Héctor González-Iglesias

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS

AbstractLaser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn. A normalisation strategy was also carried out to correct matrix effects, lack of tissue homogeneity and/or instrumental drifts using a thin gold film deposited on the sample surface. Quantitative images of cryo-sections of human eye lenses analysed by LA-ICP-MS revealed a homogeneous distribution of Fe, Cu and Zn in the nuclear region and a slight increase in Fe concentration in the outer cell layer (i.e. lens epithelium) at the anterior pole. These results were assessed also by isotope dilution mass spectrometry, and Fe, Cu and Zn concentrations determined by ID-ICP-MS in digested samples of lenses and lens capsules. FigureDepth profiling analysis and quantitative imaging analyses of Fe, Cu and Zn in eye lens sections by LA-ICP-MS using matrix-matched laboratory standards for external calibration and 197Au+ as internal standard

Comparative proteomic study in serum of patients with primary open-angle glaucoma and pseudoexfoliation glaucoma.

UNLABELLED Alterations in the sera proteins between patients with Primary Open-Angle Glaucoma (POAG), Pseudoexfoliation Glaucoma (PEXG), and healthy controls were identified through a proven approach utilizing equalization of high-abundance serum proteins with ProteoMiner™, two-dimensional fluorescent difference gel electrophoresis (2D-DIGE), MALDI-TOF/TOF, and nanoLC-MS-MS. Quantitative immunoassays of the 17 most-differentially-altered proteins identified in this analysis confirmed that they were also over expressed in the intact serum of newly recruited glaucoma patients. Overall, this report identifies a panel of candidates for glaucoma biomarkers and supports their further validation in large population studies. Additionally, functional pathway analysis of these candidate proteins suggested that they are part of a network linked to regulating immune and inflammatory-related processes. The data have been deposited to the ProteomeXchange with identifier PXD000198. BIOLOGICAL SIGNIFICANCE POAG and PEXG are major causes of age-related blindness in the world; however, treatment can be very effective if they are identified early on in the progression. Genetic linkage studies can only explain a limited number of cases, suggesting that these forms of glaucoma are multigenic in nature. Other important factors, such as modifier genes, epigenetic influences, environmental and dietary agents, and inflammatory and oxidative effects are also believed to affect the development of these diseases. The characterization of metabolic and/or proteins changes, for example in bodily fluids, before the clinical manifestation of glaucoma is of considerable relevance for its early diagnosis. In the present work, identification of over-expressed proteins in serum of glaucoma patients (POAG and PEXG) linked to immune and inflammatory processes supports the finding that changes in these pathways also manifest systemically in patients with these pathologies. This study provides a new basis to validate the identified proteins as biomarkers of glaucoma in a large-scale-multiplexed screening in sera.

The stoichiometric transition from Zn6Cu1-Metallothionein to Zn7-Metallothionein underlies the up-regulation of Metallothionein (MT) expression: quantitative analysis of MT-metal load in eye cells

Background: Metallothioneins (MTs) and immune responses are induced by exogenous zinc. Results: MTs are abundant and differentially expressed in human ocular tissues. A bioanalytical hybrid technique provided absolute measurements of MT-metal loads in cultured cells. Conclusion: Zinc stimulated the transition of Zn6Cu1-MT to Zn7-MT and blocked proinflammatory cytokines in cultured eye cells. Significance: Zn7-MT species may confer protective antioxidative effect. We examined the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based analysis. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concentration of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were determined by mass spectrometry. The total concentration of MTs was 0.24 ± 0.03 μm and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 μm. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 μm. The average metal stoichiometry of MTs was Zn6Cu1-MT, and after exposure to the different treatments, it changed to Zn7-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide molecular and analytical evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.

Nutritional iron supplementation studies based on enriched 57Fe, added to milk in rats, and isotope pattern deconvolution‐ICP‐MS analysis

Enriched stable iron isotopes in combination with isotope pattern deconvolution and ICP‐MS have been used to study the absorption and bioavailability of iron from supplemented formula milk administrated to lactating rats. The use of two enriched stable isotope tracers, one as the metabolic tracer (here 57Fe) and the other (54Fe) as quantitation tracer, is shown to provide quantitative data about endogenous and exogenous (supplemented) total Fe distribution in rat feces, urine, red blood cells (RBCs), serum, liver, and kidney. The proposed analytical methodology was validated using reference materials (serum, urine, and liver) spiked with both 54Fe and 57Fe. Quantitative information about iron absorption/bioavailability and/or metabolism can be obtained from the amounts of endogenous and exogenous iron found in the tissues and fluids analyzed, and about its kinetic after 2 weeks of iron supplementation. The obtained results are discussed in terms of iron exchanged and its half‐life in lactating rats and the observed iron levels in serum, RBCs, liver, and kidney comparing nonsupplemented rats and maternal feed rats.

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

To elucidate the potential role of single‐nucleotide polymorphisms (SNPs) in complement factor H (CFH) gene in Northern Spanish patients with age‐related macular degeneration (AMD).

Quantitative distribution of Zn, Fe and Cu in the human lens and study of the Zn–metallothionein redox system in cultured lens epithelial cells by elemental MS

The human lens is constantly subjected to exogenous and endogenous stressors, leading to oxidative cellular damage and, with time, to cataract formation. Metallothioneins (MTs) are a group of important enzymes that use metals (i.e., Zn and Cu) to protect tissues from the deleterious effects of free radicals associated with oxidative stress. This work combines elemental mass spectrometry with bio-analytical methodologies to determine (i) the total amount, the quantitative speciation and the cellular distribution of trace elements (i.e., Zn, Fe, and Cu) in human lenses and their corresponding capsules; and (ii) the effects of “exogenous” metal (i.e., 68ZnSO4, isotopically enriched in 68Zn) and stressor (i.e., IL-1α) on the zinc–MT redox system in cultured human lens epithelial cells (HLEsv) in vitro. Of all the elements analyzed, Zn was the most abundant, and it was equally present in both the capsule (9.7 ± 2.5 μg g−1 tissue) and the lens (9.5 ± 1.2 μg g−1 tissue). In contrast, Fe was found to be more than 6-fold more abundant in the capsule (1.6 ± 0.4 μg g−1) than in the lens (0.2 ± 0.1 μg g−1). Zinc in the lens is mainly associated with high molecular mass proteins, whereas in the capsule it is mostly bound to low and medium molecular mass proteins. The localization of Zn, Cu and MTs in the lens showed their preferential co-distribution in the lens epithelial cell layer, underneath the anterior capsule. Exogenous Zn is capable of inducing a stoichiometric change in MT proteins from Zn3–MT to Zn7–MT within lens epithelial cells in vitro, which may be related to their antioxidant capacity.

Iron bioavailability from supplemented formula milk: effect of lactoferrin addition

PurposeIn this work, the absorption and/or bioavailability of iron from two chemical species, 57Fe-Lf (apo-lactoferrin) complex and 57FeSO4 at low and high dose, and in Lf excess were investigated in lactating wistar rats.MethodsThe methodology used is based on the use of stable isotopes in combination with the approach “isotope pattern deconvolution” and ICP-MS for detection. This approach provides quantitative information about exogenous (57Fe) and endogenous iron (natFe) distribution in fluids and tissues in the iron-supplemented rat groups.ResultsThe observed results with supplemented rats were compared with those found in rats receiving maternal feeding. Interestingly, differences were found between groups in iron for transport and storage compartments, but not in the functional one, depending upon the dose of iron administered and the chemical species.ConclusionConsidering the results obtained, supplementation with iron salts in excess of Lf appears to be the best way of iron supplementation of formula milk.

Elemental and molecular mass spectrometry for integrated selenosugar speciation in liver and kidney tissues of maternal feeding and supplemented rats

The development of methods assessing the nutritional value and metabolism of selenium are of growing interest. In this work, the integrated used of a methodology based on HPLC-isotope pattern deconvolution (IPD)-ICP-MS and a molecular tandem mass spectrometric technique, such as HPLC-APCI-MS/MS, in the selected reaction monitoring (SRM) mode, was applied to quantify and identify the selenosugar SeGalNAc in liver and kidney tissues of lactating rats fed with formula milk supplemented with 77selenite. The SeGalNAc levels found in liver and kidney of maternal feeding rats (kidney 23 ± 3 ng g−1; liver 26 ± 3 ng g−1) were much higher than those found in supplemented (kidney 9.9 ± 0.3 ng ng−1; liver 10 ± 4 ng g−1) and non-supplemented rats (kidney 3.4 ± 0.5 ng g−1; liver 4 ± 1 ng g−1). The percentage of exogenous SeGalNAc for the supplemented group in kidney and liver reached 32 ± 1% and 30 ± 10%, respectively. Conversely, the percentage of exogenous selenium in high molecular weight selenospecies reached values higher than 58%. Thereby, most exogenous selenium seems to be incorporated into the synthesis of selenoproteins, indicating that the turnover rates are different for the different species and their synthesis might occur in different tissue compartments. Finally, the identification of SeGalNAc was confirmed in liver and, for the first time to our knowledge, in the kidney cytosol of maternal feeding and supplemented rats. Overall, we expect that the judicious use of elemental and molecular mass spectrometry tools to obtain integrated quantitative Se speciation information might help to expand our knowledge of selenium metabolism.

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

BackgroundLOXL1 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.MethodsBlood 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 χ2 test with Bonferroni and Pearson corrections. Logistic regression analyses were permormed using Sigmaplot v11. Haplotypes frequencies were performed using HaploView 4.0.ResultsSequencing 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.ConclusionsSNPs 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.

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.