Elevated selenium and lead concentrations in amyotrophic lateral sclerosis cerebrospinal fluid provide clues to ALS pathogenesis

Elevated selenium and lead concentrations in amyotrophic lateral sclerosis cerebrospinal fluid provide clues to ALS pathogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elevated selenium and lead concentrations in amyotrophic lateral sclerosis cerebrospinal fluid provide clues to ALS pathogenesis Julia Smirnova, Andra Noormägi, Elina Berntsson, Robert A. Harris, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6839641/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron degeneration and muscle atrophy, ultimately leading to death through respiratory failure. Environmental factors, including metal exposure, seem to contribute to ALS pathogenesis. Elevated concentrations of metals such as Pb, Se, and Cd have been detected in ALS cerebrospinal fluid (CSF) and blood from ALS patients, providing clues to possible pathogenesis. Here we conducted a detailed analysis of CSF and blood plasma samples collected by ultraclean techniques from seven ALS patients and seven healthy controls. Inductively coupled plasma mass spectrometry (ICP-MS) was used for metal measurements. Significantly higher concentrations of 208 Pb and 78 Se (p < 0.01), and lower concentrations of 75 As and 55 Mn (p < 0.05), were found in ALS CSF but not in ALS blood plasma. Additionally, ALS patients displayed increased concentrations of 60 Ni in blood plasma relative to controls (p < 0.05). Elevated 208 Pb/ 206 Pb and 206 Pb/ 207 Pb ratios were observed in ALS CSF, possibly tracing sources of lead exposure. Samples were also analyzed using size-exclusion chromatography coupled with ICP-MS to monitor the distribution of metals between different protein carriers. The combined findings support the hypothesis that metal exposure contributes to the pathogenesis of ALS. Cellular & Molecular Neuroscience ALS exposure ICP-MS isotopes lead metals SEC Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights 208 Pb and 78 Se were significantly elevated in ALS CSF but not in plasma 75 As and 55 Mn were significantly reduced in ALS CSF but not in plasma Lead isotope ratio data suggest varied Pb exposure sources in ALS Introduction Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disease of the motor neuron system, invariably leading to death from weakness and atrophy of voluntary muscles and the diaphragm. The time from diagnosis to death is about 3 years in most cases (Al-Chalabi and Hardiman, 2013 ), but longer survival has also been described (Weber et al., 2012 ). Research during recent decades has unveiled more than 30 ALS-associated genes, among them SOD1, C9ORF72, FUS and TARDBP (Suzuki et al., 2023 ). Disease progression is associated with the aggregation of proteins such as SOD1, C9orf72, FUS, or TDP-43 (Koski et al., 2021 ; Duranti and Villa, 2022 ; Min et al., 2024 ; Wu et al., 2024 ; Mengistu et al., 2025 ). About 90% of the ALS cases are sporadic, indicating a role for environmental factors in the pathogenesis (Fang et al., 2015 ; Vasta et al., 2023b). Accordingly, it has been suggested that ALS could be associated with exposure to pesticides (Kamel et al., 2012 ), electromagnetic fields (Vasta et al., 2023a), and metals (Roos et al., 2013 ; Gunnarsson and Bodin, 2018 ). An uneven geographic distribution of ALS (Newell et al., 2022 ; Vasta et al., 2023b) lends further support to the understanding that sporadic ALS cases might have an environmental origin. ALS is often considered as a model disorder for neuronal degeneration (Riancho et al., 2019 ). Other neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinsons disease (PD), and Multiple Sclerosis (MS) overlap with ALS to some extent, and overlap cases with clinical features from one or several of these diagnoses within the same patient exist (Vrillon et al., 2021 ; Spencer, 2022 ; De Marchi et al., 2023 ). Knowledge gained about possible environmental contributions to ALS pathogenesis can therefore also be used to gain a better understanding of the degenerative processes leading to AD (Aaseth et al., 2016 ), PD (Aaseth et al., 2018 ) and MS (Astrom and Roos, 2022 ). ALS is characterized by the degeneration of anterior horn cells in the spinal cord. Muscle atrophy and denervation are prominent findings, and quantitative electromyography is necessary for proper diagnosis to exclude other causes of muscle atrophy, like various myopathies (Dabby et al., 2001 ). Autopsy studies of ALS cases reveal sclerosis of the corticospinal tracts (Clarke and Jackson, 1867 ; El Mendili et al., 2019 ) and in some cases, degeneration of the cerebral frontal lobes (Pineda et al., 2024 ). The underlying cause(s) of ALS remain uncharacterized. Several studies have indicated an association between metals and metal exposure and ALS. Levels of lead (Pb), selenium (Se) and cadmium (Cd) are significantly elevated in the blood and cerebrospinal fluid (CSF) from ALS patients (Bar-Sela et al., 2001 ; Roos et al., 2013 ; Vinceti et al., 2013 ), which links these elements to an increased ALS risk (Fang et al., 2010 ; Fang et al., 2015 ). Concentrations of other metals such as manganese (Mn), aluminum (Al), cobalt (Co), copper (Cu), zinc (Zn), vanadium (V) and uranium (U) have been reported to be elevated in ALS CSF in other studies (Roos et al., 2013 ; Garzillo et al., 2014 ; Cicero et al., 2017 ). These findings of elevated metal concentrations in ALS CSF, especially elevated Pb, have been further verified (Conradi et al., 1978 ; Ronnevi et al., 1982 ; Molina-Holgado et al., 2007 ; Vinceti et al., 2017 ). Lead and mercury (Hg) are neurotoxic metals and exposure to Pb and Hg has been proposed to contribute to ALS pathogenesis (Livesley and Sissons, 1968 ; Chancellor et al., 1993 ). Zinc has been associated with a decreased ALS risk in a study of erythrocytes (Peters et al., 2021 ), in contrast to some previous studies (Ermilova et al., 2005 ; Hozumi, 2013 ). The concentrations of Cu were lower in the CSF of ALS patients than in controls, and even lower in a spinal-onset subgroup (Chen et al., 2022 ). These observations are consistent with another study that reported lower CSF Cu concentrations in spinal-onset patients compared to bulbar patients (Patti et al., 2020 ). Environmental exposure to Se, particularly from drinking water containing inorganic Se, has been epidemiologically linked to ALS (Kilness, 1977 ). Additionally, lower levels of brain-specific selenoprotein P have been suggested as a potential risk factor for ALS (Vinceti et al., 2013 ). Accumulation of metals in brain structures has also been reported in AD and PD (Molina-Holgado et al., 2007 ). Proposed mechanisms for nerve cell degeneration in these disorders are linked to disturbances in metal homeostasis, particularly from redox-active metals such as Cu and Fe which can generate reactive oxygen species and induce oxidative stress (Aaseth et al., 2016 ; Aaseth et al., 2018 ; Cheignon et al., 2018 ; Wärmländer et al., 2019 ; Aaseth et al., 2021 ; Dusek et al., 2022 ). First-row transition metals such as Fe, Mn, Zn and Cu are cofactors to specialized proteins and enzymes in biological systems, and are involved in vital aspects of human metabolism. These metals must be readily available for cells, and their concentrations are strictly regulated. The blood-brain barrier (BBB) and the blood-CSF barrier (i.e. the choroid plexus), regulate metal transition from the vascular system into the sealed central nervous system (CNS). Barrier properties influence metal binding to CNS proteins and choroid plexus metal permeability varies between metal species. This can be broadly categorized into general choroid plexus toxicants, which damage the choroid plexus structure (for example Hg, Cd and Arsenic (As)), selective choroid plexus toxicants (Pb, Cu and Tellurium) which impair specific plexus regulatory pathways critical to brain function rather than inducing massive pathological alteration, and finally sequestered choroid plexus toxicants (Fe, Ag, Au and Zn) trapped by the choroid plexus as an essential CNS defense mechanism (Zheng, 2001 ; Zheng et al., 2003 ). Only a few studies have investigated this permeability of the blood-CSF barrier for metals by characterization of total metal concentrations in serum and CSF and by separation of metal-bound proteins by SEC coupled with ICP-MS (Nischwitz et al., 2008 ). As yet, no single metal has been identified as the sole cause of ALS. The evolving ICP-MS instrumentation techniques have produced a growing number of metal concentration measurements in ALS, although with substantial individual variation in measured metal concentrations (Koski et al., 2023 ) and sometimes with conflicting results for several metals. Sampling technique, ambient air metal particle contamination, patient contamination and cleanliness of vials all contribute to this variation (Roos, 2019 ). By calculating CSF/blood plasma metal concentration ratios it was possible to delineate 53 metal ratios that were significantly elevated in ALS patients, including Cd/Se (p = 0.031), and 16 significant ratios with Mg as the denominator, such as Mn/Mg (p = 0.005) and Al/Mg (p = 0.014) (Koski et al. 2023 ). Herein we present results from a study in which metal concentrations were measured in the CSF and blood plasma from 7 ALS cases and 7 healthy controls. Samples were collected according to an ultraclean protocol (Roos, 2019 ) to avoid metal contamination, and metal concentrations and metal isotope ratios were measured using ICP-MS. The distribution of metals across their binding protein fractions was studied by SEC paired with ICP-MS. Materials and Methods Patient sampling CSF and blood plasma from ALS patients and controls were collected under ultraclean conditions (Roos, 2019 ) in a dedicated cleanroom at the Department of Nephrology research unit M87 Huddinge Hospital (ethical permit EPN Stockholm 2014/1815-31 with informed consent from patients and controls). These paired samples were collected into and subsequently stored in multiple polytetrafluoroethylene (PTFE) (Teflon®) vials thoroughly cleaned with ultrapure nitric acid plus hydrogen peroxide followed by several rinsing steps with ultrapure water (Rodushkin et al., 2010 ). The samples were kept on ice during aliquoting and stored at -80°C pending analysis. Chemicals Ultrapure Type 2 water with a resistivity of 18.2 MΩ/cm produced in-lab with a Direct–Q UV water purification system (Merck KGaA, Darmstadt, Germany) was used for all applications. Trace-metal grade HNO 3 was purchased from Fisher Scientific UK Ltd., Leicestershire, UK. The multi-element calibration standard and the ICP-MS internal standards mix were purchased from Agilent Technologies, Santa Clara, USA. Quality control and ICP-MS calibration ClinChek control serum for trace elements (Level II RECIPE Chemicals + Instruments GmbH, Germany) was used as a reference for the measurements of metal concentrations. This control serum contains the metal isotopes 27 Al, 24 Mg, 63 Cu, 51 V, 52 Cr, 55 Mn, 56 Fe, 66 Zn, 59 Co, 60 Ni, 78 Se, 111 Cd and 75 As at known concentration ranges with median values, but contains no ranges for 208 Pb and 238 U. Control serum was digested using two different protocols. According to the first protocol, 50 µL of the ClinCheck control stock solution was diluted 40 times with 5% ultrapure HNO 3 and then incubated for 3 hours at 95°C followed by centrifugation for 5 min at 5000g (digestion protocol D1, matrix 5%). In the second protocol 50 µL of the ClinCheck control stock solution was diluted twofold with concentrated HNO 3 and incubated for 45 min at 90°C followed by the addition of 900 µL of 1% HNO 3 to get 1 mL of solution, which was subsequently stored overnight at room temperature. On the next day samples were centrifuged for 5 min at 5000g and 1 mL of sample was transferred into new vials containing 1 mL of mQ H 2 O (digestion protocol obtained from the ICP MS core lab of Hopkins University, D2, matrix 2%). Greiner CELLSTAR® 15 mL PP vials were prewashed with 5% HNO 3 for 3 days and dried for storage. An Agilent 7800 series ICP-MS instrument (Agilent Technologies, Santa Clara, USA) coupled with Agilent SPS-4 autosampler was operated in either Helium mode or NoGas mode for the quantification of 15 selected isotopes: 27 Al, 24 Mg, 63 Cu, 51 V, 52 Cr, 55 Mn, 56 Fe, 66 Zn, 59 Co, 60 Ni, 78 Se, 111 Cd, 206 Pb, 207 Pb, 208 Pb, 238 U and 75 As. The internal standard for 27 Al, 24 Mg, 63 Cu, 51 V, 52 Cr, 55 Mn, 59 Co, 60 Ni and 56 Fe was 45 Sc, for 66 Zn, for 75 As and 78 Se was 72 Ge, for 111 Cd was 115 In, and for 206 Pb, 207 Pb, 208 Pb and 238 U was 209 Bi (5188–6525, ICP-MS internal standard mix 1 µg/mL in 2% HNO 3 , Agilent Technologies). The ICP-MS instrument was calibrated using a multielement calibration standard solution (8500–6940, Agilent Technologies) in the range of 0.050–100 ppb in 2–5% (depending on the matrix of the corresponding digestion protocol) trace metal grade HNO 3 . All samples were measured in triplicate. The Agilent MassHunter 4.4 software version C.01.04 was used for instrument control and data acquisition. The instrument was operated under general matrix working mode with RF power 1550 W, nebulizer gas flow 1.03 L/min, auxiliary gas flow 0.90 L/min and plasma gas flow 15 L/min with nebulizer MicroMist. Daily optimization of ICP-MS working conditions was performed with a tuning solution of 1 µg/L 7 Li, 59 Co, 89 Y, 140 Ce, and 205 Ti. GraphPad Prism 10 was used for data visualization and statistical analysis. A p-value of less than 0.05 was considered statistically significant. Limits of detection (DLs) and linearity (R² values) for all measured metal isotopes, determined from calibration curves under each digestion protocol (D1 and D2), are presented in Supplementary Table S1 Determination of total metal concentrations in CSF and plasma CSF and blood plasma samples were thawed on ice and vortexed. All samples were handled under a clean laminar flow hood by pipette tips pretreated with 5% HNO 3 . Samples were digested using protocol D1 and diluted 40 times with 5% HNO 3 and incubated for 3 hours at 95°C followed by centrifugation for 5 min at 5000g. Greiner CELLSTAR® 15 mL PP tubes were treated according to the protocol described in section 2.3. Limits of detection (DLs) and linearity (R² values) for all measured metal isotopes, determined from calibration curves for CSF and blood plasma samples, are presented in Supplementary Table S1. The GraphPad Prism 10 software was used for data visualization and statistical analysis. Hyphenated size exclusion chromatography ICP-MS For separation of proteins in representative CSF and blood plasma samples, SEC was performed using an Agilent Infinity HPLC system with a 1260 series µ-degasser, a 1200 series capillary pump, a Micro WPS autosampler and a 1200 series MWD VL detector directly coupled to an Agilent 7800 series ICP-MS instrument (Agilent Technologies, Santa Clara, USA). A Superdex 200 10/300 GL (10–600 kDa) SEC column was operated with 200 mM NH 4 HNO 3 , pH 7.5, at a flow rate of 0.4 µL/min. The mobile phase was prepared daily and de-metallated using a CHELEX pre-column. Demetallation of the SEC column was performed by injecting 10 mM EDTA under standardized conditions after each run. The SEC column was calibrated with human serum albumin (HSA), ceruloplasmin (CP), superoxide dismutase (Cu/Zn-SOD1) and metallothionein (Cu-MT3) at different concentrations. The vial containing 2 times-diluted CSF samples, or 4 times-diluted blood plasma sample, was stored on ice and 40 µL were transferred into the liquid chromatography autosampler before measurement. Cesium acetate was added to each sample at a final concentration of 10 µM to use 132 Cs as the internal standard. Two injections for each sample were performed: one in He mode and another in NoGas mode. The instrument was tuned with Tune Solution in the respective mode prior measurement. Chromatograms of 27 Al, 24 Mg, 63 Cu, 51 V, 52 Cr, 55 Mn, 56 Fe, 66 Zn, 59 Co, 60 Ni, 78 Se, 111 Cd, 75 As 208 Pb and 238 U isotopes were monitored. Peak integration and quantification of the intensities of all monitored isotopes were performed by normalization to the 132 Cs peak value. The data was analyzed and visualized using the Origin 9 Pro software. Results The measured metal isotope concentrations in CSF and blood plasma are depicted in Figs. 1 and 3 , while concentrations with subtracted blanks are presented in Table 1 . From this data, it is apparent that 208 Pb and 78 Se were significantly elevated in CSF (p < 0.01), but not in the blood plasma of ALS cases when compared to controls. 60 Ni was significantly elevated in blood plasma (p < 0.05) but not in CSF of ALS patients when compared to controls. Concentrations of 75 As and 55 Mn were significantly lower in the CSF (p < 0.05) but not in the blood plasma of ALS cases compared to controls. For 27 Al, 111 Cd, 59 Co, 52 Cr, 63 Cu, 56 Fe, 24 Mg, 206 Pb, 207 Pb, 238 U, 51 V and 66 Zn, no significant differences between ALS patients and controls were detected, neither in CSF nor in blood plasma. Table 1 Mean metal concentrations (± SD, blanks subtracted) in paired CSF and blood plasma samples from ALS patients and controls. Metal concentration ratios Q CSF/Plasma are presented and compared to Q CSF/Serum data from earlier studies (1) ( Nischwitz, Berthele et al. 2008 ) and (2) (Michalke, Berthele et al.). Q CSF/Plasma was considered statistically significant at *p < 0.05, **p < 0.01; 55 Mn p = 0.0052 , 208 Pb p = 0.0002 (***). CSF Blood plasma ALS ( n = 7) Controls ( n = 7) ALS ( n = 5) Controls ( n = 6) ALS Control (1) (2) Element Mean ± SD, ppb Mean ± SD, ppb Mean ± SD, ppb Mean ± SD, ppb Q CSF/plasma Q CSF/plasma Q CSF/serum Q CSF/serum 25Mg 16751 ± 490 15909 ± 997 14144 ± 1169 13643 ± 877 1.15 1.20 1.3 27Al 145 ± 14 144 ± 12 125 ± 16 134 ± 16 1.10 1.15 51V 0.17 ± 0.06 0.20 ± 0.12 0.37 ± 0.06 0.38 ± 0.08 0.38 0.56 52Cr 5.1 ± 3.6 5.9 ± 4.5 4.6 ± 1.3 5.5 ± 1.6 0.98 1.37 55Mn 0.75 ± 0.11 0.99 ± 0.20 1.15 ± 0.29 1.06 ± 0.14 0.64 0.97 0.7 0.37 56Fe 33.9 ± 10.8 50.7 ± 30.6 788 ± 104 708 ± 408 0.04 0.12 0.02 0.016 59Co 0.05 ± 0.02 0.09 ± 0.05 0.30 ± 0.07 0.49 ± 0.29 0.19 0.12 60Ni 0.66 ± 0.09 0.69 ± 0.12 1.67 ± 0.42 1.16 ± 0.24 0.40 0.62 63Cu 12.0 ± 2.3 12.1 ± 3.0 970 ± 205 936 ± 190 0.01 0.01 0.02 0.012 66Zn 23.4 ± 2.4 24.6 ± 1.2 722 ± 99 748 ± 62 0.03 0.03 0.03 0.026 75As 0.54 ± 0.11 1.00 ± 0.51 2.6 ± 1.5 10.4 ± 11.5 0.26 0.25 82Se 3.6 ± 0.5 2.7 ± 0.5 111 ± 43 98 ± 21 0.04 0.03 111Cd 0.020 ± 0.005 0.036 ± 0.027 0.097 ± 0.048 0.088 ± 0.027 0.25 0.47 206Pb 0.15 ± 0.08 0.15 ± 0.08 0.62 ± 0.19 0.48 ± 0.08 0.25 0.28 207Pb 0.14 ± 0.05 0.17 ± 0.06 0.69 ± 0.17 0.61 ± 0.11 0.20 0.31 208Pb 6.3 ± 0.6 2.7 ± 1.8 6.2 ± 0.7 5.7 ± 1.9 1.05 0.38 238U - 0.001 ± 0.004 0.15 ± 0.02 0.14 ± 0.02 The Q CSF/plasma ratios were calculated for all metals except 238 U (Table 1 ). Q CSF/plasma ratios were calculated individually for every sample, and mean values were determined for ALS patients and healthy controls. P-values were calculated from unpaired T-tests, comparing ALS cases and control groups. In the case of 208 Pb the ALS Q CSF/plasma ratio is significantly higher (p = 0.0002) than the controls ratio. In the case of 55 Mn the ALS ratio is significantly lower (p = 0.0052) than the controls ratio. Individual 206 Pb, 207 Pb and 208 Pb concentrations are presented in Table 2 , along with the ratios 206 Pb/ 207 Pb and 208 Pb/ 206 Pb. For the CSF samples, the 208 Pb/ 206 Pb ratio was higher, but not significantly so in ALS cases compared to controls (Table 2 ). The 206 Pb/ 207 Pb ratio was also somewhat higher, but not significantly higher, in the ALS cases. For the blood plasma samples no significant differences in Pb isotope ratios were observed between ALS cases and controls (Table 2 ). Table 2 Individual 206 Pb, 207 Pb and 208 Pb concentrations in paired CSF and blood plasma samples from ALS patients and controls. Mean values and lead i sotope ratios 206 Pb/ 207 Pb and 208 Pb/ 206 Pb are also presented. CSF Blood plasma Sample 206 Pb, ppb 207 Pb, ppb 208 Pb, ppb 206 Pb/ 207 Pb 208 Pb/ 206 Pb 206 Pb, ppb 207 Pb, ppb 208 Pb, ppb 206 Pb/ 207 Pb 208 Pb/ 206 Pb Control 1 0.26 0.25 0.00 1.02 0.00 0.55 0.81 3.10 0.67 5.68 Control 2 0.11 0.26 0.93 0.41 8.79 0.47 0.57 4.90 0.83 10.44 Control 3 0.17 0.21 2.06 0.83 11.78 0.53 0.48 4.82 1.09 9.16 Control 4 0.06 0.11 3.57 0.52 63.73 0.52 0.64 5.51 0.81 10.55 Control 5 0.12 0.15 3.94 0.83 31.68 0.32 0.57 8.62 0.56 27.09 Control 6 0.09 0.12 3.83 0.73 42.76 0.52 0.60 7.19 0.86 13.94 Control 7 0.26 0.12 4.75 2.15 18.11 ALS 1 0.31 0.12 6.70 2.72 21.43 0.93 0.95 6.94 0.98 7.45 ALS 2 0.10 0.20 5.94 0.51 58.18 ALS 3 0.14 0.21 6.88 0.68 47.78 0.59 0.67 6.79 0.88 11.50 ALS 4 0.08 0.15 7.08 0.57 83.29 0.41 0.50 6.06 0.82 14.88 ALS 5 0.14 0.10 6.31 1.38 43.97 0.53 0.61 6.00 0.87 11.35 ALS 6 0.11 0.08 5.61 1.46 49.55 0.64 0.72 5.33 0.89 8.35 ALS 7 0.16 0.09 5.44 1.78 33.15 Mean, ppb Controls 0.15 0.17 2.73 0.93 25.26 0.48 0.61 5.69 0.80 12.81 ALS 0.15 0.14 6.28 1.30 48.19 0.62 0.69 6.22 0.89 10.71 To evaluate the digestion protocols and ICP MS performance, quality control using serum ClinChek control for trace elements Level II was performed. No significant differences in measured metal concentrations between the two digestion protocols were observed (Table 3). However, the measured concentrations of 27 Al and 52 Cr were significantly above the control range, which suggests external introduction or interference. The results for 27 Al and 52 Cr were therefore excluded from further discussion. Sample protein fractionation was achieved by Size-Exclusion Chromatography and targeted 15 metal isotopes ( 27 Al, 24 Mg, 63 Cu, 51 V, 52 Cr, 55 Mn, 56 Fe, 66 Zn, 59 Co, 60 Ni, 78 Se, 111 Cd, 75 As 208 Pb and 238 U). The chromatograms for 63 Cu, 66 Zn, 56 Fe, and 52 Cr displayed peaks corresponding to high-molecular-weight (HMW) and/or low-molecular-weight (LMW) protein species. The distribution between HMW and LMW protein peaks varied between metal ions and sample types (CSF vs blood plasma), and chromatograms of samples from ALS cases and controls exhibited minor variations in the number of peaks and their distribution (Fig. 4 ). Column calibration identified ceruloplasmin as the primary 63 Cu-binding species in ALS blood plasma, while 63 Cu in ALS CSF was predominantly associated with LMW species. 66 Zn was distributed between both fractions, with the major LMW fraction in blood plasma and a LMW 66 Zn-bound fraction was detected in the CSF chromatograms. The 56 Fe chromatograms for CSF and blood plasma both exhibited two peaks with the same retention time, where one protein peak likely represents transferrin and the other peak likely corresponds to LMW proteins. The area of the transferrin peak was substantially lower in CSF samples than in the blood plasma samples. 52 Cr in the blood plasma was bound to a protein with a retention time similar to that of HSA, whereas in the CSF 52 Cr was only detected in the LMW fraction. 78 Se was evident as a protein-bound peak at extremely low concentrations but only in plasma samples, and was therefore excluded from further discussion. An example of a non-detectable species under our LC ICP-MS conditions is illustrated by the chromatogram for 208 Pb. Discussion Established risk factors for ALS include male gender (PMID: 25054277), advanced age and specific mutations for a subset of patients (Pasinelli and Brown, 2006 ; Logroscino et al., 2008 ; Andersen and Al-Chalabi, 2011 ). Environmental factors in general and metal exposure in particular have emerged as potential risk factors for ALS and for other neurodegenerative diseases (Felmus et al., 1976 ; Roos, 2013 ; Wang et al., 2014 ; Gunnarsson and Bodin, 2018 ). ALS has been attributed a long preclinical period (Eisen et al., 2014 ) and the complexity of metal measurements and challenges inherent in relating such measurements to environmental exposures during the lifetime of an ALS patient are acknowledged. This study used an ultraclean sampling techniques and measurements of metal concentrations by ICP-MS for precise quantification of metals, and we detected deviations in ALS patients. We here discuss in detail the implications of our findings, especially the significantly elevated concentrations of Pb and Se in CSF from ALS patients, and explore their broader relevance to ALS pathogenesis in the light of possible environmental exposures. Elevated lead concentrations in ALS body fluids Lead exposure has long been implicated as a possible cause of ALS and is implicated in other neurodegenerative diseases as well due to its well-documented detrimental effects on neuronal integrity, contribution to oxidative stress, disruption of synaptic signalling and promotion of protein misfolding and aggregation (Johnson and Atchison, 2009 ; Roos, 2013 ; Wallin et al., 2017 ). Associations between Pb exposure and ALS risk have previously been reported (Kamel et al., 2006 ; Yu et al., 2024 ). Some research has suggested a direct connection between Pb exposure and ALS, yet other studies have not been able to corroborate that (Callaghan et al., 2011 ). In this study we observed significantly elevated 208 Pb concentrations (Table 2 ) in ALS CSF compared to healthy controls (p < 0.01). These findings align with the results of a thorough meta-analysis which studied 29 high-quality articles narrowed down from 4234 articles studying metals in ALS body fluids (Kamalian et al., 2023 ). At the meta-level, ALS whole blood Pb was elevated by 2.88 µg/L (p = 0.006), and ALS CSF Pb was elevated by 0.21 µg/L (p = 0.04). This concords with the findings of the present study and strongly supports the notion that Pb contributes to ALS pathogenesis. Occupational exposure is a well-documented risk factor for Pb accumulation (Roos et al., 2006 ; Johnson and Atchison, 2009 ; Wang et al., 2014 ; Gunnarsson and Bodin, 2018 ; Farace et al., 2020 ; Olowoyo et al., 2024 ). A meticulous meta-analysis of occupational risk factors for ALS concluded that ALS risk was statistically significantly elevated for occupational exposures to metals (especially Pb), chemicals (especially pesticides), and excessive physical work (Gunnarsson and Bodin, 2018 ). Many older pesticides are Pb derivatives, such as the widely used lead arsenate (US_EPA, 2004 ; Delistraty and Yokel, 2014 ). Thus, the conclusion that pesticides are a risk factor for ALS can to some extent be extrapolated to occupational Pb exposure as well (Gunnarsson and Bodin, 2018 ). Lead is present in all aspects of occupational life where dermal Pb exposure exists (Askin and Volkmann, 1997 ), and occupationally Pb-exposed workers also receive respiratory Pb exposure from Pb-contaminated cigarettes (Dykeman et al., 2002 ; Wallin et al., 2017 ). More than a hundred occupations involving Pb exposure have been identified (Nordberg and Costa, 2021 ), where leaded paint, backyard recycling of used lead-acid batteries (Ericson et al., 2016 ) and ammunition work are prominent Pb sources (Nordberg and Costa, 2021 ). Documented occupational Pb exposure linked to an elevated ALS risk include military service occupations (Seals et al., 2016 ; Peters et al., 2017 ), construction workers (Fang et al., 2009 ; Dickerson et al., 2018 ), mechanical workers (Park et al., 2005 ; Sutedja et al., 2009 ), gasoline station forecourt attendants (Wärmländer et al., 2025 ), precision tool manufacturers, and glass, pottery, and tile workers (Peters et al., 2017 ), leather workers (Buckley et al., 1983 ) and tanners (Chio et al., 1991 ; Bhuiyan et al., 2011 ). Farmers also show an elevated ALS risk (Bale, 1975 ; Rosati et al., 1977 ), possibly because farm workers are exposed to pesticides and insecticides (Breland and Currier, 1967 ; Greenberg et al., 1979 ). In our study no significant differences in plasma Pb concentrations were determined between ALS cases and controls, but elevated 208 Pb concentrations were evident in ALS CSF (Table 2 ), suggesting that Pb accumulation in ALS may be a CNS-specific phenomenon. This pattern is consistent with a potential lack of protection from the BBB and/or blood-CSF barrier i.e. the choroid plexus, which could facilitate the preferential accumulation of Pb in the CNS while maintaining near-normal plasma levels. Previous studies have shown that Pb disrupts tight junctions in barrier systems due to its ability to substitute for calcium ions (Ca 2+ ), contributing to selective Pb 2+ BBB permeability and high Pb concentrations in the brain (Zheng, 2001 ; Wang et al., 2007 ; Sanders et al., 2009 ). Isotopic analysis of Pb: Insights into sources and mechanisms To better understand the environmental origins of Pb exposure in ALS, we analysed Pb isotopic ratios in both CSF and plasma samples. The 206 Pb/ 207 Pb and 208 Pb/ 206 Pb ratios were altered in ALS patients compared to controls, suggesting differences in Pb sources or metabolic handling. These ratios serve as geochemical fingerprints, as the natural abundance of Pb isotopes is influenced by radioactive decay pathways in geological systems (Cheng and Hu, 2010 ; Morton-Bermea et al., 2011 ). For instance, 206 Pb and 207 Pb derive from uranium decay, while 208 Pb is a decay product of 232 Th (Aberg et al., 2001 ; Kulikov et al., 2017 ). Variations in these ratios between ALS patients and controls may reflect occupational or environmental exposure to Pb sources with distinct isotopic signatures. Lead isotope ratios offer significant advantages due to their stability in physical, chemical, and biological processes. They are minimally affected by isotope fractionation, making them reliable for tracing the origins of Pb across disciplines such as archaeology, agriculture and environmental science. However, recent findings suggest that Pb isotopic fingerprints in biological samples can vary significantly between different tissues and fluids such as blood, urine and hair (Liu et al., 2007 ; Liu et al., 2009 ). This complicates a direct identification of environmental Pb sources. Isotope ratios may shift due to biological fractionation, a poorly understood phenomenon influenced by absorption, metabolism and redistribution factors (Wu et al., 2012 ). Notably, fractionation thresholds exist whereby isotopic ratios behave differently at varying blood Pb levels. For example, fractionation appears to become abnormal at high Pb concentrations, thereby altering the differences between biological samples and the Pb source (Wu et al., 2012 ). Metal exposure in ALS seems to be varied, complex and individually unique (Roos, 2013 ). Occupational exposure to several neurotoxic metals has been linked to ALS (Roos et al., 2013 ; Gunnarsson and Bodin, 2018 ) and here we report that Pb and Se, both with neurotoxic properties, are elevated in ALS CSF. Isotopic fingerprints, with the caveats outlined above, might be useful for tracing possible sources of metal exposure in ALS, using lessons learned from other neurodegenerative diseases where the geochemical origin of metal exposures has been outlined (Astrom and Roos, 2022 ). Selenium and its role in ALS Selenium, an essential trace element, exhibits a dual nature in human health: It is essential for the functioning of antioxidant enzymes but is neurotoxic at higher concentrations. Herein significantly higher Se concentrations were recorded in ALS CSF (p < 0.01), while plasma Se showed no significant differences between ALS patients and controls. This suggests that Se accumulation in CSF may be a phenomenon linked to ALS. A role for Se in ALS pathogenesis is in line with the findings of a meta-analysis in which a 4.26 µg/L elevation of serum/plasma Se levels in ALS patients (p = 0.02) was reported (Kamalian et al., 2023 ). Consumption of drinking water containing ≥ 1µg/L of inorganic selenium has been associated with a relative risk of 5.4 (95% confidence interval 1.1–26) for ALS after adjusting for confounding factors (Vinceti et al., 2010 ; Vinceti et al., 2016 ; Vinceti et al., 2019 ). Excessive Se exposure, particularly in its inorganic forms, has been implicated in oxidative stress, mitochondrial dysfunction and apoptosis, all molecular mechanisms relevant to ALS pathogenesis (Simpson et al., 2003 ; D'Amico et al., 2013 ). The absence of elevated plasma Se concentrations in our study suggests that Se dysregulation in ALS is confined to the CNS. The neurotoxic effects of Se may emanate from its ability to oxidize thiol-containing proteins and to facilitate the translocation of copper/zinc superoxide dismutase (SOD1) into mitochondria, thereby disrupting cellular function (Filippini et al., 2018 ). The level of Se toxicity likely depends on its chemical form, as inorganic and organic species present distinctly different biological properties (Vinceti et al., 2018 ). Additionally, Se-CSF (behind the BBB) concentrations are independent from Se-serum (before the BBB) concentrations, suggesting the possibility of an independent Se regulation in the CNS (Michalke et al., 2009 ). Metal-driven inflammation in ALS Evidence exists for a low-grade systemic inflammatory reaction in ALS (Keizman et al., 2009 ). The erythrocyte sedimentation rate is often normal in ALS patients at an early stage, but increases slowly over time, correlating with disease severity (Keizman et al., 2009 ). It is unknown to what extent the increased concentrations of Pb and Se contribute to the observed inflammation in ALS, but for other metals potential mechanisms have been proposed. For example, loss of cuproprotein function due to a combination of unbound copper and ROS that can either initiate and/or accelerate protein aggregation has been claimed to be central to ALS pathology. Copper homeostasis within motor neurons is disrupted in ALS patient spinal cords as well as in the SOD1G93A mouse model (Williams et al., 2016 ; Hilton et al., 2024 ). This could result in toxicity caused by the excess of redox active copper that is free to form harmful reactive oxygen species (ROS) that can cause damage through lipid peroxidation and DNA damage (Sauzeat et al., 2018 ; Sheykhansari et al., 2018 ), or by functional deficiency. The promotion of protein aggregation in neurodegenerative diseases is a general pathological characteristic, and both Pb and Hg salts increase TDP-43 aggregation in vitro (Ash et al., 2019 ). Cadmium competitively displaces zinc from its binding site in SOD1, which leads to protein folding defects and inactivation of the enzyme while simultaneously generating ROS (Huang et al., 2006 ; Oggiano et al., 2021 ). As a general pathological mechanism, protein aggregation leads to metal ion deficiency and functional loss of metal-based enzymes, which leads to more aggregation in a self-perpetuating cycle. Metal ratios in ALS Beyond Pb and Se, our study identified lower concentrations of arsenic (As) and manganese (Mn) in ALS CSF, with no significant differences in plasma levels. This contradicts the hypothesis that Mn toxicity contributes to ALS (Roos et al., 2012 ; Roos et al., 2021 ). Ratios between metal concentrations in CSF and in plasma (Q CSF/plasma ) were calculated to better understand the distribution of metal ions between CSF and blood plasma and to provide insight into BBB permeability. A higher Q CSF/plasma suggests that metal ions readily cross the BBB and accumulate in CSF, potentially contributing to neurotoxicity. In ALS patients, 208 Pb exhibited a significantly higher Q CSF/plasma , indicating that Pb accumulates in CSF, suggesting an ALS-related alteration in Pb transport or clearance mechanisms. However, despite elevated Se concentrations in ALS CSF, the Q CSF/plasma for 82 Se was not significantly increased. This could imply that the observed Se elevation originates from a systemic increase in plasma Se, rather than enhanced transport across the BBB, or that selenium homeostasis in the brain is tightly regulated by selenoproteins, preventing disproportionate accumulation in the CSF. Additionally, 55 Mn exhibited a significantly lower Q CSF/plasma in ALS, suggesting restricted Mn transport into the CSF or increased Mn efflux from the CSF, which may reflect ALS-related disturbances in Mn metabolism. No significant difference was observed for 75 As, indicating that arsenic homeostasis in the CSF is maintained despite systemic exposure. Metal ratios between CSF and blood serum have previously been reported for various metals. However, direct comparison with our Q CSF/plasma values is not possible, as plasma and serum vary in protein composition and because of differences in study design. Serum lacks fibrinogen, a key plasma protein that can bind metal ions, potentially altering the distribution of metals between compartments. The elevated plasma Ni concentrations observed in ALS patients (p < 0.05) require further investigation. Nickel induces oxidative stress, neuronal DNA damage, and protein misfolding and aggregation (Guo et al., 2019 ; Berntsson et al., 2023 ). The role of Ni in ALS pathogenesis remains to elucidate. Childhood exposure to Ni and Cr, and co-exposure to Ni, Cr, Sr, and other metals, has recently been linked to adult-onset ALS (Figueroa-Romero et al., 2020 ). Genotoxic effects of metals in ALS Most monogenetic forms of familial ALS (fALS) do not develop in utero or in childhood but instead appear later in adulthood. Nonetheless, fALS typically present at a younger age compared to sporadic ALS (sALS). Mathematical modeling, adapted from cancer research, suggests that the development of ALS is a multistep process requiring a sequence of six separate genetic or environmental events to happen within an individual genome. This modeling indicates that environmental factors contribute to the development of fALS just as they do for sALS (Al-Chalabi et al., 2014 ). Lead, Se and Ni, along with other redox-active metals, are known to induce genotoxic effects, including DNA damage and impaired DNA repair mechanisms (Rusov et al., 1996 ; Guo et al., 2019 ; Nagaraju et al., 2022 ). Lead interferes with DNA repair enzymes, while excessive Se can generate reactive oxygen species which damage mitochondrial and nuclear DNA (Valdiglesias et al., 2010 ). These genotoxic pathways align closely with the mechanisms of motor neuron degeneration observed in ALS, underscoring the need for further exploration of these effects. Strengths and limitations A key strength of this study is the ultraclean protocol employed during sample collection (Roos, 2019 ) and the minimization of contamination during sample handling and digestion, enhancing measurement reliability. The polypropylene vials used in this study were acid-washed with ultrapure nitric acid to avoid metal contamination. We suggest using PTFE vials (Teflon®) instead of polypropylene vials for laboratory work in future studies of metals in ALS to eliminate metal contamination from the plastic vials. The ICP-MS measurements were performed in helium mode [He] to reduce polyatomic interferences (e.g. Argon based interferences for As, Fe and Cr). No gas [No Gas] mode was used for the Pb isotopes, whose polyatomic interferences are minimal, leveraging the sensitivity for Pb. Quality control with ClinCheck Level II serum confirmed that 208 Pb concentrations were within reported ranges, supporting the choice of [No Gas] mode for that element. These strengths of the current study are limited by the small number of ALS and control samples. Future perspectives This study highlights the critical role of environmental metal exposures in ALS pathogenesis and opens several avenues for future research. The following actions are suggested: Trace exposure sources: Pb isotope analysis should be expanded to identify specific environmental and occupational sources of metal exposure in ALS patients. Explore mechanisms of metal dysregulation: Investigate the molecular mechanisms underlying selective metal accumulation in the CNS and their interaction with ALS-specific genetic vulnerabilities. Longitudinal studies: Perform prospective studies tracking metal exposure, accumulation, and disease progression to establish causal relationships and time-dependent effects. Use a wide range of samples: Include hard tissues samples along with CSF and blood plasma/serum, taken under ultraclean conditions simultaneously from the same individual. Explore protein-bound metal distribution patterns: Use LC ICP-MS as a powerful tool to separate protein fractions. Conclusion Exposure to neurotoxic metals seem to contribute to ALS pathogenesis. 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J Neurol Sci 433:120021 Sutedja NA, Fischer K, Veldink JH, van der Heijden GJ, Kromhout H, Heederik D, Huisman MH, Wokke JJ, van den Berg LH (2009) What we truly know about occupation as a risk factor for ALS: a critical and systematic review. Amyotroph Lateral Scler 10(5–6):295–301 Suzuki N, Nishiyama A, Warita H, Aoki M (2023) Genetics of amyotrophic lateral sclerosis: seeking therapeutic targets in the era of gene therapy. J Hum Genet 68(3):131–152 US_EPA (2004) Historic Arsenical Pesticide Research: US Environmental Protection Agency, Office of Pesticide Programs: https://semspub.epa.gov/work/05/259803.pdf Valdiglesias V, Pasaro E, Mendez J, Laffon B (2010) In vitro evaluation of selenium genotoxic, cytotoxic, and protective effects: a review. Arch Toxicol 84(5):337–351 Vasta R, Callegaro S, Grassano M, Canosa A, Cabras S, Di Pede F, Matteoni E, De Mattei F, Casale F, Salamone P, Mazzini L, De Marchi F, Moglia C, Calvo A, Chiò A, Manera U 2023a. Exposure to electromagnetic fields does not modify neither the age of onset nor the disease progression in ALS patients. Amyotroph Lateral Scler Frontotemporal Degeneration 24(3–4):343–346 Vasta R, Callegaro S, Sgambetterra S, Cabras S, Di Pede F, De Mattei F, Matteoni E, Grassano M, Bombaci A, De Marco G, Fuda G, Marchese G, Palumbo F, Canosa A, Mazzini L, De Marchi F, Moglia C, Manera U, Chiò A, Calvo A 2023b. Presymptomatic geographical distribution of ALS patients suggests the involvement of environmental factors in the disease pathogenesis. J Neurol 270(11):5475–5482 Vinceti M, Bonvicini F, Rothman KJ, Vescovi L, Wang F (2010) The relation between amyotrophic lateral sclerosis and inorganic selenium in drinking water: a population-based case-control study. Environ Health 9:77 Vinceti M, Solovyev N, Mandrioli J, Crespi CM, Bonvicini F, Arcolin E, Georgoulopoulou E, Michalke B (2013) Cerebrospinal fluid of newly diagnosed amyotrophic lateral sclerosis patients exhibits abnormal levels of selenium species including elevated selenite. Neurotoxicology 38:25–32 Vinceti M, Ballotari P, Steinmaus C, Malagoli C, Luberto F, Malavolti M, Rossi PG (2016) Long-term mortality patterns in a residential cohort exposed to inorganic selenium in drinking water. Environ Res 150:348–356 Vinceti M, Filippini T, Mandrioli J, Violi F, Bargellini A, Weuve J, Fini N, Grill P, Michalke B (2017) Lead, cadmium and mercury in cerebrospinal fluid and risk of amyotrophic lateral sclerosis: A case-control study. J Trace Elem Med Biol 43:121–125 Vinceti M, Filippini T, Wise LA (2018) Environmental Selenium and Human Health: an Update. Curr Environ Health Rep 5(4):464–485 Vinceti M, Filippini T, Malagoli C, Violi F, Mandrioli J, Consonni D, Rothman KJ, Wise LA (2019) Amyotrophic lateral sclerosis incidence following exposure to inorganic selenium in drinking water: A long-term follow-up. Environ Res 179(Pt A):108742. Vrillon A, Deramecourt V, Pasquier F, Magnin E, Wallon D, Lozeron P, Bouaziz-Amar E, Paquet C (2021) Association of Amyotrophic Lateral Sclerosis and Alzheimer's Disease: New Entity or Coincidence? A Case Series. J Alzheimers Dis 84(4):1439–1446 Wallin C, Sholts SB, Österlund N, Luo J, Jarvet J, Roos PM, Ilag L, Gräslund A, Wärmländer SKTS (2017) Alzheimer's disease and cigarette smoke components: effects of nicotine, PAHs, and Cd(II), Cr(III), Pb(II), Pb(IV) ions on amyloid-beta peptide aggregation. Sci Rep 7(1):14423 Wang MD, Gomes J, Cashman NR, Little J, Krewski D (2014) A meta-analysis of observational studies of the association between chronic occupational exposure to lead and amyotrophic lateral sclerosis. J Occup Environ Med 56(12):1235–1242 Wang Q, Luo W, Zheng W, Liu Y, Xu H, Zheng G, Dai Z, Zhang W, Chen Y, Chen J (2007) Iron supplement prevents lead-induced disruption of the blood-brain barrier during rat development. Toxicol Appl Pharmacol 219(1):33–41 Weber M, Neuwirth C, Thierbach J, Schweikert K, Czaplinski A, Petersen J, Jung HH, Birve A, Marklund SL, Andersen PM (2012) ALS patients with SOD1 mutations in Switzerland show very diverse phenotypes and extremely long survival: Table 1. J Neurol Neurosurg Psychiatry 83(3):351–353 Williams JR, Trias E, Beilby PR, Lopez NI, Labut EM, Bradford CS, Roberts BR, McAllum EJ, Crouch PJ, Rhoads TW, Pereira C, Son M, Elliott JL, Franco MC, Estevez AG, Barbeito L, Beckman JS (2016) Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the Copper-Chaperone-for-SOD. Neurobiol Dis 89:1–9 Wu J, Liu D, Xie Q, Wang J (2012) Biological fractionation of lead isotopes in Sprague-Dawley rats lead poisoned via the respiratory tract. PLoS ONE 7(12):e52462 Wu J, Wu J, Chen T, Cai J, Ren R (2024) Protein aggregation and its affecting mechanisms in neurodegenerative diseases. Neurochem Int 180:105880 Wärmländer SKTS, Österlund N, Wallin C, Wu J, Luo J, Tiiman A, Jarvet J, Gräslund A (2019) Metal binding to the amyloid-beta peptides in the presence of biomembranes: potential mechanisms of cell toxicity. J Biol Inorg Chem 24(8):1189–1196 Wärmländer SKTS, Tshoni UA, Olowoyo JO, Koski L, Kobyana AS, Lion NG, Mugivhisa LL, Roos PM (2025) Occupational lead exposure in gasoline station forecourt attendants and other occupations in relation to ALS (amyotrophic lateral sclerosis) risk. https://doi.org/10.5281/zenodo.15192465 . Zenodo Preprint Yu W, Yu F, Li M, Yang F, Wang H, Song H, Huang X (2024) Quantitative association between lead exposure and amyotrophic lateral sclerosis: a Bayesian network-based predictive study. Environ Health 23(1):2 Zheng W (2001) Toxicology of choroid plexus: special reference to metal-induced neurotoxicities. Microsc Res Tech 52(1):89–103 Zheng W, Aschner M, Ghersi-Egea JF (2003) Brain barrier systems: a new frontier in metal neurotoxicological research. Toxicol Appl Pharmacol 192(1):1–11 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6839641","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467741094,"identity":"3a6e7934-0b86-468e-a736-2a2b399cfb27","order_by":0,"name":"Julia Smirnova","email":"","orcid":"","institution":"Department of Chemistry and Biotechnology, Tallinn University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Smirnova","suffix":""},{"id":467741095,"identity":"450e3a4f-3965-40f0-8cde-d79720451954","order_by":1,"name":"Andra Noormägi","email":"","orcid":"","institution":"Department of Chemistry and Biotechnology, Tallinn University of Technology, 19086 Tallinn, Estonia","correspondingAuthor":false,"prefix":"","firstName":"Andra","middleName":"","lastName":"Noormägi","suffix":""},{"id":467742768,"identity":"4c9f9d29-b8c2-4228-b12d-76c746de9975","order_by":2,"name":"Elina Berntsson","email":"","orcid":"","institution":"Chemistry Section, Stockholm University","correspondingAuthor":false,"prefix":"","firstName":"Elina","middleName":"","lastName":"Berntsson","suffix":""},{"id":467742769,"identity":"3f527329-e2d1-45e3-8bcf-7e7976b57584","order_by":3,"name":"Robert A. Harris","email":"","orcid":"","institution":"Department of Clinical Neuroscience, Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"A.","lastName":"Harris","suffix":""},{"id":467742770,"identity":"8224e6cc-aac8-42c6-9ed5-bc58616bbdc7","order_by":4,"name":"Sebastian Wärmländer","email":"","orcid":"","institution":"Chemistry Section, Stockholm University","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Wärmländer","suffix":""},{"id":467742771,"identity":"344acbbe-09d9-4c45-93b9-2b195f5d7119","order_by":5,"name":"Astrid Gräslund","email":"","orcid":"","institution":"Chemistry Section, Stockholm University","correspondingAuthor":false,"prefix":"","firstName":"Astrid","middleName":"","lastName":"Gräslund","suffix":""},{"id":467742772,"identity":"6e749eb9-1f89-427a-ab7a-03763d944813","order_by":6,"name":"Peep Palumaa","email":"","orcid":"","institution":"Department of Chemistry and Biotechnology, Tallinn University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Peep","middleName":"","lastName":"Palumaa","suffix":""},{"id":467742773,"identity":"723e8675-d4c6-4e4b-b46d-48de40f5acb1","order_by":7,"name":"Per M. Roos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDADfh7GBhK1SPaQrMXgDLEqzdl7HzBXVNjYG5853MD4o4IILZY9xw0Yz5xJS9x2trGBmYcYqwxupDEwNrYdTjA7z9jAzNhGjJb7z8Ba7I37GRsYf/4jyhY2sBbGDbyNDQy8DURosexJYzjYAPTLjDMHGw7zHCNCizn7McaHDcAQ4+9Jf/jwRw0xDgPiAzDOAZzK0LWMglEwCkbBKMAPABE+NLhMkkiSAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Clinical Neuroscience, Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Per","middleName":"M.","lastName":"Roos","suffix":""}],"badges":[],"createdAt":"2025-06-06 21:54:35","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6839641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6839641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84394629,"identity":"e374796c-3860-4954-b970-78193a95c387","added_by":"auto","created_at":"2025-06-11 12:17:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95319,"visible":true,"origin":"","legend":"\u003cp\u003eMetal concentrations in CSF samples from 7 ALS patients and 7 controls, and comparisons with blanks. All samples and blanks were treated with 5% HNO\u003csub\u003e3\u003c/sub\u003e at 95 °C for 3 hours. *p\u0026lt;0.05, **p\u0026lt;0.01. \u003csup\u003e55\u003c/sup\u003eMn p=0.014, \u003csup\u003e78\u003c/sup\u003eSe p=0.0034, \u003csup\u003e208\u003c/sup\u003ePb p=0.0003, \u003csup\u003e238\u003c/sup\u003eU p=0.029, \u003csup\u003e75\u003c/sup\u003eAs p=0.036.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/9aaa38fee640495acc9fefcc.png"},{"id":84394327,"identity":"57be04a5-49cf-4dca-a720-e767527c2284","added_by":"auto","created_at":"2025-06-11 12:09:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92458,"visible":true,"origin":"","legend":"\u003cp\u003eMetal concentrations in blood plasma samples from 5 ALS patients and 6 controls, and comparisons with blanks. All samples and blanks were treated with 5% HNO\u003csub\u003e3\u003c/sub\u003e at 95 °C for 3 hours. *p\u0026lt;0.05, **p\u0026lt;0.01. \u003csup\u003e60\u003c/sup\u003eNi p\u0026lt;0.030.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/c6cc6b0626612deb152de52f.png"},{"id":84394303,"identity":"0c551c8f-0a0b-49b9-8c83-402bd116d5f6","added_by":"auto","created_at":"2025-06-11 12:09:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109748,"visible":true,"origin":"","legend":"\u003cp\u003eMetal concentrations of the ClinChek Control sample for trace elements Level II (Ref:8880-8882), and comparisons with blanks. The blanks and ClinCheck sample were prepared using two different digestion protocols, D1 and D2, explained in the Materials and Methods section. The dotted lines represent control ranges for each metal isotope according to the ClinCheck Control data sheet.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/1d98fe82935264b819b0111f.png"},{"id":84394317,"identity":"99b0f8d5-cfec-4012-bfd5-c25b1fe37c20","added_by":"auto","created_at":"2025-06-11 12:09:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220140,"visible":true,"origin":"","legend":"\u003cp\u003eSize exclusion chromatographic (SEC) separation of the peaks for Cu, Fe, Cr, Zn, Pb, and Se in blood plasma and CSF samples from ALS patients. Top panel: mass calibration of the column with human serum albumin (HSA) (dimer 133 kDa, monomer 66.5 kDa), ceruloplasmin (CP) 132 kDa, Cu/Zn-SOD1 31.6 kDa, Cu-metallothionein-3 (Cu-MT3) 6.9 kDa. Conditions: Superdex 200 10/30 GL column, elution buffer 200 mM NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e, pH 7.5. The plasma samples were diluted 4 times, and the CSF samples were diluted 2 times.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/91f3d160a24b91348591ed6b.jpg"},{"id":84394635,"identity":"488d7c51-a4f7-47b6-97e4-2b707f0f6316","added_by":"auto","created_at":"2025-06-11 12:17:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1592695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/1f16660a-f741-4e2b-a3d7-8687506e0a0c.pdf"},{"id":84394319,"identity":"423c33a5-203a-4f7e-b962-e739f917d4a3","added_by":"auto","created_at":"2025-06-11 12:09:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19160,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6839641/v1/ea9f353bc90e98c2dda8cd2c.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eElevated selenium and lead concentrations in amyotrophic lateral sclerosis cerebrospinal fluid provide clues to ALS pathogenesis\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n\u003cli\u003e\u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e78\u003c/sup\u003eSe were significantly elevated in ALS CSF but not in plasma\u003c/li\u003e\n\u003cli\u003e\u003csup\u003e75\u003c/sup\u003eAs and \u003csup\u003e55\u003c/sup\u003eMn were significantly reduced in ALS CSF but not in plasma\u003c/li\u003e\n\u003cli\u003eLead isotope ratio data suggest varied Pb exposure sources in ALS\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eAmyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disease of the motor neuron system, invariably leading to death from weakness and atrophy of voluntary muscles and the diaphragm. The time from diagnosis to death is about 3 years in most cases (Al-Chalabi and Hardiman, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), but longer survival has also been described (Weber et al., \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Research during recent decades has unveiled more than 30 ALS-associated genes, among them \u003cem\u003eSOD1, C9ORF72, FUS\u003c/em\u003e and \u003cem\u003eTARDBP\u003c/em\u003e (Suzuki et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Disease progression is associated with the aggregation of proteins such as SOD1, C9orf72, FUS, or TDP-43 (Koski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Duranti and Villa, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Min et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mengistu et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). About 90% of the ALS cases are sporadic, indicating a role for environmental factors in the pathogenesis (Fang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vasta et al., 2023b). Accordingly, it has been suggested that ALS could be associated with exposure to pesticides (Kamel et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), electromagnetic fields (Vasta et al., 2023a), and metals (Roos et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). An uneven geographic distribution of ALS (Newell et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vasta et al., 2023b) lends further support to the understanding that sporadic ALS cases might have an environmental origin.\u003c/p\u003e \u003cp\u003eALS is often considered as a model disorder for neuronal degeneration (Riancho et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Other neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease (AD), Parkinsons disease (PD), and Multiple Sclerosis (MS) overlap with ALS to some extent, and overlap cases with clinical features from one or several of these diagnoses within the same patient exist (Vrillon et al., \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Spencer, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; De Marchi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Knowledge gained about possible environmental contributions to ALS pathogenesis can therefore also be used to gain a better understanding of the degenerative processes leading to AD (Aaseth et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), PD (Aaseth et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and MS (Astrom and Roos, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ALS is characterized by the degeneration of anterior horn cells in the spinal cord.\u003c/p\u003e \u003cp\u003eMuscle atrophy and denervation are prominent findings, and quantitative electromyography is necessary for proper diagnosis to exclude other causes of muscle atrophy, like various myopathies (Dabby et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Autopsy studies of ALS cases reveal sclerosis of the corticospinal tracts (Clarke and Jackson, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1867\u003c/span\u003e; El Mendili et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and in some cases, degeneration of the cerebral frontal lobes (Pineda et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The underlying cause(s) of ALS remain uncharacterized.\u003c/p\u003e \u003cp\u003eSeveral studies have indicated an association between metals and metal exposure and ALS. Levels of lead (Pb), selenium (Se) and cadmium (Cd) are significantly elevated in the blood and cerebrospinal fluid (CSF) from ALS patients (Bar-Sela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Roos et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vinceti et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which links these elements to an increased ALS risk (Fang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Fang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Concentrations of other metals such as manganese (Mn), aluminum (Al), cobalt (Co), copper (Cu), zinc (Zn), vanadium (V) and uranium (U) have been reported to be elevated in ALS CSF in other studies (Roos et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Garzillo et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cicero et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These findings of elevated metal concentrations in ALS CSF, especially elevated Pb, have been further verified (Conradi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Ronnevi et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Molina-Holgado et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Vinceti et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Lead and mercury (Hg) are neurotoxic metals and exposure to Pb and Hg has been proposed to contribute to ALS pathogenesis (Livesley and Sissons, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Chancellor et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZinc has been associated with a decreased ALS risk in a study of erythrocytes (Peters et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), in contrast to some previous studies (Ermilova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hozumi, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The concentrations of Cu were lower in the CSF of ALS patients than in controls, and even lower in a spinal-onset subgroup (Chen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These observations are consistent with another study that reported lower CSF Cu concentrations in spinal-onset patients compared to bulbar patients (Patti et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Environmental exposure to Se, particularly from drinking water containing inorganic Se, has been epidemiologically linked to ALS (Kilness, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Additionally, lower levels of brain-specific selenoprotein P have been suggested as a potential risk factor for ALS (Vinceti et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccumulation of metals in brain structures has also been reported in AD and PD (Molina-Holgado et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Proposed mechanisms for nerve cell degeneration in these disorders are linked to disturbances in metal homeostasis, particularly from redox-active metals such as Cu and Fe which can generate reactive oxygen species and induce oxidative stress (Aaseth et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Aaseth et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cheignon et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Aaseth et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dusek et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). First-row transition metals such as Fe, Mn, Zn and Cu are cofactors to specialized proteins and enzymes in biological systems, and are involved in vital aspects of human metabolism. These metals must be readily available for cells, and their concentrations are strictly regulated. The blood-brain barrier (BBB) and the blood-CSF barrier (i.e. the choroid plexus), regulate metal transition from the vascular system into the sealed central nervous system (CNS). Barrier properties influence metal binding to CNS proteins and choroid plexus metal permeability varies between metal species. This can be broadly categorized into general choroid plexus toxicants, which damage the choroid plexus structure (for example Hg, Cd and Arsenic (As)), selective choroid plexus toxicants (Pb, Cu and Tellurium) which impair specific plexus regulatory pathways critical to brain function rather than inducing massive pathological alteration, and finally sequestered choroid plexus toxicants (Fe, Ag, Au and Zn) trapped by the choroid plexus as an essential CNS defense mechanism (Zheng, \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Only a few studies have investigated this permeability of the blood-CSF barrier for metals by characterization of total metal concentrations in serum and CSF and by separation of metal-bound proteins by SEC coupled with ICP-MS (Nischwitz et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs yet, no single metal has been identified as the sole cause of ALS. The evolving ICP-MS instrumentation techniques have produced a growing number of metal concentration measurements in ALS, although with substantial individual variation in measured metal concentrations (Koski et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and sometimes with conflicting results for several metals. Sampling technique, ambient air metal particle contamination, patient contamination and cleanliness of vials all contribute to this variation (Roos, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By calculating CSF/blood plasma metal concentration ratios it was possible to delineate 53 metal ratios that were significantly elevated in ALS patients, including Cd/Se (p\u0026thinsp;=\u0026thinsp;0.031), and 16 significant ratios with Mg as the denominator, such as Mn/Mg (p\u0026thinsp;=\u0026thinsp;0.005) and Al/Mg (p\u0026thinsp;=\u0026thinsp;0.014) (Koski et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHerein we present results from a study in which metal concentrations were measured in the CSF and blood plasma from 7 ALS cases and 7 healthy controls. Samples were collected according to an ultraclean protocol (Roos, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to avoid metal contamination, and metal concentrations and metal isotope ratios were measured using ICP-MS. The distribution of metals across their binding protein fractions was studied by SEC paired with ICP-MS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient sampling\u003c/h2\u003e \u003cp\u003eCSF and blood plasma from ALS patients and controls were collected under ultraclean conditions (Roos, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in a dedicated cleanroom at the Department of Nephrology research unit M87 Huddinge Hospital (ethical permit EPN Stockholm 2014/1815-31 with informed consent from patients and controls). These paired samples were collected into and subsequently stored in multiple polytetrafluoroethylene (PTFE) (Teflon\u0026reg;) vials thoroughly cleaned with ultrapure nitric acid plus hydrogen peroxide followed by several rinsing steps with ultrapure water (Rodushkin et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The samples were kept on ice during aliquoting and stored at -80\u0026deg;C pending analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003eUltrapure Type 2 water with a resistivity of 18.2 MΩ/cm produced in-lab with a Direct\u0026ndash;Q UV water purification system (Merck KGaA, Darmstadt, Germany) was used for all applications. Trace-metal grade HNO\u003csub\u003e3\u003c/sub\u003e was purchased from Fisher Scientific UK Ltd., Leicestershire, UK. The multi-element calibration standard and the ICP-MS internal standards mix were purchased from Agilent Technologies, Santa Clara, USA.\u003c/p\u003e\n\u003ch3\u003eQuality control and ICP-MS calibration\u003c/h3\u003e\n\u003cp\u003eClinChek control serum for trace elements (Level II RECIPE Chemicals\u0026thinsp;+\u0026thinsp;Instruments GmbH, Germany) was used as a reference for the measurements of metal concentrations. This control serum contains the metal isotopes \u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e51\u003c/sup\u003eV, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e55\u003c/sup\u003eMn, \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e66\u003c/sup\u003eZn, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e60\u003c/sup\u003eNi, \u003csup\u003e78\u003c/sup\u003eSe, \u003csup\u003e111\u003c/sup\u003eCd and \u003csup\u003e75\u003c/sup\u003eAs at known concentration ranges with median values, but contains no ranges for \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e238\u003c/sup\u003eU. Control serum was digested using two different protocols. According to the first protocol, 50 \u0026micro;L of the ClinCheck control stock solution was diluted 40 times with 5% ultrapure HNO\u003csub\u003e3\u003c/sub\u003e and then incubated for 3 hours at 95\u0026deg;C followed by centrifugation for 5 min at 5000g (digestion protocol D1, matrix 5%). In the second protocol 50 \u0026micro;L of the ClinCheck control stock solution was diluted twofold with concentrated HNO\u003csub\u003e3\u003c/sub\u003e and incubated for 45 min at 90\u0026deg;C followed by the addition of 900 \u0026micro;L of 1% HNO\u003csub\u003e3\u003c/sub\u003e to get 1 mL of solution, which was subsequently stored overnight at room temperature. On the next day samples were centrifuged for 5 min at 5000g and 1 mL of sample was transferred into new vials containing 1 mL of mQ H\u003csub\u003e2\u003c/sub\u003eO (digestion protocol obtained from the ICP MS core lab of Hopkins University, D2, matrix 2%). Greiner CELLSTAR\u0026reg; 15 mL PP vials were prewashed with 5% HNO\u003csub\u003e3\u003c/sub\u003e for 3 days and dried for storage. An Agilent 7800 series ICP-MS instrument (Agilent Technologies, Santa Clara, USA) coupled with Agilent SPS-4 autosampler was operated in either Helium mode or NoGas mode for the quantification of 15 selected isotopes: \u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e51\u003c/sup\u003eV, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e55\u003c/sup\u003eMn, \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e66\u003c/sup\u003eZn, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e60\u003c/sup\u003eNi, \u003csup\u003e78\u003c/sup\u003eSe, \u003csup\u003e111\u003c/sup\u003eCd, \u003csup\u003e206\u003c/sup\u003ePb, \u003csup\u003e207\u003c/sup\u003ePb, \u003csup\u003e208\u003c/sup\u003ePb, \u003csup\u003e238\u003c/sup\u003eU and \u003csup\u003e75\u003c/sup\u003eAs. The internal standard for \u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e51\u003c/sup\u003eV, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e55\u003c/sup\u003eMn, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e60\u003c/sup\u003eNi and \u003csup\u003e56\u003c/sup\u003eFe was \u003csup\u003e45\u003c/sup\u003eSc, for \u003csup\u003e66\u003c/sup\u003eZn, for \u003csup\u003e75\u003c/sup\u003eAs and \u003csup\u003e78\u003c/sup\u003eSe was \u003csup\u003e72\u003c/sup\u003eGe, for \u003csup\u003e111\u003c/sup\u003eCd was \u003csup\u003e115\u003c/sup\u003eIn, and for \u003csup\u003e206\u003c/sup\u003ePb, \u003csup\u003e207\u003c/sup\u003ePb, \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e238\u003c/sup\u003eU was \u003csup\u003e209\u003c/sup\u003eBi (5188\u0026ndash;6525, ICP-MS internal standard mix 1 \u0026micro;g/mL in 2% HNO\u003csub\u003e3\u003c/sub\u003e, Agilent Technologies). The ICP-MS instrument was calibrated using a multielement calibration standard solution (8500\u0026ndash;6940, Agilent Technologies) in the range of 0.050\u0026ndash;100 ppb in 2\u0026ndash;5% (depending on the matrix of the corresponding digestion protocol) trace metal grade HNO\u003csub\u003e3\u003c/sub\u003e. All samples were measured in triplicate. The Agilent MassHunter 4.4 software version C.01.04 was used for instrument control and data acquisition. The instrument was operated under general matrix working mode with RF power 1550 W, nebulizer gas flow 1.03 L/min, auxiliary gas flow 0.90 L/min and plasma gas flow 15 L/min with nebulizer MicroMist. Daily optimization of ICP-MS working conditions was performed with a tuning solution of 1 \u0026micro;g/L \u003csup\u003e7\u003c/sup\u003eLi, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e89\u003c/sup\u003eY, \u003csup\u003e140\u003c/sup\u003eCe, and \u003csup\u003e205\u003c/sup\u003eTi. GraphPad Prism 10 was used for data visualization and statistical analysis. A p-value of less than 0.05 was considered statistically significant. Limits of detection (DLs) and linearity (R\u0026sup2; values) for all measured metal isotopes, determined from calibration curves under each digestion protocol (D1 and D2), are presented in Supplementary Table S1\u003c/p\u003e\n\u003ch3\u003eDetermination of total metal concentrations in CSF and plasma\u003c/h3\u003e\n\u003cp\u003eCSF and blood plasma samples were thawed on ice and vortexed. All samples were handled under a clean laminar flow hood by pipette tips pretreated with 5% HNO\u003csub\u003e3\u003c/sub\u003e. Samples were digested using protocol D1 and diluted 40 times with 5% HNO\u003csub\u003e3\u003c/sub\u003e and incubated for 3 hours at 95\u0026deg;C followed by centrifugation for 5 min at 5000g. Greiner CELLSTAR\u0026reg; 15 mL PP tubes were treated according to the protocol described in section 2.3. Limits of detection (DLs) and linearity (R\u0026sup2; values) for all measured metal isotopes, determined from calibration curves for CSF and blood plasma samples, are presented in Supplementary Table S1. The GraphPad Prism 10 software was used for data visualization and statistical analysis.\u003c/p\u003e\n\u003ch3\u003eHyphenated size exclusion chromatography ICP-MS\u003c/h3\u003e\n\u003cp\u003eFor separation of proteins in representative CSF and blood plasma samples, SEC was performed using an Agilent Infinity HPLC system with a 1260 series \u0026micro;-degasser, a 1200 series capillary pump, a Micro WPS autosampler and a 1200 series MWD VL detector directly coupled to an Agilent 7800 series ICP-MS instrument (Agilent Technologies, Santa Clara, USA). A Superdex 200 10/300 GL (10\u0026ndash;600 kDa) SEC column was operated with 200 mM NH\u003csub\u003e4\u003c/sub\u003eHNO\u003csub\u003e3\u003c/sub\u003e, pH 7.5, at a flow rate of 0.4 \u0026micro;L/min. The mobile phase was prepared daily and de-metallated using a CHELEX pre-column. Demetallation of the SEC column was performed by injecting 10 mM EDTA under standardized conditions after each run. The SEC column was calibrated with human serum albumin (HSA), ceruloplasmin (CP), superoxide dismutase (Cu/Zn-SOD1) and metallothionein (Cu-MT3) at different concentrations. The vial containing 2 times-diluted CSF samples, or 4 times-diluted blood plasma sample, was stored on ice and 40 \u0026micro;L were transferred into the liquid chromatography autosampler before measurement. Cesium acetate was added to each sample at a final concentration of 10 \u0026micro;M to use \u003csup\u003e132\u003c/sup\u003eCs as the internal standard. Two injections for each sample were performed: one in He mode and another in NoGas mode. The instrument was tuned with Tune Solution in the respective mode prior measurement. Chromatograms of \u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e51\u003c/sup\u003eV, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e55\u003c/sup\u003eMn, \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e66\u003c/sup\u003eZn, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e60\u003c/sup\u003eNi, \u003csup\u003e78\u003c/sup\u003eSe, \u003csup\u003e111\u003c/sup\u003eCd, \u003csup\u003e75\u003c/sup\u003eAs \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e238\u003c/sup\u003eU isotopes were monitored. Peak integration and quantification of the intensities of all monitored isotopes were performed by normalization to the \u003csup\u003e132\u003c/sup\u003eCs peak value. The data was analyzed and visualized using the Origin 9 Pro software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe measured metal isotope concentrations in CSF and blood plasma are depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, while concentrations with subtracted blanks are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. From this data, it is apparent that \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e78\u003c/sup\u003eSe were significantly elevated in CSF (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but not in the blood plasma of ALS cases when compared to controls. \u003csup\u003e60\u003c/sup\u003eNi was significantly elevated in blood plasma (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in CSF of ALS patients when compared to controls. Concentrations of \u003csup\u003e75\u003c/sup\u003eAs and \u003csup\u003e55\u003c/sup\u003eMn were significantly lower in the CSF (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in the blood plasma of ALS cases compared to controls. For \u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e111\u003c/sup\u003eCd, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e206\u003c/sup\u003ePb, \u003csup\u003e207\u003c/sup\u003ePb, \u003csup\u003e238\u003c/sup\u003eU, \u003csup\u003e51\u003c/sup\u003eV and \u003csup\u003e66\u003c/sup\u003eZn, no significant differences between ALS patients and controls were detected, neither in CSF nor in blood plasma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eMean metal concentrations (\u0026plusmn;\u0026thinsp;SD, blanks subtracted) in paired CSF and blood plasma samples from ALS patients and controls. Metal concentration ratios Q\u003c/em\u003e\u003csub\u003e\u003cem\u003eCSF/Plasma\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eare presented and compared to Q\u003c/em\u003e\u003csub\u003e\u003cem\u003eCSF/Serum\u003c/em\u003e\u003c/sub\u003e \u003cem\u003edata from earlier studies (1) (\u003c/em\u003eNischwitz, Berthele et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) \u003cem\u003eand (2) (Michalke, Berthele et al.). Q\u003c/em\u003e\u003csub\u003e\u003cem\u003eCSF/Plasma\u003c/em\u003e\u003c/sub\u003e \u003cem\u003ewas considered statistically significant at *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01;\u003c/em\u003e \u003csup\u003e\u003cem\u003e55\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMn p\u0026thinsp;=\u0026thinsp;0.0052\u003c/em\u003e, \u003csup\u003e\u003cem\u003e208\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePb p\u0026thinsp;=\u0026thinsp;0.0002 (***).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBlood plasma\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eALS (\u003cem\u003en\u003c/em\u003e = 7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControls (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALS (\u003cem\u003en\u003c/em\u003e = 5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eControls (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eALS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eQ\u003csub\u003eCSF/plasma\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003csub\u003eCSF/plasma\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eQ\u003csub\u003eCSF/serum\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eQ\u003csub\u003eCSF/serum\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25Mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16751\u0026thinsp;\u0026plusmn;\u0026thinsp;490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15909\u0026thinsp;\u0026plusmn;\u0026thinsp;997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14144\u0026thinsp;\u0026plusmn;\u0026thinsp;1169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13643\u0026thinsp;\u0026plusmn;\u0026thinsp;877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27Al\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e145\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e144\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e125\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e134\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e51V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52Cr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e55Mn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.64\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.97\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e56Fe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.7\u0026thinsp;\u0026plusmn;\u0026thinsp;30.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e788\u0026thinsp;\u0026plusmn;\u0026thinsp;104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e708\u0026thinsp;\u0026plusmn;\u0026thinsp;408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e59Co\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60Ni\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e63Cu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e970\u0026thinsp;\u0026plusmn;\u0026thinsp;205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e936\u0026thinsp;\u0026plusmn;\u0026thinsp;190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e66Zn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e722\u0026thinsp;\u0026plusmn;\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e748\u0026thinsp;\u0026plusmn;\u0026thinsp;62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e75As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e82Se\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e111\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e98\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e111Cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.020\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.097\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.088\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e206Pb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e207Pb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e208Pb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e238U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Q\u003csub\u003eCSF/plasma\u003c/sub\u003e ratios were calculated for all metals except \u003csup\u003e238\u003c/sup\u003eU (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Q\u003csub\u003eCSF/plasma\u003c/sub\u003e ratios were calculated individually for every sample, and mean values were determined for ALS patients and healthy controls. P-values were calculated from unpaired T-tests, comparing ALS cases and control groups. In the case of \u003csup\u003e208\u003c/sup\u003ePb the ALS Q\u003csub\u003eCSF/plasma\u003c/sub\u003e ratio is significantly higher (p\u0026thinsp;=\u0026thinsp;0.0002) than the controls ratio. In the case of \u003csup\u003e55\u003c/sup\u003eMn the ALS ratio is significantly lower (p\u0026thinsp;=\u0026thinsp;0.0052) than the controls ratio.\u003c/p\u003e \u003cp\u003eIndividual \u003csup\u003e206\u003c/sup\u003ePb, \u003csup\u003e207\u003c/sup\u003ePb and \u003csup\u003e208\u003c/sup\u003ePb concentrations are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, along with the ratios \u003csup\u003e206\u003c/sup\u003ePb/\u003csup\u003e207\u003c/sup\u003ePb and \u003csup\u003e208\u003c/sup\u003ePb/\u003csup\u003e206\u003c/sup\u003ePb. For the CSF samples, the \u003csup\u003e208\u003c/sup\u003ePb/\u003csup\u003e206\u003c/sup\u003ePb ratio was higher, but not significantly so in ALS cases compared to controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003csup\u003e206\u003c/sup\u003ePb/\u003csup\u003e207\u003c/sup\u003ePb ratio was also somewhat higher, but not significantly higher, in the ALS cases. For the blood plasma samples no significant differences in Pb isotope ratios were observed between ALS cases and controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIndividual\u003c/em\u003e \u003csup\u003e206\u003c/sup\u003ePb, \u003csup\u003e207\u003c/sup\u003ePb \u003cem\u003eand\u003c/em\u003e \u003csup\u003e208\u003c/sup\u003ePb \u003cem\u003econcentrations in paired CSF and blood plasma samples from ALS patients and controls. Mean values and lead i\u003c/em\u003esotope ratios \u003csup\u003e206\u003c/sup\u003ePb/\u003csup\u003e207\u003c/sup\u003ePb and \u003csup\u003e208\u003c/sup\u003ePb/\u003csup\u003e206\u003c/sup\u003ePb are also presented.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003eBlood plasma\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e207\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e208\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb/\u003c/p\u003e \u003cp\u003e\u003csup\u003e207\u003c/sup\u003ePb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003csup\u003e208\u003c/sup\u003ePb/\u003c/p\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003csup\u003e207\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003csup\u003e208\u003c/sup\u003ePb, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb/\u003c/p\u003e \u003cp\u003e\u003csup\u003e207\u003c/sup\u003ePb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003csup\u003e208\u003c/sup\u003ePb/\u003c/p\u003e \u003cp\u003e\u003csup\u003e206\u003c/sup\u003ePb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e 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colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e13.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.15\u003c/p\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e11.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e83.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e11.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean, ppb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e12.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the digestion protocols and ICP MS performance, quality control using serum ClinChek control for trace elements Level II was performed. No significant differences in measured metal concentrations between the two digestion protocols were observed (Table\u0026nbsp;3). However, the measured concentrations of \u003csup\u003e27\u003c/sup\u003eAl and \u003csup\u003e52\u003c/sup\u003eCr were significantly above the control range, which suggests external introduction or interference. The results for \u003csup\u003e27\u003c/sup\u003eAl and \u003csup\u003e52\u003c/sup\u003eCr were therefore excluded from further discussion.\u003c/p\u003e \u003cp\u003eSample protein fractionation was achieved by Size-Exclusion Chromatography and targeted 15 metal isotopes (\u003csup\u003e27\u003c/sup\u003eAl, \u003csup\u003e24\u003c/sup\u003eMg, \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e51\u003c/sup\u003eV, \u003csup\u003e52\u003c/sup\u003eCr, \u003csup\u003e55\u003c/sup\u003eMn, \u003csup\u003e56\u003c/sup\u003eFe, \u003csup\u003e66\u003c/sup\u003eZn, \u003csup\u003e59\u003c/sup\u003eCo, \u003csup\u003e60\u003c/sup\u003eNi, \u003csup\u003e78\u003c/sup\u003eSe, \u003csup\u003e111\u003c/sup\u003eCd, \u003csup\u003e75\u003c/sup\u003eAs \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e238\u003c/sup\u003eU). The chromatograms for \u003csup\u003e63\u003c/sup\u003eCu, \u003csup\u003e66\u003c/sup\u003eZn, \u003csup\u003e56\u003c/sup\u003eFe, and \u003csup\u003e52\u003c/sup\u003eCr displayed peaks corresponding to high-molecular-weight (HMW) and/or low-molecular-weight (LMW) protein species. The distribution between HMW and LMW protein peaks varied between metal ions and sample types (CSF \u003cem\u003evs\u003c/em\u003e blood plasma), and chromatograms of samples from ALS cases and controls exhibited minor variations in the number of peaks and their distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Column calibration identified ceruloplasmin as the primary \u003csup\u003e63\u003c/sup\u003eCu-binding species in ALS blood plasma, while \u003csup\u003e63\u003c/sup\u003eCu in ALS CSF was predominantly associated with LMW species. \u003csup\u003e66\u003c/sup\u003eZn was distributed between both fractions, with the major LMW fraction in blood plasma and a LMW \u003csup\u003e66\u003c/sup\u003eZn-bound fraction was detected in the CSF chromatograms. The \u003csup\u003e56\u003c/sup\u003eFe chromatograms for CSF and blood plasma both exhibited two peaks with the same retention time, where one protein peak likely represents transferrin and the other peak likely corresponds to LMW proteins. The area of the transferrin peak was substantially lower in CSF samples than in the blood plasma samples. \u003csup\u003e52\u003c/sup\u003eCr in the blood plasma was bound to a protein with a retention time similar to that of HSA, whereas in the CSF \u003csup\u003e52\u003c/sup\u003eCr was only detected in the LMW fraction. \u003csup\u003e78\u003c/sup\u003eSe was evident as a protein-bound peak at extremely low concentrations but only in plasma samples, and was therefore excluded from further discussion. An example of a non-detectable species under our LC ICP-MS conditions is illustrated by the chromatogram for \u003csup\u003e208\u003c/sup\u003ePb.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEstablished risk factors for ALS include male gender (PMID: 25054277), advanced age and specific mutations for a subset of patients (Pasinelli and Brown, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Logroscino et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Andersen and Al-Chalabi, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Environmental factors in general and metal exposure in particular have emerged as potential risk factors for ALS and for other neurodegenerative diseases (Felmus et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Roos, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ALS has been attributed a long preclinical period (Eisen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and the complexity of metal measurements and challenges inherent in relating such measurements to environmental exposures during the lifetime of an ALS patient are acknowledged.\u003c/p\u003e \u003cp\u003eThis study used an ultraclean sampling techniques and measurements of metal concentrations by ICP-MS for precise quantification of metals, and we detected deviations in ALS patients. We here discuss in detail the implications of our findings, especially the significantly elevated concentrations of Pb and Se in CSF from ALS patients, and explore their broader relevance to ALS pathogenesis in the light of possible environmental exposures.\u003c/p\u003e\n\u003ch3\u003eElevated lead concentrations in ALS body fluids\u003c/h3\u003e\n\u003cp\u003eLead exposure has long been implicated as a possible cause of ALS and is implicated in other neurodegenerative diseases as well due to its well-documented detrimental effects on neuronal integrity, contribution to oxidative stress, disruption of synaptic signalling and promotion of protein misfolding and aggregation (Johnson and Atchison, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Roos, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Associations between Pb exposure and ALS risk have previously been reported (Kamel et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Some research has suggested a direct connection between Pb exposure and ALS, yet other studies have not been able to corroborate that (Callaghan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study we observed significantly elevated \u003csup\u003e208\u003c/sup\u003ePb concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in ALS CSF compared to healthy controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings align with the results of a thorough meta-analysis which studied 29 high-quality articles narrowed down from 4234 articles studying metals in ALS body fluids (Kamalian et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). At the meta-level, ALS whole blood Pb was elevated by 2.88 \u0026micro;g/L (p\u0026thinsp;=\u0026thinsp;0.006), and ALS CSF Pb was elevated by 0.21 \u0026micro;g/L (p\u0026thinsp;=\u0026thinsp;0.04). This concords with the findings of the present study and strongly supports the notion that Pb contributes to ALS pathogenesis.\u003c/p\u003e \u003cp\u003eOccupational exposure is a well-documented risk factor for Pb accumulation (Roos et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Johnson and Atchison, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Farace et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Olowoyo et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A meticulous meta-analysis of occupational risk factors for ALS concluded that ALS risk was statistically significantly elevated for occupational exposures to metals (especially Pb), chemicals (especially pesticides), and excessive physical work (Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Many older pesticides are Pb derivatives, such as the widely used lead arsenate (US_EPA, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Delistraty and Yokel, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, the conclusion that pesticides are a risk factor for ALS can to some extent be extrapolated to occupational Pb exposure as well (Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Lead is present in all aspects of occupational life where dermal Pb exposure exists (Askin and Volkmann, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), and occupationally Pb-exposed workers also receive respiratory Pb exposure from Pb-contaminated cigarettes (Dykeman et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). More than a hundred occupations involving Pb exposure have been identified (Nordberg and Costa, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), where leaded paint, backyard recycling of used lead-acid batteries (Ericson et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and ammunition work are prominent Pb sources (Nordberg and Costa, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Documented occupational Pb exposure linked to an elevated ALS risk include military service occupations (Seals et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Peters et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), construction workers (Fang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Dickerson et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), mechanical workers (Park et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sutedja et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), gasoline station forecourt attendants (W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), precision tool manufacturers, and glass, pottery, and tile workers (Peters et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), leather workers (Buckley et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and tanners (Chio et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Bhuiyan et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Farmers also show an elevated ALS risk (Bale, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Rosati et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), possibly because farm workers are exposed to pesticides and insecticides (Breland and Currier, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Greenberg et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1979\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study no significant differences in plasma Pb concentrations were determined between ALS cases and controls, but elevated \u003csup\u003e208\u003c/sup\u003ePb concentrations were evident in ALS CSF (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that Pb accumulation in ALS may be a CNS-specific phenomenon. This pattern is consistent with a potential lack of protection from the BBB and/or blood-CSF barrier i.e. the choroid plexus, which could facilitate the preferential accumulation of Pb in the CNS while maintaining near-normal plasma levels. Previous studies have shown that Pb disrupts tight junctions in barrier systems due to its ability to substitute for calcium ions (Ca\u003csup\u003e2+\u003c/sup\u003e), contributing to selective Pb\u003csup\u003e2+\u003c/sup\u003e BBB permeability and high Pb concentrations in the brain (Zheng, \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sanders et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIsotopic analysis of Pb: Insights into sources and mechanisms\u003c/h2\u003e \u003cp\u003eTo better understand the environmental origins of Pb exposure in ALS, we analysed Pb isotopic ratios in both CSF and plasma samples. The \u003csup\u003e206\u003c/sup\u003ePb/\u003csup\u003e207\u003c/sup\u003ePb and \u003csup\u003e208\u003c/sup\u003ePb/\u003csup\u003e206\u003c/sup\u003ePb ratios were altered in ALS patients compared to controls, suggesting differences in Pb sources or metabolic handling. These ratios serve as geochemical fingerprints, as the natural abundance of Pb isotopes is influenced by radioactive decay pathways in geological systems (Cheng and Hu, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Morton-Bermea et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For instance, \u003csup\u003e206\u003c/sup\u003ePb and \u003csup\u003e207\u003c/sup\u003ePb derive from uranium decay, while \u003csup\u003e208\u003c/sup\u003ePb is a decay product of \u003csup\u003e232\u003c/sup\u003eTh (Aberg et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kulikov et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Variations in these ratios between ALS patients and controls may reflect occupational or environmental exposure to Pb sources with distinct isotopic signatures.\u003c/p\u003e \u003cp\u003eLead isotope ratios offer significant advantages due to their stability in physical, chemical, and biological processes. They are minimally affected by isotope fractionation, making them reliable for tracing the origins of Pb across disciplines such as archaeology, agriculture and environmental science. However, recent findings suggest that Pb isotopic fingerprints in biological samples can vary significantly between different tissues and fluids such as blood, urine and hair (Liu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This complicates a direct identification of environmental Pb sources. Isotope ratios may shift due to biological fractionation, a poorly understood phenomenon influenced by absorption, metabolism and redistribution factors (Wu et al., \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Notably, fractionation thresholds exist whereby isotopic ratios behave differently at varying blood Pb levels. For example, fractionation appears to become abnormal at high Pb concentrations, thereby altering the differences between biological samples and the Pb source (Wu et al., \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetal exposure in ALS seems to be varied, complex and individually unique (Roos, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Occupational exposure to several neurotoxic metals has been linked to ALS (Roos et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gunnarsson and Bodin, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and here we report that Pb and Se, both with neurotoxic properties, are elevated in ALS CSF. Isotopic fingerprints, with the caveats outlined above, might be useful for tracing possible sources of metal exposure in ALS, using lessons learned from other neurodegenerative diseases where the geochemical origin of metal exposures has been outlined (Astrom and Roos, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSelenium and its role in ALS\u003c/h2\u003e \u003cp\u003eSelenium, an essential trace element, exhibits a dual nature in human health: It is essential for the functioning of antioxidant enzymes but is neurotoxic at higher concentrations. Herein significantly higher Se concentrations were recorded in ALS CSF (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while plasma Se showed no significant differences between ALS patients and controls. This suggests that Se accumulation in CSF may be a phenomenon linked to ALS. A role for Se in ALS pathogenesis is in line with the findings of a meta-analysis in which a 4.26 \u0026micro;g/L elevation of serum/plasma Se levels in ALS patients (p\u0026thinsp;=\u0026thinsp;0.02) was reported (Kamalian et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsumption of drinking water containing\u0026thinsp;\u0026ge;\u0026thinsp;1\u0026micro;g/L of inorganic selenium has been associated with a relative risk of 5.4 (95% confidence interval 1.1\u0026ndash;26) for ALS after adjusting for confounding factors (Vinceti et al., \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Vinceti et al., \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Vinceti et al., \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Excessive Se exposure, particularly in its inorganic forms, has been implicated in oxidative stress, mitochondrial dysfunction and apoptosis, all molecular mechanisms relevant to ALS pathogenesis (Simpson et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; D'Amico et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe absence of elevated plasma Se concentrations in our study suggests that Se dysregulation in ALS is confined to the CNS. The neurotoxic effects of Se may emanate from its ability to oxidize thiol-containing proteins and to facilitate the translocation of copper/zinc superoxide dismutase (SOD1) into mitochondria, thereby disrupting cellular function (Filippini et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The level of Se toxicity likely depends on its chemical form, as inorganic and organic species present distinctly different biological properties (Vinceti et al., \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, Se-CSF (behind the BBB) concentrations are independent from Se-serum (before the BBB) concentrations, suggesting the possibility of an independent Se regulation in the CNS (Michalke et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMetal-driven inflammation in ALS\u003c/h2\u003e \u003cp\u003eEvidence exists for a low-grade systemic inflammatory reaction in ALS (Keizman et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The erythrocyte sedimentation rate is often normal in ALS patients at an early stage, but increases slowly over time, correlating with disease severity (Keizman et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is unknown to what extent the increased concentrations of Pb and Se contribute to the observed inflammation in ALS, but for other metals potential mechanisms have been proposed. For example, loss of cuproprotein function due to a combination of unbound copper and ROS that can either initiate and/or accelerate protein aggregation has been claimed to be central to ALS pathology. Copper homeostasis within motor neurons is disrupted in ALS patient spinal cords as well as in the SOD1G93A mouse model (Williams et al., \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hilton et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This could result in toxicity caused by the excess of redox active copper that is free to form harmful reactive oxygen species (ROS) that can cause damage through lipid peroxidation and DNA damage (Sauzeat et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sheykhansari et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or by functional deficiency. The promotion of protein aggregation in neurodegenerative diseases is a general pathological characteristic, and both Pb and Hg salts increase TDP-43 aggregation \u003cem\u003ein vitro\u003c/em\u003e (Ash et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cadmium competitively displaces zinc from its binding site in SOD1, which leads to protein folding defects and inactivation of the enzyme while simultaneously generating ROS (Huang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Oggiano et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a general pathological mechanism, protein aggregation leads to metal ion deficiency and functional loss of metal-based enzymes, which leads to more aggregation in a self-perpetuating cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMetal ratios in ALS\u003c/h2\u003e \u003cp\u003eBeyond Pb and Se, our study identified lower concentrations of arsenic (As) and manganese (Mn) in ALS CSF, with no significant differences in plasma levels. This contradicts the hypothesis that Mn toxicity contributes to ALS (Roos et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Roos et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ratios between metal concentrations in CSF and in plasma (Q\u003csub\u003eCSF/plasma\u003c/sub\u003e) were calculated to better understand the distribution of metal ions between CSF and blood plasma and to provide insight into BBB permeability. A higher Q\u003csub\u003eCSF/plasma\u003c/sub\u003e suggests that metal ions readily cross the BBB and accumulate in CSF, potentially contributing to neurotoxicity. In ALS patients, \u003csup\u003e208\u003c/sup\u003ePb exhibited a significantly higher Q\u003csub\u003eCSF/plasma\u003c/sub\u003e, indicating that Pb accumulates in CSF, suggesting an ALS-related alteration in Pb transport or clearance mechanisms. However, despite elevated Se concentrations in ALS CSF, the Q\u003csub\u003eCSF/plasma\u003c/sub\u003e for \u003csup\u003e82\u003c/sup\u003eSe was not significantly increased. This could imply that the observed Se elevation originates from a systemic increase in plasma Se, rather than enhanced transport across the BBB, or that selenium homeostasis in the brain is tightly regulated by selenoproteins, preventing disproportionate accumulation in the CSF.\u003c/p\u003e \u003cp\u003eAdditionally, \u003csup\u003e55\u003c/sup\u003eMn exhibited a significantly lower Q\u003csub\u003eCSF/plasma\u003c/sub\u003e in ALS, suggesting restricted Mn transport into the CSF or increased Mn efflux from the CSF, which may reflect ALS-related disturbances in Mn metabolism. No significant difference was observed for \u003csup\u003e75\u003c/sup\u003eAs, indicating that arsenic homeostasis in the CSF is maintained despite systemic exposure.\u003c/p\u003e \u003cp\u003eMetal ratios between CSF and blood serum have previously been reported for various metals. However, direct comparison with our Q\u003csub\u003eCSF/plasma\u003c/sub\u003e values is not possible, as plasma and serum vary in protein composition and because of differences in study design. Serum lacks fibrinogen, a key plasma protein that can bind metal ions, potentially altering the distribution of metals between compartments.\u003c/p\u003e \u003cp\u003eThe elevated plasma Ni concentrations observed in ALS patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) require further investigation. Nickel induces oxidative stress, neuronal DNA damage, and protein misfolding and aggregation (Guo et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Berntsson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The role of Ni in ALS pathogenesis remains to elucidate. Childhood exposure to Ni and Cr, and co-exposure to Ni, Cr, Sr, and other metals, has recently been linked to adult-onset ALS (Figueroa-Romero et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGenotoxic effects of metals in ALS\u003c/h2\u003e \u003cp\u003eMost monogenetic forms of familial ALS (fALS) do not develop in utero or in childhood but instead appear later in adulthood. Nonetheless, fALS typically present at a younger age compared to sporadic ALS (sALS). Mathematical modeling, adapted from cancer research, suggests that the development of ALS is a multistep process requiring a sequence of six separate genetic or environmental events to happen within an individual genome. This modeling indicates that environmental factors contribute to the development of fALS just as they do for sALS (Al-Chalabi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLead, Se and Ni, along with other redox-active metals, are known to induce genotoxic effects, including DNA damage and impaired DNA repair mechanisms (Rusov et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nagaraju et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lead interferes with DNA repair enzymes, while excessive Se can generate reactive oxygen species which damage mitochondrial and nuclear DNA (Valdiglesias et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These genotoxic pathways align closely with the mechanisms of motor neuron degeneration observed in ALS, underscoring the need for further exploration of these effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStrengths and limitations\u003c/h2\u003e \u003cp\u003eA key strength of this study is the ultraclean protocol employed during sample collection (Roos, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and the minimization of contamination during sample handling and digestion, enhancing measurement reliability. The polypropylene vials used in this study were acid-washed with ultrapure nitric acid to avoid metal contamination. We suggest using PTFE vials (Teflon\u0026reg;) instead of polypropylene vials for laboratory work in future studies of metals in ALS to eliminate metal contamination from the plastic vials. The ICP-MS measurements were performed in helium mode [He] to reduce polyatomic interferences (e.g. Argon based interferences for As, Fe and Cr). No gas [No Gas] mode was used for the Pb isotopes, whose polyatomic interferences are minimal, leveraging the sensitivity for Pb. Quality control with ClinCheck Level II serum confirmed that \u003csup\u003e208\u003c/sup\u003ePb concentrations were within reported ranges, supporting the choice of [No Gas] mode for that element. These strengths of the current study are limited by the small number of ALS and control samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFuture perspectives\u003c/h2\u003e \u003cp\u003eThis study highlights the critical role of environmental metal exposures in ALS pathogenesis and opens several avenues for future research. The following actions are suggested:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eTrace exposure sources: Pb isotope analysis should be expanded to identify specific environmental and occupational sources of metal exposure in ALS patients.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eExplore mechanisms of metal dysregulation: Investigate the molecular mechanisms underlying selective metal accumulation in the CNS and their interaction with ALS-specific genetic vulnerabilities.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eLongitudinal studies: Perform prospective studies tracking metal exposure, accumulation, and disease progression to establish causal relationships and time-dependent effects.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eUse a wide range of samples: Include hard tissues samples along with CSF and blood plasma/serum, taken under ultraclean conditions simultaneously from the same individual.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eExplore protein-bound metal distribution patterns: Use LC ICP-MS as a powerful tool to separate protein fractions.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eExposure to neurotoxic metals seem to contribute to ALS pathogenesis. Lead and selenium concentrations are elevated in ALS cerebrospinal fluid and should be further investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis study was supported by the Estonian Research Council grant (PRG 1289) to P.P. and by grants from the Kamprad Research Foundation, grant #20200063, the Ulla-Carin Lindquist Foundation for ALS Research, grant #8024247192, the ALS Bone Metals Donation Karolinska Institutet, grant #K825871153 and the Magnus Bergvall foundation, grant #202104517 to P.M.R.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAaseth J, Alexander J, Bjorklund G, Hestad K, Dusek P, Roos PM, Alehagen U (2016) Treatment strategies in Alzheimer's disease: a review with focus on selenium supplementation. 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Toxicol Appl Pharmacol 192(1):1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Karolinska Institute","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ALS, exposure, ICP-MS, isotopes, lead, metals, SEC","lastPublishedDoi":"10.21203/rs.3.rs-6839641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6839641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron degeneration and muscle atrophy, ultimately leading to death through respiratory failure. Environmental factors, including metal exposure, seem to contribute to ALS pathogenesis. Elevated concentrations of metals such as Pb, Se, and Cd have been detected in ALS cerebrospinal fluid (CSF) and blood from ALS patients, providing clues to possible pathogenesis. Here we conducted a detailed analysis of CSF and blood plasma samples collected by ultraclean techniques from seven ALS patients and seven healthy controls. Inductively coupled plasma mass spectrometry (ICP-MS) was used for metal measurements. Significantly higher concentrations of \u003csup\u003e208\u003c/sup\u003ePb and \u003csup\u003e78\u003c/sup\u003eSe (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and lower concentrations of \u003csup\u003e75\u003c/sup\u003eAs and \u003csup\u003e55\u003c/sup\u003eMn (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), were found in ALS CSF but not in ALS blood plasma. Additionally, ALS patients displayed increased concentrations of \u003csup\u003e60\u003c/sup\u003eNi in blood plasma relative to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Elevated \u003csup\u003e208\u003c/sup\u003ePb/\u003csup\u003e206\u003c/sup\u003ePb and \u003csup\u003e206\u003c/sup\u003ePb/\u003csup\u003e207\u003c/sup\u003ePb ratios were observed in ALS CSF, possibly tracing sources of lead exposure. Samples were also analyzed using size-exclusion chromatography coupled with ICP-MS to monitor the distribution of metals between different protein carriers. The combined findings support the hypothesis that metal exposure contributes to the pathogenesis of ALS.\u003c/p\u003e","manuscriptTitle":"Elevated selenium and lead concentrations in amyotrophic lateral sclerosis cerebrospinal fluid provide clues to ALS pathogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 12:08:34","doi":"10.21203/rs.3.rs-6839641/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12215e6f-0b6a-4e92-9d85-77cc2dd3b8e7","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49668100,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2025-06-11T12:08:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 12:08:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6839641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6839641","identity":"rs-6839641","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}