Unveiling compositional images of specific proteins in individual cells by LA-ICP-MS: Labelling with ruthenium red and metal nanoclusters.

BACKGROUND: Recent biological studies have demonstrated that changes can occur in the cellular genome and proteome due to variations in cell volume. Therefore, it is imperative to take cell volume into account when analyzing a target protein. This consideration becomes especially critical in experimental models involving cells subjected to different treatments. Failure to consider cell volume could obscure the studied biological phenomena or lead to erroneous conclusions. However, quantitative imaging of proteins within cells by LA-ICP-MS is limited by the lack of methods that provide the protein concentration (protein mass over cell volume) rather than just protein mass within individual cells. RESULTS: The combination of a metal tagged immunoprobe with ruthenium red (RR) labelling enables the simultaneous analysis of a specific protein and the cell volume in each cell analyzed by LA-ICP-(Q)MS. The results indicate that the CYP1B1 concentration exhibits a quasi-normally distribution in control ARPE-19 cells, whereas AAPH-treated cells reveal the presence of two distinct cell groups, responding and non-responding cells to an in vitro induced oxidative stress. The labelling of the membrane with RR and the measurement of Ru mass in each cell by LA-ICP-MS offers higher precision compared to manually delimitation of the cell perimeter and eliminates the risk of biased information, which can be prone to inter-observer variability. The proposed procedure is fast and minimizes errors in cell area assignment and offers the possibility to carry out a faster data treatment approach if just relative volumes are compared, which can be advantageous for specific applications. SIGNIFICANCE AND NOVELTY: This work presents an innovative strategy to directly study the distribution and concentration of proteins within individual cells by LA-ICP-MS. This method employs ruthenium red as a cell volume marker and Au nanoclusters (AuNCs) tagged immunoprobes to label the protein of interest. Furthermore, the proposed labelling strategy enables rapid data processing, allowing for the calculation of relative concentrations and thus facilitating the comparison across large datasets. As a proof-of-concept, the concentration of the CYP1B1 protein was quantified in ARPE-19 cells under both control and oxidative stress conditions.

Elemental mass spectrometry to study metallo-transcriptomic changes during the in vitro degeneration of the retinal pigment epithelium.

Trace elements play crucial roles in cellular biology. Their improper homeostasis may contribute to the progress of eye diseases, exacerbated during ageing. The retinal pigment epithelium (RPE) is progressively deteriorated during age-related neurodegeneration and metal homeostasis may be compromised. In this study, elemental mass spectrometry (MS) was combined with cellular and molecular biology techniques to identify changes in trace elements during the in vitro degeneration of human RPE cells. Cells were collected at 21, 91, and 133 days and processed for RNA sequencing; Ca, Na, P, Mg, and Cu quantification by flow injection analysis and inductively coupled plasma-MS; and protein analysis by immunocytochemistry. Four-month-old RPE cultures showed depigmentation, impaired barrier function, and antioxidant protection, manifesting signs of epithelial-to-mesenchymal transition. Na and P significantly increased in the cytosol of degenerated RPE cells (from 15 ± 20 to 13495 ± 638 ng·µg-1 and from 30.6 ± 9.5 to 116.8 ± 16.8 ng·µg-1, respectively). Mg decreased in both the cytosol and insoluble fraction of cells (from 2.83 ± 0.40 to 1.58 ± 0.56 ng·µg-1 and from 247.57 ± 11.06 to 30 ± 8 ng·g-1, respectively), while P and Cu decreased in the insoluble fraction after 133 days in culture (from 9471 ± 1249 to 4555 ± 985 ng·µg-1 and from 2251 ± 79 to 1054 ± 235 ng·g-1, respectively), along with changes in metal-dependent antioxidant enzymes and Cu transporters. This RPE model reflected metal homeostatic changes, providing additional perspectives on effects of metal regulation during ageing.

Determination of endogenous trace elements in extracellular vesicles secreted by an in vitro model of human retinal pigment epithelium under oxidative stress conditions using ICP-MS.

The determination of endogenous Fe, Cu and Zn in exosomes (<200 nm extracellular vesicles) secreted by an in vitro model of the human retinal pigment epithelium (HRPEsv cell line) was carried out by inductively coupled plasma - mass spectrometry (ICP-MS). Results for cells treated with 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) inducing oxidative stress (OS) conditions were compared with non-treated (control) cells in order to evaluate possible differences in the metal composition between both groups. Three sample introduction systems were tested for ICP-MS analysis: a micronebulizer and two single cell nebulization systems (as total consumption set-ups), being found one of the single cell systems (operating in bulk mode) as the most suitable. Two protocols for the isolation of exosomes from cell culture media were investigated based on differential centrifugation and precipitation with a polymer-based reagent. Transmission electron microscopy measurements showed smaller and more homogeneous sizes (15-50 nm versus 20-180 nm size range) together with a higher particle concentration for exosomes purified by precipitation compared to differential centrifugation. However, it was observed that the contribution of polymer-based protocol to the Fe, Cu and Zn blank was significant as compared to the differential centrifugation protocol. Therefore, considering the low concentrations of the evaluated endogenous elements in exosomes from the HRPEsv cell line, the polymer-based precipitation method was discarded. When comparing metal levels in samples from control versus OS-treated HRPEsv cells, results for Fe and Cu were statistically similar. However, upregulation of Zn was found during OS conditions (11 versus 34 µg L-1 in control and OS-treatment, respectively), showing Zn depletion through secretory activity induced by OS, underlying the antioxidant ability of RPE cells.

Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·µg-1 at 10 days to 0.062 ± 0.043 ng·µg-1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL-1 at 10 days to 0.056 ± 0.023 ng·mL-1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development.

Multiple heart-cutting two dimensional liquid chromatography and isotope dilution tandem mass spectrometry for the absolute quantification of proteins in human serum.

We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KH2PO4 at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (recoveries from 98 to 113%). Precisions in the range of 1-9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays.

Candidate Glaucoma Biomarkers: From Proteins to Metabolites, and the Pitfalls to Clinical Applications.

Glaucoma is an insidious group of eye diseases causing degeneration of the optic nerve, progressive loss of vision, and irreversible blindness. The number of people affected by glaucoma is estimated at 80 million in 2021, with 3.5% prevalence in people aged 40-80. The main biomarker and risk factor for the onset and progression of glaucoma is the elevation of intraocular pressure. However, when glaucoma is diagnosed, the level of retinal ganglion cell death usually amounts to 30-40%; hence, the urgent need for its early diagnosis. Molecular biomarkers of glaucoma, from proteins to metabolites, may be helpful as indicators of pathogenic processes observed during the disease's onset. The discovery of human glaucoma biomarkers is hampered by major limitations, including whether medications are influencing the expression of molecules in bodily fluids, or whether tests to validate glaucoma biomarker candidates should include human subjects with different types and stages of the disease, as well as patients with other ocular and neurodegenerative diseases. Moreover, the proper selection of the biofluid or tissue, as well as the analytical platform, should be mandatory. In this review, we have summarized current knowledge concerning proteomics- and metabolomics-based glaucoma biomarkers, with specificity to human eye tissue and fluid, as well the analytical approach and the main results obtained. The complex data published to date, which include at least 458 different molecules altered in human glaucoma, merit a new, integrative approach allowing for future diagnostic tests based on the absolute quantification of local and/or systemic biomarkers of glaucoma.

Antioxidant Defenses in the Human Eye: A Focus on Metallothioneins.

The human eye, the highly specialized organ of vision, is greatly influenced by oxidants of endogenous and exogenous origin. Oxidative stress affects all structures of the human eye with special emphasis on the ocular surface, the lens, the retina and its retinal pigment epithelium, which are considered natural barriers of antioxidant protection, contributing to the onset and/or progression of eye diseases. These ocular structures contain a complex antioxidant defense system slightly different along the eye depending on cell tissue. In addition to widely studied enzymatic antioxidants, including superoxide dismutase, glutathione peroxidase, catalase, peroxiredoxins and selenoproteins, inter alia, metallothioneins (MTs) are considered antioxidant proteins of growing interest with further cell-mediated functions. This family of cysteine rich and low molecular mass proteins captures and neutralizes free radicals in a redox-dependent mechanism involving zinc binding and release. The state of the art of MTs, including the isoforms classification, the main functions described to date, the Zn-MT redox cycle as antioxidant defense system, and the antioxidant activity of Zn-MTs in the ocular surface, lens, retina and its retinal pigment epithelium, dependent on the number of occupied zinc-binding sites, will be comprehensively reviewed.

Multiplex bioimaging of proteins-related to neurodegenerative diseases in eye sections by laser ablation - Inductively coupled plasma - Mass spectrometry using metal nanoclusters as labels.

Simultaneous determination of proteins with micrometric resolution is a significant challenge. In this study, laser ablation (LA) inductively coupled plasma - mass spectrometry (ICP-MS) was employed to quantify the distribution of proteins associated to the eye disease age-related macular degeneration (AMD) using antibodies labelled with three different metal nanoclusters (MNCs). PtNCs, AuNCs and AgNCs contain hundreds of metal atoms and were used to detect metallothionein 1/2 (MT1/2), complement factor H (CFH) and amyloid precursor protein (APP) in retina, ciliary body, retinal pigment epithelium (RPE), choroid and sclera from human cadaveric eye sections. First, the labelling of MNCs bioconjugated primary antibodies (Ab) was optimised following an immunolabelling protocol to avoid the non-specific interaction of MNCs with the tissue. Then, the LA and ICP-MS conditions were studied to obtain high-resolution images for the simultaneous detection of the three labels at the same tissue section. A significant signal amplification was found when using AuNCs, AgNCs and PtNCs labelled Ab of 310, 723 and 1194 respectively. After the characterisation of MNCs labelled immunoprobes, the Ab labelling was used for determination of MT1/2, CFH and APP in the RPE-choroid-sclera, where accumulation of extracellular deposits related to AMD was observed. Experimental results suggest that this method is fully suitable for the simultaneous detection of at least three different proteins.

Association Study of MTHFR Polymorphisms with Nonarteritic Anterior Ischemic Optic Neuropathy in a Spanish Population.

INTRODUCTION: Nonarteritic anterior ischemic optic neuropathy (NAION), painless loss of central and/or peripheral vision, is a multifactorial disease caused by insufficient blood flow through the posterior ciliary arteries to the optic nerve head. Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene, triggering hyperhomocysteinemia as a consequence of a decreased activity of the codified enzyme, have been considered to be among the risk factors of NAION. OBJECTIVE: The main aim was to study the association of the most common MTHFR genetic polymorphisms C677T and A1298C with NAION in a Spanish population. METHODS: In this case-control study, the association of the most common MTHFR polymorphisms was investigated in 94 unrelated native Spanish patients diagnosed with NAION and 204 healthy controls. Two single nucleotide polymorphisms located in the MTHFR gene, C677T (rs1801133) and A1298C (rs1801131), were analyzed by DNA sequencing and TaqMan assays. RESULTS: The allelic and genotypic frequencies of the MTHFR variants obtained in the NAION group were not significantly different when compared with the control group. A higher frequency of the C677T/A1298C genotype, codifying the nonmutated MTHFR form, was obtained in control subjects (11.27%) compared to NAION patients (4.26%), suggesting a protective effect of the wild-type protein, although this result was not conclusive considering the obtained confidence interval (CI) (95% CI: 0.13-1.06). Study of additional clinical factors including hypertension, diabetes mellitus, and dyslipidemia showed no association with a higher risk of NAION. Conversely, the clinical history of heart or cerebrovascular diseases was significantly higher in NAION patients compared to controls. Over the world, risk variants of the MTHFR gene are highly frequent, excluding African black populations, indicating a racial influence. CONCLUSIONS: The MTHFR variants did not significantly increase the risk of suffering from NAION. However, considering that individuals with at least one of the risk variants have the MTHFR enzyme with decreased activity, it cannot be ruled out that these mutations are relevant for the development of NAION in a subgroup of the population with other specific characteristics. These may include high plasma levels of homocysteine along with nutritional deficiencies including low folate or vitamin B12 and the combination of systemic and local risk factors.

Systemic Alterations of Immune Response-Related Proteins during Glaucoma Development in the Murine Model DBA/2J.

Animal models of glaucoma, a neurodegenerative disease affecting the retina, offer the opportunity to study candidate molecular biomarkers throughout the disease. In this work, the DBA/2J glaucomatous mouse has been used to study the systemic levels of several proteins previously identified as potential biomarkers of glaucoma, along the pre- to post-glaucomatous transition. Serum samples obtained from glaucomatous and control mice at 4, 10, and 14 months, were classified into different experimental groups according to the optic nerve damage at 14 months old. Quantifications of ten serum proteins were carried out by enzyme immunoassays. Changes in the levels of some of these proteins in the transition to glaucomatous stages were identified, highlighting the significative decrease in the concentration of complement C4a protein. Moreover, the five-protein panel consisting of complement C4a, complement factor H, ficolin-3, apolipoprotein A4, and transthyretin predicted the transition to glaucoma in 78% of cases, and to the advanced disease in 89%. Our data, although still preliminary, suggest that disease development in DBA/2J mice is associated with important molecular changes in immune response and complement system proteins and demonstrate the utility of this model in identifying, at systemic level, potential markers for the diagnosis of glaucoma.

The Use of Vitamins and Coenzyme Q10 for the Treatment of Vascular Occlusion Diseases Affecting the Retina.

Nutritional supplementation with antioxidants and vitamins is widely recommended in the treatment of vascular disorders affecting the retina, although there is insufficient evidence on its effectiveness. The vitamin-like compound coenzyme Q10 (CoQ10) is a nutritional supplement of current interest to treat neurodegenerative diseases. Here, we report a retrospective clinical case series study of 48 patients diagnosed with retinal vascular diseases, including non-arteritic ischemic optic neuropathy (NAION), retinal artery occlusion (RAO), and homonymous hemianopia or quadrantanopia following stroke, treated with oral supplementation with CoQ10 (100 mg per day) and vitamins. Patient follow-up was performed using the Humphrey field analyzer and 30-2 testing algorithm to determine the visual field index (VFI) and progression rates. All treated patients showed positive VFI progression rates per year: +11.5 ± 15% for NAION patients (n = 18), +22 ± 17% for RAO patients (n = 7), +9.3 ± 10.5% for hemianopia/quadrantanopia patients (n = 10), and +11 ± 21% for patients with other conditions (n = 13). The interruption of CoQ10 supplementation in one patient resulted in a pronounced decrease of the VFI, which was partially recovered when treatment was restored. This study supports the role of CoQ10 as a nutritional therapeutic agent for vascular diseases affecting the retina. Owing to decreased VFI after interruption of CoQ10, its beneficial effects may be reversible.

The association study of lipid metabolism gene polymorphisms with AMD identifies a protective role for APOE-E2 allele in the wet form in a Northern Spanish population.

PURPOSE: To elucidate the potential role of eleven single nucleotide polymorphisms (SNPs) in the most relevant lipid metabolism genes in Northern Spanish patients with age-related macular degeneration (AMD). METHODS: A case-control study of 228 unrelated native Northern Spanish patients diagnosed with AMD (73 dry and 155 wet) and 95 healthy controls was performed. DNA was isolated from peripheral blood and genotyped for the SNPs APOE rs429358 and rs7412; CTEP rs3764261; LIPC rs10468017 and rs493258; LPL rs12678919; ABCA1 rs1883025; ABCA4 rs76157638, rs3112831 and rs1800555; and SCARB1 rs5888, using TaqMan probes. An additional association study of ε2, ε3 and ε4 major isoforms of APOE gene with AMD has been carried out. RESULTS: The allele and genotype frequencies for each of the eleven sequence variants in the lipid metabolism genes did not show significant differences when comparing AMD cases and controls. Statistical analysis revealed that APOE-ε2 carrier genotypes were less frequently observed in patients with wet AMD compared to controls (5.8% versus 13.7%, respectively: p = 3.28 × 10-2 ; OR = 0.42, 95% CI: 0.19-0.95). The frequency of the allele T of rs10468017 (LIPC gene) was lower in dry AMD cases compared to controls (15.8 versus 27.9%, respectively: p = 8.4 × 10-3 OR = 0.57, 95% CI: 0.33-0.98). CONCLUSIONS: Our results suggest a protective role for APOE-ε2 allele to wet AMD in the Northern Spanish population.

Association study of high-frequency variants of MTHFR gene with retinal vein occlusion in a Spanish population.

Background: To study the association of the most common methylenetetrahydrofolate reductase (MTHFR) genetic polymorphisms C677T and A1298C with retinal vein occlusion (RVO) in a Spanish population. Methods: Case-control study involving 359 subjects, 183 unrelated native Spanish patients diagnosed with RVO, distributed in central or branch RVO, and 176 healthy controls. Two SNPs located in the gene MTHFR, C677T (rs1801133) and A1298C (rs1801131) were analyzed by DNA sequencing and TaqMan assays. Results: A high prevalence of the MTHFR variants T and C of the SNP C677T and A1298C, respectively, was observed in our population. Specifically, 88.07% of controls and 85.25% of RVO patients have at least one of these variants. However, the prevalence of these variants was not significantly different when comparing RVO patients and controls. The variant T of C677T was identified in 60.65% of RVO patients and 59.10% of control subjects, while the variant C of A1298C was present in 46.45% of RVO patients and 51.14% of controls. No association of dyslipidemia, diabetes mellitus, glaucoma, thyroid disease and renal disease with RVO was observed, while hypertension was significantly higher in the RVO patients (p < .0001). Conclusions: The MTHFR variants, T of C677T and C of A1298C, did not significantly increase the risk of suffering RVO in a Spanish population and therefore additional risk factors are contributing to the onset of the disease.

Fluorescent silver nanoclusters as antibody label in a competitive immunoassay for the complement factor H.

Silver nanoclusters (AgNCs) were investigated as labels for the development of a fluoroimmunoassay for the complement factor H (CFH). The reductive one-pot synthesis of AgNCs using lipoic acid as a ligand was optimized by varying the concentration of NaBH4, the temperature and the reaction time. The average diameter and crystal structure of the AgNCs (which display red fluorescence) were determined by HR-TEM. The silver concentration was quantified by ICP-MS. Labelling of the antibody against CFH with AgNCs was optimized. The antibody was labeled with the AgNCs without compromising the recognition capabilities of the antibody. A competitive fluoroimmunoassay was then developed. Fluorescence is measured at excitation/emission maxima of 430/660 nm. The assay has a 0.4 ng mL-1 detection limit and a linear range that extends from 1.2 to 23 ng mL-1. The results compare favorably with those obtained by a commercial ELISA kit. The method was applied to the determination of CFH in spiked human serum. Graphical abstract Schematic presentation of the method for the determination of complement factor H (CFH) protein in human blood by using a CFH-antibody labelled with fluorescent silver nanoclusters.

Isotopically Enriched Tracers and Inductively Coupled Plasma Mass Spectrometry Methodologies to Study Zinc Supplementation in Single-Cells of Retinal Pigment Epithelium in Vitro.

Mass spectrometry-based techniques, such as inductively coupled plasma mass spectrometry (ICPMS) and laser ablation (LA) ICPMS, combined with an isotope pattern deconvolution mathematical tool are proposed for a better understanding of supplementation studies in cultured cells. An in vitro model of human retinal pigment epithelium (HRPEsv) cells was treated with different concentrations (0-150 µm Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with t68ZnSO4 or t70Zn-gluconate alone and in combination (1:1 molar ratio) were investigated to evaluate the exogenous contribution and distribution of Zn in the treated cells. In order to obtain not only the Zn concentration for a cell population (mineralized cells) but also single cell information about the contribution of exogenous Zn and their distribution within micrometer cells structures, LA-ICPMS was employed to directly analyze cryopreserved cells. natZn, t68Zn, and t70Zn molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being t68Zn and t70Zn molar fractions close to 1 in the cell nuclei. Under the selected experimental conditions tested (24 h treatments), no significant differences were obtained in the Zn distribution depending on its chemical form.

The Zinc-Metallothionein Redox System Reduces Oxidative Stress in Retinal Pigment Epithelial Cells.

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques. The Zn-MT system was induced in an in vitro model of RPE cells and determined by elemental mass spectrometry with enriched isotopes and mathematical calculations. Induced-oxidative stress was quantified using fluorescent probes. We observed that 25, 50 or 100 µM of zinc induced Zn-MT synthesis (1.6-, 3.6- and 11.9-fold, respectively), while pre-treated cells with zinc (25, 50, and 100 µM) and subsequent 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) treatment increased Zn-MT levels in a lesser extent (0.8-, 2.1-, 6.1-fold, respectively), exerting a stoichiometric transition in the Zn-MT complex. Moreover, AAPH treatment decreased MT levels (0.4-fold), while the stoichiometry remained constant or slightly higher when compared to non-treated cells. Convincingly, induction of Zn-MT significantly attenuated oxidative stress produced by free radicals' generators. We conclude that the stoichiometry of Zn-MT plays an important role in oxidative stress response, related with cellular metal homeostasis.

Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies.

The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., 68ZnSO4), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.

Metallothionein polymorphisms in a Northern Spanish population with neovascular and dry forms of age-related macular degeneration.

BACKGROUND: To elucidate the potential role of single nucleotide polymorphisms (SNPs) in the metallothionein (MT) genes in Northern Spanish patients with age-related macular degeneration (AMD). METHODS: A total of 130 unrelated Northern Spanish natives diagnosed with AMD (46 dry, 35 neovascular, and 49 mixed) and 96 healthy controls, matched by age and ethnicity, were enrolled in a case-control study. DNA was isolated from peripheral blood and genotyped for 14 SNPs located at 5 MT genes (MT1A: rs11076161, rs 11640851, rs8052394, and rs7196890; MT1B: rs8052334, rs964372, and rs7191779; MT1M: rs2270836 and rs9936741; MT2A: rs28366003, rs1610216, rs10636, and rs1580833; MT3: rs45570941) using TaqMan probes. The association study was performed using the HaploView 4.0 software. RESULTS: The allelic and genotypic frequencies analysis revealed that rs28366003 at MT2A gene is significantly associated with dry AMD. The frequency of genotype AG was significantly higher in dry AMD than in control cases (p = 2.65 × 10-4; AG vs. AA) conferring more than ninefold increased risk to dry AMD (OR = 9.39, 95% CI: 2.11-41.72), whereas the genotype AA confers disease protection (OR = 0.82, 95% CI: 0.71-0.95). No statistically significant differences were observed between AMD subjects and controls in the rest of the 14 SNPs analyzed. CONCLUSIONS: The present study is the first to investigate the potential association of SNPs at MT genes with susceptibility to AMD. We found a significant association of SNP rs28366003 at MT2A gene with susceptibility to the dry form of AMD in a Northern Spanish population.

Bioimaging of metallothioneins in ocular tissue sections by laser ablation-ICP-MS using bioconjugated gold nanoclusters as specific tags.

An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma - mass spectrometry (ICP-MS). Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 µm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 µm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method. Graphical abstract Gold nanoclusters (AuNCs) conjugated to a primary specific antibody serve as a label for amplified bioimaging of metallothioneins (MTs) by laser ablation coupled to inductively coupled plasma - mass spectrometry (ICP-MS) in human ocular tissue sections.

LOXL1 gene variants and their association with pseudoexfoliation glaucoma (XFG) in Spanish patients.

BACKGROUND: LOXL1 gene is the most important genetic risk factor known so far for pseudoexfoliation glaucoma (XFG). Our purpose was to evaluate the potential association of individual genetic variants of the lysyl oxidase-like 1 (LOXL1) gene and haplotypes with XFG in Spanish patients. METHODS: Blood samples were collected from a total of 105 Spanish patients with XFG and 200 healthy controls. The entire LOXL1 gene along with the promoter, coding and non-coding regions including the 5'- and 3'-untranslated regions, were sequenced using next-generation sequencing in 99 XFG patients. SNPs rs16958477 (promoter), rs1048661 (exon 1), rs3825942 (exon 1), rs2165241 (intron 1) and rs3522 (exon 7) in LOXL1 were genotyped by restriction fragment-length polymorphism (RFLP) in all Spanish control participants and in six additional XFG patients, and a case-control association study was performed. Comparisons of the allelic and genotypic frequencies were performed using standard χ(2) test with Bonferroni and Pearson corrections. Logistic regression analyses were permormed using Sigmaplot v11. Haplotypes frequencies were performed using HaploView 4.0. RESULTS: Sequencing of the LOXL1 gene in XFG participants identified a total of 212 SNPs, of which 49 exhibited allelic frequencies with significant differences between cases and controls, and 66 were not previously described. The allele frequencies of SNPs rs16958477, rs1048661, rs3825942, rs2165241, were significantly associated with an increased risk for XFG, however the SNP rs3522 was not. The haplotype frequencies of SNPs rs16958477, rs1048661, rs3825942 and rs2165241 and their association with XFG indicated that the CGGT haplotype, containing all four risk alleles, and the AGGT haplotype, which carries the protective allele of rs16958477 and three risk alleles of the other three SNPs, were significantly associated with XFG (p = 4.5×10(-6), and p = 8.8×10(-6)), conferring more than 2-fold increased disease susceptibility. CONCLUSIONS: SNPs of the LOXL1 gene are associated with XFG in the Spanish population. This information adds new support to the distinct risk association frequencies of LOXL1 alleles with XFG in Western European and Asian populations. Identification and validation of additional SNPs along the entire LOXL1 gene of XFG cases may provide insightful information on their potential role in the pathogenesis of this disease.