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Title: Določevanje aktivnosti AChE encima v človeškem serumu z uporabo FIA-TLS metode : Magistrsko delo Authors: ID Korte, Dorota (Mentor) More about this mentor... ID Jerkič, Andrej (Author) Files: Andrej_Jerkic.pdf (2,51 MB) MD5: 725D980752AA2A0503E10A0F796857DC   Language: Slovenian Work type: Master's thesis Typology: 2.09 - Master's Thesis Organization: FZO - Faculty of Environmental Sciences Abstract: Acetilholinesteraza (AChE) in butirilholinesteraza (BChE) sta encima, odgovorna za razgradnjo nevrotransmitorja acetilholina oziroma butirilholina. Encima spadata v skupino holinesteraz in imata poleg razgradnje nevrotransmiterjev pomembno vlogo v telesu kot indikatorja za različne bolezni ter zastrupitve z organofosfati in drugimi kemikalijami. Cilji magistrskega dela so razviti občutljivo metodo za določevanje aktivnosti AChE encima v človeškem serumu, določiti celokupno aktivnost AChE ter BChE encimov v vzorcih človeškega seruma neznanih pacientov z uporabo FIA-TLS tehnike ter izmeriti aktivnost AChE v človeškem serumu z uporabo inhibitorja BChE. Opravili smo meritve kinetike pretvorbe acetiltioholina v 5-tio-2-nitrobenzoat (TNB), na podlagi katerih smo izračunali specifično aktivnost AChE encima in aktivnosti različnih njegovih redčitev ter izvedli spektralno karakterizacijo proučevanih vzorcev. Vzorce AChE encima smo pripravili v raztopini 5,5'DITIO-bis (2-nitrobenzoični) kislini (reagent) in raztopini acetiltioholin jodida (substrat). Ugotovili smo, da je absorpcijski vrh za raztopine AChE pri valovni dolžini približno 412 nm. Z meritvami absorpcijskega spektra različnih redčitev AChE smo ugotovili, da absorbanca hitreje narašča v vzorcih z višjo koncentracijo AChE, ker se je povečevala aktivnost encima. Ugotovili smo tudi, da z nižanjem molarnosti DTNB iz 0,50 mM na 0,05 mM lahko znižamo LOD metode zaradi znižanja vrednosti signala slepega vzorca. Tega ne moremo trditi pri znižanju koncentracije DTNB iz 0,05 mM na 0,025 mM zaradi nepopolne reakcije med acetilholinom (ACh), 5-tio-2-nitrobenzoatom (TNB) in acetilholinesterazo (AChE). Metodo FIA-TLS smo optimizirali z uporabo daljše mešalne zanke ter s povečanjem moči vzbujevalnega žarka. Z optimizacijo FIA-TLS sistema nam je uspelo izmeriti vrednosti TLS signala vzorcev z nižjo koncentracijo AChE encima (70 μV) v primerjavi z neoptimiziranim TLS sistemom (110 μV). Izračunana vrednost LOD UV-Vis metode znaša 3,6 mU/ml, medtem ko za FIA-TLS metodo znaša 0,6 mU/ml. V prvem sklopu meritev smo izmerili 14 vzorcev krvnega seruma. Aktivnosti celotne ChE v teh vzorcih znašajo med 2118 mU/ml in 15172 mU/ml, povprečna aktivnost BChE in AChE v vzorcih znaša 4561 mU/ml, mediana meritev pa znaša 2964 mU/ml. V drugem sklopu meritev smo merili 19 vzorcev krvnega seruma z in brez dodatka BChE inhibitorja. Aktivnosti vzorcev brez BChE inhibitorja (celotne ChE) znašajo med 1642 mU/ml in 5958 mU/ml s povprečno vrednostjo 2910 mU/ml in mediano 2228 mU/ml, medtem ko aktivnosti istih vzorcev z dodanim inhibitorjem (samo AChE) znašajo od 519 mU/ml do 1118 mU/ml s povprečno vrednostjo 822 mU/ml in mediano 816 mU/ml. Po dodatku BChE se je aktivnost ChE v vzorcih v povprečju zmanjšala za 3,5-krat oziroma od 55 % do 85 %. Keywords: Acetilholinesteraza, butirilholinesteraza, FIA-TLS, aktivnost AChE encima, inhibicija BChE encima Place of publishing: Nova Gorica Year of publishing: 2018 PID: 20.500.12556/RUNG-4209-86d2dbea-5d7f-85a4-1a0f-d1d38da926ba  COBISS.SI-ID: 5275131  NUK URN: URN:SI:UNG:REP:4SKNKCZ2 Publication date in RUNG: 30.11.2018 Views: 4712 Downloads: 173 Metadata:     Cite this work Plain text BibTeX EndNote XML EndNote/Refer RIS ABNT ACM Ref AMA APA Chicago 17th Author-Date Harvard IEEE ISO 690 MLA Vancouver : Copy citation

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Secondary language

Language:	English
Title:	Determination of AChE activity in human serum by the use of FIA-TLS : Master’s thesis
Abstract:	Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes which belongs to a group of cholinesterases. Both enzymes are responsible for catalyzing the hydrolysis of the cholinergic neurotransmitters like acetylcholine and butyrilcholine. Thus, play important role in a body as indicators for different diseases and poisoning with organophosphates. The goals of the work are to develop a sensitive method for determination of AChE activity in human serum to determine the total AChE and BChE activity in human serum samples from unknown patients by the use of FIA-TLS technique, as well as to measure the AChE activity in serum samples by using BChE inhibitor. AChE samples were prepared with the reagent 5, 5’DITHIO-bis (2-nitrobenzoic) acid (DTNB) and substrate acetylthiocholine iodide. By UV-Vis spectrophotometry the maximum absorption of AChE standard solutions at wavelength of 412 nm was determined. Measurement of AChE solution spectra showed, that the absorbance increase is faster in case of samples with higher concentrations of AChE. By increasing the AChE concentration in the samples, their activity also increases. It was found, that with lowering the molarity of DTNB from 0.50 mM to 0.05 mM the LOD of the method can be significantly decreased, because of decreasing the value of the blank signal. This is not valid when decreasing the DTNB concentration from 0.05 mM to 0.025 mM because the reaction of acetylthiocholine (Ach), 5, 5’DITHIO-bis (2-nitrobenzoic) acid (DTNB) and acetilholinesterase (AChE) is not completed. Furthermore, FIA-TLS method was optimized by using longer mixing and increased power of the excitation laser beam. Calibration curve for optimized FIA-TLS method was constructed and LOD was calculated. By the use of optimized FIA-TLS system it was possible to measure nearly twice lower signals (~70 μV) than by not-optimized system (~110 μV). Calculated LOD for UV-Vis spectrophotometry was 3.6 mU/ml, whereas 0.6 mU/ml for FIA-TLS method. In the first part of FIA-TLS measurement, total activity of 14 serum samples were determined. It was found, that the enzymes activity varies from 2118 mU/ml to 15172 mU/ml. Overall average BChE and AChE activity in serum samples determined in our work was 4561 mU/ml with the median value of 2964 mU/ml. In the second part of work, FIA-TLS measurements of 19 serum samples with and without BChE inhibitor were performed. Enzyme activity without BChE inhibitor (total ChE) varies from 1642 mU/ml in 5958 mU/ml with average activity of 2910 mU/ml and median value of 2228 mU/ml. Activity of the same 19 samples with added BChE inhibitor (only AChE) varies from 519 mU/ml to 1118 mU/ml with average activity of 822 mU/ml and median value of 816 mU/ml. By addition of BChE inhibitor, average activity of ChE samples decrease for 3.5x. Decrease in ChE activity after addition of BChE inhibitor varies from 55% to 85 %.
Keywords:	Acetylcholinesterase, Butyrilcholinesterase, FIA-TLS, AChE activity, BChE inhibitor

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