Cerebrospinal Fluid Iron-Ferritin Ratio as a Potential Progression Marker for Parkinson's Disease.

Longitudinal PD CSF samples were subjected to ICP-MS and the total amount of iron and other bioelements was quantified. Additionally, ferritin and protein biomarkers of neurodegeneration were measured. Over time, mean iron levels significantly increased while levels of ferritin decreased.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III)).

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 µg/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 µg/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was <3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Assessment of copper, iron, zinc and manganese status and speciation in patients with Parkinson's disease: A pilot study.

BACKGROUND: The objective of this pilot study was to assess iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) status (hair, serum, and urine) and speciation (serum) in Parkinson's disease (PD) patients. METHODS: A pilot study involving a total of 27 subjects (13 PD patients, 14 controls) was performed. Serum, urine, and hair metal content was assessed using ICP-MS. Speciation analysis of Cu, Zn, Fe, and Mn was performed using a hybrid HPLC-ICP-MS system. RESULTS: Group comparisons did not reveal any significant group difference in serum Cu, Zn, Fe, and Mn total metal level between PD patients and controls. Speciation analysis revealed a significant decrease in Cu/ceruloplasmin copper in association with elevation of low-molecular weight species (amino acids)-bound copper. It is proposed that in PD, binding of Cu(II) ions to ceruloplasmin is reduced and free copper ions coordinate with low molecular weight ligands. The level of Mn-albumin complexes in PD patients was more than 4-fold higher as compared to the respective value in the control group. The observed difference may be considered as a marker of redistribution between high and low molecular weight ligands. CONCLUSIONS: Metal speciation is significantly affected in serum of PD-patients. These findings are indicative of the potential role of metal metabolism and PD pathogenesis, although the exact mechanisms of such associations require further detailed studies.

Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS).

Neuronal iron dyshomeostasis occurs in multiple neurodegenerative diseases. Changes in the Fe(II)/Fe(III) ratio toward Fe(II) is closely related to oxidative stress, lipid peroxidation, and represents a hallmark feature of ferroptosis. In particular for body fluids, like cerebrospinal fluid (CSF), reliable quantitative methods for Fe(II)/(III) redox-speciation analysis are needed to better assess the risk of Fe(II)-mediated damage in brain tissue. Currently in the field of metallomics, the most direct method to analyze both iron species is via LC-ICP-MS. However, this Fe(II)/(III) speciation analysis method suffers from several limitations. Here, we describe a unique method using capillary electrophoresis (CE)-ICP-MS for quantitative Fe(II)/(III) speciation analysis that can be applied for cell lysates and biofluid samples. Compared to LC, CE offers various advantages: (1) Capillaries have no stationary phase and do not depend on batch identity of stationary phases; (2) Replacement of aged or blocked capillaries is quick with no performance change; (3) Purge steps are effective and short; (4) Short sample analysis time. The final method employed 20 mM HCl as background electrolyte and a separation voltage of +25 kV. In contrary to the LC-method, no complexation of Fe-species with pyridine dicarboxylic acid (PDCA) was applied, since it hampered separation. Peak shapes and concentration detection limits were improved by combined conductivity-pH-stacking achieving 3 µg/L detection limit (3σ) at 13 nL injection volume. Calibrations from LOD-150 µg/L were linear [r 2 [Fe(II)] = 0.9999, r 2 [Fe(III)] = 0.9951]. At higher concentrations Fe(II) curve flattened significantly. Measurement precision was 3.5% [Fe(II) at 62 µg/L] or 2.2% [Fe(III) at 112 µg/L] and migration time precision was 2% for Fe(III) and 3% for Fe(II), each determined in 1:2 diluted lysates of human neuroblastoma cells. Concentration determination accuracy was checked by parallel measurements of SH-SY5Y cell lysates with validated LC-ICP-MS method and by recovery experiments after standard addition. Accuracy (n = 6) was 97.6 ± 3.7% Fe(III) and 105 ± 6.6%Fe(II). Recovery [(a) +33 µg/L or (b) +500 µg/L, addition per species] was (a): 97.2 ± 13% [Fe(II)], 108 ± 15% [Fe(III)], 102.5 ± 7% (sum of species), and (b) 99±4% [Fe(II)], 101 ± 6% [Fe(III)], 100 ± 5% (sum of species). Migration time shifts in CSF samples were due to high salinity, but both Fe-species were identified by standard addition.

Manganese causes neurotoxic iron accumulation via translational repression of amyloid precursor protein and H-Ferritin.

For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD-like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose- and time-dependently blocks the protein translation of amyloid precursor protein (APP) and heavy-chain Ferritin (H-Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H-Ferritin are post-transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5'-untranslated regions (5'-UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5'-UTR-activity of APP and H-Ferritin, presumably via increased iron responsive proteins-iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe2+ -specific probes (RhoNox-1 and IP-1) and ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS), we show that loss of the protective axis of APP and H-Ferritin resulted in unchecked accumulation of redox-active ferrous iron (Fe2+ ) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn-induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn-mediated suppression of APP and H-Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn-induced neurotoxicity is partly attributable to the translational inhibition of APP and H-Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.