1. TLS Spectrometry & Detection in Liquid Chromatography, FIA and Microfluidic SystemsMladen Franko, predavanje na tuji univerzi Opis: This presentation provides theoretical backround for thermal lens spectrometry (TLS) and TLS microscopy (TLM) and describes the basic instrumentation for TLS and TLM measurements. Presented applications of TLS and TLM include detection in HPLC (carotenoids, bilirubin, biliverdin), FIA (alergens, Ag, Ag+, phytoplankton and cyanobacterial pigments, organophospate pesticides) and TLM detection in microfluidic systems (Cr(VI), microcystin, NGAL, study of diffusion proceses)
Ključne besede: Spekrometry TLS, TLM, HPLC, FIA, microfluidics, bilirubin, biliverdin, carotenoids, alergens, mikrocystin, pigments, organophosphate pesticides, NGAL, Ag, Cr(VI), diffusion
Objavljeno v RUNG: 16.09.2016; Ogledov: 6207; Prenosov: 6
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2. Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial CellsLovro Žiberna, Mitja Martelanc, Mladen Franko, Sabina Passamonti, 2016, izvirni znanstveni članek Opis: Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with
cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular
diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships.
Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric
detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To
confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a
high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass
spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5
pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular
bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by
up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis.
Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in
the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant
properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and
may thus be a dynamic factor of the endothelial function.
Ključne besede: Antioxidants, bilirubin, endothelial cells
Objavljeno v RUNG: 07.07.2016; Ogledov: 5822; Prenosov: 409
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3. 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, izvirni znanstveni članek Opis: 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.
Ključne besede: Bilirubin, biliverdin, serum, HPLC, TLS
Objavljeno v RUNG: 29.03.2016; Ogledov: 6736; Prenosov: 0
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