Ugotavljanje vpliva S-adenozilmetionina na aktivnost tiopurin-S-metiltransferaze v eritrocitih zdravih preiskovancev : diplomska naloga

Lorbek, Gregor (author), Karas Kuželički, Nataša (mentor), Milek, Miha (co-mentor)

Genetski polimorfizmi encima tiopurin-S-metiltransferaze (TPMT) imajo velik vpliv na toksičnost tiopurinskih učinkovin. Posamezniki, ki so homozigotni ali heterozigotni za mutirani alel, imajo znižano aktivnost TPMT, zato posledično pride do manjše deaktivacije tiopurinov, kar lahko vodi v pojav življenjsko nevarnih neželenih učinkov. Genotipizacija mutiranih alelov TPMT predstavlja razmeroma enostaven in hiter farmakogenetski test, na osnovi katerega lahko posamezniku določimo optimalno terapijo. Uporabo takšnega testiranja v klinični praksi omejuje dejstvo, da ujemanje med genotipom in fenotipom TPMT ni popolno. Na modelnih celičnih linijah poleg genotipa TPMT na encimsko aktivnost vpliva tudi koncentracija S-adenozilmetionina (SAM), ki je kosubstrat za TPMT, obenem pa stabilizira nativno strukturo proteina. V tej raziskavi smo v eritrocitih zdravih preiskovancev z metodo tekočinske kromatografije visoke ločljivosti izmerili koncentracijo SAM in določili aktivnost TPMT. V procesu določanja aktivnosti TPMT smo sprva v kromatogramih naleteli na pojav neznane spojine, ki je onemogočala kvantifikacijo produktov encimske reakcije, vendar smo metodo uspešno optimizirali. S statistično metodo multiple linearne regresije smo dokazali vpliv SAM na aktivnost TPMT v eritrocitih. Posamezniki z nižjo koncentracijo SAM so imeli nižjo aktivnost TPMT, medtem ko so imeli tisti z višjo koncentracijo SAM tudi višjo aktivnost TPMT. Domnevamo, da gre za posttranslacijsko uravnavanje aktivnosti preko vezave SAM in posledične stabilizacije TPMT. Vpliv SAM je bil majhen (β = 0,119), vendar statistično značilen (p = 0,026). Ujemanje med genotipom in aktivnostjo TPMT je bilo 91,8-odstotno. Nekoliko manjše je bilo v skupini posameznikov s srednjo aktivnostjo (80-odstotno) v primerjavi s posamezniki z visoko aktivnostjo (97,2-odstotno). Neskladnosti med genotipom in fenotipom TPMT nismo uspeli pojasniti z razlikami v koncentracijah SAM, najverjetneje zaradi majhnega števila preiskovancev v skupinah z neujemanjem med genotipom in aktivnostjo TPMT. Naša raziskava nakazuje, da bi bil lahko SAM oziroma polimorfizmi v genih, ki so vpleteni v njegovo biosintezo, potencialni novi biološki označevalci pri individualizaciji terapije s tiopurini.

• Language: Slovenian
• Keywords: tiopurini tiopurin-S-metiltransferaza S-adenozilmetionin merjenje koncentracij RP-HPLC
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [G. Lorbek]
• Year: 2010
• Number of pages: VIII, 67 f.
• PID: 20.500.12556/RUL-121337
• UDC: 61
• COBISS.SI-ID: 2812273
• Publication date in RUL: 05.10.2020

Secondary language

• Language: English
• Title: The influence of S-adenosylmethionine on the activity of thiopurine-S-methyltransferase in erythrocytes of healthy individuals

Abstract

The genetic polymorphisms of thiopurine-S-methyltransferase (TPMT) have a great impact on the toxicity of thiopurine drugs. Homozygous or heterozygous individuals for mutant alleles exhibit decreased TPMT activity, consequently leading to lower deactivation of thiopurines, which can cause life threatening side effects. Genotyping analysis of TPMT mutant alleles represents a relatively simple and rapid pharmacogenetic test for the determination of an optimal dose for a specific patient. Clinical application of TPMT genotyping is somewhat limited by the fact that the TPMT genotype to phenotype correlation is incomplete. Apart from the impact of genotype on TPMT activity, S-adenosylmethionine (SAM) has been reported to positively correlate with TPMT activity in cancer cell lines. SAM is a cosubstrate for TPMT but also stabilizes the native structure of the protein. In the present study, we measured the concentration of SAM and TPMT activity in erythrocytes of healthy individuals using high-performance liquid chromatography. Formation of an unknown compound was observed in the obtained chromatograms, which obstructed the quantification of the TPMT reaction specific methylated product, but the method was optimized successfully. Using multiple linear regression we determined the influence of SAM on the erythrocyte TPMT activity. The individuals with lower concentrations of SAM had lower TPMT activity compared to individuals with higher concentrations of SAM that had higher TPMT activity. This is most probably due to posttranslational regulation of TPMT activity by the binding of SAM and consequent stabilization of TPMT. The effect of SAM was relatively modest (β = 0.119) but nevertheless significant (p = 0.026). TPMT genotype to phenotype correlation was 91.8% with lower concordance in the group with intermediate activity (80%) compared to the group with high activity (97.2%). The rate of discordance could not be ascribed to differences in the concentrations of SAM, probably due to small number of individuals in genotype to phenotype discordance groups. Our study indicates the potential application of SAM or polymorphisms of enzymes, participating in its biosynthesis, as potential novel biomarkers in the individualization of thiopurine therapy.

• Keywords: Thiopurine-S-methyltransferase S-adenosylmethionine genotype to phenotype correlation multiple linear regression high-performance liquid chromatography