Vadim Yanshole (Presenter)
International Tomography Center
Bio: Vadim Yanshole obtained M.Sc. (Hons) at the Novosibirsk State University (Novosibirsk, Russia, 2001-2007). In 2011 he received Ph.D. in chemical physics. Since 2007 he is working at the ITC SB RAS (Novosibirsk, Russia) in the fields of proteomics, metabolomics and mass-spectrometry; now he is the leading specialist in the institute in these fields. He is experienced in MALDI-TOF and high-resolution LC-MS techniques, and their use in the biological applications. Current research interest is mainly devoted to quantitative metabolomic profiling of the different human tissues and liquids, especially eye constituents – lens, aqueous humor, and cornea. Recent investigations include the development of the method for spatial distribution of the metabolites in biological tissues.
Authorship: Vadim V. Yanshole (1,2), Lyudmila V. Yanshole (1,2), Semen O. Tamara (1), Yuri P. Tsentalovich (1,2)
(1) International Tomography Center SB RAS, Novosibirsk, Russia (2) Novosibirsk State University, Novosibirsk, Russia
| Short Abstract The transparency of the eye lens depends on the integrity of lens proteins – crystallins – which may undergo numerous PTMs throughout the lifespan and cause cataracts. The protection of the lens cells is mainly governed by metabolites; most of them are synthesized in the lens epithelium or enter the lens through the epithelial layer from the surrounding aqueous humor. The study of the metabolite composition and spatial distribution inside the lens may help to establish the mechanisms of metabolite transport in the lens. Current report provides the data on the metabolomic profiles of normal and cataractous human lenses and spatial metabolite distribution inside the lens. The concentration of more than 50 metabolites in the lens cortex and nucleus and spatial distribution of 34 metabolites along the optical and equatorial axes has been determined with the use of NMR and HPLC-MS methods. |
Long Abstract
Introduction
Cataract, or clouding of the eye lens, is the leading cause of vision impairment in the world. The lens tissue has a very specific structure: it does not have a vascular system, and the lens proteins – crystallins – do not turnover throughout the lifespan. The transparency of the eye lens depends on the integrity of crystallins which may undergo numerous posttranslational modifications throughout the lifespan. The protection of lens proteins is provided by various small molecules – metabolites, which diffuse inside the lens from the aqueous humor or are synthesized in the lens epithelial layer. The further metabolite transport inside the lens is proposed to be driven by passive diffusion or active Na+ ion currents, but the contribution of each mechanism is still unknown. Therefore, the study of changes in the metabolite composition of a cataractous lens as compared to a normal lens may elucidate the possible mechanisms responsible for the cataract formation.
Methods
Quantitative metabolomic profiles of normal and cataractous human lenses were obtained with the combination of two methods – high-frequency 1H nuclear magnetic resonance (NMR) and ion-pairing high-performance liquid chromatography with high-resolution ESI-q-TOF mass-spectrometric detection (LC-MS) methods. NMR-based quantification was achieved with the use of one internal standard for all metabolites, while LC-MS quantification required the construction of the calibration curves for each metabolite under study using the commercially-available chemical standards. For the measurements of the metabolites spatial distribution a specific method has been developed: the tissue was cut into slices on cryotome along the axis of interest, and the slices were further quantitatively analyzed by LC-MS.
Results
The quantitative content of more than fifty metabolites has been determined in this work for normal aged and cataractous human lenses. The most abundant metabolites in the normal lens are myo-inositol, lactate, creatine, glutathione, glutamate, and glucose.
For the majority of metabolites, their levels in the lens cortex and nucleus are similar, with the few exceptions including antioxidants and ultraviolet (UV) filters: the concentrations of glutathione, ascorbate and NAD in the lens nucleus decrease as compared to the cortex, while the levels of the secondary UV filters formed from primary UV filters in redox processes increase. That confirms that the lens core is metabolically inert, and the metabolic activity in the lens nucleus is mostly restricted by protection from the oxidative stress caused by UV irradiation, UV filter spontaneous decomposition, or other factors.
Spatial distribution of 34 metabolites along the optical and equatorial axes of the human lens has been determined. For the majority of metabolites, the homogeneous distribution has been observed. That suggests that the rate of the metabolite transformation in the lens is low due to the general metabolic passivity of the lens fiber cells. However, the redox processes are active in the lens; as a result some metabolites including antioxidants, demonstrate the “nucleus-depleted” type of distribution, whereas secondary UV filters show the “nucleus-enriched” type. The metabolite concentrations at the lens poles and equator are similar for all metabolites under study.
Conclusions
The combination of NMR and LC-MS methods turned out to be very fruitful and reliable for metabolite identification and quantification. Comprehensive quantitative metabolic profiles of the human eye lens have been acquired for the first time. The obtained data can be used for the analysis of changes in the lens chemical composition occurring with age and with the cataract development. The concentric pattern of the “nucleus-depleted” and “nucleus-enriched” distributions testifies that the metabolite distribution inside the lens is mostly governed by a passive diffusion, relatively free along the fiber cells and retarded in the radial direction across the cells. No significant difference in the metabolite distribution patterns between the normal and cataractous human lenses was found.
References & Acknowledgements:
This work was supported by RSF (Project 14-14-00056), RFBR (Project 14-03-00027), and the Grant of the President of RF (project MK-5367.2015.3)
| Description | Y/N | Source |
| Grants | yes | RSF, RFBR, Grant of the President of RF |
| Salary | yes | ITC SB RAS |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |