En människas metabolom består av en rad olika småmolekyler, s.k.
metaboliter. Exempel på metaboliter kan vara aminosyror, vitaminer och sockerarter. Sammansättningen av en människas metabolom påverkas dels av det genetiska arvet, men också av allt en kropp kan tänkas utsättas för såsom mat, dryck, gifter och sjukdomar. Genom att studera förändringar i metabolomet, s.k. metabolomik, kan man få en uppfattning om vad som försiggår i kroppen. Informationen från en metabolomikstudie kan således användas för att till exempel försöka förstå mekanismen bakom en sjukdom eller för att få en inblick i hur en viss kost påverkar kroppen. För att öka chansen att upptäcka en eventuell förändring i metabolomet har de flesta mätmetoder inom metabolomik utvecklats för att kunna bestämma så många metaboliter som möjligt. Tyvärr har denna prioritering oftast gjorts utan reflektion över vilken kvalité varje enskild metabolit mäts med.
Selektivitet är en parameter som är av stor vikt när det kommer till mätkvalitet.
Selektivitet skulle kunna liknas vid ett mått på andelen störande ämnen (interferenser) vid bestämning av en metabolit. Trots denna viktiga aspekt negligeras oftast selektivitet inom metabolomikforskning. Problematiken med att åsidosätta selektivitet skulle kunna liknas vid att man var ute efter att väga en gammelgädda i en sjö, men att man inte reflekterar över att man vid invägningen av gäddan samtidigt har en brax eller mört på vågen. Denna avhandling avser att belysa betydelsen av selektivitet samt att utveckla en metod för att kunna mäta selektivitet i metabolomik.
För att minimera effekten av interferenser på mätningen av metaboliter är det brukligt att prover förbehandlas innan själva mätningen utförs. Eftersom de flesta metabolomikmetoder har utvecklats för att detektera så många metaboliter som möjligt används oftast väldigt generella förbehandlingar av provet. Det leder till att en förhållandevis stor andel av interferenser är kvar i provet vid mätning. I avhandlingen studeras flera olika förberedelsertekniker och dess effekt på selektivitet. Genomgående upptäcktes en rad interferenser störa bestämningen av metaboliter som ett resultat av en alltför dålig selektivitet. Denna effekt kan ha stor betydelse i en metabolomikstudie då en observerad mätförändring hos en molekyl de facto kan vara en sekundär effekt av en förändring i en interferens. Till exempel visade det sig att salthalten i proverna hade stor inverkan på mätningarna och effekten har visats kunna ge
en upp till 30 ggr förändring i respons hos metaboliter. Detta kan i en metabolomikstudie vara förödande då det sedan länge är känt att salthalten i t.ex. urin tenderar att fluktuera både mellan individer och tid på dygnet.
En metabolomikstudie består av en rad steg där följande oftast är inkluderade:
generering av prover, provupparbetning, mätning, dataanalys och tolkning av resultat. Varje steg i flödet kan utföras på olika sätt, vilket i sin tur kommer att ha mer eller mindre påverkan på metabolitpresentationen av metabolomet.
Därför behövs det bra tillvägagångsätt för att kunna utvärdera olika metoder för varje enskilt steg. I avhandlingen presenteras ett nytt och unikt tillvägagångsätt för att bestämma selektivitet i metabolomikstudier.
Tillvägagångsättet går ut på att det för varje enskild metabolit beräknas en kvot som anger hur stor andel den aktuella metabolitens respons utgör av den totala responsen vid just den mätningen (dvs. en fisks viktsandel av den totala vikten på vågen). Ett värde på 1 betyder hög selektivitet då det bara är en metabolit som mäts vid just den tidpunkten. Vid ett värde nära 0 så gäller det omvända och metaboliten i fråga mäts uppenbarligen med en väldigt låg selektivitet. Detta värde skulle därmed kunna liknas vid ett mått på hur hög kvalitet varje enskild metabolit kan mätas med. Tillvägagångsättet kan därför vara användbart vid jämförelser mellan metoder och hjälpa till att välja den mest lämpliga.
Eftersom varje metabolomikstudie oftast är unik kan man anta att interferenserna i stor utsträckning också är unika. Det är därför författarens uppfattning att generiska metoder bör undvikas och varje steg i metabolomikflödet bör optimeras med avseende på nyckelmetaboliter och förväntade interferenser i det studerade provet. Selektivitet bör betraktas som en viktig parameter i metabolomikstudier eftersom dess länk till mätkvalitet är tydligare än när man t.ex. mäter antalet detekterbara metaboliter. En allmänt förbättrad selektivitet inom metabolomikfältet bör leda till ökad kvalitet inom fältet. Detta skulle kunna leda till att färre falska positiva/negativa resultat genereras vilket i sin tur skulle spara både tid och resurser.
Acknowledgments
The work of this thesis was carried out at the Division of Analytical Pharmaceutical Chemistry at Uppsala University, Sweden. I would like to express my sincere gratitude and appreciation to the following persons, I wouldn’t have managed without you;
First of all I would like to thank my main supervisor prof. Curt Pettersson for recruiting and guiding me throughout these years. I am very grateful for your straightforward manner and for always having time for even the smallest things. Even though I’ve been part of teams throughout my entire life, it is not until now I understand the true concept of being part of a team. I would also like to thank prof. Torbjörn Arvidsson, my co-supervisor, for always staying positive and for always trying to see things from another perspective.
All former and present colleagues:
Mikael Engskog and Jakob Haglöf, thanks for all the stimulating discussions. It has been a real pleasure and a privilege being part of the same research group for more than 6 years. Ylva Hedeland, thank you for all advices, both technology and teaching related; I wouldn’t have managed without them. Mikael Hedeland, thank you for being a role model when it comes to critical thinking, a true inspiration source. The present members of the A-team; Alfred and Annelie, thanks for the friendship during these years, it wouldn’t have been the same without you guys. I have truly valued our relaxed discussions, both the scientific and the non-scientific ones, and I’m really looking forward to our next trip. The former members of the A-team;
Alexander and Axel, thanks for climbing the ‘teaching ladder’ together. I still remember the first course we supervised together, it was insane. Victoria, thanks for being an inspiration source when it comes to orderliness and, even though I try, I will never reach your caliber. Olle, thanks for all tips and chats over these years, I have enjoyed them all. Ida, thanks for valid tips during our group meetings and special thanks for not participating in the ‘Blodomloppet 2016’, I really needed to ‘win’ something that year. Kristian, my roommate, thanks for pleasant company, nice chats and for the quick fixes with my computer and Excel. To all the other current and former colleagues at the division, thanks for making this trip enjoyable and for all the help I have
received. Also big thanks to all other colleagues at the department, for assistance and for the lunch and coffee breaks during these years.
This thesis would not have been possible without the expertise and contribution from my co-authors, all of whom I greatly thank. I would also like to thank the current and former employees at the Medical product agency that I unfortunate don’t meet every day. Torgny, Kalle, Birgit, Andreas, Ahmad and Monika, thanks for all the tips and for always staying positive.
Special thanks to Ian McEwen, for inspiring me to go into research and introducing me to NMR spectroscopy.
Smålands nation and The Swedish Academy of Pharmaceutical Sciences (Apotekarsocieteten), thanks for traveling grants and scholarships enabling me to participate in international courses and conferences.
Friends and family;
Åke, thanks for all weird topics we have discussed during these years and for being a good friend. I still enjoy your company, even though we have swapped the bouldering hall, our playground, to a playground for children. Jonas, thanks for once in a while reminding me the importance of having a ‘semla’
or ‘smågodis’. Big thanks to all friends outside the university, helping me to relax and enjoy life. Lars-Göran and Berit Nilsson, thanks for all support during these years and for welcoming me into your family with open arms.
My parents Lisa and Lars, and my siblings Nina, Josefin and Freja, thanks for support and for all wide discussions at the kitchen table; they have enlarged my perspective and built my character.
Finally I would like to thank the most important ones in my life, Ellinor, Myra and Ture. Thanks for making my worst days feel much better and for giving me the energy to pull through. Ellinor, it’s an understatement when I say that I wouldn't have managed without you. Our family is the best reason for looking forward and I love you with all of my heart.
Albert,
Uppsala, April 2017
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