Use of Quantitative Metabolomics for Investigation of Age-Related Nuclear Cataracts Vadim Yanshole (Presenter) International Tomography Center SB RAS Presenter 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 metabolomic analysis of lens of different animal species.

Authors: Vadim V. Yanshole (1,2), Lyudmila V. Yanshole (1,2), Olga A. Snytnikova (1,2), Kudryavtsev I.S. (3), Yuri P. Tsentalovich (1,2)
(1) International Tomography Center SB RAS, Novosibirsk, Russia (2) Novosibirsk State University, Novosibirsk, Russia (3) Novosibirsk Regional Clinical Bureau of Forensic Medical Examination, Novosibirsk, Russia

A cataract (clouding of the lens) is the most common cause of vision declining of older people. Unlike most other human tissues, the lens has specific structure to be transparent: firstly, the lens consists mainly of fiber cells without organelles, and secondly, it lacks blood vessels. The protection of the lens is mainly provided by metabolites; most of them are synthesized in the lens epithelium or enter the lens through the epithelial layer from the surrounding aqueous humor (AH). Therefore, changes in the metabolome of the lens and AH may help to establish the molecular mechanisms of the cataract onset.
Current report provides the data analysis of changes in the metabolomic profiles of human eye lens and AH under the development of cataracts. The concentration of more than 80 metabolites in the lens and AH have been determined with the combined use of LC-MS, LC-OD and NMR methods.

Introduction

The incoming information received by a person comes mainly from the sight. Visual disturbance inevitably affects the quality of life. Therefore, diseases associated with a visual impairment are among the most pressing problems in modern medicine. According to WHO, a cataract (clouding of the lens) develops in more than half of people over 65 years of age and become the most common cause of decline and loss of vision in older people.

Metabolomics is one of the fastest growing branches of modern systems biology and biochemistry; it finds its application for the study of a wide range of human pathologies. The development of pathological processes can lead to significant changes in the metabolomic profile of a tissue. Unlike most other human tissues, the lens has very specific structure to be transparent: firstly, the lens consists mainly of fiber cells without organelles, and secondly, it lacks blood vessels. The protection of the lens is mainly provided by metabolites; most of them are synthesized in the lens epithelium or enter the lens through the epithelial layer from the surrounding aqueous humor (AH). Comparison of the metabolomic compositions of lenses and AH taken from patients with age-related nuclear cataract with lenses and AH taken from human cadavers without cataract can shed light onto molecular mechanisms underlying onset of cataract.

Methods

Quantitative metabolomic profiles of eye tissue extracts (lens, AH) were obtained with the combination of three methods – high-frequency 1H nuclear magnetic resonance (NMR) and ion-pairing high-performance liquid chromatography with optical (LC-OD) and high-resolution ESI-q-TOF mass-spectrometric detection (LC-MS) methods. NMR-based quantification was achieved with the use of one internal standard (DSS) for all metabolites under study, while LC-MS quantification required the construction of the calibration curves for each metabolite under study using the commercially available chemical standards.

The molecular UV filters of kynurenine family (total 12 compounds) were quantified by the UV absorption measured by the DAD cell during the same LC-MS runs (LC-OD). Assuming that the extinction coefficients at 360 nm are equal for all the kynurenine-like compounds, the calibration curve, common for the majority of UV filters, was plotted based on the HPLC UV peak area versus kynurenine concentration.

Results

The concentrations of more than 80 metabolites were determined for four groups of samples: lenses and AH from cataract patients and lenses and AH from human cadavers. In cataractous lens the most abundant metabolites are (in descending order): myo-inositol, lactate, acetate, glutamate, glutathione; in AH – lactate, glucose, glutamine, alanine, valine. The concentrations of nucleotides, UV filters, antioxidants and some other metabolites in the lens are much higher than that in AH, while the levels of glucose and hydroxybutyrates are lower. The concentrations of the majority of metabolites in non-cataractous post-mortem samples of both lens and AH are higher than that in samples from the cataract patients.

Conclusions & Discussion

The combination of LC-MS, LC-OD and NMR methods turned out to be very fruitful and reliable for metabolite identification and quantification. Our metabolomic data confirm the hypothesis that although the age-related cataract usually manifests itself as the opacification of the lens nucleus, the initial site of the cataract onset might be the lens epithelial layer. The most important for the lens protection metabolites – antioxidants, UV filters, osmolytes – are synthesized in the lens epithelial cells. The reduced levels of these metabolites were found in the cataractous lenses; that indicates that the cataract development may originate from the dysfunction of the lens epithelial cells. The increase in the concentrations in non-cataractous post-mortem tissues for other metabolites corresponds to the post-mortem processes.

The LC-MS measurements were supported by the Russian Scientific Foundation (Project 18-73-10014); NMR measurements were supported by the Russian Foundation for Basic Research (Projects 17-03-00656, 18-34-00137); sample preparation was supported by the Siberian Branch of the Russian Academy of Sciences (Project 0333-2018-0009)