María Luisa Fernández-Sánchez

Organised surfactant assemblies in analytical atomic spectrometry

Abstract The use of surfactant-based organised assemblies in analytical atomic spectroscopy is extensively and critically reviewed along three main lines: first, the ability of organised media to enhance detection of atomic spectroscopic methods by favourable manipulation of physical and chemical properties of the sample solution second, the extension of separation mechanisms by resorting to organised media and third a discussion of synergistic combinations of liquid chromatography separations and atomic detectors via the use of vesicular mobile phases. Changes in physical properties of sample solutions aspirated in atomic spectrometry by addition of surfactants can be advantageously used in at least four different ways: (i) to improve nebulisation efficiency; (ii) to enhance wettability of solid surfaces used for atomisation; (iii) to improve compatibility between aqueous and organic phases; and (iv) to achieve good dispersion of small particles in “slurry” techniques. Controversial results and statements published so far are critically discussed. The ability of surfactant-based organised assemblies, such as micelles and vesicles, to organise reactants at the molecular level has also been applied to enhance the characteristics of chemical generation of volalite species of metals and semi-metals (e.g., hydride or ethylide generation of As, Pb, Cd, Se, Sn, and cold vapour Hg generation) used in atomic methods. Enhancements in efficiency/transport of volatile species, increases in the reaction kinetics, stabilisation of some unstable species and changes in the selectivity of the reactions by surfactants are dealt with. Non-chromatographic cloud-point separations to design pre-concentration procedures with subsequent metal determination by atomic methods are addressed along with chromatographic separations of expanded scope by addition of surfactants to the conventional aqueous mobile phases of reversed-phase high-performance liquid chromatography. Finally, the synergistic effect of using vesicles to improve both the separation capabilities of reversed-phase HPLC and the detectability of atomic detectors by on-line vesicular hydride generation is described. In particular, the possible separation mechanisms responsible for micellar and vesicular mobile phases in reversed-phase chromatographies are analysed and compared. The possible effect of modification of stationary phases by monomers of the surfactants should also be taken into account. The application of such on-line couplings to develop new hybrid approaches to tackle modern problems of trace element speciation for As, Hg, Se, and Cd completes this revision of the present interface between analytical atomic spectroscopy and surfactant-based organised assemblies.

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.

Selenium levels and Glutathione peroxidase activity in the plasma of patients with type II diabetes mellitus

Selenium, an essential trace element, is involved in the complex system of defense against oxidative stress through selenium-dependent glutathione peroxidases (GPx) and other selenoproteins. Because of its antioxidant properties, selenium or its selenospecies at appropriate levels could hinder oxidative stress and so development of diabetes. In this vein, quantitative speciation of selenium in human plasma samples from healthy and diabetic patients (controlled and non-controlled) was carried out by affinity chromatography (AF) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) and isotope dilution analysis (IDA). Similarly, it is well known that patients with diabetes who exhibit poor control of blood glucose show a decreased total antioxidant activity. Thus, we evaluated the enzymatic activity of GPx in diabetic and healthy individuals, using the Paglia and Valentine enzymatic method, observing a significant difference (p<0.05) between the three groups of assayed patients (healthy (n=24): 0.61±0.11U/ml, controlled diabetic (n=38): 0.40±0.12U/ml and non-controlled diabetic patients (n=40): 0.32±0.09U/ml). Our results show that hyperglycemia induces oxidative stress in diabetic patients compared with healthy controls. What is more, glycation of GPx experiments demonstrated that it is the degree of glycation of the selenoenzyme (another species of the Se protein) what actually modulates its eventual activity against ROS in type II diabetes mellitus patients.

Total zinc quantification by inductively coupled plasma-mass spectrometry and its speciation by size exclusion chromatography–inductively coupled plasma-mass spectrometry in human milk and commercial formulas: Importance in infant nutrition

This paper summarises results of zinc content and its speciation in human milk from mothers of preterm and full-term infants at different stages of lactation and from synthetic formula milks. Human milk samples (colostrum, 7th, 14th, and 28th day after delivery) from Spanish and Brazilian mothers of preterm and full-term infants (and also formula milks) were collected. After adequate treatment of the sample, total Zn was determined, while speciation analysis of the Zn was accomplished by size exclusion chromatography coupled online with the ICP-MS. It is observed that total zinc content in human milk decreases continuously during the first month of lactation, both for preterm and full term gestations. All infant formulas analysed for total Zn were within the currently legislated levels. For Zn speciation analysis, there were no differences between preterm and full term human milk samples. Moreover Zn species elute mainly associated with immunoglobulins and citrate in human milk whey. Interestingly the speciation in formula milk whey turned out to be completely different as the observed Zn(2+) was bound almost exclusively to low molecular weight ligands (citrate) and only comparatively very low amounts of the metal appeared to be associated with higher mass biomolecules (e.g. proteins).

Quantitative bioimaging of Ca, Fe, Cu and Zn in breast cancer tissues by LA-ICP-MS

In recent years, several studies have shown that concentrations of essential trace elements naturally present in breast tissues (e.g. Ca, Fe, Cu and Zn) may be significantly increased in breast cancer tissues. This is not surprising because essential elements are responsible for a great number of metabolic and biological processes. The essential trace elements may play some major functions in life: stabilizers, elements of structure, elements for hormonal function and cofactors in enzymes. In any case, the role of trace elements in breast cancer is complex, because it affects many types of molecules, cells and tissues. The combination of analytical and immunehistochemical assays is crucial for better understanding of the role of essential trace elements in promoting tumor growth and migration. Bioimaging analytical techniques with adequate spatial resolution are today of crucial interest to investigate the spatial distribution of trace elements and correlate them with histological aspects in breast tissues. In this vein, in this particular study the application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used for the first time to investigate the actual distribution of the essential trace bioelements (Ca, Fe, Cu and Zn) in breast cancer tissues, and its possible application for tumor diagnostic and prognostic purposes. As has been demonstrated in this study, the levels of Ca, Fe, Cu and Zn in the tumor area are significantly higher than the levels found in the non-tumor one, as well as, a heterogeneous distribution of the investigated metals.

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.

Speciation and isotope pattern deconvolution for inductively coupled plasma-mass spectrometry quantitative studies of mineral metabolism and supplementation

Human breast milk can be considered as “ideal” food for the correct development of newborn babies and, for those that are not breast-fed, formula milk has to be used instead. Ideally, the composition of such formula milk preparations should closely resemble that of maternal human milk. Considerable differences between both in the total content of trace elements such as Fe, Cu, Se, Zn, and I and in their chemical form in both milk types have been demonstrated. Speciation analysis in milk whey was carried out first by high-performance liquid chromatography (HPLC) with inductively coupled plasma-mass spectrometry (ICP-MS) elemental detection and showed that the observed element distribution patterns were very different in the investigated human and formula milks. Using complementary molecular mass techniques (i.e., MALDI-TOF), the identity and chemical characterization of some biomolecules (e.g., protein) with which metals are associated in each fraction was also established (by a typical heteroatom-tagged proteomics protocol). Attempts to assess the nutritional value of elemental supplements in formula milk with the aid of quantitative chemical speciation, using stable isotopes in combination with ICP-MS and isotope pattern deconvolution (IPD), proved to be successful to differentiate and quantify endogenous (natural) and exogenous (supplemented) Se or Fe trace levels. In particular, the application of such ICP-MS based techniques to study Se bioavailability from formula milk and metabolism in Se-supplemented lactating rats is discussed in detail. Quantification of selenospecies of endogenous (natural) and exogenous (supplement) Se in rat’s urine is demonstrated and relevant information on possible Se biotransformations and its final catabolism from such results is discussed.

In vivo study of the effect of lactoferrin on iron metabolism and bioavailability from different iron chemical species for formula milk fortification

Iron fortification in infant formulas is a common practice for providing iron to newborns in order to avoid its deficiency (anemia). Depending on the physicochemical species used, its bioavailability might be insufficient to meet iron requirements. In this vein, the influence of Lactoferrin (Lf) presence on iron bioavailability in 2‐week‐old wistar rats fed with formula milk fortified with 57Fe(III)2‐Lf or 57Fe(II)SO4 (in presence of Lf) using quantitative speciation (by HPLC‐ICP‐MS) and Isotope Pattern Deconvolution (IPD) is studied here. Results obtained were compared among fortifiers and also with the maternal group. In RBCs, iron was mainly bound to hemoglobin in all the assayed groups in the same extent. Regarding serum samples, several iron‐proteins were observed (such as transferrin and albumin). In both samples, iron content in the fractions studied was similar in all groups compared and exogenous 57Fe incorporation of intaked iron was always above 50%, showing no significative differences between physicochemical forms but related to the dose administered. Regarding iron stores (liver) the group fed with formula milk fortified with the higher dose of 57FeSO4 in presence of Lf presented the highest values of total iron even superior than those found in the maternal group, and also the highest exogenous (57Fe) incorporation. In conclusion, it was proved that iron fortification is required to ensure proper iron levels in all body compartments. No significative differences were observed between different physicochemical species when iron is administered at low doses. However, higher iron doses lead to a greater incorporation in all the iron‐proteins studied.

MMP-11 as a biomarker for metastatic breast cancer by immunohistochemical-assisted imaging mass spectrometry

MMP-11 is a member of the matrix metalloproteinase family (MMPs) which are overexpressed in cancer cells, stromal cells and the adjacent microenvironment. The MMP protein family encompasses zinc-dependent endopeptidases that degrade the extracellular matrix (ECM), facilitating the breakdown of the basal membrane and matrix connective tissues. This function is believed to be important in cancer development and metastasis. This paper investigated a gold nanoparticle-based immunohistochemical assay to visualise the distribution of MMP-11 in human breast cancer tissues from eight patients with and without metastases by employing laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The expression of MMP-11 was increased and more heterogeneous in metastatic specimens compared to non-metastatic tumour samples. These findings demonstrate that imaging breast tumours by LA-ICP-MS may be a useful tool to aid the prognosis and treatment of breast cancer. As an example, samples of two patients are presented who were diagnosed with matching characteristics and grades of breast cancer. Although both patients had a similar prognosis and treatment, only one developed metastases.