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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.
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Bilberry (Vaccinium myrtillus) is a commercially important wild berry species, which accumulates high amounts of polyphenols, particularly anthocyanins, in the skin and flesh. Whilst a number of studies have quantified these phytochemicals in intact ripe bilberry fruit, we extend the current knowledge by investigating the spatial distribution of anthocyanin-associated polyphenols in fruit tissue, and study their links with primary metabolism during ripening. To address this, we used LC-MS and mass spectrometry imaging to measure and map primary and secondary metabolites in fruit. Correlation analysis showed that five sugars displayed strong positive correlations with anthocyanin accumulation, whereas all amino acids were negatively correlated. The accumulation patterns of polyphenols correlated in fruit skin and flesh, but altered with development. Finally, spatial segmentation analysis revealed that the chemical signatures of ripening first appear at defined regions under the skin and rapidly expand to encompass the entire fruit at the eating-ripe stage.
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Vaccinium myrtillus , Antocianinas , Frutas/química , Polifenóis/análiseRESUMO
Ocular drug absorption after eye drop instillation has been widely studied, but partitioning phenomena and spatial drug distribution are poorly understood. We investigated partitioning of seven beta-blocking drugs in corneal epithelium, corneal stroma, including endothelium and conjunctiva, using isolated porcine tissues and cultured human corneal epithelial cells. The chosen beta-blocking drugs had a wide range (-1.76-0.79) of n-octanol/buffer solution distribution coefficients at pH 7.4 (Log D7.4). In addition, the ocular surface distribution of three beta-blocking drugs was determined by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) after their simultaneous application in an eye drop to the rabbits in vivo. Studies with isolated porcine corneas revealed that the distribution coefficient (Kp) between the corneal epithelium and donor solution showed a positive relationship and good correlation with Log D7.4 and about a 50-fold range of Kp values (0.1-5). On the contrary, Kp between corneal stroma and epithelium showed an inverse (negative) relationship and correlation with Log D7.4 based on a seven-fold range of Kp values. In vitro corneal cell uptake showed a high correlation with the ex vivo corneal epithelium/donor Kp values. Partitioning of the drugs into the porcine conjunctiva also showed a positive relationship with lipophilicity, but the range of Kp values was less than with the corneal epithelium. MALDI-IMS allowed simultaneous detection of three compounds in the cornea, showed data in line with other experiments, and revealed uneven spatial drug distribution in the cornea. Our data indicate the importance of lipophilicity in defining the corneal pharmacokinetics and the Kp values are a useful building block in the kinetic simulation models for topical ocular drug administration.
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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.
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Marcação por Isótopo/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biologia Computacional/instrumentação , Biologia Computacional/métodos , Desenho de Equipamento , Glucose/análise , Glucose/metabolismo , Humanos , Marcação por Isótopo/instrumentação , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentaçãoRESUMO
Huntington's disease (HD) is a fatal disorder associated with germline trinucleotide repeat expansions in the HTT gene and characterised by striatal neurodegeneration. No efficacious interventions are available for HD, highlighting a major unmet medical need. The molecular mechanisms underlying HD are incompletely understood despite its monogenic aetiology. However, direct interactions between HTT and membrane lipids suggest that lipidomic perturbations may be implicated in the neuropathology of HD. In this study, we employed matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) to generate a comprehensive, unbiased and spatially resolved lipidomic atlas of the caudate nucleus (CN) in human post-mortem tissue from neurologically normal (n = 10) and HD (n = 13) subjects. Fourier transform-ion cyclotron resonance mass spectrometry and liquid chromatography-tandem mass spectrometry were used for lipid assignment. Lipidomic specialisation was observed in the grey and white matter constituents of the CN and these features were highly conserved between subjects. While the majority of lipid species were highly conserved in HD, compared to age-matched controls, CN specimens from HD cases in our cohort spanning a range of neuropathological grades showed a lower focal abundance of the neuroprotective docosahexaenoic and adrenic acids, several cardiolipins, the ganglioside GM1 and glycerophospholipids with long polyunsaturated fatty acyls. HD cases showed a higher focal abundance of several sphingomyelins and glycerophospholipids with shorter monosaturated fatty acyls. Moreover, we demonstrate that MALDI-IMS is tractable as a primary discovery modality comparing heterogeneous human brain tissue, provided that appropriate statistical approaches are adopted. Our findings support further investigation into the potential role of lipidomic aberrations in HD.
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Núcleo Caudado/metabolismo , Núcleo Caudado/patologia , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Idoso , Cromatografia Líquida/métodos , Estudos de Coortes , Feminino , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem/métodosRESUMO
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.
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The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens, and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream), and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours. In contrast, both uniform and spatially defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three-dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.
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Olho/diagnóstico por imagem , Olho/metabolismo , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Humor Aquoso/diagnóstico por imagem , Humor Aquoso/metabolismo , Peixes , Liofilização , Glutationa/análise , Camundongos Endogâmicos C57BL , Fosfolipídeos/análise , Coelhos , Ratos Wistar , Corpo Vítreo/diagnóstico por imagem , Corpo Vítreo/metabolismoRESUMO
Lens is the avascular tissue in the eye between the aqueous humor and vitreous. Drug binding to the lens might affect ocular pharmacokinetics, and the binding may also have a pharmacological role in drug-induced cataract and cataract treatment. Drug distribution in the lens has been studied in vitro with many compounds; however, the experimental methods vary, no detailed information on distribution between the lens sublayers exist, and the partition coefficients are reported rarely. Therefore, our objectives were to clarify drug localization in the lens layers and establish partition coefficients for a wide range of molecules. Furthermore, we aimed to illustrate the effect of lenticular drug binding on overall ocular drug pharmacokinetics. We studied the distribution of 16 drugs and three fluorescent dyes in whole porcine lenses in vitro with imaging mass spectrometry and fluorescence microscopy techniques. Furthermore, we determined lens/buffer partition coefficients with the same experimental setup for 28 drugs with mass spectrometry. Finally, the effect of lenticular binding of drugs on aqueous humor drug exposure was explored with pharmacokinetic simulations. After 4 h, the drugs and the dyes distributed only to the outermost lens layers (capsule and cortex). The lens/buffer partition coefficients for the drugs were low, ranging from 0.05 to 0.8. On the basis of the pharmacokinetic simulations, a high lens-aqueous humor partition coefficient increases drug exposure in the lens but does not significantly alter the pharmacokinetics in the aqueous humor. To conclude, the lens seems to act mainly as a physical barrier for drug distribution in the eye, and drug binding to the lens affects mainly the drug pharmacokinetics in the lens.
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Corantes Fluorescentes/farmacocinética , Cristalino/efeitos dos fármacos , Absorção Ocular/fisiologia , Preparações Farmacêuticas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/metabolismo , Área Sob a Curva , Soluções Tampão , Corantes Fluorescentes/química , Cristalino/metabolismo , Microscopia de Fluorescência , Peso Molecular , Absorção Ocular/efeitos dos fármacos , Concentração Osmolar , Preparações Farmacêuticas/química , Suínos , Distribuição Tecidual , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
Tryptophan-derived UV filters are predominantly found in the lenses of primates and humans. While protective against UV radiation, aging alters the complement and spatial distributions of human lens UV filters, and a role for UV filters has been suggested in age-related cataract formation. To establish how the spatial distributions of UV filters change in normal human lens aging, matrix assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) was utilised to map the locations and relative abundance of multiple UV filters simultaneously. Frozen human lenses were cryosectioned axially, and the 20⯵m-thick sections coated with MALDI matrix via robotic sprayer and analysed using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS. While signal for many UV filters was detected throughout the lenses, signal intensity was generally highest in the central (embryonic) nucleus and decreased uniformly in outer (foetal, juvenile, adult) nuclear and cortical regions, and many UV filter signals declined with age. In contrast, two antioxidant-conjugated UV filters (Cys-3-OHKG and GSH-3-OHKG) were restricted to the lens nucleus and their relative signal increased with increasing lens age. The enhanced spatial resolution of MALDI-IMS over manual trephine dissection techniques and its multiplex capability allowed the spatial relationships between lens UV filters to be established and explored in relation to aging. Together these results confirmed that the complement of UV filters in each lens is dynamic and undergoes significant age-related changes. In the future, this information could be used to compare with other lens biomolecule changes to better understand the lens aging process and age-related cataract formation.
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Envelhecimento/fisiologia , Cristalinas/metabolismo , Cristalino/metabolismo , Raios Ultravioleta , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Fourier , Glucosídeos/metabolismo , Glutationa/metabolismo , Humanos , Cinurenina/metabolismo , Núcleo do Cristalino/metabolismo , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The human subventricular zone (SVZ) has a defined cytological and neurochemical architecture, with four constituent laminae that act in concert to support its neurogenic activity. Lipidomic specialisation has previously been demonstrated in the neurologically normal human SVZ, with enrichment of functionally important lipid classes in each lamina. The SVZ is also responsive to neurodegenerative disorders, where thickening of the niche and enhanced proliferation of resident cells were observed in Huntington's disease (HD) brains. In this study, we hypothesised lipidomic changes in the HD SVZ. Using matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry, this analysis shows differences in the lipidomic architecture in the post-mortem Vonsattel grade III cases. Relative to matched, neurologically normal specimens (N = 4), the lipidomic signature of the HD SVZ (N = 4) was characterized by loss of sulfatides and triglycerides in the myelin layer, with an ectopic and focal accumulation of sphingomyelins and ceramide-1-phosphate observed in this lamina. A striking loss of lipidomic patterning was also observed in the ependymal layer, where the local abundance of phosphatidylinositols was significantly reduced in HD. This comprehensive spatially resolved lipidomic analysis of the human HD SVZ identifies alterations in lipid architecture that may shed light on the mechanisms of SVZ responses to neurodegeneration in HD. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
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Doença de Huntington/patologia , Ventrículos Laterais/metabolismo , Ventrículos Laterais/patologia , Metabolismo dos Lipídeos , Adulto , Idoso , Autopsia , Feminino , Análise de Fourier , Humanos , Lipídeos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Monoéster Fosfórico Hidrolases , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingomielinas , Sulfoglicoesfingolipídeos , TriglicerídeosRESUMO
The subventricular zone is a key site of adult neurogenesis and is also implicated in neurodegenerative diseases and brain cancers. In the subventricular zone, cell proliferation, migration and differentiation of nascent stem cells and neuroblasts are regulated at least in part by lipids. The human subventricular zone is distinctly layered and each layer contains discrete cell types that support the processes of neuroblast migration and neurogenesis. We set out to determine the lipid signatures of each subventricular layer in the adult human brain (n = 4). We utilised matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry and liquid chromatography-mass spectrometry to characterise the lipidome of the subventricular zone, with histology and microscopy used for identifying anatomical landmarks. Our findings showed that the subventricular zone was rich in sphingomyelins and phosphatidylserines but deficient in phosphatidylethanolamines. The ependymal layer had an abundance of phosphatidylinositols, whereas the myelin layer was rich in sulfatides and triglycerides. The hypocellular layer showed enrichment of sphingomyelins. No discrete lipid signature was seen in the astrocytic ribbon. The biochemical functions of these lipid classes are consistent with the localisation we observed within the SVZ. Our study may, therefore, shed new light on the role of lipids in the regulation of adult neurogenesis.
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Ventrículos Laterais/metabolismo , Metabolismo dos Lipídeos , Células-Tronco Neurais/metabolismo , Neurogênese , Adulto , Astrócitos/metabolismo , Diferenciação Celular , Proliferação de Células , Epêndima/metabolismo , Humanos , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esfingomielinas/metabolismo , Substância Gelatinosa/metabolismo , Triglicerídeos/metabolismoRESUMO
Azines are important in many extraterrestrial environments, from the atmosphere of Titan to the interstellar medium. They have been implicated as possible carriers of the diffuse interstellar bands in astronomy, indicating their persistence in interstellar space. Most importantly, they constitute the basic building blocks of DNA and RNA, so their chemical reactivity in these environments has significant astrobiological implications. In addition, N and O atoms are widely observed in the ISM and in the ionospheres of planets and moons. However, the chemical reactions of molecular anions with abundant interstellar and atmospheric atomic species are largely unexplored. In this paper, gas-phase reactions of deprotonated anions of benzene, pyridine, pyridazine, pyrimidine, pyrazine, and s-triazine with N and O atoms are studied both experimentally and computationally. In all cases, the major reaction channel is associative electron detachment; these reactions are particularly important since they control the balance between negative ions and free electron densities. The reactions of the azine anions with N atoms exhibit larger rate constants than reactions of corresponding chain anions. The reactions of azine anions with O atoms are even more rapid, with complex product patterns for different reactants. The mechanisms are studied theoretically by employing density functional theory; spin conversion is found to be important in determining some product distributions. The rich gas-phase chemistry observed in this work provides a better understanding of ion-atom reactions and their contributions to ionospheric chemistry as well as the chemical processing that occurs in the boundary layers between diffuse and dense interstellar clouds.
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We have studied the gas-phase reactions of CF(+) with 24 neutral species. Reaction rate constants and product branching fractions are measured at 298 K using a flowing afterglow-selected ion flow tube. Experimental work is supported by computational chemistry calculations to provide insight into the reactivity of classes of neutral molecules. Reactions of CF(+) with small triatomic species and oxygen-containing organic molecules produce the stable molecule CO. The product branching fractions are discussed, and the potential energy surfaces for a few representative reactions are examined. CF(+) is highly reactive with complex molecules and will likely be destroyed in dense environments in the interstellar medium. However, the lack of reactivity with small diatomic molecules will likely enable its survival in diffuse regions.