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Nutzen-Risiko-Bewertung von Mineralstoffen und Spurenelementen

 | 
Andrea Hartwig
, 
Beate Köberle
, 
Bernhard Michalke

Vortragsreihe

A new manganese biomonitoring concept for Mn exposure assessment based on Mn speciation

B. Michalke, M. Lucio, B. Kanawati et A. Berthele

Résumé

For humans Mn is an essential trace element, but at higher doses a neurotoxic metal. Chronic Mn exposure is affecting the central nervous system. Occupational Mn overexposure leads to an accumulation in the brain and has been shown to cause progressive, permanent, neurodegenerative damage with syndromes similar to idiopathic Parkinsonism. Mn is transported by an active mechanism across neural barriers (NB) finally into the brain, but to date modes of Mn neurotoxic action are poorly understood. This paper investigates the relevant Mn-carrier species which are responsible for a widely uncontrolled transport across NB. Mn speciation in paired serum/cerebrospinal fluid (CSF) samples was performed by size exclusion chromatography – inductively coupled plasma – dynamic reaction cell – mass spectrometry (SEC-ICP-DRC-MS) and capillary zone electrophoresis (CZE) coupled to ICP-DRC-MS in an 2D approach for clear identification. The Mn-species from the different sample types were interrelated, and correlation coefficients were calculated.
In serum, protein-bound Mn-species like Mn-transferrin (Mn-Tf) had important influence on total Mn in serum if Mntotal was less than 1.5 µg/L, but above 1.6 µg/L the serum-Mntotal concentration was correlated with increasing Mn-citrate (Mn-Cit) concentration. Correlations between serum Mn species and CSF showed that Mntotal from CSF was correlated to Mn-Cit from serum above 1.6 µg/L Mntotal in serum.
Statistical models discriminated the samples in two groups where CSF samples were either correlated to Mntotal and Mn-Cit (samples with serum Mntotal > 1.5 µg/L) or correlated to Mn-TF (samples with serum Mntotal < 1.5 µg/L).
We conclude that elevated Mn-Citserum could be a valuable marker for increased total Mn in CSF (and brain). It could be used as a marker for elevated risk of Mn-dependent neurological disorders in occupational health.

Entrées d'index

Note de l’éditeur

Running title: Mn-speciation for Mn-biomonitoring

Texte intégral

Introduction

1Mn is an essential trace element required for normal growth, development and cellular homeostasis [1]. Mn is a required cofactor of several enzymes necessary for neuronal and glial cell function, as well as enzymes involved in neurotransmitter synthesis and metabolism [2, 3, 4]. However, excessive Mn exposure can accumulate in the brain causing severe neurological disorder similar to Parkinson´s disease (PD) [5]. Studies from occupational health after Mn exposure showed a limitation of mental ability, proceeding to psychotic phase with reduction of psychomotoric coordination, damage of the extrapyramidal nervous system and finally leading to symptoms similar to Parkinson´s disease [6, 7, 8].

2In a series of investigations we previously showed that different Mn-species are present in human serum and cerebrospinal fluid (CSF) [9, 10, 11, 12]. In serum mainly Mn-transferrin (Mn-Tf) was found, aside from small amounts of Mn-citrate. Contrary, in CSF mainly low molecular mass (LMM) Mn-species were found, where Mn-citrate was identified as the major Mn-species [11]. Nischwitz et al. investigated paired serum and CSF samples [12] showing that total Mn and Mn-Tf were downgraded across neural barriers (NB) but Mn-citrate was enriched. Also Yokel et al. [13] and Aschner et al. [14] found Mn-citrate to be facilitated transported across NB when performing perfusion experiments in rat brains.

3Thus, Mn-speciation targeted to Mn-citrate in human serum could provide a Mn-biomarker for Mn-exposure. Up to now a validated biomarker for Mn exposure is not available: Total Mn determination in blood or serum often cannot distinguish between non-exposed and exposed persons [15], and renal Mn excretion being below 1% of total Mn excretion is consequently not suitable as biomarker [16]. Therefore, in this paper significant relation-ships between Mn-species from serum and CSF were elucidated. Identification of Mn-compounds was provided using an orthogonal speciation scheme based on analysis of the samples by SEC ICP-DRC-MS, followed from analysis of SEC characterized fractions by CE-ICP-DRC-MS. Pearson´s relationships between serum and CSF Mn species were calculated.

Figure 1: Scheme of environmental-and bio-monitoring: For avoiding exposure effects (diseases) monitoring should be as early as possible (left side of the scheme). In case of Mn, simple metal analysis in bodyfluids does not result in clear differentiation between exposed and non-exposed persons. Therefore, biomonitoring must include metabolized Mn species (secondary Mn species) and their specific pathways: Mn speciation may help to improve efficiency of differentiation of exposed from non-exposed persons (from Angerer, modified).

Experimental

Chemicals

4From Sigma-Aldrich, Deisenhofen, Germany: Blue dextran: 2000 kDa, α-2­macroglobuline: 609 kDa, arginase: 107 kDa, transferrin: 78 kDa, albumin: 68,5 kDa, β-lactoglobuline: 36,5 kDa, lysozyme: 14,3 kDa, Metallothionein: 7 kDa, L-thyroxine: 777 Da, N,N´-bis(t-BOC)-l-cystine: 440,5 Da, citric acid: 192,5 Da, inorganic MnCl2.

5From Merck, Darmstadt, Germany: TRIS, HNO3, HCl (suprapure), NH4-acetate (NH4Ac), acetic acid (HAc) and TSK SEC-gel (230 – 450 mesh). From Air-Liquide, Gröbenzell, Germany: Argonliqu and NH3.

Standards, samples and sample preparation

6Mn-protein stock standards were prepared by dissolving the powder of each compound in 10 mL TRIS-HCl buffer (10 mM, pH 7.4). Stock solution of MnCl2 was prepared by dissolving 100 mg/L (related to Mn). Mn-citrate stock solution was prepared by mixing a solution of 1 g/L citrate with a MnCl2 solution (5 mg/L) using a ratio of 4+1 (v:v), resulting in a Mn-citrate stock concentration of 1 mg Mn/L. Mn-albumin and Mn-transferrin stock solutions were prepared in analogy by mixing 1 g/L protein solution with 5 mg/L MnCl2 solution (4+1, each), resulting in 1 mg Mn/L for each compound. Stock solutions were aliquoted and stored in the dark at -20°C. Working solutions were prepared daily by appropriate dilution with TRIS-HCl, 10 mM, pH 7.4.

7Serum samples were collected from healthy persons as described in [12] and stored at 4°C in a refrigerator. CSF samples were taken from non Mn exposed persons at the Dept. of Neurology, Technical University of Munich, following standard clinical procedures. The CSF samples were aliquoted and stored at -20°C. Before analysis, the CSF samples were thawed slowly at 4 °C and analyzed immediately.

SEC Parameters

8SEC was performed with a Knauer 1100 Smartline inert Series gradient HPLC system and two serially installed SEC columns: Biobasic 300 mesh column (300x8mm ID, Thermo, separation range 700 - 5 kDa) serially connected to a 550x10 mm ID Kronlab ECO column filled with TSK-HW40S (separation range 100 -2000 Da). Tris-HAc (10 mM, pH 7.4) + 250 mM NH4Ac was used as the eluent at a flow-rate of 0.75 mL/min.

9For additional identification of Mn-species by CE-ICP-DRC-MS, the SEC column effluent was fractionated in 2.5 min intervals with a ‘‘Fraction Collector 100’’ (Pharmacia, Freiburg, Germany).

Capillary zone electrophoresis coupled to ICP-DRC-MS

10A "Biofocus 3000" capillary electrophoresis system (BioRad, Munich, Germany) was used as the CE device at a temperature of 20°C. The capillary (120 cm x 50 µm ID, non-coated) was from CS-Chromatographie Service GmbH (Langerwehe, Germany).

11TRIS (10 mM, adjusted to pH 8.0 with HAc) buffer was the background electrolyte (BE). For sample stacking a buffer sandwich was injected consisting of 160 nL Na-acetate as leading electrolyte (LE), 60 nL sample, and 235 nL terminating electrolyte (TE, = BE/H2O (1:100)). The applied voltage was +28 kV. The hyphenation was described earlier [11].

ICP-MS parameters

12A Nexlon ICP-MS, Perkin Elmer (Sciex, Toronto, Canada) with dynamic reaction cell capability was employed for on-line determination of 55Mn in the graphic mode.

13For SEC coupling the ICP-introduction was managed with a Meinhard nebulizer whilst for CE-coupling a Micromist nebulizer was installed. The RF power was 1250 W, the plasma gas was 15 L Ar/min. The nebulizer gas was optimized and finally set to 0.98 (Meinhard) or 1.02 (Micromist) mL Ar/min. The dwell time was 500 ms for SEC coupling but 100 ms for CE coupling. The dynamic reaction cell (DRC) was operated using NH3 for the DRC gas, finally at a flow rate of 0.58 ml/min. DRC band pass (q) was 0.45.

Results and Discussion

SEC-separation of Mn-species in serum and CSF

14Mn-proteins were separated from LMM Mn-compounds in serum and CSF samples. The main part of Mn in serum is associated with proteins, whilst in CSF the main Mn fraction is associated with LMM Mn-species, predominantly with the citrate fraction. This was confirming our previous findings [10, 11].

15Mn species were additionally investigated with CE-ICP-DRC-MS in 2.5 min fractions from SEC separation (two-dimensional analysis). For example figure 2 demonstrates the Mn-electropherogram of the citrate SEC fraction from CSF. The comparison with standard solutions containing ά-2-macroglobulin, Mn-citrate and inorganic Mn, standard match is found for Mn-citrate but no match was seen for ά-2-macroglobulin. However, the latter was not suspected in this citrate SEC fraction.

Figure 2: CE-ICP-DRC-MS analysis of the Mn-citrate SEC fraction from CSF and three standard Mn-species for comparison. Standard matches are observed for Mn-citrate and traces of inorganic Mn. The peak at 10.5 min relates to Mn-malate (standard match not shown).

16Based on the above described 2D approach the Mn-peaks in SEC chromategrams were assigned to the main Mn-species eluting at respective retention times for quantification [17] and calculating Pearson´s relationships.

Pearson´s relationships

17Mn-species concentrations from serum and CSF were interrelated and Pearson´s correlation coefficients were calculated. Only few relationships had reasonable r2 values indicating correlation, which were dependent on the total Mn concentration in serum.

18The total Mn concentration in serum was correlated to the serum Mn-Tf fraction when total Mn was < 1.5 µg/L. Above 1.5 µg/L correlation was excluded. Contrary, no correlation was seen for Mn-citrate vs. total Mn in serum below 1.5 µg/L, but a pronounced correlation was found above 1.5 µg/L total Mnserum.

19Therefore we conclude that above a total Mnserum concentration of 1.5 µg/L Mn-citrate seems to be the most important Mn-species in serum, being responsible for the elevated Mntotal concentration. The findings from serum are also reflected in correlations between serum vs. CSF Mn species: for total Mnserum concentrations below 1.5 µg/L, total MnCSF concentrations were correlated mainly with Mn-Tf from serum, but above 1.5 µg/L (total Mnserum) the total MnCSF concentration was predominantly correlated to Mn-citrate in serum.

20This result is specifically important because it indicates that Mn-citrate is the main Mn-species in CSF when total MnCSF or total Mnserum concentration is elevated. Furthermore, elevated Mn-citrateserum and Mn-citrateCSF concentrations are directly correlated. This can be used for estimating the Mn-citrateCSF concentration from the Mn-citrateserum concentration. This fact could be used for biomonitoring purposes, as CSF usually is not simply available (only after strict neurological indication at the hospital) in contrast to serum.

Statistical evaluation

21Two statistical techniques have been applied in order to retrieve useful information whether there is a differentiation between sample pairs.

22First we applied an orthogonal partial least square discriminant analysis (OPLS/O2PLS-DA) dividing the dataset in two classes and studying the dependence of the variables Y1 = total Mn, Y2 = Mn-Tf fraction (peak 45-47) and Y3 = Mn-citrate fraction (peak 55-57) and studying which are the responsible variables for the groups’ separation. The goodness of the fit and the prediction were expressed by these two indexes respectively, R²(Y) was 0.85 and Q²(cum) = 0.54 (the maximum value for both is 1), as seen in figure 3.

23The loading Biplot expresses this relation between the variables “total Mn in CSF”, “Mn-TF in CSF”, “Mn-citrate in CSF” and the observation in the dataset of paired samples. Here it is seen that “Mn-Tf” is more related to “class 1 members”, i.e. members of the violet group which have total Mnserum < 1.5 µg/L whilst “total MnCSF ” and “Mn-citrateCSF ” are positively related to each other and with the “class 2 members”, i.e. members of the orange group having total Mnserum > 1.5 µg/L.

Figure 3: OPLS/O2PLS-DA model: It visualizes the two groups differences. Here it is shown that Mn-Tf is more related with the violet group having total Mn in serum < 1.5 µg/L whilst total MnCSF and Mn-citrateCSF are positively related to each other and with the orange group, having total Mn in serum > 1.5 µg/L.

24We applied also canonical discriminant analysis. The class variable has been set up in the canonical discriminant analysis as dependent variable.

25Summarized from both statistical evaluations we get a confirmation of Pearson´s relationships calculated above: When total Mn in serum is above 1.5 µg/L, then “total Mn in CSF” and “Mn-citrate in CSF” are higher and CSF samples out of these sample pairs are differentiated from CSF samples from sample pairs where total Mnserum is below 1.5 µg/L.

Conclusion

26A set of paired serum and CSF samples has been investigated with two 2D approaches for Mn speciation. The established SEC-ICP-DRC-MS method allowed smooth separation of important Mn species, whilst CE-ICP-DRC-MS provided improved species identification. The interrelation of Mn-species from both sample types revealed correlations from serum Mn­species and CSF Mntotal or CSF Mn-species. As the most important result we found CSF Mntotal or CSF Mn-Cit to be correlated with serum Mn-Cit when serum Mntotal was (slightly elevated) > 1.5 µg/L, but serum Mntotal and CSF Mntotal were more correlated to serum Mn-Tf when serum Mntotal was < 1.5 µg/L.

Bibliographie

References

[1] Erikson KM, Syversen T, Aschner J, Aschner M (2005) Interaction between excessive manganese-exposure and dietary iron-deficiency in neuro-degeneration. Toxicol Pharmacol 19: 415 – 421.

[2] Erikson KM, Schner M (2003) Manganese neurotoxicity and glutamat-GABA interaction. Neurochem Int 43: 475 – 480.

[3] Butterworth J (1986) Changes in nine enzyme markers for neurons, glia, and endothelial cells in agonal state and Huntington’s disease caudate nucleus. J Neurochem 47: 583–587.

[4] Hurley LS, Keen CL. In: Underwood E, Mertz W, editors. Manganese in trace elements in human health and animal nutrition. New York: Academic Press; 1987. p. 185–225.

[5] Aschner M, Guilarte TR, Schneider JS, Zheng W (2007) Manganese: Recent advances in understanding its transport and neurotoxicity. Toxicol Appl Pharmacol 221: 131–47.

[6] R.T. Ingersoll, E.B. Montgomery and H.V. Aposhian, NeuroToxicology, 20 (2-3) (1999) 467-476.

[7] Cerosimo MG, Koller WC (2006) The diagnosis of manganese-induced Parkinsonism. Neurotoxicology, 27: 340-346.

[8] Ordonez-Librado JL, Guiterrez-Valdez AL, Colin-Baranque L, Anaya-Martinez V, Diaz-Beech P, Avila-Costa MR (2008) Manganese inhalation as a Parkinson disease model. Neuroscience, 155: 7 – 11.

[9] Michalke B (2004) Manganese speciation using capillary electrophoresis-ICP-mass spectrometry. J. Chrom A 1050: 69 – 76.

[10] Quintana M, Klouda AD, Gondikas A, Ochsenkühn-Petropoulou M, Michalke B (2006) Analysis of Size Characterized Manganese Species from Liver Extracts using Capillary Zone Electrophoresis coupled to Inductively Coupled Plasma Mass Spectrometry (CZE-ICP-MS). Analytica Chimica Acta 573-574: 172-180.

[11] Michalke B, Berthele A, Mistriotis P, Ochsenkühn-Petropoulou M, Halbach S, Manganese Speciation in Human Cerebrospinal Fluid using Capillary Zone Electrophoresis coupled to Inductively Coupled Plasma Mass Spectrometry, Electrophoresis, 2007, 28: 1380 – 1386.

[12] Nischwitz V, Berthele A, Michalke B (2008) Speciation analysis of selected metals and determination of their total contents in paired serum and CSF samples: An approach to investigate the permeability of the human blood-CSF-barrier, Analytica Chimica Acta, 627/2: 258-269.

[13] Yokel RA, Crossgrove JS (2004) Health Effects Institute Research Report 119: 1-84.

[14] Yokel RA, Crossgrove RS (2004) Manganese Toxicokinetics at the Blood Brain Barrier. Health Effects Institute research Report 119: 1 – 84.

[15] Smith D, Gwiazda R, Bowler R, Roels H, Park R, Taicher C, Lucchini R (2007) Biomarkers of Mn exposure in humans. Am J Ind Med.

[16] Saric M (1986) Handbook on the Toxicity of Metals, Vol II, Specific Metals, New York, Elsevier Science Publishing Co: 354 – 386.

[17] Michalke B, Lucio M, Kanawati B, Berthele A, Manganese speciation in paired serum and CSF samples using SEC-DRC-ICP-MS and CE-DRC-ICP-MS, Anal Bioanal Chemistry, Published on-line 2013, DO1: 10.1007/s00216-012-6662-7.

Table des illustrations

Légende Figure 1: Scheme of environmental-and bio-monitoring: For avoiding exposure effects (diseases) monitoring should be as early as possible (left side of the scheme). In case of Mn, simple metal analysis in bodyfluids does not result in clear differentiation between exposed and non-exposed persons. Therefore, biomonitoring must include metabolized Mn species (secondary Mn species) and their specific pathways: Mn speciation may help to improve efficiency of differentiation of exposed from non-exposed persons (from Angerer, modified).
URL http://books.openedition.org/ksp/docannexe/image/153/img-1.jpg
Fichier image/jpeg, 224k
Légende Figure 2: CE-ICP-DRC-MS analysis of the Mn-citrate SEC fraction from CSF and three standard Mn-species for comparison. Standard matches are observed for Mn-citrate and traces of inorganic Mn. The peak at 10.5 min relates to Mn-malate (standard match not shown).
URL http://books.openedition.org/ksp/docannexe/image/153/img-2.jpg
Fichier image/jpeg, 224k
Légende Figure 3: OPLS/O2PLS-DA model: It visualizes the two groups differences. Here it is shown that Mn-Tf is more related with the violet group having total Mn in serum < 1.5 µg/L whilst total MnCSF and Mn-citrateCSF are positively related to each other and with the orange group, having total Mn in serum > 1.5 µg/L.
URL http://books.openedition.org/ksp/docannexe/image/153/img-3.jpg
Fichier image/jpeg, 64k

Auteurs

Helmholtz Center Munich – German Research Center for Environmental Health, Research Unit Analytical BioGeo Chemistry, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
Email: bernhard.michalke@helmholtz-muenchen.de

Helmholtz Center Munich – German Research Center for Environmental Health, Research Unit Analytical BioGeo Chemistry, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

Helmholtz Center Munich – German Research Center for Environmental Health, Research Unit Analytical BioGeo Chemistry, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

Department of Neurology, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany

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