Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Applications of stable isotopes in MALDI imaging: current approaches and an eye on the future.

Matrix-assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) is now an established imaging modality with particular utility in the study of biological, biomedical and pathological processes. In the first instance, the use of stable isotopically labelled (SIL) compounds in MALDI-IMS has addressed technical barriers to increase the accuracy and versatility of this technique. This has undoubtedly enhanced our ability to interpret the two-dimensional ion intensity distributions produced from biological tissue sections. Furthermore, studies using delivery of SIL compounds to live tissues have begun to decipher cell, tissue and inter-tissue metabolism while maintaining spatial resolution. Here, we review both the technical and biological applications of SIL compounds in MALDI-IMS, before using the uptake and metabolism of glucose in bovine ocular lens tissue to illustrate the current limitations of SIL compound use in MALDI-IMS. Finally, we highlight recent instrumentation advances that may further enhance our ability to use SIL compounds in MALDI-IMS to understand biological and pathological processes. Graphical Abstract.

Mapping glucose metabolites in the normal bovine lens: Evaluation and optimisation of a matrix-assisted laser desorption/ionisation imaging mass spectrometry method.

The spatial resolution of microdissection-based analytical methods to detect ocular lens glucose uptake, transport and metabolism are poor, whereas the multiplexing capability of fluorescence microscopy-based approaches to simultaneously detect multiple glucose metabolites is limited in comparison with mass spectrometry-based methods. To better understand lens glucose transport and metabolism, a more highly spatially resolved technique that maintains the fragile ocular lens tissue is required. In this study, a sample preparation method for matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) analysis of ocular lens glucose uptake and metabolism has been evaluated and optimised. Matrix choice, tissue preparation and normalisation strategy were determined using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS of bovine lens tissue and validation performed using gas chromatography-MS. An internal standard was applied concurrently with N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC) matrix to limit cracking of the fresh frozen lens tissue sections. MALDI IMS data were collected at a variety of spatial resolutions to detect both endogenous lens metabolites and stable isotopically labelled glucose introduced by ex vivo lens culture. Using this approach, initial steps in important metabolic processes that are linked to diabetic cataract formation were spatially mapped in the bovine lens. In the future, this method can be applied to study the dynamics of glucose uptake, transport and metabolic flux to aid in the study of diabetic lens cataract pathophysiology.

Age-dependent changes in glutathione metabolism pathways in the lens: New insights into therapeutic strategies to prevent cataract formation-A review.

Ocular tissues possess a robust antioxidant defence system to minimize oxidative stress and preserve tissue structure and function. Glutathione (GSH) is a powerful antioxidant and in the lens exists at unusually high concentrations. However, with advancing age, GSH levels deplete specifically in the lens centre initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering and age-related nuclear cataract. However, antioxidant supplementation has been shown to be ineffective in preventing or delaying cataract indicating that a better understanding of the delivery, uptake and metabolism of GSH in the different regions of the lens is required. This information is essential for the development of scientifically informed approaches that target the delivery of GSH to the lens nucleus, the region of the lens most affected by age-related cataract.

FTICR mass spectrometry-based multivariate analysis to explore distinctive metabolites and metabolic pathways: A comprehensive bioanalytical strategy toward time-course metabolic profiling of Thymus vulgaris plants responding to drought stress.

In this research, metabolic profiling/pathways of Thymus vulgaris (thyme) plant were assessed during a water deficit stress using an FTICR mass spectrometry-based metabolomics strategy incorporating multivariate data analysis and bioinformatics techniques. Herein, differences of MS signals in specific time courses after water deficit stress and control cases without any timing period were distinguished significantly by common pattern recognition techniques, i.e., PCA, HCA-Heatmap, and PLS-DA. Subsequently, the results were compared with supervised Kohonen neural network (SKN) ones as a non-linear data visualization and capable mapping tool. The classification models showed excellent performance to predict the level of drought stress. By assessing variances contribution on the PCA-loadings of the MS data, the discriminant variables related to the most critical metabolites were identified and then confirmed by ANOVA. Indeed, FTICR MS-based multivariate analysis strategy could explore distinctive metabolites and metabolic pathways/profiles, grouped into three metabolism categories including amino acids, carbohydrates (i.e., galactose, glucose, fructose, sucrose, and mannose), and other metabolites (rosmarinic acid and citrate), to indicate biological mechanisms in response to drought stress for thyme. It was achieved and approved through the MS signals, genomics databases, and transcriptomics factors to interpret and predict the plant metabolic behavior. Eventually, a comprehensive pathway analysis was used to provide a pathway enrichment analysis and explore topological pathway characteristics dealing with the remarkable metabolites to demonstrate that galactose metabolism is the most significant pathway in the biological system of thyme.

A giant ureteral stone in a 32-year-old man: a case report.

Giant ureteral calculi are defined as stones greater than 5 cm in length or circumference. These giant calculi can cause blockage of the ureter, dilation of the kidney and also decreased kidney function if not treated in time. The patient in this report presented with complaints of bilateral episodic pain of the bilateral lumbar region. Kidney, ureter and bladder (KUB) X-ray test showed a large bilateral ureteral stone about 14 cm in length and 106 g weight in the left ureter and 3 cm longitudinal diameter in the right ureter and also a staghorn stone in the left upper collecting system. Thereafter, the ureteric calculi were managed successfully using the combination of open and endoscopic techniques.

Evaluation of Thin-Layer Chromatography-Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Imaging for Visualization of Crude Oil Interactions.

A light oil was separated into four chromatographic fractions that serve as proxy for SARA fractions. The fractions were (semi)quantified on a rod by TLC-flame ionization detection and characterized on a plate with laser desorption ionization-mass spectrometry imaging (TLC-LDI-MS). Comparisons of (semi)quantitative TLC-FID and qualitative TLC-LDI-MS results showed that LDI-MS was most sensitive for detection of molecules in the polar P1 fraction, and, to some extent, for the aromatics fraction, while no signal was observed for the most polar P2 and saturates fractions. Based on these results, limits of the compositional space, as observed by the laser ionization technique, were evaluated. The molecular speciation between and within the spots of the aromatics and the P1 fractions were analyzed and interpreted in terms of oil-SiO2 versus oil-solvent interactions, as a function of molecular characteristics such as DBE, aromaticity (H/C ratio), heteroatom content, degree of alkylation, and shielding of heteroatoms. In addition, the high oil loading resulted in an interesting bifurcation of the aromatics spot, which implies that oil-oil interactions can be enforced and studied in the TLC model system.

Mechanism, decomposition pathway and new evidence for self-healing of manganese oxides as efficient water oxidizing catalysts: new insights.

The electrochemical water-oxidation reaction usually requires a catalyst to reduce the overpotential and Earth-abundant catalysts, like MnO2, are attracting much attention. Here we use chemometric analysis, EPR and UV-Vis spectroscopies to track Mn(II) and MnO4(-) byproducts to the reaction of a MnO2 film in the presence of cerium(IV) ammonium nitrate. Permanganate ion is involved in at least two key reactions: it may oxidize water to O2 or can combine with Mn(II) to remake MnO2 solid. We propose mechanisms for water oxidation and present a self-healing process for this reaction.