Application of high-performance liquid chromatography combined with ultra-sensitive thermal lens spectrometric detection for simultaneous biliverdin and bilirubin assessment at trace levels in human serumMitja Martelanc
, Lovro Žiberna
, Sabina Passamonti
, Mladen Franko
, 2016, original scientific article
Abstract: We present the applicability of a new ultra sensitive analytical method for the simultaneous determination of biliverdin and bilirubin in human serum. The method comprises isocratic reversed-phase(RP) C18 high-performance liquid chromatography (HPLC) and thermal lens spectrometric detection (TLS)based on excitation by a krypton laser emission line at 407 nm. This method enables the separation of IX-α biliverdin and IX-α bilirubin in 11 min. with limit of detection (LOD) and limit of quantitation (LOQ) for
biliverdin of 1.2 nM and 3 nM, and 1nM and 2.8 nM for bilirubin,respectively.In addition, a step-gradient elution was set up, by changing the mobile phase composition, in order to further enhance the sensitivity for bilirubin determination with LOD and LOQ of 0.5 nM and 1.5 nM, respectively. In parallel, an isocraticHPLC-DAD method was developed for benchmarking against HPLC-TLS methods. The LOD
and LOQ forbiliverdin were 6 nM and 18 nM, and 2.5 nM and 8nM for bilirubin,respectively. Additionally, both isocratic methods were applied for measuring biliverdin and free bilirubin in human serum samples (from 2 male and 2 female healthy donors). Combining isocratic HPLC method with TLS
detector was crucial for first ever biliverdin determination in serum together with simultaneous free bilirubin determination. We showed for the first time the concentration ratio of free bilirubin versus unbound biliverdin in human serum samples.
Found in: ključnih besedah
Keywords: Bilirubin, biliverdin, serum, HPLC, TLS
Published: 29.03.2016; Views: 3120; Downloads: 0
Fulltext (964,28 KB)
THE ROLE OF SPECT/CT SCINTIGRAPHY IN LOCALIZATION OF PARATHYROID ADENOMASSebastijan Rep
, 2016, master's thesis
Abstract: Parathyroid scintigraphy is based on the different accumulation and distribution of radiopharmaceutical 99mTc-MIBI (MIBI) in parathyroid and adjacent tissues. Different scintigraphic techniques can be used: subtraction scintigraphy is a combination of MIBI and 99mTc-pertehnetate (pertehnetate) scintigraphy; SPECT can be performed after intravenous injection of MIBI or in the combination with low-dose computerized tomography (SPECT/CT) which allows precise anatomical localization of enlarged parathyroid glands (EPG).
The objectives of our study were: to assess diagnostic performance of preoperative localization of EPG (adenomas and hyperplasia) of the early, delayed and combined SPECT/CT and of subtraction scintigraphy in patients with biochemically proven primary hyperparathyroidism (PHP); to determine whether size of EPG, serum parathyroid hormone (PTH), calcium (Ca) and phosphate (P) correlate with successful localization; to assess radiation exposure of patients with early and late SPECT/CT and subtraction scintigraphy
The study included 41 patients in whom early, delay SPECT/CT and subtraction scintigraphy were performed. For all scintigraphic methods sensitivity, specificity, positive predictive value, negative predictive value and accuracy were calculated. Receiver Operating Characteristic analysis was used to express relationship between scintigraphy results and size of the EPG and serum Ca, P and PTH levels. ImPACT CT Patient Dosimetry Calculator and weighting factors for radiopharmaceuticals (MIBI and pertechnetate) were used for calculating patient expose dose.
Sensitivity and specificity were 51.1% and 98.3 for subtraction scintigraphy, 65.1% and 97.5% for early SPECT/CT and 65.1% and 95.5% for delay SPECT/CT. In combined early and delay SPECT/CT sensitivity and specificity were 74.4% and 95.1%.Only size of the EPG correlated with successful localization in our study (AUC of 0.791-0.810). The highest average ED per patient had subtraction scintigraphy (7.37±0.40 mSv), which was statistically significant higher in comparison to combined early and delayed SPECT/CT (6.81±0.44 mSv).
The most sensitive and most accurate method of preoperative localization of EPG in patients with PHP is combined early and delayed SPECT/CT. Only size of the EPG correlates with successful localization. The highest radiation exposure is after subtraction scintigraphy.
Found in: ključnih besedah
Summary of found: ...(PHP); to determine whether size of EPG, serum parathyroid hormone (PTH), calcium (Ca) and phosphate...
Keywords: Enlarged parathyroid glands, early SPECT/CT, delayed SPECT/CT, combined SPECT/CT, subtraction scintigraphy, 99mTc-MIBI, 99mTc-pertechnetate, effective dose, serum parathyroid hormone, calcium, phosphate
Published: 03.10.2016; Views: 3790; Downloads: 183
Fulltext (1,03 MB)
DOLOČANJE ACETILHOLINESTERAZE V HUMANIH KRVNIH VZORCIHTjaša Birsa
, 2018, master's thesis
Abstract: V strokovni literaturi zasledimo encim acetilholinesterazo (AChE) v različnih raziskavah s področja okoljevarstva, medicine in veterine. Zmanjšana encimska aktivnost je lahko indikator onesnaženosti okolja z organofosfatnimi in karbamatnimi pesticidi, policikličnimi aromatskimi ogljikovodiki ter težkimi kovinami. Spremembe v encimski aktivnosti AChE pa so tudi biološki označevalec nekaterih nevrodegenerativnih bolezni pri človeku.
V človeškem telesu se encim AChE primarno nahaja v živčnem sistemu, najdemo pa ga tudi v krvi, zlasti v membranah eritrocitov. V krvi je v večji meri prisoten tudi encim butirilholinesteraza (BChE), ki ravno tako spada v družino holinesteraz. Naš cilj je bil razviti metodo, ki bi omogočala specifično merjenje encimske aktivnosti AChE v okviru celotnih holinesteraz v krvnem serumu.
Za merjenje encimske aktivnosti smo uporabili Ellmanovo metodo. Količino obarvanih produktov reakcije smo določali s spektroskopijo s toplotnimi lečami TLS in z UV-VIS spektrofotometrijo. Z različnimi koncentracijami čistega encima AChE smo najprej določili mejo zaznavnosti obeh metod (LOD). Detekcija s TLS je bila bolj občutljiva, saj je bil LOD 3,13-krat manjši kot pri detekciji z UV-VIS. Potem smo izmerili encimsko aktivnost celotnih holinesteraz AChE in BChE v serumskih vzorcih zdravih ljudi. Izračunana encimska aktivnost holinesteraz se je v analiziranih vzorcih gibala med 3004 mU mL-1 in 5698 mU mL-1. Po dodatku specifičnega inhibitorja BChE se je celotna encimska aktivnost znižala za najmanj 60 %, kar kaže, da večina holinesteraz v krvnem serumu pripada BChE. Preizkusili smo tudi metodo enojne imunske precipitacije, vendar se je le-ta izkazala za neuspešno pri ločevanju posameznih holinesteraz iz serumskih vzorcev.
Na podlagi naših rezultatov smo prišli do zaključka, da je za merjenje encimske aktivnosti posameznih holinesteraz AChE in BChE v krvnem serumu najprimernejša uporaba specifičnih inhibitorjev, vendar bi bilo to metodo potrebno še dodatno optimizirati.
Found in: ključnih besedah
Summary of found: ...acetilholinesteraza, krvni serum, nevrodegenerativne bolezni, spektroskopija s toplotnimi lečami, UV-VIS...
Keywords: acetilholinesteraza, krvni serum, nevrodegenerativne bolezni, spektroskopija s toplotnimi lečami, UV-VIS spektrofotometrija, imunska precipitacija.
Published: 07.11.2018; Views: 2058; Downloads: 107
Fulltext (1,65 MB)
The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture.Quaglino Daniela
, Tiozzo Roberta
, Paolinelli Devincenzi Chiara
, Annovi Giulia
, Boraldi Federica
, 2008, original scientific article
Abstract: The effect of serum deprivation on proliferating cells is well known, in contrast its role on primary
cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explore
the response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normal
human dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of
10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastin
synthesis) was evaluated morphologically and biochemically. Moreover, the protein profile was
investigated by 2-DE and differentially expressed proteins were identified by MS. Serum withdrawal
caused cell shrinkage but did not significantly modify the total cell number. ROS production,
as evaluated by the dihydroethidium (DH2) probe, was increased after serum deprivation,
whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in the
absence of serum. By proteome analysis, 41 proteins appeared to significantly change their
expression, the great majority of protein changes were related to the cytoskeleton, the stress response
and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cell
culture can adapt themselves to environmental changes, without significantly altering cell viability,
at least after a few days of treatment, even though serum withdrawal represents a stress
condition capable to increase ROS production, to influence cell metabolism and to interfere with
cell behaviour, favouring the expression of several age-related features.
Found in: ključnih besedah
Keywords: Dermal fibroblasts / Primary cell culture / ROS production / Serum withdrawal
Published: 22.07.2019; Views: 1132; Downloads: 0
Fulltext (462,68 KB)
Metabolic dysregulation in vitamin E and carnitine shuttle energy mechanisms associate with human frailtyJames Nazroo
, Royston Goodacre
, Neil Pendleton
, Frederick Wu
, Iain White
, Bram Vanhoutte
, Gindo Tampubolon
, Alan Marshall
, Krisztina Mekli
, Zahra Rattray
, Tarani Chandola
, Caroline Johnson
, Drupad Trivedi
, Yun Xu
, Nicholas Rattray
, 2019, original scientific article
Abstract: Global ageing poses a substantial economic burden on health and social care costs. Enabling a greater proportion of older people to stay healthy for longer is key to the future sustainability of health, social and economic policy. Frailty and associated decrease in resilience plays a central role in poor health in later life. In this study, we present a population level assessment of the metabolic phenotype associated with frailty. Analysis of serum from 1191 older individuals (aged between 56 and 84 years old) and subsequent longitudinal validation (on 786 subjects) was carried out using liquid and gas chromatography-mass spectrometry metabolomics and stratified across a frailty index designed to quantitatively summarize vulnerability. Through multivariate regression and network modelling and mROC modeling we identified 12 significant metabolites (including three tocotrienols and six carnitines) that differentiate frail and non-frail phenotypes. Our study provides evidence that the dysregulation of carnitine shuttle and vitamin E pathways play a role in the risk of frailty.
Found in: ključnih besedah
Summary of found: ...metabolic phenotype associated with frailty. Analysis of serum from 1191 older individuals (aged between 56...
Keywords: metabolomics, frailty, ageing, LC-MS, serum
Published: 08.11.2019; Views: 1011; Downloads: 36
Fulltext (2,35 MB)