Fluorimetrijska metoda za mjerenje aktivnosti tripeptidil peptidaze 1 u homogenatu leukocita

master's thesis

• Title (english): Fluorimetric method for measuring activity of the tripeptidyl peptidase 1 in the leukocyte homogenate
• Author: Nikolina Blažević
• Mentor: Ksenija Fumić (mentor)
• Committee member: Ksenija Fumić (predsjednik povjerenstva)
• Committee member: Petra Turčić (član povjerenstva)
• Committee member: Marija Grdić Rajković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2019-07-08, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Neuronalne ceroidne lipofuscinoze (NCL) su nasljedne, progresivno degenerativne bolesti koje prvenstveno utječu na
mozak i mrežnicu. One se smatraju lizosomalnim poremećajima skladištenja (LSD), zbog njihovog karakterističnog
nakupljanja autofluorescentnih ceroidnih lipopigmenata, podjedinica C mitohondrijskih ATP sintetaza ili
sfingolipidnih aktivatorskih proteina A i D u lizosomima većine stanica. Klasični kasni infantilni oblik NCL je
uzrokovan mutacijama u CLN2 genu koji kodira lizosomalnu tripeptidil peptidazu 1. Klasični kasni infantilni fenotip
CLN2 bolesti ima predvidljiv klinički tijek obilježen epilepsijom i brzim padom psihomotoričkih sposobnosti.
Najčešći početni simptomi su kašnjenje razvoja govora i napadaji, koji se obično počinju pojavljivati u dobi između 2 i
4 godine.
Rana dijagnoza CLN2 (neuronalna ceroidna lipofuscinoza tipa 2) bolesti je presudna kako bi se osigurala optimalna
skrb za bolesnike i njihove obitelji. CLN2 bolest je moguće dijagnosticirati mjerenjem aktivnosti enzima TPP1. TPP1
enzim je pepstatin-nestabilna lizosomska serin egzopeptidaza s optimalnom in vitro aktivnošću u kiselom pH.
Razvijena je rekombinantna terapija za liječenje CLN2, cerliponaza alfa, koja je najučinkovitija kod primjene nakon
pojave prvih simptoma bolesti.
Cilj rada je uvođenje metode za mjerenje aktivnosti enzima tripeptidil peptidaze 1 u uzorku leukocita izoliranih iz
pune krvi.
In house fluorimetrijska metoda određivanja aktivnosti enzima tripeptidil peptidaze 1 u leukocitima izoliranim iz
EDTA krvi pokazala se kao pouzdana metoda za postavljanje dijagnoze CLN2. Takva se metoda može koristiti u
selektivnoj laboratorijskoj dijagnostici nakon postavljene kliničke sumnje na CLN2. Za primjenu u rutinskoj
laboratorijskoj dijagnostici potrebno je proširiti ispitivanje referentnog intervala na većem broju uzoraka, prvenstveno
djece do pete godine života, jer su oni ciljana populacija za buduću CLN2 dijagnostiku. Međutim, zbog brojnih
ograničenja fluorimetrijske metode, kao prva metoda probira preporučuje se metoda mjerenja aktivnosti TPP1 iz
uzorka suhe kapi krvi na filter papiru.

Abstract (english)

The neuronal ceroid lipofuscinoses (NCLs) are inherited progressive degenerative diseases that primarily affect the
brain and retina. They are considered lysosomal storage disorders (LSD), because of their characteristic accumulation
of autofluorescent ceroid lipopigments, subunit c of mitochondrial ATP synthase or sphingolipid activator proteins A
and D in lysosomes of most cells. The classic late infantile onset NCL is caused by mutations in CLN2 gene which
encodes lysosomal tripeptidyl peptidase 1. The classic late-infantile phenotype of CLN2 disease has a predictable
clinical course marked by epilepsy and rapid psychomotor decline. The most common initial symptoms are language
delay and seizures, which typically begin to manifest between the ages of two to four years.
Early diagnosis of CLN2 disease is crucial to ensure optimum care for patients and their families. CLN2 disease can
be diagnosed by measuring the activity of the enzyme TPP1. TPP1 enzyme is a pepstatin-insensitive lysosomal serine
exopeptidase with optimal in vitro activity at acidic pH. Recombinant therapy for the treatment of CLN2 has been
developed, cerliponase alpha, which is most effective when is applied after the first symptoms of the disease.
The aim of the study was to introduce methods for measuring the activity of the enzyme tripeptidyl peptidase 1 in a
sample of leukocytes isolated from full blood.
In house fluorimetric method of determining the activity od the enzyme tripeptidyl peptidase 1 in leukocytes isolated
from EDTA blood has proven to be a reliable method for diagnosing CLN2. Such a method can be used in selective
laboratory diagnostics after clinical suspected CLN2. For routine laboratory diagnostics, it is necessary to extend the
reference interval test on a large number of samples, primarily children up to the age of five, as they are a target
population for future CLN2 diagnostics. However, due to the numerous limitations of the fluorimetric method, the
method of measurement of TPP1 activity from the dry blood sample on filter paper is recommended as the first
method of screening.

• Keywords: neuronalna ceroidna lipofuscinoza tipa 2; tripeptidil peptidaza 1; Battenova bolest
• Keywords (english): neuronal ceroid lipofuscinosis type 2; tripeptidyl peptidase 1; Batten disease
• Language: croatian
• URN:NBN: urn:nbn:hr:163:125241
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-02-14 14:18:44