Mariola Olkowicz (Presenter)
Poznan University of Life Sciences
Bio: I am a Doctor of Chemical Sciences. My first professional work was as analyst position in the Pomeranian Science and Technology Park. In terms of my job were: participation in research projects, supervision on the functionality of analytical background, implementation of the orders of analytical services (development, validation of analytical methods), reporting of research results, documentation keeping in accordance with ISO 9001 standards and the principles of Good Laboratory Practice (GLP). After two years of work in the Science and Technology Park I started to work at the Poznan University of Life Sciences as a lecturer, where I was employed for one year. For the next three years I worked as postdoctoral scientist within the TEAM program at the Medical University of Gdansk where performed research tasks in the project: “Nucleotides in pathology, diagnosis and therapy of heart disease”. Since a few months I am employed as adjunct at Department of Biotechnology of Poznan University of Life Sciences, where I deal with application of LC/MS technique in biomedical research.
Authorship: Mariola Olkowicz (1,2), Janusz Debski (3), Patrycja Jablonska (1), Ewa Kaniewska (1,4), Michal Dadlez (3), Ryszard T. Smolenski (1,4)
(1) Medical University of Gdansk, Gdansk, Poland; (2) Poznan University of Life Sciences, Poznan, Poland; (3) Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; (4) Heart Science Centre, Imperial College at Harefield Hospital, Harefield, U.K
| Short Abstract To evaluate the potential relationship between aortic valve stenosis and amino acid (AA) metabolome, a new LC–MS/MS method has been developed and validated for simultaneous determination of AA in human plasma. The optimized method enabled to comprehensively quantify 43 target metabolites using an off-line sample pre-treatment method, followed by reversed-phase ion-pair liquid chromatography (Surveyor, Thermo Scientific) coupled on-line to triple quadrupole mass spectrometry (TSQ Vantage, Thermo) via a positive electrospray ionization source. We found several changes in AA concentrations and their ratios such as ornithine : arginine and Fischer ratio in AS group compared with those in the control. |
Long Abstract
Calcific aortic valve stenosis (CAVS) is the most common reason for aortic valve replacement and its prevalence is thought to increase in our ageing population. While CAVS was initially believed to be a passive, degenerative process, it is now well recognized that the progression of calcification in aortic stenosis is actively regulated. Recent studies revealed an association between CAVS and cardiovascular risk factors for atherosclerosis but therapies such as statin therapies fail to delay or reverse the pathology. Deeper understanding of the pathophysiology is therefore required to identify appropriate preventive measures. Metabolomics has recently emerged as effective platform for the non-biased analysis of complex metabolic patterns associated with pathologies such as aortic stenosis, and in combination with the proteomics may reveal information that has never been accessible before. Amino acids and related metabolites (AA) are particularly relevant due to its diverse metabolic and regulatory functions.
To evaluate the potential relationship between aortic valve stenosis and AA metabolome, a new LC–MS/MS method has been developed and validated for simultaneous determination of AA in human plasma. The optimized method enabled to comprehensively quantify 43 target metabolites using an off-line sample pre-treatment method, followed by reversed-phase ion-pair liquid chromatography (Surveyor, Thermo Scientific) coupled on-line to triple quadrupole mass spectrometry (TSQ Vantage, Thermo) via a positive electrospray ionization (ESI) source. The analytes were extracted from plasma samples by protein precipitation and then separated on a Synergi Hydro-RP column in a linear acetonitrile gradient. The detection was performed in multiple reaction-monitoring mode. The limits of quantitation for the analytes determined were within 0.025–0.75 µmol/L, while the absolute recoveries of the metabolites from plasma were all more than 85%.
The method developed was applied for analysis of plasma from patients with aortic stenosis (n = 47) and healthy age-matched controls (n = 18). We found several changes in AA concentrations and AA concentration ratios such as ornithine : arginine ratio and the molar ratio of branched-chain AA to aromatic AA (Fischer ratio) in AS group compared with those in the control. The most pronounced differences were observed in urea cycle-related AA (i.e., arginine, citrulline, and histidine) and BCAA-related AA. In the AS group, amounts of arginine, citrulline and ornithine decreased (89.4±5.7 vs. 67.0±4.9 µM, 57.1±3.8 vs. 44.0±2.5 µM and 42.8±3.4 vs. 36.1±3.1 µM, respectively), whereas those for histidine and branched chain AA were elevated 2-3 folds and by 45-60%, respectively. The contents of asymmetric dimethylarginine (ADMA) and its monomethylated derivative (NMMA), recognized as potential biomarkers of NO-associated endothelial dysfunction in cardiovascular diseases were also increased by 25-30% in aortic stenosis compared to control group. In the AS group, the Fischer ratio and four specific AAs, ie, methionine, tyrosine, 1-methylhistidine, and histidine had significant correlation with BNP, LV ejection fraction and acute phase proteins (CRP, haptoglobin, alpha-1-antitrypsin), respectively.
In conclusion, plasma AA profiling identified several correlations of specific AA patterns with valve pathology that may deliver potential new biomarkers for early disease detection, clinical diagnosis, and therapy.
References & Acknowledgements:
This research was supported by TEAM programme of Foundation for Polish Science (TEAM/2011-8/7).
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