Xanthine Oxidoreductase Kev Ua Haujlwm hauv Plateletpoor Thiab Rich Plasma Raws Li Qhov Ntsuas Oxidative Stress Indicators Hauv Cov Neeg Mob Yuav Tsum Tau Kho Lub Raum Hloov Kho

Tom ntej: Xanthine oxidoreductase(XOR) yog hydroxylase enzyme koom nrog hauv cov metabolism ntawm purines. Kev ua XOR tuaj yeem sib txawv: homodimer protein tuaj yeem hloov mus rau hauv ob hom isoforms XD (antioxidant) thiab XO (prooxidant). Oxidative stress thiab infammation uas nrogmob raum mob(CKD), lim ntshav, thiab hloov lub raum, ua rau platelet activation. Txoj kev tshawb no tsom los txiav txim siab qhov cuam tshuam ntawm kev siv lub raum hloov kho rau xanthine oxidoreductase thiab nws cov haujlwm isoform.

Cov ntaub ntawv thiab cov txheej txheem: Cov pab pawg tshawb fawb muaj 117 tus neeg mob, muab faib ua 4 pawg: hemodialysis - 30 cov neeg mob, peritoneal dialysis - 30 cov neeg mob, cov neeg mob hloov lub raum - 27, thiab kev kho kom zoo {{5} } cov neeg mob. Pawg tswj hwm muaj 30 tus neeg ua haujlwm noj qab haus huv. Cov txiaj ntsig: Qhov sib txawv tseem ceeb tau pom hauv XOR kev ua hauv platelet-poor plasma (PPP) hauv cov pab pawg kawm (p=0.001). Muaj kev sib raug zoo ntawm hom kev kho lub raum hloov ntawm tag nrho cov oxidoreductase isoforms hauv PPP (p<0.001 all isoforms) and XD (p=0.008), XO (p<0.001) in platelet-rich plasma (PRP). A relationship was observed between the activity of all oxidoreductase isoforms in PPP and PRP, the type of renal replacement therapy the duration of dialysis, and the age of patients. Theua rau mob raum mobkuj tau tshwm sim hauv qhov sib txawv hauv XD thiab XO kev ua hauv PPP.

Cov ntsiab lus: Hom kev hloov lub raum siv rau hauv cov neeg mob CKD, hnub nyoog ntawm cov neeg mob, lub sijhawm ntawm kev lim ntshav, CKD ua rau, thiab theem ntawm kev loj hlob cuam tshuam rau kev ua haujlwm ntawm XOR thiab nws cov isoforms.

Ntsiab lus: Xanthine oxidoreductase, Platelets, Lub raum hloov kho,Mob raum mob, Antioxidant enzymes

Nws yuav siv sij hawm ntev npaum li cas rau CISTANCHE ua haujlwm?

Keeb kwm

Xanthine oxidoreductase (XOR) yog hydroxylase enzyme koom nrog hauv cov metabolism ntawm purines. Nws catalyzes oxidation ntawm hypoxanthine rau xanthine thiab xanthine rau uric acid (UA). Cov haujlwm XOR tuaj yeem sib txawv: homodimer protein tuaj yeem hloov mus rau hauv ob qhov sib txawv isoforms. Xanthine dehydrogenase (XD) tau nthuav tawm feem ntau hauv cov ntaub so ntswg noj qab haus huv, thiab xanthine oxidase (XO) yog tsim los ntawm kev hloov pauv tom qab XD, oxidation cysteine ​​residues nrog rau kev txwv tsis pub muaj protein ntau, ua lub luag haujlwm tseem ceeb hauv cov hlwb thiab cov ntaub so ntswg thaum raug mob [1-3. ]. Cov dej num ntawm isoforms hais tawm tsam ib leeg [4]. XOR ua nyob rau hauv lub xub ntiag ntawm NAD + raws li ib tug dehydrogenase, thiab nrog molecular oxygen raws li ib tug oxidase. Lub peev xwm ntawm XOR kom hloov pauv sai ntawm antioxidant rau oxidant, nyob rau hauv ntau hom kev puas tsuaj ntawm cov ntaub so ntswg, yog ib qho tseem ceeb rau lub cev tiv thaiv kab mob sai sai, muaj txiaj ntsig zoo, piv txwv li, kab mob los yog kab mob fungal [5].

Cov tshuaj tiv thaiv nruab nrab ntawm xanthine dehydrogenase (XDO) thiab cov pa oxygen tuaj yeem cuam tshuam nrog ob qho tib si NAD + thiab O2 tab sis nthuav tawm ntau dua affinity rau NAD + [6]. Qhov nruab nrab isoform no tsis tau raug cais tawm, tab sis qhov kev txiav txim siab ntawm nws cov haujlwm ua kom yooj yim rau kev taug qab ntawm kev hloov pauv ntawm XD rau XO [7].

XOR cov dej num hauv ntau yam kab mob tau raug tshawb xyuas dav. Yog vim li cas rau qhov kev txaus siab nyob rau hauv no enzyme yog lub dualism ntawm nws cov kev ua: lub peev xwm los tsim antioxidants, thiab ntawm qhov tod tes, reactive oxygen hom creation. Kev nce hauv XOR kev ua haujlwm tshwm sim hauv cov kab mob xws li kab mob siab kab mob siab, kis kab mob mononucleosis, kab mob autoimmune, mob ntsws, schizophrenia, thiab ntshav qab zib hom II. Qhov nce hauv XOR kev ua haujlwm kuj tseem pom nyob rau hauv cov ntshav ntawm cov neeg mob tom qab lub raum lossis lub siab hloov pauv [8]. Nws tau pom tias cov noob coding rau XOR tuaj yeem ua lub luag haujlwm rau lub raum maturation, thiab adipogenesis hauv ob lub raum, thiab tuaj yeem tiv thaiv kev hloov pauv ntawm cov hlwb epithelial rau hauv cov ntaub so ntswg mesenchymal [9]. Txawm li cas los xij, vim peb qhov kev paub zoo tshaj plaws, tsis muaj cov ntaub ntawv tshaj tawm uas tau tshawb xyuas XOR kev ua hauv platelet-poor plasma (PPP) thiab platelet-nplua nuj plasma (PRP) hauv cov neeg mob uas tau kho lub raum hloov. Qhov tseem ceeb ntawm kev ua XOR hauv pawg neeg mob no yog cuam tshuam nrog kev ua kom platelet nce ntxiv, uas yog tshwm sim los ntawm oxidative stress thiab infammation uas nrog rau mob raum (CKD), lim ntshav, thiab hloov lub raum. Tsis tas li ntawd, thaum lub sij hawm lim ntshav thiab hloov khoom nruab nrog cev, cov ntaub so ntswg thiab cov hlab ntsha puas lawm, thiab cov platelets yog cov frst hlwb kom ncav cuag qhov chaw ntawm cov ntaub so ntswg puas tsuaj, koom tes rau thawj theem ntawm cov txheej txheem inflammatory thiab kho [10].

PRP thiab PPP yog cov feem ntawm cov ntshav plasma nrog cov platelet sib txawv. Cov ntsiab lus platelet ntawm PRP thiab PPP yog platelets / ml thiab platelets / ml, feem. PPP thiab PRP tau txais los ntawm kev rov centrifuging thiab ntxuav tag nrho cov ntshav ntawm tib neeg ntawm qhov sib txawv centrifugal ceev [11, 12].

PPP, raws li ib tug centrifugation byproduct ntawm anticoagulated ntshav, muaj ib tug qis platelet concentration tshaj li cov ntshav ib txwm. Cov khoom tseem ceeb ntawm PPP yog fibrinogen, fibronectin, thiab thrombin. Cov teebmeem lom ntawm PPP yog koom nrog hauv hemostasis thiab coagulation, ua raws li cov cell txuas vector, thiab txhawb nqa mitosis ntawm fibroblasts thiab epithelial hlwb [13]. Txawm hais tias PPP tsis muaj zog hauv platelets li PRP, nws tau pom tias PPP tuaj yeem txhawb nqa kev loj hlob ntawm tes thiab muaj sia nyob. PPP txhawb kev kho qhov txhab-txuas nrog cell ua haujlwm thiab ua kom cov cell tsiv teb tsaws thiab kev loj hlob ntawm fibroblasts [14, 15]. Platelet-nplua nuj plasma muaj ntau cov platelets ntau dua li cov ntshav plasma (kwv yees li 150–400 × 103 hlwb / dL). Nws yog ib qho ntawm feem ntau txhais ntawm PRP hauv cov ntaub ntawv [16]. Txog niaj hnub no, cov kev tshawb fawb txwv tsis pub muaj XOR thiab nws cov haujlwm isoform hauv platelet-nplua nuj lossis cov ntshav tsis zoo hauv cov neeg mob uas mob raum mob. Tan et al. pom cov hlwb puas los ntawm reactive oxygen hom (ROS) (nyob rau hauv cov ntaub ntawv ntawm lub raum mob hloov kho) "kua" XOR isoforms, ua rau kom nce qib enzyme hauv ntshav [17]. Cov txheej txheem no piav qhia txog kev ua haujlwm qis ntawm XOR thiab nws cov isoforms hauv platelets piv rau PPP. Yog li, oxidoreductases activincity hauv PPP thiab PRP yog qhov txaus siab heev, vim tias lawv tuaj yeem pab nthuav tawm cov txheej txheem ntawm tes uas tshwm sim txhua hnub, sis. Cov kev tshawb fawb zoo li no kuj tseem tuaj yeem hais qhia ncaj qha txog qhov hnyav ntawm oxidative kev nyuaj siab hauv pawg neeg mob no.

Berry et al. piav qhia ntxiv tias xanthine oxidoreductase tau faib rau hauv daim siab, cov hnyuv me, cov qog mammary, thiab cov hlwb endothelial. Cov txheej txheem subcellular localization tau pom tias muaj xanthine oxidoreductase ob qho tib si hauv cytoplasm thiab ntawm cov cell membranes. Lawv kuj qhia tau hais tias cov ntshav xanthine oxidasereductase tuaj yeem yog vim xanthine oxidoreductase shading los ntawm cell membranes los yog xau los ntawm cytoplasm. Qhov no yog ib qho ntawm cov laj thawj uas peb sim cov haujlwm ntawm XOR hauv PPP thiab PRP kom paub qhov txawv ntawm cov enzyme kev ua hauv platelets thiab lwm cov qe ntshav uas muaj nyob hauv cov ntshav [18]. Raws li kev ua haujlwm ntawm cov enzymes antioxidant, peb tuaj yeem txiav txim siab seb hom kev kho lub raum hloov pauv tsawg dua yuav ua rau tus neeg mob muaj kev ntxhov siab oxidative. Vim yog ob qho xwm txheej ntawm XOR, kev nkag siab ntawm kev sib raug zoo ntawm hom kev kho lub raum hloov thiab kev ua haujlwm ntawm XOR isoforms tuaj yeem nthuav dav thiab pab tau zoo hauv kev xaiv hom kev kho raum hloov.

Cov ntaub ntawv thiab cov txheej txheem

Kev pom zoo thiab kev tso cai ncaj ncees

Lub Bioethical Commission ntawm Pomeranian Medical University hauv Szczecin tau pom zoo cov kev tshawb fawb ua tiav (tsis muaj KB-0012/36/11). Txhua tus neeg koom nrog, suav nrog cov neeg ua haujlwm pab dawb noj qab haus huv hauv pawg tswj hwm, tau raug ceeb toom txog lub hom phiaj thiab kev ua haujlwm ntawm txoj kev tshawb fawb thiab tau tso cai pub dawb rau cov qauv thiab tshaj tawm cov ntaub ntawv tshwm sim.

Pab pawg kawm

Muaj 147 tus neeg koom nrog: pawg tswj hwm ntawm 30 tus neeg ua haujlwm noj qab haus huv (NK), thiab 117 tus neeg mob uas muaj kab mob raum tsis zoo (CKD) tuaj koom Nephrology, Transplanetology, thiab Internal Diseases Clinic ntawm Pomeranian Medical University hauv Szczecin. Te cov neeg mob tau muab faib ua 4 pawg raws li kev kho mob uas lawv tau txais: 30 cov neeg mob ua ntej thiab tom qab hemodialysis (HD A thiab HD B): 30 cov neeg mob tau txais peritoneal dialysis (PD); 27 cov neeg mob ua ntej thiab tom qab hloov lub raum (5-7 hnub tom qab kev phais) (TE, TE A); 30 cov neeg mob tau txais kev kho mob (CT) (CKD theem 2–5). Cov poj niam txiv neej, hnub nyoog, lub sijhawm ntawm kev lim ntshav, ua rau thiab theem ntawm cov kab mob hauv lub raum ntev, thiab creatinine concentration hauv kev kuaj thiab tswj pawg tau muab rau hauv Cov Lus 1 thiab 2.

Cov qauv

Cov ntshav kuaj (K2EDTA (8 ml), 3.8% trisodium citrate (9: 1; v / v), thiab ntshav (8 ml)) tau kos los ntawm txhua tus neeg koom nrog kev kawm. Hemodialyzed cov neeg mob ntshav tau kos los ntawm lawv cov fistula arteriovenous; peripheral venipuncture tau siv rau txhua tus neeg koom nrog. Cov qauv coj los ntawm cov neeg mob hemodialysis ua ntej (HD A) thiab li 10 feeb tom qab lub twj tso kua mis nres (HD B). Hloov cov ntshav cov neeg mob tau sau ua ntej hloov pauv (TE) thiab 5-7 hnub tom qab kev phais (TE A). Cov neeg mob uas raug xaiv rau pawg TE tsis koom nrog pawg ntawm hemodialysis lossis peritoneal dialysis cov neeg mob hauv txoj kev tshawb no. Lawv yog cov neeg mob tsim nyog rau kev hloov pauv los ntawm thoob plaws tebchaws Poland. Feem ntau ntawm cov neeg mob uas hloov lub raum mus rau lub raum tau ua ntej modeanalysis. K2EDTA thiab cov ntshav txhaws tau centrifuged ntawm 2600 rpm rau 10 min ntawm 20 degree kom tau cov ntshav thiab ntshav, raws li. Kom tau txais platelet-nplua nuj plasma (PRP) thiab platelet-poor plasma (PPP), cov ntshav sau nrog citrate tau centrifuged nyob rau hauv cov xwm txheej ntawm 1100 rpm rau 10 min ntawm 20 degree. Qhov tshwm sim PRP raug xa mus rau lub raj tshiab thiab centrifuged ntawm 6000 rpm rau 10 min ntawm 20 degree: platelet-plasma tsis zoo (PPP) tau pauv mus rau ib lub raj cais; cov platelet pellet tau yaug ob zaug thiab muab tshem tawm hauv Tyroda tsis kam (pH7.4). Plasma, ntshav, PPP, thiab PRP tau khov ntawm −80 degree kom txog thaum qhov kev ntsuam xyuas tau ua tiav. Hauv cov neeg mob hemodialysis, cov ntshav tau sau ua ntej kev tswj hwm heparin thiab tom qab kev lim ntshav ntev li 4-5 teev ntawm qhov nruab nrab (heparin ib nrab-lub neej - 4 h) txhawm rau tshem tawm cov kev cuam tshuam ntawm heparin ntawm XOR kev ua haujlwm.

Xanthine oxidoreductase kev ua haujlwm hauv platelet-poor plasma thiab platelets

Kev txiav txim siab tau ua nrog Perkin Elmer UV / VIS Lambda 40P spectrophotometer. Kev hloov pauv tau raug kaw ntawm 340nm (XD) thiab 302nm (XDO, XO) rau 5min ntawm 30 degree. Cov kev ua enzyme tau ntsuas raws li kev tsim ntawm uric acid thiab NADH (nce hauv A340 thiab A302) thiab qhia hauv mU × mL−1 (milliunits ib milliliter). Kev ua haujlwm enzymatic tau suav nrog, suav nrog tus nqi pib ntawm cov tshuaj tiv thaiv. Kev tsim cov uric acid tau ntsuas ntawm 302nm (isoforms XDO thiab XO) vim tias nws cov absorbance tseem siab nyob ntawd, qhov kev hloov hauv NAD + concentration tsis pab. Thee extinction coefficient rau NADH + H + ε340=6.22 × 103 [L∙mol−1 cm−1] tau siv los xam cov kev ua ntawm isoforms ntawm xanthine oxidoreductase NADH + H +: ε302=2.30 × 103 [ L∙mol−1 cm–1] [7, 19–22].

Table 1 Cov yam ntxwv ntawm cov neeg mob hemodialysis (HD), peritoneal dialysis (PD) kho conservatively (CKD), raum hloov (TE), thiab tswj pawg (C) koom nrog hauv txoj kev tshawb no (txhais tau tias ± SD)

P * - qhov tseem ceeb ntawm qhov sib txawv ntawm HD, PD thiab CKD pawg, TE thiab C pes tsawg Fisher xeem rau qhov zoo sib xws; rau quantitative variables - ib-txoj kev ANOVA thiab; P ** - qhov tseem ceeb ntawm cov ntaub ntawv sib txawv ntawm HD, PD thiab CKD pawg thiab TE pes tsawg Fisher test rau qhov zoo sib xws rau qhov sib txawv ntawm qhov sib txawv ntawm ib-txoj kev ANOVA lossis; DM - mob ntshav qab zib nephropathy; HA - kub siab; GIK - glomerular infammation raum; ADPKD - polycystic raum kab mob tau txais autosomal dominant; NS - tsis muaj qhov sib txawv ntawm qhov tseem ceeb.

Table 2 Cov yam ntxwv ntawm cov neeg mob hemodialysis (B - ua ntej HD, A - tom qab), peritoneal dialysis (PD) kho conservatively (CKD) ua ntej thiab tom qab hloov lub raum (TE B thiab TE A) thiab pawg tswj hwm (NK) koom nrog hauv kawm (txhais tau tias ± SD)

P * - qhov tseem ceeb ntawm qhov sib txawv ntawm HD A, HD B, PD thiab CKD pawg, TE thiab C rau ntau qhov sib txawv - Kruskal Wallis ANOVA, one-way ANOVA lossis Student's t-test P ** - statistical significance for differents between HD A , HD B, PD thiab CKD thiab TE pab pawg rau Kruskal Wallis's ANOVA quantitative variables lossis ANOVA one-way analysis Kt/V - dialysis index (volume fraction V purified by clearance K at time t) NS tsis muaj kev sib raug zoo ntawm cov ntaub ntawv.

Kev txheeb xyuas kev txheeb xyuas txhawm rau txheeb xyuas kev faib tawm, KS xeem (Kolmogorov-Smirnov) tau siv, uas nyob rau hauv rooj plaub ntawm qee qhov kev hloov pauv (kev ua haujlwm ntawm XD thiab XDO isoforms hauv PRP) tau pom qhov tsis zoo ntawm qhov tsis sib xws. Exact Fisher thiab Chi-square xeem tau siv los txheeb xyuas cov ntaub ntawv ntau. Siv Student's t-test and ANOVA analysis for univariate systems, qhov sib txawv ntawm qhov sib txuam (paired) thiab tsis sib xws (unpaired) variables raug soj ntsuam nyob rau hauv cov ntaub ntawv ntawm cov variables nrog ib tug ib txwm faib. Nyob rau hauv cov ntaub ntawv ntawm cov variables uas tsis yog-ib txwm faib, Kruskal-Wallis ANOVA tsom xam tau ua los soj ntsuam qhov sib txawv ntawm cov tsis, nrog rau cov Mann-Whitney U nonparametric xeem rau unpaired cov ntaub ntawv los yog Wilcoxon rau cov ntaub ntawv ua ke. Ib tug linear ntau regression qauv tau siv los txiav txim qhov multifactor kev ntsuam xyuas ntawm kev sib raug zoo ntawm cov tsis tau kawm. Kev txheeb xyuas cov txiaj ntsig tau ua tiav siv Statistica 12 (StatSoft).