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Eva lu a tion o f D ia lys is T rea tm en t in U rem ic Pa tie n ts by G e l F iltra tion o f Se rumJesus O s ad a ,1 T e od o sl o G sa,2 C a rm e n S an z,4 I sabe l M lllan ,3 and Ju lIo B ote lla4A g roup o f subs tan ces o f m o lecu la r m asses be tw een 3 0 0an d 1 5 0 0 D a have been found to b e to x ic m e tabo lites inp a tien ts w ith u rem ia . W e de te rm ined th e concen tra tio n inse rum o f th ese m o lecu les in the fo llow ing g roups o f pa tien ts :tw o hem od ia ly zed g roups (on e w ith cuprophane and th eo th e r w ith po lyac ry lo n itn le d ia ly ze rs ), o ne group trea ted w ithcon tinuous am bu la to ry p e riton ea l d ia lys is , o ne group o fnond ia ly zed azo tem ic p atien ts , and one con tro l g roup o fh ea lth y p e rsons . U ltra filtra tes o f the sub jec ts se ra w erefrac tion a ted on Sephadex G -15 fo llow ed by ion -exch angech rom a tog raphy . E lu a tes w ere m on ito red by abso rban ce a t2 5 4 and 206 nm . Pa rtia lly cha rac te rized p eaks P1 and P2 ,o b ta ined by ge l filtra tio n , co rre la ted w ith the concen tra tio n o fc rea tin in e in s e r u m ; th eir c on ce ntra tio ns w ere s ig nific an tly (P

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I,. nm- In nm

I

Pa t ien t groupCuprophanePolyacry lonitnieCAPDNDAPH e al th y c o nt ro ls

n M ea n (a n d ran ge ), m m /m L of serum6 15 (5 -20 ) #{1 76 } 7 (4 ...1#{216})a.b.c6 12 (5-17)a.b.c 6 (4 ...8)a.b.c6 4 (1 ...9)a.d.e 3 (1-4) 6 3 (l....5)a.d.e I ( o .7 -. 2. 5) 7 0 (0 -0 ) 0 (0 -0 )

206 nm- 2o 4 nm

ox ito c in [M r lo s e D a ]

SO SO 70 soT ime

A A

CL IN IC A L CHEM ISTRY , Vo l. 36 , N o . 11 , 1 9 9 0 1 9 0 7

in 3 h (12 ) . C oncen tration s w ere ex pre ssed as m illim eter o fp eak he igh t p er m illiliter o f se rum .

S ta tis tica l a na ly sis . The Sh ap iro -W ilk tes t was appliedto estab lish th e b ehav ior o f d istr ibu t ions. W henever th eSh ap iro -W ilk te st rejec ted th e hypo th esis o f n orm al d istr i-bu tion , or w hen th e B artlett te st for hom ogen e ity o f vari-an ces w a s sign if ican tly d iffe ren t, w e calcu lated th e ov era llsign if icance of d iffe rences w ith th e K ruska l-W allis ( o n e -w ay analysis of variance) te st. If th e d ifferences w eresig n ifican t, w e th en tested th e d iffe ren ces b etw een theg ro ups pa ir-w ise , u sing th e Mann -Wh i tn ey U-tes t (13 ) .D iffe ren ces w ere con side red nonsign ifican t when P >0 .05 .

A ssoc ia tion be tw een va riab les w as asse ssed by Sp ea r -m ans rank-o rde r co rre latio n co eff icien t (r .) (14 ) . Precisionstu d ie s w ere ca rr ied ou t by dup lica te ana lysis o f samp le s ind ifferen t day s and resu lts w ere ex pres sed as CV (% ) .Resu l t s

F igu res 1 an d 2 show th e ch rom atog raph ic p rofiles ob -t amed by g el if itra tion an d ion -ex change ch rom atog raphy ,respect ive ly . G el filtra tion of urem ic se rum yie lded five orsix peaks at 254 m u. In con tra st, no peak ap peared inhea lthy con tro ls (d a ta not shown ). A t 206 m u, th e ch ro -m a tog ram show ed peaks in bo th g roup s. P eak s P 1 and P2 ,w ith re ten tion tim es of 39 and 50 m iii, r e sp ective ly , w ereco llected an d fu rthe r p roces sed by ion -exchang e chrom a-tog raphy . The pro file o f u rem ic se rum a t 254 u rn show ed 11peaks by io n -ex ch an ge chrom ato grap hy , w he reas no peakscou ld be de tec te d at th is w ave length in sera from hea lth ysub jects . T h e r ep ro du ci bi li ty (be tw een -run C V ) o f r e te n ti ontim es for th e p eak s w a s 6% an d 8% in g el if itra tion andion -exchang e ch rom a tograp hy , resp ectiv ely .

Analy tica l prec ision (C V ) w as < 16% . T o calcu late th isC V , w e u sed resu l ts of dup lica te ana lyses on d ifferent days ,31 pairs of da ta for each p eak . T he m eans and rang es fo reach p eak (m m lm L ) w ere as fo llow s: P 1 at 254 am , 6. 8(0 -20 ); P1 a t 206 m u, 28(0 -67 ); P 2 at 254 am , 3 .4 (0 -10 ); P 2at 206 m u, 30(5 .5 -56 ); P 5 at 206 m u , 9 .5 (0 -23 ); an d P 6 at254 am , 5 .3 ( 0- 18 .5 ).

T able 1 show s th e va lu es fo r p eak s 1 and 2 o bt ai ne d byge l f i l t rat ion , as monito red at 254 m u. The h igh est va lu eswere observed in th e h emod ia ly zed patients (cup rophanean d po lyacry lon i t r i le) , w ith no sta tis tica l va ria tio n b e -tw een th e va lu es ob tained by th ese tw o d iffe ren t p ro ce -

F ig . 1 . C hrom a tog ra ph ic pro file ob ta ine d by g e l filtra tio n from aurem ic se ru mW e a p p lie d 0 .5 m L o f u ltra flltra te to S e p ha de x G -1 5 a nd e lu ted it w ith 0 .01m o l/ L T rI s- H CI b uffe r, p H 8.6 , a t a flo w rate of 1 0 m L / h . T he e lu ate s w erem on ito red a t 2 5 4 a nd 20 6 nm . F rac tion s b etw ee n 1 0 00 a nd 15 0 0 D a(accord ing to the o xy toc in e lu tlon pe ak ) w ere co lle cte d

P.akb

f lm .F ig . 2 . C hro ma to gra ph ic p ro file obta ined b y io n-e xc ha ng e c hro ma -

tog raph y o f a u rem ic serumW e used d le thy lam lnoe thy l - Sephadex A-25 , eq u ilib ra ted w ith 0 .01 m oL /LTns K C I buffer , p H 8 .6 , a nd e lu ted with th e s am e bu ffe r bu t w ith a cont i nuousio nic s tren gth gra die nt to N aC I, 1 m o l/L F low ra te w as 10 mI/h

Tab le 1 . C oncen tra tIons o f P eaks Ob ta Ined by G elF iltra tIon M on Ito red at 2 5 4 nm

Peek P1 P eak P2

P

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40 -

30 -

20 -.

-jE 6 0 -JE

10 10 -i0 0

I-C NDAP C A P D AN CU I-C NDAP CAPD AN (UF ig . 3 . M ean (and SD ) c on ce ntra tio n o f P 1 (left) an d P2 ( r ight ) by ge l f iltra tion and m easured a t 206 nm (c f . , Tab le 1 , at 25 4 nm )AN , polyacrylonl ti l le; CAPO , c on tin uo us a mb ula to ry p erito ne al dialysis; CU , cuprophane; HC , hea l thy c on tr ols ; a nd NDAP , non dia ly zed az ote mic pat i en ts .S t at is t ic a l a n a ly s is wa s by M an n-W h ftn ey s U -te st:

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Tab le 3 . S ign Iflcan t (P < 0 .01 ) Rank-O rde r Correlat loSp ea rman . r,

n C oeff lc lents

Creet in lne P1 P 2 ,P 5,

206 nmP 1,254 nm54 nm 206 nm54 nm 206 nm

Crea t in ine - 0.71 0.63 0 .62 0 .57 0 .76 0 .61C rea tin ine c lea ran ce -0 .74 -0 .85 -0 .85 -0 .75 -0 .7 -0 .76 -0 .77

tio n s, w h ich w ou ld resu l t in in creased po rosity o f th em emb ra n e (21 ) , or to a m inor m etabo lic a ltera tion ( 20 ) . F orwha tev er rea son , C A PD appea rs to rem ove n eu ro tox icm idd le m olecu les m or e e ff icie ntly than any o th er typ e ofd ia lysis ( 3, 7 ,2 1- 24 ).

The d ifferen t types o f h em od ia ly sis sy stem s s tud iedsh ow ed no d iffe ren ces w ith respect to th e hand lin g ofm idd le m o lecu le s , as w as a lso foun d by Asab a (15) . O urre su lts con trad ic t p rev io us repo rts reg ard in g th e b eh av iorof po lyac ry lon itr ile m em b rane de riv ed from in v itro stud iesw ith B 12 as an exam p le o f a m idd le m olecu le. T he sim ilar -ity o f resu lts in tw o d iffe re nt p op ula tion s rega rd ing m idd lemo lecu l e behav ior in d ifferen t dia lysis pro ced ure s und er-sco res th e v a lid ity o f th is m e tho d fo r con tro l of th e con cen -tra tio n s o f th ese metabo l i tes in sera o f azo tem ic p atien ts .N ond ia lyzed azo tem ic patien ts sh ow the low es t va lu es o fm idd le m o lecu le s in u rem ic pa tien ts , w h ich m akes sen sew hen one co nside rs tha t th is g rou p h as the h ig hest m eanfo r c rea tin in e c lea ran ce (8 m L m in ), com pared w ith th eC A PD group (3 m L /m in ), th e po lyacry lon i t r i le-d ialyzedgrou p (1 m L fm in) or th e cup rop hane -d ia lyzed group (0.8mLm in ) , an d that th e con cen tra tio n o f m idd le mo lecu l e scon side red as a who le ( as d ete rm in ed by ge l f iltra tion ) isinv erse ly as soc ia ted w ith c rea tin in e c lea ran ce (T ab le 3 ) .M idd le m o lecu le s ap pa ren tly a re c lose ly re la ted to therem a in ing ren al func tion , a s repo rted by Asab a e t a l. ( 25 )and by our g ro up pre viously ( 26 ) . Thu s th e con cen tra tion ofm idd le m olecu le s ha s been fo und to b e a ss oc ia te d w ith th eseveri ty of sym p tom s in nond ia lyzed azo tem ic p atien ts (27 ).P e rhaps th e g lom eru lus filte rs the se su bstances as easily asl ow -mo lecu l a r -mass so lu tes.

Wh en w e con sid er th e v a lu es in a ll s tud ied groups ofp eak s P 5 and P 6 ob tained b y i on -e xc ha ng e ch romatogra-phy , in com parison w ith th e va lues o f peak P 1 (ob tained byge l f i l t rat ion ) , al l exh ib i t th e same behav io r (T able s 1 and2 and Figures 3 an d 5). T h ere fo re , for an in it ia l es tim ate o fth e c on ce ntra tio n of th e m idd le m ole cu les , ge l- if itratio nch romatography wou ld b e ad equate , d esp ite its b e in g anonspecif ic ana ly tical tech n iqu e, g iven tha t the u ltraf il-tra te con tain s a lso ca rboh ydra tes , o rgan ic acids, am in o

I ac ids , and o th er u ltra v io le t-ab sorb in g so lu tes (2 8 , 29) .A ltho ugh b io ch em ica lly su ch an ana ly sis w ou ld b e u nsa t-is fac to ry , e sp ec ia lly g iven tha t p eak 7 c is compo sed ofsev e r a l different m idd le m olecu le s (30 , 31 ), c lin ically th isapproach m ay be accep tab le . U rem ic n eu rop a thy ha s am ultifacto ria l e tio lo gy , and , as p rev iou sly repo rted by ou rg rou p , m oto r conduc t ion veloci ty is associated w ith m idd lem olecu le s a s a who le ( 26 ) . In p rac tica l te rm s, th is ab br ev i-a ted techn ique fo r an aly zin g m id d le m olecu le s is qu iteapp licab le in th e c lin ica l se tting b ecause of th e s im plif ic a-t ion as w ell a s low er cost and accep tab le in fo rm atio n . T h isw ill p rov ide c lin ica l p ractitio ne rs a use fu l m e tho d fo rmonito r ing con cen tration s o f m idd le mo lecu l e s in p at ie nt swith u rem ia .

W e espec ially th an k D r. C ueille fo r h is gene rous g ift of material

fo r th e b42 p eak an d D rs. O rdo vas and K am fo r the ir c ritic alrev isio n of this m anu scrip t . W e are a lso g ra te fu l to M iss A u roraOsad a for h er he lp in d raw ing graph ics and to M iss Rosel le Obrienfo r h er h e lp in prepa rin g th is m anusc rip t. T h is i nv e st ig a ti on w assuppor ted by a gran t aw arded by F ISS ( INSALUD) .Re fe rences1. B ab b A L, F ar r e l PC , U velli D A , e t a l. H em odia lyz er ev alu ationby examinat ion o f s ol ut e m o lecu la r sp ec tra. T ran s A m Soc A rtifIn tern O rga ns 19 72 ;1 8:9 8-1 05 .2 . A sab a H . A ccum u latio n and exc re tion o f m idd le m olecu le s . ClinN eph ro l 198 3 ; 1 9 :11 6-23 .3 . B abb A , Popov ich R , C hris toph er T , e t a l. T he gen es is o f th esqu are m e te r-ho ur h ypo thesis . T rans A m Soc A rtif In te rn O rgans1971 ;17 :81 -91 .4 . F aquer P , M an N -K , C ueille G , e t a l. Imp ro v ed separation andquan t i f icat ion of th e m idd le molecu le b4 2 in u rem ia . C lin C hem1983;29:703-7 .5 . K opple JD . N itrog en m etabo lism . In : Mass ry SG , G lassock R J,eds . T extb ook of n eph ro logy , V o l. 2 . B a ltim ore: W illiam s & W il-k ins , 1989 ;1238-47 .6 A saba H , Bergstrom J , F #{ 25 2} rs t, et a l. T he effect of rena lt ransp lan tat ion on m idd le mo lecu l e s in p la sm a and urine . C linN ep hr ol 1 97 7;8 :3 29 -3 4.7. Menyh a r t z J, Gro f J. M any h ithe r to un know n pep tid es a rep rin cip al c on stitu en t of u rem ic m idd le m olecu le s . C lin C hem1981;27:1712 .-6 .8. Schoo ts AC , Gerlag PGG , M u ld er AW , e t a l. Liqu id -ch romato -g ra ph ic p ro filin g of so lu tes in serum o f u rem ic p at ie nt s u nd er go in gh em od ia ly sis and ch ron ic am bu lato ry p eriton ea l d ialy sis . H ighcon centra tion of p seudour id in e in C A PD patien ts. Clin Chem1988;34:91-7 .9. C am pbe ll R A . O the r u rem ic to x ins . O p . ci t . (ref . 5) : 1144-53 .10 . Schoo ts AC , D*jkstra JB , R ingo ir SM G , e t a l. A re the c la ssicalm arke rs su ffic ien t to d esc r ibe u rem ic so lu te accum u latio n ind ia lyzed pa tien ts? C lin Chem 1988 ;34 :1022-9 .it H enry R I, C annon D C , W ink elm an JW . C lin ica l chem istry .P rin c ip le s and techn ic s, 2nd ed . M ary lan d : H arp e r & R ow Pub lish -e rs , I nc ., 1 97 4:1 54 1-2 .12 . Fu rst P , Z im m erm an L , B erg strom J. Determinat ion o f e nd og -en o u s m idd le m o lecu le s in no rm al an d u rem ic body flu ids. C linN ep hr ol 1 97 6;5 :1 78 -8 8.13 . S iege l S , C as te lla n NJ. In : A nke r JD , e d. N o n- pa ra m et ri cs ta tis tics for th e be ha vio ra l sc ien ce s, 2nd ed . N ew Y ork : McG r awHill , 1988 .14 . Soka l R R , R oh lf F J . In C otte r S , ed . B iom etry . T he princip lesand prac tice o f sta t istic s in b io log ica l re search , 2nd ed . N ew Y ork :W H Freem an and C o ., 1981 .15 . A aaba H . U rem ic m idd le m o lecu le s. A ccum u la tion , rena lexcre tion and el iminat ion by ex tra -co rpo real treatm en t. S cand JU ro l N ep hro l 1982;67(Suppfl:1 -65 .16 . Bergstrom J, FO rs t P , Z im m erm an L . U rem ic m idd le m o le -cu les ex ist an d a re b io log ica lly ac tive . C lin N ep hr ol 1 97 9;1 1:2 29 -38 .17 . Nav ar ro J, Contreras P , T ouka in e JL , F rey ria A M , L ate r R ,T raeg e r J. Effect of m idd le m o lecu le s on immuno lo g i ca l functions.A rtif O rg an s 1 98 0; 4( Su pp l): 76 -8 1.18 . C ue ille G . M ise en ev idence e t ev alu a tion des m oyenn esm olecu le s de Ia tu lle de la v itam ins B -12 p r# {233}sen tesd ana le sliqu id es b io lo g iqu es d e su je ts no rm aux e t d e p atie nts uremiques. JCh roma to g r 1978;146:5&-65 .19 . M erola E , D eu lo fen R , C om pany X , e t al. E va luac iO n de Iacromatograf la de in te rcam bio iO n ico p ars la d e term inac iO n dem edianas m olecu la s . Q u im C lin 1 985 ;4 :17 7-S1 .20 . Berg s t rOm J, A saba H , Fu rst P , L ind ho lm B . M idd le m o lecu le s

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in ch ron ic u rem ic patients trea ted w ith co n tinu ous ambu la to ryp e riton ea l d ia lysis . P en t D ia l B u ll 1983 ;3 :S7-9 .2i . Farre ll PC , R ande rso n D H. M em brane permeab i l i ty in longterm C A PD . T ran s A m Soc A rtif In tern O rg ans 198026 :19 7-2 00 .22 . B abb A l, Johansen PJ , S trau d M J, e t a l. Bi .d i rec t i ona l per -meab il i ty o f the hum an pe rito neum to m id d le m o lecu le s . P roc E urD ia l T ransp lan t A ssoc 1973 ;10 :247-57 .23 Schoo ts AC , M ikke rs FEP , C laessens H A , e t a l. Character iza-t ion o f urem ic m id d le m olecu lar frac tion by g a s c h ro m a to g ra p hy ,mas s sp ec trom etry , iso tachophore sis and liq u id ch rom ato grap hy .Cli i i Ch em 1982 ;28 :45 -9 .24 . S choots A C , M ikke ra F , C ram ers C , e t a l. U rem ic tox ins an dth e e lusive m idd le m olecu le s . N eph ro n 1984 ;3 8 :1 -8 .25 . A saba H , F flrs t P , O u lee R , e t a l. T h e effect of hemodialysia onendogen ous m idd le mo lecu l e s in u rem ic pa t ien ts . C lin N ephro l1979;11 :257 -66 .26 . Botel la J , G ea T , Sanz -G u aja rdo D , e t a l. B lood lev els o f m id d lem olecu le s and th eir effec ts on m oto r n e rve con duc tion v e loc ity .

A rtif O rg an s 1 98 0;4 (S up pl):1 51 -5 .2 7 . A saba H , A lvestran d A , B erg strom J , F (fr st P , Y ah iel V .U rem ic m idd le m olecu le s in n on-d ia lyz ed a zo te mic pat ien t s: re la -tio n to symp toms an d clin ica l b io chem istr ies , C h in N ephro l1982 ;17 :90-5 .28 . W ills W R . U rem ic to x ins an d th eir e ffect on in termed iaryme tab o l i sm . C lin C he m 19 85;31 :5 -13 .29 . Nav ar ro J, C on tre ras P , Tourain e JL , e t a l. A re m id d l em ole cu le s re sp on sib le for tox ic ph enom ena in ch ro n ic rena l fa il-u re ? N eph ro n 1982;32 :301-7 .30 . Z im me rm an L, FUrs t P, Bergstrom J, JO rn va ll H . A ne wglycine con tain ing compound w ith a b locked am in o gro up fromurem ic b ody flu ids. C lin N eph ro l 1980;14 : 107 - 11 .31 . Z im m e rm an L , JO nnva ll H , B ergstrom J, FUrs t P, SjO va ll J .Character izat ion of a doub le con jugate in u rem ic bo dy flu ids.G lu cu ro nid ate d o -h yd ro xy be nz oy lg ly cin e. FEBS Le t t 1981;129:237-40 .