Evaluation of the Potential of Single Particle ICP-MS for the Accurate Measurement of the Number Concentration of AuNPs of Different Sizes and Coatings.

Single particle inductively coupled plasma-mass spectrometry (spICP-MS) is an emerging technique that is capable of simultaneous measurement of the size and number concentration of metal-containing nanoparticles (NPs) at environmentally relevant levels. Although spICP-MS is widely applied to different fields, challenges remain in obtaining accurate and consistent particle number concentration (PNC) measurements. This paper presents, for the first time, a rigorous assessment of spICP-MS capabilities for measuring the PNC of gold NP (AuNP) suspensions of different sizes and coatings. The calibration of spICP-MS was accomplished with the National Institute of Standards and Technology (NIST) AuNP reference material (RM) 8013. The comparability of both spICP-MS direct and derived determination of PNC and reference PNC derived based on the mean particle size or the particle size distribution obtained by different reference sizing techniques was first assessed for NIST AuNP RM 8012, nominal diameter 30 nm. To enable a proper assessment of the accuracy of the spICP-MS results, a comprehensive estimation of the expanded uncertainty for PNC determination was carried out. Regardless of NP size or coating, a good agreement (90-110%) between spICP-MS direct determination of PNC and reported PNCs was obtained for all of the suspensions studied only when reliable in-house Au mass fractions and thorough mean particle size determinations were included in the calculation of the derived PNCs. The use of the particle size distribution over the mean size to derive PNCs resulted in larger differences for materials with a low contribution (<2%) of smaller NPs (30 nm), materials with a higher polydispersity (100 nm), or materials with two distinct subpopulations of particles (60 nm), regardless of NP coating.

Expression of tyrosine hydroxylase isoforms and phosphorylation at serine 40 in the human nigrostriatal system in Parkinson's disease.

Tyrosine hydroxylase is the key enzyme controlling the synthesis of the catecholamines including dopamine. The breakdown of dopamine into toxic compounds has been suggested to have a key role in the degeneration of the dopaminergic neurons in Parkinson's disease. Humans are unique in containing four isoforms of tyrosine hydroxylase, but understanding of the role of these isoforms under normal conditions and in disease states is limited. The aim of this work was to determine the level and distribution of the four human isoforms in tissues from healthy controls and patients with Parkinson's disease. The results show that isoform 1 and isoform 2 are the major tyrosine hydroxylase isoforms in human brain, but that tyrosine hydroxylase isoform 2 is more abundant in the substantia nigra than the tyrosine hydroxylase isoform 1. The two minor isoforms, isoform 3 and isoform 4, are expressed at a proportionally higher level in the terminal field regions (caudate and putamen) compared to the substantia nigra. There was a selective loss of tyrosine hydroxylase isoform 1 in Parkinson's disease compared to age-matched controls and a corresponding increase in the proportion of tyrosine hydroxylase isoform 2. Phosphorylation of serine 40 was significantly increased in caudate, putamen and ventral tegmental area, but not in the substantia nigra, in Parkinson's disease brain. These results show a selective sparing of tyrosine hydroxylase isoform 2 in Parkinson's disease. Isoform 2 exhibits a reduced capacity for activation compared to isoform 1, which may account for the selective sparing of cells expressing isoform 2 in Parkinson's disease. Surviving neurons in Parkinson's disease brain exhibit a substantial increase in tyrosine hydroxylase phosphorylation consistent with a compensatory mechanism of increased dopamine synthesis in the terminal field regions.

Validation of Single Particle ICP-MS for Routine Measurements of Nanoparticle Size and Number Size Distribution.

Single particle inductively coupled plasma-mass spectrometry (spICP-MS) is an emerging technique capable of simultaneously measuring nanoparticle size and number concentration of metal-containing nanoparticles (NPs) at environmental levels. single particle ICP-MS will become an established measurement method once the metrological quality of the measurement results it produces have been proven incontrovertibly. This Article presents the first validation of spICP-MS capabilities for measuring mean NP size and number size distribution of gold nanoparticles (AuNPs). The validation is achieved by (i) calibration based on the consensus value for particle size derived from six different sizing techniques applied to National Institute of Standards and Technology (NIST) Reference Material (RM) 8013; (ii) comparison with high-resolution scanning electron microscopy (HR-SEM) used as a reference method, which is linked to the International System of Units (SI) through a calibration standard characterized by the NIST metrological atomic force microscope; and (iii) evaluation of the uncertainty associated with the measurement of the mean particle size to enable comparison of the spICP-MS and HR-SEM methods. After establishing HR-SEM and spICP-MS measurement protocols, both methods were used to characterize commercial AuNP suspensions of three different sizes (30, 60, and 100 nm) with four different coatings and surface charge at pH 7. Single particle ICP-MS measurements (corroborated by HR-SEM) revealed the existence of two distinct subpopulations of particles in the number size distributions for four of the 60 nm commercial suspensions, a fact that was not apparent in the measurement results supplied by the vendor using transmission electron microscopy. This finding illustrates the utility of spICP-MS for routine characterization of commercial AuNP suspensions regardless of size or coating.

Development of a kelp powder (Thallus laminariae) Standard Reference Material.

A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder (Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K1, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K1, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. Graphical Absract Arsenic species and isomers of vitamin K1 were determined in the development of SRM 3232 Kelp Powder (Thallus laminariae).

Overcoming challenges in single particle inductively coupled plasma mass spectrometry measurement of silver nanoparticles.

Single particle ICP-MS has evolved rapidly as a quantitative method for determining nanoparticle size and number concentration at environmentally relevant exposure levels. Central to the application of spICP-MS is a commonly used, but not rigorously validated, calibration approach based on the measured transport efficiency and the response of ionic standards. In this work, we present a comprehensive and systematic study of the accuracy, precision and robustness of spICP-MS using the rigorously characterized reference material (RM) 8017 (Polyvinylpyrrolidone Coated Nominal 75 nm Silver Nanoparticles), recently issued by the National Institute of Standards and Technology (NIST). We report for the first time, statistically significant differences in frequency-based and size-based measures of transport efficiency with NIST RM 8013 Gold Nanoparticles and demonstrate that the size-based measure of transport efficiency is more robust and yields accurate results for the silver nanoparticle RM relative to TEM-based reference values. This finding is significant, because the frequency-based method is more widely applied. Furthermore, we demonstrate that the use of acidified ionic standards improves measurement of ICP-MS Ag response, but does not degrade the accuracy of the results for AgNP suspensions in water or various other diluents. Approaches for controlling AgNP dissolution were investigated and are shown to effectively improve particle stability in dilute suspensions required for spICP-MS analysis, while minimally affecting the measured intensity and allowing for more robust analysis. This study is an important and necessary advancement toward full validation and adoption of spICP-MS by the broader research community. Graphical abstract Measurement challenges in spICP-MS analysis.

Development of two fine particulate matter standard reference materials (<4 µm and <10 µm) for the determination of organic and inorganic constituents.

Two new Standard Reference Materials (SRMs), SRM 2786 Fine Particulate Matter (<4 µm) and SRM 2787 Fine Particulate Matter (<10 µm) have been developed in support of the US Environmental Protection Agency's National Ambient Air Quality Standards for particulate matter (PM). These materials have been characterized for the mass fractions of selected polycyclic aromatic hydrocarbons (PAHs), nitrated PAHs, brominated diphenyl ether (BDE) congeners, hexabromocyclododecane (HBCD) isomers, sugars, polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners, and inorganic constituents, as well as particle-size characteristics. These materials are the first Certified Reference Materials available to support measurements of both organic and inorganic constituents in fine PM. In addition, values for PAHs are available for RM 8785 Air Particulate Matter on Filter Media. As such, these SRMs will be useful as quality control samples for ensuring compatibility of results among PM monitoring studies and will fill a void to assess the accuracy of analytical methods used in these studies. Graphical Abstract Removal of PM from filter for the preparation of SRM 2786 Fine Particulate Matter.

Lysosomal-associated membrane protein 2 isoforms are differentially affected in early Parkinson's disease.

Lysosomes are the primary catabolic compartment for the degradation of intracellular proteins through autophagy. The presence of abnormal intracellular α-synuclein-positive aggregates in Parkinson's disease (PD) indicates that the degradative capacity of lysosomes is impaired in PD. Specific dysfunction of chaperone-mediated autophagy (CMA) in PD is suggested by reductions in the CMA membrane receptor, lysosomal-associated membrane protein (LAMP) 2A, although whether LAMP2A is the only LAMP2 isoform affected by PD is unknown. Messenger RNA (mRNA) and protein expression of all three LAMP2 isoforms was assessed in brain extracts from regions with and without PD-related increases in α-synuclein in autopsy samples from subjects in the early pathological stage of PD (n = 9), compared to age- and postmortem delay-matched controls (n = 10). In the early stages of PD, mRNA expression of all LAMP2 isoforms was not different from controls, with LAMP2B and LAMP2C protein levels also unchanged in PD. The selective loss of LAMP2A protein directly correlated with the increased levels of α-synuclein and decreased levels of the CMA chaperone heat shock cognate protein 70 in the same PD samples, as well as with the accumulation of cytosolic CMA substrate proteins. Our data show that LAMP2 protein isoforms are differentially affected in the early stages of PD, with LAMP2A selectively reduced in association with increased α-synuclein, and suggests that dysregulation of CMA-mediated protein degradation occurs before substantial α-synuclein aggregation in PD.

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson's disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson's disease. We investigated relationships between Parkinson's disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson's disease without GBA1 mutations. Brain regions with and without a Parkinson's disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson's disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson's disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson's disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson's disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson's disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson's disease pathology.

Capabilities of single particle inductively coupled plasma mass spectrometry for the size measurement of nanoparticles: a case study on gold nanoparticles.

The increasing application of engineered nanomaterials (ENMs) in consumer and medical products has motivated the development of single-particle inductively coupled plasma mass spectrometry (spICP-MS) for characterizing nanoparticles under realistic environmental exposure conditions. Recent studies have established a set of metrological criteria and evaluated the feasibility of spICP-MS for sizing or quantifying various highly commercialized ENMs. However, less is known about the performance of spICP-MS for detecting nanoparticles with sizes greater than 80 nm. This paper presents a systematic study on spICP-MS for accurate size measurement of gold nanoparticles from 10 to 200 nm. We show that dwell time contributes significantly to the quality of data, with the optimal dwell time that limits split particle events, particle coincidences and false positives being 10 ms. A simple approach to correct for split particle events is demonstrated. We show that transient features of single particle events can be temporally resolved on a conventional quadrupole ICP-MS system using a sufficiently short dwell time (0.1 ms). We propose an intensity-size diagram for estimating the linear dynamic size range and guiding the selection of ICP-MS operating conditions. The linear dynamic size range of the ICP-MS system under standard (highest) sensitivity conditions is 10 to 70 nm but can be further extended to 200 nm by operating in less sensitive modes. Finally, the ability of spICP-MS to characterize heterogeneous forms of metal containing nanoparticles is evaluated in mixtures containing both dissolved and poly disperse nanoparticulate Au.

Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson's disease.

Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson's disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

Characterization of nanoparticles in silicon dioxide food additive.

Silicon dioxide (SiO2), in its amorphous form, is an approved direct food additive in the United States and has been used as an anticaking agent in powdered food products and as a stabilizer in the production of beer. While SiO2 has been used in food for many years, there is limited information regarding its particle size and size distribution. In recent years, the use of SiO2 food additive has raised attention because of the possible presence of nanoparticles. Characterization of SiO2 food additive and understanding their physicochemical properties utilizing modern analytical tools are important in the safety evaluation of this additive. Herein, we present analytical techniques to characterize some SiO2 food additives, which were obtained directly from manufacturers and distributors. Characterization of these additives was performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and single-particle inductively coupled plasma mass spectrometry (spICP-MS) after the food additive materials underwent different experimental conditions. The data obtained from DLS, spICP-MS, and electron microscopy confirmed the presence of nanosized (1-100 nm) primary particles, as well as aggregates and agglomerates of aggregates with sizes greater than 100 nm. SEM images demonstrated that most of the SiO2 food additives procured from different distributors showed similar morphology. The results provide a foundation for evaluating the nanomaterial content of regulated food additives and will help the FDA address current knowledge gaps in analyzing nanosized particles in commercial food additives.

Development of a Standard Reference Material for metabolomics research.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Recognizing and overcoming analytical error in the use of ICP-MS for the determination of cadmium in breakfast cereal and dietary supplements.

The potential effect of spectral interference on the accurate measurement of the cadmium (Cd) mass fraction in fortified breakfast cereal and a variety of dietary supplement materials using inductively coupled plasma quadrupole mass spectrometry was studied. The materials were two new standard reference materials (SRMs)--SRM 3233 Fortified Breakfast Cereal and SRM 3532 Calcium Dietary Supplement--as well as several existing materials--SRM 3258 Bitter Orange Fruit, SRM 3259 Bitter Orange Extract, SRM 3260 Bitter Orange-containing Solid Oral Dosage Form, and SRM 3280 Multivitamin/Multielement Tablets. Samples were prepared for analysis using the method of isotope dilution and measured using various operating and sample introduction configurations including standard mode, collision cell with kinetic energy discrimination mode, and standard mode with sample introduction via a desolvating nebulizer system. Three isotope pairs, (112)Cd/(111)Cd, (113)Cd/(111)Cd, and (114)Cd/(111)Cd, were measured. Cadmium mass fraction results for the unseparated samples of each material, measured using the three instrument configurations and isotope pairs, were compared to the results obtained after the matrix was removed via chemical separation using anion exchange chromatography. In four of the six materials studied, measurements using the standard mode with sample introduction via the desolvating nebulizer gave results for the unseparated samples quantified with the (112)Cd/(111)Cd isotope pair that showed a positive bias relative to the matrix-separated samples, which indicated a persistent inference at m/z112 with this configuration. Use of the standard mode, without the desolvating nebulizer, also gave results that showed a positive bias for the unseparated samples quantified with the (112)Cd/(111)Cd isotope pair in three of the materials studied. Collision cell/kinetic energy discrimination mode, however, was very effective for reducing spectral interference for Cd in all of the materials and isotope pairs studied, except in the multivitamin/multielement matrix (SRM 3280) where the large corrections for known isobaric interferences or unidentified interferences compromised the accuracy. For SRM 3280, matrix separation provided the best method to achieve accurate measurement of Cd.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Combining secondary ion mass spectrometry image depth profiling and single particle inductively coupled plasma mass spectrometry to investigate the uptake and biodistribution of gold nanoparticles in Caenorhabditis elegans.

Analytical techniques capable of determining the spatial distribution and quantity (mass and/or particle number) of engineered nanomaterials in organisms are essential for characterizing nano-bio interactions and for nanomaterial risk assessments. Here, we combine the use of dynamic secondary ion mass spectrometry (dynamic SIMS) and single particle inductively coupled mass spectrometry (spICP-MS) techniques to determine the biodistribution and quantity of gold nanoparticles (AuNPs) ingested by Caenorhabditis elegans. We report the application of SIMS in image depth profiling mode for visualizing, identifying, and characterizing the biodistribution of AuNPs ingested by nematodes in both the lateral and z (depth) dimensions. In parallel, conventional- and sp-ICP-MS quantified the mean number of AuNPs within the nematode, ranging from 2 to 36 NPs depending on the size of AuNP. The complementary data from both SIMS image depth profiling and spICP-MS provides a complete view of the uptake, translocation, and size distribution of ingested NPs within Caenorhabditis elegans.

Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations.

Speciation measurements of gadolinium in liposomal MRI contrast agents (CAs) are complicated by the presence of emulsifiers, surfactants, and therapeutic agents in the formulations. The present paper describes two robust, hyphenated chromatography methods for the separation and quantification of gadolinium in nanoemulsion-based CA formulations. Three potential species of gadolinium, free gadolinium ion, gadolinium chelated by diethylenetriamine pentaacetic acid, and gadolinium chelated by 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, were present in the CA formulations. The species were separated by reversed-phase chromatography (reversed phase high-performance liquid chromatography, RP-HPLC) or by high-pressure size-exclusion chromatography (HPSEC). For RP-HPLC, fluorescence detection and post-column online isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) were used to measure the amount of gadolinium in each species. Online ID-ICP-MS and species-specific isotope dilution (SID)-ICP-MS were used in combination with the HPSEC column. The results indicated that some inter-species conversions and degradation had occurred within the samples and that SID-ICP-MS should be used to provide the most reliable measurements of total and speciated gadolinium. However, fluorescence and online ID-ICP-MS might usefully be applied as qualitative, rapid screening procedures for the presence of free gadolinium ions.

Demonstrating the comparability of certified reference materials.

Certified reference materials (CRMs) enable the meaningful comparison of measurement results over time and place. When CRMs are used to calibrate or verify the performance of a measurement system, results produced by that system can be related through the CRM to well-defined, stable, and globally accessible reference(s). Properly done, this directly establishes the metrological traceability of the results. However, achieving the meaningful comparison of results from measurement systems calibrated and/or verified with different CRMs requires that the different materials truly deliver the same measurand, that is, are "the same" within stated uncertainty except for differences in the level of the analyte of interest. We here detail experimental and data analysis techniques for establishing and demonstrating the comparability of materials. We focus on (1) establishing a uniform interpretation of the common forms of CRM uncertainty statements, (2) estimating consistent measurement system response uncertainties from sometimes inconsistent experimental designs, (3) using "errors-in-variables" analysis methods to evaluate comparability studies and novel graphical tools for communicating results of the evaluation to reviewing authorities and potential CRM customers, and (4) augmenting established comparability studies with new materials using measurements provided by the certifying institution. These experimental and data analytic tools were developed in support of the Joint Committee for Traceability in Laboratory Medicine's efforts to enhance the reliability of clinical laboratory measurements and are illustrated with potassium and cholesterol measurands of clinical relevance; however, these tools can be applied to any group of materials that deliver the same nominal measurand with stated value and uncertainty.

Excessive dopamine neuron loss in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) and Parkinson's disease (PD) differ in their response to dopaminergic replacement therapies, despite having a similar degree of neuronal degeneration in the dopaminergic substantia nigra. We observed more widespread dopamine neuron loss in the extranigral A10 midbrain cell groups in PSP compared with PD. These cell groups innervate subcortical and cortical regions and may be required for adequate response to levodopa therapy.