.....,e ....s a m p l e d a n d I0t3 of a zyme in we added Then 0a.....i t e,r 3 0 s o f a g i t a t i o n the Vortex, the vials ~ we...... . . . . . . i n t h e d"ar."k a~ o m re t e~n.......... J p e r a• t u ~ r 3 0 ~ i n t o al}o P i . P ..... r e c o ~..... b ~....n:...... a ti ..... on i t h a p o e n z y .n ~ e . T t l e n t ~[........ c, | u b e s w e r e t r a n s f e r r e d t o 4 ° C a n d k e p t at t h a t te p e r a t u r e f o r 5 i n b e f o r e 1000 p l o f ! . - [ l - ! a C ] t.:~ ' rosa ..... : n e s o lu t i o n w e r e a d d e d . I e d . i a ! e l y.,. a f ..... t e . ,........... a~ d s. .t h. e y. . w.e r. e c'io....... s c "d wi tb a stopper . . . . t - : ......' a sheet of paper (4×2 c )folded ~eogtbwi i n t o t o a n d i p. . . . a l e d w i t h 8 0 g ! o f h y a m i n e h y d r o x i d e : s o l u t i o n a~,,'s {|i u s t r a t e d in F i g i. asperfor edforexactlyl5 mi,lat37°C ith c o n,,,s ,t a]x t a g i t a ti o n in a I n c u b a t i o n w', ..... s h a k i ~ g w a l e t b a t i~ e n , u~,,,,ing s: a ~ r i n g e , ? m I I-ICI w e r e i n j e e r e d t h r o u g h i he s t o p. .p. e. .r. . .t o s t o p t h e enz,,., ..... : y ....... a ric, r e a c i i o n . F o r t h e n e x t 2 h t h e and capture of iaeo ,o,,. hydroxide was performed at 20-22°C. .... t h e s h e e t s o f pap., r r e c u i o f f a n d i t r o d u c e d i n t o v i a l s c o n i a i : n i n g ] 13 m l o f s c i n ~alion licluid .... T}le* t a d :i.... o a"...... c l i:v l: t ....... y W a S d 'e r ....~"! ...... n e d i n a, ! i q u i d s c i n ! l i a t i o n s............ .......~.... ( S L 30. p e*c t........ r o n ~e~er l n t e r ~ c c h n i q u ~ Plaisir~ F-rat~ " ) , Results
A &t
]twubatioJ~ tinge at 3 7 ~C. F i g .... 2 s h o w s t, b a..... ........ t ,,he action velocity was constant b e t w e e n a a n d 30 r a i n a n d t h a t a f t e r 3 0 r a i n t h e e n d - p o i n t o f enzymatic., r e a c t i o n w,,,:as hie ......A l l m e a s u ~ m e n ~ s w e r e p e r e d w i t h oil i n c u b a t i o n ti e o f 15 r a i n . Ap tl2e i
zyme ub
ion mediu
trat
With an the
action
at tionof73.5 g t :~ in s n o t l i m i t e d b y ....t h e a p o e n z , l ~ e
40~0
1 2 , 6 !0 iS ~0
30
45
Fig .....2, E f ~ c t o f i n c u b a t i o n tinle at 37~C the M e t h s |i ....
concentr~tion. Wel~ave of i.47 r a g - ! ....
S~hst
te
~ nt
adeour
60
the
easure
of
entsusingan
ti
T h e incubaliL~n m i x t u ~ is given in
ar~oe
yn~eco~t~
ntration
T h e e n z y m a t i c a c t i v i t y ~ncrea d a~ t e a s e ~f t y r o s i n e c o J l c e n t r a t i o n in t h e a s s a y m e d i u bet e e n 0 . 7 3 5 a n d 1.471 t o o l - 1 " .... A b o v c 1.47! ~ o i . l - i , l h e e n z y m a t i c ac -t i v i t y w a s ~ a.~: t n t a i n e d c o n s t a n t a n d we h..... a .VC :,Se !~e c l e d a t y r o s i n e c o n c e n t r a t i o n o f 2,941 ~ m o i . i " ~ to a v o i d su b s t r z t e c o n c c n t ~ t i o n b e i n g a Iim i ring f a c t o r in t he z y m a ~ i c r e a c t ion u s e d ... .... ~
.
.
.
.
in"luence# ....... of E D . The fiuence of EDTA as s t u d i e d ~ r c o n c e n t r a t i o n s " ran,ingg b e t ~ e.... e n , 0 a n d 1.6 n g . ~1 ..... ~ p l a s m a ° W e h a v e o b s e r v e d a a x i m a l sti~nula. t i o n of e n z y atic a c t i v i t y ( 3 " / % ) a t 0.8 n g m l " i E D T A as c o p a r e d to the a c t i v i t y .. "~ ....: t .....)'' de m i n e d in a.......b....... s e-n....... t e of T A .... I n f l ~ n c e of l ~ q ~~lity it~ t h e pture CO,, W h a t m a n p a p e r i n ~ r t e d i n t o ~ial s t o p p e r s w a s ~ p r e g n a l e d w i t h 40, 80, i20 o r 2 0 0 / a i o f h y a m i n e h .y d rox . . ide. so. lu .ti on, . .U si. n g. a hi g h P L P c o n c e n ira ~io n o f ~'50 n o l . I - J, n o d i f:l'ere 1iec in r a d i o t i v i t y s u r e d was obser between ca p t u ~ . T h u s ~h e 8 0 t~ l o f h y a m i n e b y d r o x i d e wla icla w e re u s d i n o u r st u ~1y w e s u f f i c i e n t to k e e p all t h e f o r t d. ]n, fhte~Ice o f hepari!~, By a* ....n a' l y....z i n g s e r u m in the a b s e n c e o r in p r e s e n c e of l 0 | U. l - 3 h e p a r i n , w e o b s e r v e d th h e p a r i n n d u c e s a n ir e a s e o f i 9 . 5 % in t h e efficiency of zymatic reaction, a is t h e a n wily bl samples col !eeted i n t o vials c o n t a i n i n g h e p a r i n to o b t a i n a final c o n n t r a l i o n o f i 0 I U - 1....
teinizati ,t Jono, t h e fi ....
pro We have studied p u s i n g p e r c h l o r i c a c i d ( H C i O 4 a n d t,h e
u r e s f o r d e p leini , , n d u s i n g t 'h, ....................a-......... guta-
2:I 0
t,ion W h using the a c i d p r o c e d u r e , the excess:, - ' .... s o f t t C I O 4 will be n e u t r a l i d t o obtai n a supcrnatan t with a pH nea r 5 T h e a d d ilion o f ....... s ' p o t a s s i u m h y d r o g...e n ca r b o n a t e is a~socla led wi:th t h e a t i o n o f i n s o l u b ! e KCIO.~ 0 w h i c h is eli n............ atedi b y C¢~ [..... l h e s u p e a i a I, Wh u s i n g this t e c h n i q u e is t o o r ifugation: . . . . . ...... .t t .o ~. e .v e ~ a l k a l i n e (pI-I 8.0), prohibits PLP use h e- b u f ~:~ " ..... o f the i n c u b a t i o n m e d i u :is F u r t h e r o ~ , this p r o c e d u h u l d be a s s o c i a t e d w i t h loss. T I u s e induces: an i m p o r t a n t g a s e o u s re.~.ase, whic ~' o f c a ici utn c a r b o t~a t e s u p p I ie s a s u p e r n a t a n t w i t Ix p H 6.2, w h ici~ a i Io s en zy n ~a t i c : ~ N e .............. d e t e....~ m~nation. T h i s p c e d u r e d o e s :not i n d u c e i m p o r t a n t gae, s lease. W h c n u s i n g | h e r m a | d e p r o t e i i z a t i o n at 80 o C f o r 10 ino ttie s............. u p e r 1~"a t a n Is ~r a n d t h e p H was: after ..... I .... ~ ..... . . . . . . . . . T h e c o pari a b e l n the t o p ceriums using o r t ! C l O , w i t h n e u t l i z a t i o n o f the: s u p e r n a ~ a ~ t b y c a l c i u m c a r b o n a t e , s h o w s a higher. . . . e. f f. i c. i e. n c y with t h e r m a l d e p r o t e i n i z a l i o n (Fig. 3). i n a d d i t i o n , t h e ,::atteri' .......... p r e d tire is easily a p p l i e d t o a la series of vials. .
Cha
of the m~kod
t
p l a ~ a PLP
.
.
.
nination
F:~g0 4 s h o ....w ...... ~ a linear a~ed b y U V light p s o r to b e t w e e n 0 a n d 140 n m o ! . i ~ P L P
to a . . . . the
~sooJ 800 g
0 1020
40
6~
',00
t40
PLP ¢0ncenlta!ioa |am01 I"~t F i g :3. . .|.n. u c n c e o f tlie d e p r o i c i n i {ion p r e d u ~ o n t}~e P L P recover, ..... from dif~ren p|as PLP c ~ ....~. . t~: ........ s. e al 8 ~ .p.r. o. .~. n i ~ t i (~ 8 0 ~ C ~ r 10 rain u ~ i ..... . . . . . . . cl .. ....... w i t h ~ "~ate bL~ r p l t 5,5 I n . . ~ I .~ i c. .acid . d~~eini p g O X ~he a ~ d c s s e~ w a s te d cak~u m F i g ; 4 ~ S~and~rd c u r v e ~ r
plas
PLP deiermina~ion,
e incubatiot~
i x t u r e is g i v e n i n the
Metkods
21 t BLE I S l"ECI FI C 1| Y O F T H E E N Z Y M A ~ I C R EA {- I O N Subsiance 80
n
n tool,
i + )
as +
Pyridoxit~e Pyridoxa~inc Pyridoxan+ineophasphate 4- Py ri d ox +c aci d Pyridoxal
PLP
~t
r~i~iO~ expressed (n,no|,l
I nte r fe re n c e
...... + )
.............................................................................................................
+
4
16 2.4 0.4 0
2 3 0.6 0
+o+. , .... T h e r o v e r y ,+ ' .......v " o f P L P . R+.~++,++............ d e t e r m i n e d .at t,w d i f f e n t |,e..++..els n p l a s , n a s p i n ~ n s w,+........... +,e s u p p l e e n t e d w i t h a l h e o tica .....c o n t e n t t i o n o f 37.7 n o l * | .....) P L P ,o M e a n t v h s 95 ....l e 2.41% (r S E M ) . plas :spcc)- -+ mens were supplemented with a o f 75.4 n ~ o l . I PLP. Under c o n d i t i o n s ~ e a n r e c o v e r y w a s 89.9 _+ 3.30% ... Sp~.~+c./t);
We
have
measured
the
radioactivity
produced
by
80 n m o + | - i -+
OYri+:doxa i+n e p h o s p h a t e , p y r i d o x a m i n e , p y r i d o x i n e , p..... y ' ri d o........ x a.....! a n d 4 - p y r i d o x i c ,'icid b y... . .c.o . p a r's oi n witti t h e r a d i o a c t i v i t y p r o d u c e d by a P L P c o n c e n t r a t i o n o f 80 n ~ o l . 1- ) ( ble I). T h e r e s u l t s s h o that only pyridoxine, p y r i d o × a m i and pyridoxa~ine a n d 3%. )
~
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p h o s p h a t e .¥ie!d an i n t e r f e r e n c e r e a c t i o n
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pool of rum a l i q u o t e d in ° a n a t y z e d in i 6 d i f f e s i o v e r a 2 ....~ o n t h ' s p e r i o d ......F o r a . . . . t h e y .........: l+on fficie++t w ~0.97% . 38.98n ol . .I+ ...... A
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"+..... ~+~,+++e+ ....t tOtL E i g h t s+e e a n a l y d t h e d a y+ oP +san.+,pling a++nd 15 d a y s ....~ft+er at ~ 2 0 + C , T h e m n P L P c o n c e n t .... tion ~ a s 47°57 6.21 a n d 4 6 . 9 2 _+ 6 . 9 4 n n ~ o l - I ~1, r e s p e c l i The ean difference for paired ~0,65.4-_ 0+93 n m o i , i ......) w h i c h a s n o t signifi n t l y d i l u e n t from zero.
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Mean plasma PLP concentration as 48,8 __. 3,00 ( S E M ) n ~ ~ t . i - ) in 25 h e a l t h y i ng n o ora, . 1++s u p p l e ' a l l , / s i g n i f i c a n t f....i "i f f e r e n c e e x i s t e d s u b j e c t s tak+ e n t a t i o n , A sta t'istic bet n m e a n p l a s m a P L P c o n c e n t r a t i o n s d e t e r m i n e d in m e n ( n 13) a n d in en (n 12): 56.7 5 : 4 . 3 0 v e us 40.2 ± 2,52 n m o l . l+ i p < (1.005 ( F i g . 5). ++. u ~ ~ c p a i e n t s r e a t e d b y i n t e r m i t ten t h e m o d i a ! y s i s h a d a s i g n i f i c a n t ( F < 0 , 0 0 7 ) de+ r e a s e in ~ e a n p l a s a P L P as copared to c o n t r o l v a l u e s ( 2 2 . 2 ..... 2 . 4 7 n ~ o i , ! ..... , n 17) .... I n t ';|+e ....'p a t i e n t s n o s t a t i s l i c a I l y signi fic a n t d i f f e r e n c e w a s o b rved b e t n the sex 23.6 :t: 3,74 f o r e n .+and f o r ni w e e n ( F i g . 5) ! by 250 to 750 g ....
+ e
212:
pyridoxo~ per d a y i n d u c e d on increase in tnean p l a s m a P L P c o n c e n t r a t i o n , w h i c h as higher than t o b s e r v e d in h e a l t h y s u b j e c t s .... A f t e r 30 d a y s of s u p p l e m e n t a t i o n , c e a l r "a t :t o n o f 8 9 . 5 0 ~ | qo.59 12 tents(5~c-n~nd 7 ]iadap|as a P L P c....o n................ _
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488~30
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f:ig 5 PMs~a PLP t~ ntraiions in h e a l l h y niro|s (!eftL in non dialy d u mic ~t~ien|s with e - ~ t a. . ic. . ~ ~ i l.u ~ .~CRF,. m M . d ~ . ~ ) .....d in ~ m ~ c ~ t ~ n i s trc~tc° ....~ (t'tD, righi). T h e horizont~i corresponds to m ii plasma PLP concentration in e,,:h group and tlie hatched ar¢~= to .& SEM. Student's : test indic:ires p ¢ O.OOl ~ r ht:atthy controls vs° CP, F or vs. ! ! t ) lien ts. O a n d o d e n o!e ~ m a i e d tnale su bj eels. respec ti ty,
n O[ . ~ , W e |l......... I" ..... a v e" a | s o PLP in o b t a i n e d 'a,t ~he b e g i n n i n g or at the e n d o f several h e m o d i a l y s i s s e s s i o n s . P L P w a s | ndcte;ctable :.... ~ .... V cry t rong bi ndi ng to pi 'i',s ~ a p leins, in t h e s e s a m p l e s i :- i u.......r-c .........:lc... p a t i e n t s w i t h c h r o n i c renal failure ai ....I ~ e se n n o n - d i a r y ........
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i i
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10
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30
40
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Fig. 6~ Distribation of plasma PLP ncentrat .... ....... i ons in kidney t~anspl range if normal valu ~ u n d in 25 hCaithy coat Is.
t patients. The arrows indicate tt~e
213
hod d e p l e t i o n of p! m a P L P : 29.3 1.74 n m o l . 1- I ( p < 0 . 0 0 1 vs n t ~ l s ) . Ora! supple in the n patients with 300 d a y pyridoxol for 15 day:,.,, i uced a striking increase o f p i a P L P concet~trations to 90 _+ 129 n oi ....1 ....i. . in kidney. t r a. n s p l. a n t . patients, mean plas a P L P " .... n .... . . co; cent tion was d e c r e a s e d by c o - - p a r [ s o n i t h n o r ~ al su bj ec ts (33.81 __+3.54 n in o l . l - ~, n i 16). T h e d istr ib u tio n o f all , a l u e s is shown in Fig 6. Ttl~s Fig. de o n s l r a t that 97 subj ts had a concert tion below 40 n o l . 1 i 75 of which h a d a deficiency (belo 20 nmol - I" ). a n d p e doxicaily f o u r subj ~s h a d a high pl~ls~a P L P ............. .... con ~1 a tion: (89, 116, 238 a n d 2 5 1 n m o l . i ...... ). .
.
.
.
.
.
.
.
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Oi'scussion he e n z y m a t i c m e t h o d used in the p r e s e n t s i u d y sho s su rficient sensitivity. .lnation ....... * s".p e e t f ....1 0 a n d r e p ucibi~ity for the di e e r of pia~ma P L P n ntrationo .... in lion is always u probie b e "c a u s e of the ex~ste e o f for s of v i t a m i n in blood. Microbiological a~says yield values with ide v a r i a t i o n s and scatter b e c a u s e oi' the lack of specificity of lnicr rganis s for vitamin B6 co p o u n d s [i31° Using c h e ~ i ca m e t hods, wide va ri a t ion s res u ] t fro in i t~terreren ce s a n d i n s t a bili ty o f c o m p o u nds for ed b2~' ~he t h e i I r,eactlo)l [%21 }0 O n tl~e "........... nt ...... rary..... !he e v m a t i c tcchJ~iques a r e specific for P L P . Pias a P L P d e t e r m i n e d in o u r s l u d y for h ItbV subjects arc co p a r a b l e to those o b t a i n e d by C h a b n e r a n d Livingston [4}, b u t t h e y a r e io r than ihose o b t a i n e d by Hines a n d Love [18] w b o used apophosphoryla~,e ....... .s as a p o e n z y m e . The ai Jx p r o b i e ~ wi til P L P mea sure ent i s i t s b i n d ing t o p Ia s ~ a pro t e i n s. P L P a n d PL f o r ~ Schiff ba s ith free an~ino g ups of p r o t e i n ....In t h e e pr. s................. e n ' of protein-pr ~ a g e n t s , all the Schiff bases d o n o t b ak a n d P L P m ains b o u n d to pr ipitated proteins, at [east in par{ [71. . . . us P L P e x t r a c t i o n f ~ ~ p r o t e i n s is i n t o piete, a n d this p b l e m could ex p l a l n the di fferences in p l a s m a P L P c o n c e n t r a t i o n s r e p o r t e d in the litera!ure. P e e h l o r i ¢ acid and trichto acetic acid ° h a v e been p o s e d for d c p r o t e l' n i'z a I ion. However, the e x. . . . . . ss• use of these acids nece~itat ihe|at eli i tion of the acid exc s b y p r ipi i i o n ( K C I O 4 ) o r b y e x t el[on with e t h e r for t r ic h l o ............. ...... acetJcacido Neutraii ln~.ufficmnt . for trichioroacetic acid. which inhibils the enzy atic reaclio u d for assay. F u r t h e r ore the recovery of P L P f r o ~ trichioroacetic e x t r a c t s o f hemolysates is i n c o m p l e t e [ 7 ]. U sin g t h e ~ et hod d escri bed h e ~ d eprotei ni za, ion b y ode ra tc hea t coagu i a t io n ~: al oW. s good recoveries of P L P fro p l a s m a s a m p l e s to be ob [ ' ........ ned. P vious i n v e s t i g a t i o n s indi te the possibility of P L P deficiency n h e m e d i a ! y s i s ina~ion of p l a s m a P L P n ntrations d e m o n s t r a t e s clearly an i p o r t a n t d e c r e a s e c o ~ p a r e d ith h e a l t h y subjects. O u r results are in a g u e e a t ]th those of S t o n e et al [23]. e d ~ a s e o b s e r v e d in l~emodi.a l y s i s p a t i e n t s d s not a p p e a r to be due to a loss o f P L P into the dialysis ft uid, since P L P is st ngly bou nd protei as [26,27]~ a n d e h a v e been u n a b l e to sure a n y P L P in p l a s m a ultrafi ttrates ......F u r r i e r ore, a defie:it of similar i rend-stag,, : ha| failure n o t yet t a t e d tan is also o edin u~micpati ts by hc~ ialysiS.
++4
++
Wlien t din 8 he odialysis p a t i e n t s ith p y r i d o x ~ t h e i r p | a s a P L P ncent~tions increase a c c o r d i n g to the vita in B6 ~ e t a b o l i s p r e v i o u s l y suggested [+++,+8}+in hich pyHdoxol is c o n v e r t e d to pyr[doxoi p h o s p h a e a n d p y r i d o × a l a n d m e t a b o l i z e d to ~s a d e f e c t in P L P s y n t h e s i s f r o m p y r i d o x o | c a n be d i s c a r d e d as ~n ex p!an a i ion for P L P defici e ~ y in h e r o d ialys~s p a t leo ts. T h e d e ri ci e ncy ~ u st b e d u e to an i n c ~ a s e in et~bo!~c c l e a r a n c e o f P L P in ~ c h as d e o n s t ted by Spa n n u t h et a I [ 22} + F u rt h e rm o re + o u r re s u I t s w it!~ o ra i su pp! e e n t ~ t i o n t t h c r z p e u i i c doses of +pyridoxol+.++ widely used in h e ~ o d i a l y s i s p a t i e n t s , i n d i c a t e a'n ~ o f p l a s m a P L P concert l r a t i o n compared w: .....~th physiologic~l over o ad ..... . . n.c e n .l r ~ l i o n s . d recently by at c h is excessive vit~ t36 has b n ;++tr s K o p p | e et i [29) In k i d n e y t r a n s p l a n t p a t i e n t s ith n o r m a l or n e a r n o r ~ a t renal f u n c t i o n , ++e° a s o obscrv'ed a m n r k e d d e f i c i e n c y in p l a s m a P L P nc'entr+ations. A ~ ~ o r deficit ( b e l o w 20t~ o l + l ~ ) i s p r e s e t i n 6 5 % o f t h e p a t i e n l s s t died. ..... + t + lh¢+~i+ T e pa+|++ents +a++ +dy have a deficit o:f p l a s m a P L P + n+ ntrat++on whe+++l +++++++v+ re / +tI+cir kid+cy g+ ft .... Afte+r+ tamp i a n t a t i o a they mainta+n th+e de+tic+rat :unde+r i m u o o s u p p r e s s i v e t h e r a p y ( g c n e r a | | y zzath+ fine a n d steroids)+ It is p o s s i b l e that these d r u g s have an i n h i b i t o r y action o n pyridoxa! p h o s p h a t e kinnse in the liver+ n o :+.uch ct'fect l~as b e e n ~ p o r t e d in the l o dat+e T h i s p | a s a P L P c o u | d p. .e.r.h. a p s t h e ....very +~p t~ s t i m u | a t i o n o f alan~ne aminot activity i n d u c e d by P|+P s u p p l e e n t a t i o n i vii using pl a from r+,+n~lc t r a n s p l a n t p a t i e n t s {301 H o w e r. the c h r o n i c d e f i c i e n c y in tissue P L P +, oct+tration i n d u c e s a dr;crease e .... ' is gel+e r a fly c~n in a p. o t .r a n .s a . i n' a........ s y n t h e s i s, w h i c h . . . . . p a t i e n t s in th e• abscrl of c y+....... t o l y....t i c ~i r d i s e a s e [ 23, 31,32]..... o b s e r v e d in l~en+odiatysis T h u s .... in ~1 d e i e r m i f i n n of the sti u l a t i o n inde× o1" t ~ n s a ~ n se tivi ty b y P LPd esnoiallowt e ofthePLP In k i d n e y. . .t.r a n s p l a n t p a t i e n t s the p ! a s ~ a I'LP deficit c o u l d indic a,t....e a tissue P L P deficit. H o vcr. the lat te+r +c o u l d c o n s t i t u t e an a d v a n t a g e for s t e r o i d ~ i ~ n on target .... c"o ]S, since P L P h a s been r e p o r t e d to d e c r e a s e the f o r m a t i o n o f c o plexes betw++e C....~ sic id a n d c y t o s o l i c p t o r a n d their t r a n s f e r t o the nucl r ste icl ~ rcee p i o r 133].
R¢+~ nees ! [..... n l BL Bain JA ....WilIiams i{ L, q'he dist ribu lion of vitami n ~6 in t h e t s " of two i n b r ~ strat ins of +~ice ~ple|e and ~i~amin ~ef~ient s .....J B i ~ C h e m t962: 237: 1989~ i ~ I. 2 Sau HE. JE. ~:ke+ l, : N Jr. as en, o¢ t * nutri~ionnl status o f vitami+i B6 in the hu an ....A J Clin N v t r I972 25: 629+b42. 3 C h a u h a n MS, Dakshinamurti K+ N u o m e ! r i ¢ assay of pyrido~ai and pyridoxa| 5'.ph phate. Anal Bi tern 1979: ~ ; 426-a32, 4 Chabner B+ i v i n ton |)+ A sinlp}e c h e m i c assay for pyridoxa| O~1o~Pi+ate+ Anal Bi hem ~970: 34: 413 ....423. 5 C h a u h a n MS+ K F|uo a y of B6 vi~ame~ in ~o!ogica! |e~Mo Clan O i i m Acla 198 | M : 159+ 167. 6 qStmtJr0 Z+ T~kanash~ S: F l u o 6 m e t r i c determination of pyridoxal and pyridoxal 5 ¢- p hO ~sph~le in b~ologi 1 m~tetiAis by the reaction with ~un+de+ . . . . i + In: + M cColmtck : , DB, Wright LD, cds+ M e t h ~ s in eazym~)t~y+ i. f 8A, New York: A demie P s+ 1970:471 ~475+ 7 St+~ + SK+ Beutier | A new fluori trig me:ih e determination o f pvrido×al 5-+ h o ~ h e + Bi hi Acta : ~5-77~
8 Hender.~on HM. e conversio41 of ~ ~ d o x i n e pb.osph~e ~nm pyr oxa| ph,~:,F le in ~erto~ia B i I iem 3 i% 5; 9 5: 7 7 5 - 779. 9 Whi(e RS, Deln ey WB, Purification ~nd p ~ c r t , ~ s of vito in i hemistry t 970: 9: 4057-4064. 0 Ander~,on .... B B, art MB F u l - 3 o ~ The measurement of rum pyrido×a| by a .................. ic biliously u~i ! ~ ¢ t ~ h aci| !u s i ....2 C li ~ Pa t h o i 1970,: 2 3 ~ ......~ ... I~ ME. {of h | ~be vitamin B6 m~k° ~ic~b/o~ I $ Da~ R 26: -214~ ~2 Rab~owi~z 3C. M ~ a d y NI. S~i~ EE ...... ; ro p u t e r m / ~ ' ......~ ef p v d d o ~ ~ h L ~ s~o J B ~ ~ ; ~75: ~ - i ~ . | 3 Ark:in L Sci~!!z AS. w,[ a~ns WL. F ~ v CN. Yeasl ~ i c r o b i o l ~ / c a ! e t h o d s l~r deter ~ a l i o n of viVa.ins Pyrido×ine ...A~al . {rid Eng C h e m ~943; 15; i41-144. 14 Sundare.,~n PR. C o u s i n 1)11. Mi" ........ Of pyr~doxal phosphate u~,~ngt..~y s~a~ si si~ic.i. i~ C a n d ~ y r o s i n c ap~ afboxytase. In' McCormick DB, Wrigh! LD, eds. Methods in e n z y m o i o ~ , Vol. 18A. New York: ademic Press. 1 9 ~ : 509-512, 15 t|~,m~li A. E~ ~;~tic de|er inafion of pyrido×al ph ph~,~e in p: mz by degarhoxy|z~ion ~f I | y sine ....14C(U) ~nd a co par/son wi~h lhe !ryptophzn load lest0 Sc;~nd J (?iin [,~b invest 1967 20: !
....
!6 li~ske|| BE. ~ e ~ l EE. A~ a~~pt a ~y for pyridoxal p~ p t~ Anal Hi he i : 45: 5 6 ~ . 17 Okndz K+ FIU+i S+ w++dz M+ M ~ + y+ ef py+~d al phosphate ~++ng t~ptophan+laC+ ++ + it+ ++ topha+ e .... in: M c r ick DB .... Wrigh| LD+ ~s+ Meth s e n ~ m o i o g y . Vo| .... 18A. Ne York' < A demic Press, t 9 7 0 : 5 0 5 :0,,, I~ ilines JD, Lo DS. of serum z~d b| pyridoxa] p:hospi~le n n~r~iio~s with purified rabbi| skelelal m u s i c apopi~osphorylase b. J Lab C!in Med 1969; 7 3 : 3 4 3 ....3 19 Pierce Hi, CampBell C, Moore ~ Hillman RS. Modifications i~ the p u r i f i ~ r~bbit skeletal muscle a p o p h o s p o ~ i ~ s e b. Assay of serum phosphate levels. Biochem Med 1974; t0: 360~367. 20 A l l e n ~ a r k S, H i e | | E, [.a n.Cohn U. New mclhod f~r qua~t}~a~ive :~a~y~s of p y r i d o x ~ , l5-~p h o s phate in biological malefia!. J C h r o n ~ l o S r t978; i~6::485 ....489. 2i SF, $hane B. ~ l i ztioa o~ v i ( a ~ n B6 c o ~ unds in h n : ~ biood ~ad ur~neo C I ~ Ch/m A c ~ ~ 8 ; 21: 7 1 - ~ ° 22 C[ ......Jr. Wi,,,a oek LG0. W.a g . r C. . S~ . . WJ. . .| ~. a pta a ~ ............. f a r c e ~f pyrid~xa| 5".p ~ha~e ~n r i t z in u ie m $ C l ~ Meal i ~ ~: 2-7. 23 Stone Warn k LG, W~g r C, B6 d~icie y in n m~a......A ~ J Cli~ N u l r 19t5; 28: 24 Kleiner M J, T~ie SS, Sulii n IF, C h a ~ i j. Vitamin B6 d e f i c i e n ~ in ~ a ~ , | e ~ z n c c din|ysis patients: me|abo|ic e{ Is of repletion, A m J C!i~ Nu|F 1980: 33" 1612-1619. 25 Dobbclsiein H, K;S cr W ~ Mempel W, Gr s ~ W i ! d e H. Edcl HH. Vi|zmin B6 deficiency in ure ia and iis i,nplic ions ~ r the depression of immunc respon s. Kidney [nter~ 1974; 5' 233-239. 2.6 Lumeng L, Brashear RE, Li TK. Pyrido~ai 5'.ph phate in plasma; s o u s e .... protein-binding and ceilu~a~ | nsport J Lab Clia Med i974; 84: 3 ~ - 3 ~ 3 . 27 A ~ d e r s B N e ~ m a r k PAo Ra fins M, (3teen R P s z binding of v i t ~ i ~ B6 compounds. Na~u ( ~) i :2~: ° 28 S n ~ l H~skd] ~Eo T ~ tabo|~ . . . . of vi|~m~n 1t6 pr B~. 197| ° 2 ! ....7! 29 KoppIe jl'). u ~ KC, n k r a n ~ M.t' ~ a~o ily r ~ i ~ l ~ a ~ ~ r p idox/ae s ~ ~ l e m e n l s in ch nic i fa/t~tre0 K ~ n e y i 98!; 694 .....7 ~ .... 30 Laconr ~, V a ~ u l t A , Deg F, Brunezu M. K ~ i s Baitly M. de in vitro de p h osphale calzly~ique d ~ ~ ses dans une . . . . . . . • .. e pyridoxal dans la d~ter i n a t i de puiation de t nsplanl~s r6naux. Clan Chim ,&eta 1982; 120: | - ! 2 .... 3t Wa k L G , "St one .... , W J. Wagner . . C. . D ~.r ~ ~. d a ~ a r ! a ! e a m i n o t r a n s ~ f a s e (SOOT) livity } n rum of u ~ m i e patients. Cli n Chem 1974; 2 0 : [ 2 i 3-1216. 32 Wolf P L Wii[iams D , plon N, utsoa AS. Low aspartate lrans~m!n~se activity in serum of paiients u n d e ~ o i n g c h r o n ~ hemodial~'s~s. Ciin Che 1972; ~8: 567-568, L i l w a ~ G. Modn!ator a~d lhe glue or~icoid recep| . Trends B ocher, i 979; 2 i 2 ~ .