{"id":2006,"date":"2020-05-05T18:45:33","date_gmt":"2020-05-05T16:45:33","guid":{"rendered":"https:\/\/www.newslab.sk\/nove-pristupy-k-diagnostike-lyzozomovych-ochoreni-na-baze-stanovenia-volnych-oligosacharidov-v-moci\/"},"modified":"2020-05-06T09:41:18","modified_gmt":"2020-05-06T07:41:18","slug":"new-approaches-in-the-diagnosis-of-lysosomal-storage-disorders-based-on-the-determination-of-free-oligosaccha-rides-in-urine","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/new-approaches-in-the-diagnosis-of-lysosomal-storage-disorders-based-on-the-determination-of-free-oligosaccha-rides-in-urine\/","title":{"rendered":"New approaches in the diagnosis of lysosomal storage disorders based on the determination of free oligosaccha- rides in urine"},"content":{"rendered":"

*<\/strong>All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.<\/strong><\/span><\/p>\n

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\u00davod<\/strong><\/p>\n

Napriek odhadovan\u00e9mu predpokladu, \u017ee zriedkav\u00fdmi ochoreniami trp\u00ed na Slovensku a\u017e 300 000 \u013eud\u00ed(1), molekulov\u00e1 podstata t\u00fdchto ochoren\u00ed je mnohokr\u00e1t unik\u00e1tna, a preto ich diagnostika vy\u017eaduje maxim\u00e1lne personalizovan\u00fd pr\u00edstup. Lyzoz\u00f3mov\u00e9 ochorenia (z angl. Lysosomal Storage Disorders \u2013 LSD) tvoria \u0161irok\u00fa skupinu zriedkav\u00fdch dedi\u010dn\u00fdch metabolick\u00fdch ochoren\u00ed, v r\u00e1mci ktorej ich rozde\u013eujeme na:<\/p>\n

A.) poruchy v \u0161tiepen\u00ed a degrad\u00e1cii glyk\u00e1nov a glykoprote\u00ednov (napr. alfa- a beta-manozid\u00f3za, sialid\u00f3za), glykolipidov (napr. GM1 gangliozid\u00f3za, Gaucherova a Fabryho choroba), glykozaminoglyk\u00e1nov (napr. MPS I-III) a mukopolysacharidov (napr. MPS IV), B.) poruchy v \u0161tiepen\u00ed a degrad\u00e1cii lipidov \u2013 sfingolipidov (napr. Niemann Pick typu A alebo B) a triacylglyceridov, C.) poruchy v degrad\u00e1cii prote\u00ednov a D.) poruchy lyzoz\u00f3mov\u00fdch transportn\u00fdch prote\u00ednov (napr. mukolipid\u00f3zy III a IV)(2).<\/p>\n

Jednotliv\u00e9 LSD s\u00fa sprev\u00e1dzan\u00e9 hromaden\u00edm nedegradovan\u00fdch produktov v tkaniv\u00e1ch a glykokonjug\u00e1tov v mo\u010di v d\u00f4sledku nedostato\u010dnej aktivity lyzoz\u00f3mov\u00fdch degrada\u010dn\u00fdch enz\u00fdmov (hydrol\u00e1z). Niektor\u00e9 z nich, napr. vo\u013en\u00e9 oligosacharidy obsiahnut\u00e9 v mo\u010di (z angl. free oligosaccharides \u2013 FOS), maj\u00fa charakteristick\u00fd profil, \u0161pecifick\u00fd pre dan\u00e9 ochorenie. FOS mo\u017eno detegova\u0165 r\u00fdchlou, presnou a neinvaz\u00edvnou diagnostickou met\u00f3dou \u2013 tandemovou hmotnostnou spektrometriou MALDI-TOF\/TOF, ktor\u00e1 presne definuje \u0161trukt\u00farne zlo\u017eenie nahromaden\u00fdch oligosacharidov v mo\u010di a predstavuje alternat\u00edvu tradi\u010dn\u00fdch skr\u00edningov\u00fdch techn\u00edk, ako je met\u00f3da tenkovrstvovej chromatografie (TLC), ktorej identifik\u00e1cia metabolitov spo\u010d\u00edva v porovn\u00e1van\u00ed pohyblivosti FOS nach\u00e1dzaj\u00facich sa vo vzorke so \u0161tandardnou zmesou oligosacharidov s 1 \u2013 9 hex\u00f3zov\u00fdmi jednotkami. T\u00e1to met\u00f3da je vhodn\u00fdm prvotn\u00fdm skr\u00edningov\u00fdm pr\u00edstupom, v pr\u00edpade hrani\u010dn\u00e9ho, resp. pozit\u00edvneho v\u00fdsledku je v\u0161ak \u010fal\u0161ia charak- teriz\u00e1cia pozorovan\u00e9ho profilu esenci\u00e1lna. Na rozl\u00ed\u0161enie, \u010di kumulovan\u00e9 FOS poch\u00e1dzaj\u00fa zo stravy alebo s\u00fa tvoren\u00e9 patologick\u00fdmi, \u0161pecifick\u00fdmi metabolitmi, predstavuje vhodn\u00e9 vyu\u017eitie vysokorobustn\u00e1 MALDI TOF\/TOF anal\u00fdza.<\/p>\n

LSD ochorenia s\u00fa typick\u00e9 multisyst\u00e9mov\u00fdmi klinick\u00fdmi pr\u00edznakmi s trvalou progresiou v d\u00f4sledku neust\u00e1leho hromadenia nedegradovan\u00e9ho substr\u00e1tu. Niektor\u00e9 pr\u00edznaky sa m\u00f4\u017eu spo\u010diatku javi\u0165 ako nev\u00fdznamn\u00e9, resp. typick\u00e9 pre in\u00fa \u010di be\u017enej\u0161iu chorobu (napr\u00edklad vracanie, hypoglyk\u00e9mia, hypot\u00f3nia, letargia, k\u0155\u010de, hluchota, v\u0161eobecn\u00e9 neprospievanie, psychomotorick\u00e1 retard\u00e1cia) a m\u00f4\u017eu sa prejavi\u0165 v akomko\u013evek veku, od fet\u00e1lneho obdobia a\u017e po senium. Kombinovan\u00e1 prevalencia LSD predstavuje a\u017e 1 : 5 000 \u017eivonaroden\u00fdch(3), a preto je ich v\u010dasn\u00e1 a spo\u013eahliv\u00e1 diagnostika esenci\u00e1lnym faktorom ovplyv\u0148uj\u00facim \u017eivot pacientov a ich rod\u00edn. Oddelenie glykobiol\u00f3gie Chemick\u00e9ho \u00fastavu SAV\u00a0 v spolupr\u00e1ci s Centrom dedi\u010dn\u00fdch metabolick\u00fdch por\u00fach N\u00daDCH v Bratislave zaviedlo a optimalizovalo analytick\u00fa met\u00f3du na stanovenie profilov vo\u013en\u00fdch oligosacharidov v mo\u010di, ktor\u00e1 v porovnan\u00ed s tradi\u010dn\u00fdmi konven\u010dn\u00fdmi met\u00f3dami disponuje vysokou presnos\u0165ou, \u0161pecificitou, citlivos\u0165ou a m\u00f4\u017ee by\u0165 pou\u017eit\u00e1 okrem prvotn\u00e9ho diagnostick\u00e9ho pr\u00edstupu aj v pr\u00edpade monitorovania \u00fa\u010dinnosti terapie.<\/p>\n

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Materi\u00e1l a met\u00f3da<\/strong><\/p>\n

Vzorky mo\u010du odobrat\u00e9 od pacientov s r\u00f4znymi lyzoz\u00f3mov\u00fdmi ochoreniami (beta-manozid\u00f3za, alfa-manozid\u00f3za, r\u00f4zne typy mukopolysacharid\u00f3z, mukolipid\u00f3za, gangliozid\u00f3za I. a II. typu, sialid\u00f3za) a glykogen\u00f3zou II. typu (Pompeho choroba), resp. od zdrav\u00fdch fyziologick\u00fdch jedincov ako negat\u00edvnych kontrol, boli uchov\u00e1van\u00e9 pri \u201320 \u00b0C a\u017e do ich spracovania. Rieden\u00fd mo\u010d bol po odstr\u00e1nen\u00ed nerozpustn\u00e9ho sedimentu lyofilizovan\u00fd a takto vysu\u0161en\u00e9 vzorky sa derivatizovali permetyl\u00e1ciou (PerMe) s j\u00f3dmet\u00e1nom v z\u00e1saditom prostred\u00ed NaOH, ktor\u00e9ho nadbytok bol n\u00e1sledne odstr\u00e1nen\u00fd extrakciou s chloroformom. Pripraven\u00e9 PerMe FOS sa po vysu\u0161en\u00ed rekon\u0161tituovali do 50 % metanolu a v pomere 1 : 1 s matricou 2,5-dihydroxybenzoovou kyselinou sa naniesli na MALDI platni\u010dku. Anal\u00fdza PerMe FOS sa uskuto\u010dnila na tandemovom hmotnostnom spektrometri (UltrafleXtreme MALDI TOF\/TOF, Bruker Daltonics, MA, USA) v reflektr\u00f3novom pozit\u00edvnom i\u00f3novom m\u00f3de. Postanalytick\u00e1 interpret\u00e1cia nameran\u00fdch spektier bola spracovan\u00e1 softv\u00e9rom GlycoWorkbench a ProteinScape 3. 0.<\/p>\n

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V\u00fdsledky<\/h2>\n

Anal\u00fdzou permetylovan\u00fdch FOS boli stanoven\u00e9 charakteristick\u00e9 mo\u010dov\u00e9 profily pre 7 vybran\u00fdch LSD. V spektr\u00e1ch FOS kontroln\u00fdch vzoriek (zdrav\u00e9 kontroly) bolo pozorovan\u00fdch nieko\u013eko \u0161tandardn\u00fdch vo\u013en\u00fdch oligosacharidov, ktor\u00e9 s\u00fa pr\u00edtomn\u00e9 medzi v\u0161etk\u00fdmi vekov\u00fdmi skupinami, najm\u00e4 m\/z 838,40, 879,43, 1 240,61 a 1 274,62. V spektr\u00e1ch novorodencov na materskom mlieku sa potvrdili \u0161pecifick\u00e9 FOS poch\u00e1dzaj\u00face z matersk\u00e9ho mlieka. Z\u00edskan\u00e9 v\u00fdsledky anal\u00fdz profilov FOS s\u00fa v zhode s d\u00e1tami publikovan\u00fdmi v litera- t\u00fare(4,5).<\/p>\n

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FOS reprezentat\u00edvnych profilov LSD (obr\u00e1zok 1)<\/em><\/strong>, z\u00edskan\u00e9 anal\u00fdzou MALDI TOF\/TOF permetylovan\u00fdch oligosacharidov obsiahnut\u00fdch v mo\u010di pacientov, predstavuj\u00fa typick\u00e9 zlo\u017eenie oligosacharidov vypl\u00fdvaj\u00face z nefunk\u010dnosti alebo ni\u017e\u0161ej aktivity \u0161pecifick\u00fdch lyzoz\u00f3mov\u00fdch hydrol\u00e1z:<\/p>\n