Connecting inorganic mercury and lead measurements in blood to dietary sources of exposure that may impact child development.

Pre-natal and post-natal chemical exposures and co-exposures from a variety of sources including contaminated air, water, soil, and food are common and associated with poorer birth and child health outcomes. Poor diet is a contributing factor in the development of child behavioral disorders. Child behavior and learning can be adversely impacted when gene expression is altered by dietary transcription factors such as zinc insufficiency or deficiency or by exposure to toxic substances permitted in our food supply such as mercury, lead, or organophosphate pesticide residue. Children with autism spectrum disorder and attention deficit hyperactivity disorders exhibit decreased or impaired PON1 gene activity which is needed by the body to metabolize and excrete neurotoxic organophosphate pesticides. In this current review we present an updated macroepigenetic model that explains how dietary inorganic mercury and lead exposures from unhealthy diet may lead to elevated blood mercury and/or lead levels and the development of symptoms associated with the autism and attention deficit-hyperactivity disorders. PON1 gene activity may be suppressed by inadequate dietary calcium, selenium, and fatty acid intake or exposures to lead or mercury. The model may assist clinicians in diagnosing and treating the symptoms associated with these childhood neurodevelopmental disorders. Recommendations for future research are provided based on the updated model and review of recently published literature.

Quantification of persistent organic pollutants in dietary supplements using stir bar sorptive extraction coupled with GC-MS/MS and isotope dilution mass spectrometry.

In this work, we describe a method developed to quantify persistent organic pollutants (POPs) including polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in dietary supplement samples using stir-bar sorptive extraction (SBSE)-GC-MS/MS-isotope dilution mass spectrometry (IDMS). This method enables accurate, precise, and sensitive quantification of POPs in plant-extract based dietary supplement products commercially available in the United States. When compared with calibration curves, IDMS provided more accurate and precise measurements. The mean error of measurements using this method was 7.24% with a mean RSD of 8.26%. The application of GC-MS/MS enabled approximately two-order-of-magnitude lower limit of quantifications compared with GC-MS. 12 commercially available plant-extract based dietary supplement samples were analysed using this method. PAHs including naphthalene, acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene were detected in most of the products and had average concentrations over 1 ng/g. OCPs were detected less frequently than PAHs in these products, and none of the OCPs had mean concentrations over 1 ng/g. The mean toxin concentration of each product was calculated, and the highest value was 3.20 ng/g. These results were compared with existing guidelines and none of the analytes in the samples were found to be above the daily allowable limits.

Chronic exposure to xenobiotic pollution leads to significantly higher total glutathione and lower reduced to oxidized glutathione ratio in red blood cells of children with autism.

Analyses of reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (tGSH) in red blood cell samples from 30 children diagnosed with autism and 30 age, gender, and socioeconomic status matched controls were undertaken. The children's ages ranged from 2 to 9. Samples were obtained from subjects residing in Western Pennsylvania, an area of the United States greatly affected by high levels of mercury deposition and airborne PM 2.5 particulates. Liquid chromatography - mass spectrometry was utilized by following EPA Method 6800 for sample analyses. The children with autism had a significantly lower mean red blood cell (RBC) reduced to oxidized glutathione ratio (GSH/GSSG) compared to the control children (p = 0.025). In addition, compared to the controls, the children with autism had significantly higher RBC tGSH values (p = 0.0076) and GSH values (p = 0.022). These results suggest that exposure to toxic elements may prompt compensatory increases in production of GSH in children with autism in environments higher in toxins. The compensation did not fully correct the anti-oxidant properties of exposure to xenobiotics as demonstrated by the significantly lower GSH/GSSG in children with autism compared to controls. Out of a set of glutathione biomarkers, GSH/GSSG may best determine the degree of compensation for oxidative stress in children with autism.

Ultra-trace level speciated isotope dilution measurement of Cr(VI) using ion chromatography tandem mass spectrometry in environmental waters.

The reliable analysis of highly toxic hexavalent chromium, Cr(VI), at ultra-trace levels remains challenging, given its easy conversion to non-toxic trivalent chromium. This work demonstrates a novel analytical method to quantify Cr(VI) at low ngL(-1) concentration levels in environmental water samples by using speciated isotope dilution (SID) analysis and double-spiking with Cr(III) and Cr(VI) enriched for different isotopes. Ion chromatography tandem mass spectrometry (IC-MS/MS) was used for the analysis of Cr(VI) as HCrO4(-) → CrO3(-). Whereas the classical linear multipoint calibration (MPC) curve approach obtained a method detection limit (MDL) of 7ngL(-1) Cr(VI), the modified SID-MS method adapted from U. S. EPA 6800 allowed for the quantification of Cr(VI) with an MDL of 2ngL(-1) and provided results corrected for Cr(VI) loss occurred after sample collection. The adapted SID-MS approach proved to yield more accurate and precise results than the MPC method, allowed for compensation of Cr(VI) reduction during sample transportation and storage while eliminating the need for frequent external calibration. The developed method is a complementary tool to routinely used inductively-coupled plasma (ICP) MS and circumvents typically experienced interferences.

Mean serum-level of common organic pollutants is predictive of behavioral severity in children with autism spectrum disorders.

Autism spectrum disorders (ASD), and their pathogenesis, are growing public health concerns. This study evaluated common organic pollutant serum-concentrations in children, as it related to behavioral severity determined by rating scales and the Autism Diagnostic Observation Schedule (ADOS). Thirty children, ages 2-9, with ASD and thirty controls matched by age, sex, and socioeconomic status were evaluated using direct blood serum sampling and ADOS. Pooling concentrations of all studied pollutants into a single variable yielded cohort-specific neurobehavioral relationships. Pooled serum-concentration correlated significantly with increasing behavioral severity on the ADOS in the ASD cohort (p = 0.011, r = 0.54), but not controls (p = 0.60, r = 0.11). Logistic regression significantly correlated mean pollutant serum-concentration with the probability of diagnosis of behaviorally severe autism, defined as ADOS >14, across all participants (odds ratio = 3.43 [95% confidence: 1.14-10.4], p = 0.0287). No specific analyte correlated with ADOS in either cohort. The ASD cohort displayed greater quantitative variance of analyte concentrations than controls (p = 0.006), suggesting a wide range of detoxification functioning in the ASD cohort. This study supports the hypothesis that environmental exposure to organic pollutants may play a significant role in the behavioral presentation of autism.

Evaluation of Dietary Supplement Contamination by Xenobiotic and Essential Elements Using Microwave-Enhanced Sample Digestion and Inductively Coupled Plasma-Mass Spectrometry.

Dietary supplements were analyzed by evaluating the elemental content in six widely consumed products manufactured by four well-known companies. The elements included the neurotoxic and carcinogenic elements cadmium, mercury, aluminum, lead, arsenic, and antimony, as well as the essential elements zinc, selenium, chromium, iron, and copper, which were often not listed as ingredients on the product labels. Contamination from either xenobiotic or essential elements was found in all samples analyzed. The samples were prepared using US Environmental Protection Agency (EPA) Method 3052, microwave-enhanced digestion. The resulting digests were analyzed by Inductively Coupled Plasma-Mass Spectrometry based on EPA Method 6020B. The analytical protocols were validated by analyzing a multivitamin standard reference material, the National Institute of Standards and Technology Standard Reference Material 3280. The application of EPA standard methods demonstrated their utility in making accurate and precise measurements in complex matrices with multiple ingredients and excipients. In the future, the use of these methods could provide a uniform quality assurance protocol that can be implemented along with other industry guidelines to improve the production of dietary supplements.

Overview of 'Omics Technologies for Military Occupational Health Surveillance and Medicine.

Systems biology ('omics) technologies are emerging as tools for the comprehensive analysis and monitoring of human health. In order for these tools to be used in military medicine, clinical sampling and biobanking will need to be optimized to be compatible with downstream processing and analysis for each class of molecule measured. This article provides an overview of 'omics technologies, including instrumentation, tools, and methods, and their potential application for warfighter exposure monitoring. We discuss the current state and the potential utility of personalized data from a variety of 'omics sources including genomics, epigenomics, transcriptomics, metabolomics, proteomics, lipidomics, and efforts to combine their use. Issues in the "sample-to-answer" workflow, including collection and biobanking are discussed, as well as national efforts for standardization and clinical interpretation. Establishment of these emerging capabilities, along with accurate xenobiotic monitoring, for the Department of Defense could provide new and effective tools for environmental health monitoring at all duty stations, including deployed locations.

A cleanroom sleeping environment's impact on markers of oxidative stress, immune dysregulation, and behavior in children with autism spectrum disorders.

BACKGROUND: An emerging paradigm suggests children with autism display a unique pattern of environmental, genetic, and epigenetic triggers that make them susceptible to developing dysfunctional heavy metal and chemical detoxification systems. These abnormalities could be caused by alterations in the methylation, sulfation, and metalloprotein pathways. This study sought to evaluate the physiological and behavioral effects of children with autism sleeping in an International Organization for Standardization Class 5 cleanroom. METHODS: Ten children with autism, ages 3-12, slept in a cleanroom for two weeks to evaluate changes in toxin levels, oxidative stress, immune dysregulation, and behavior. Before and after the children slept in the cleanroom, samples of blood and hair and rating scale scores were obtained to assess these changes. RESULTS: Five children significantly lowered their concentration of oxidized glutathione, a biomarker of oxidative stress. The younger cohort, age 5 and under, showed significantly greater mean decreases in two markers of immune dysregulation, CD3% and CD4%, than the older cohort. Changes in serum magnesium, influencing neuronal regulation, correlated negatively while changes in serum iron, affecting oxygenation of tissues, correlated positively with age. Changes in serum benzene and PCB 28 concentrations showed significant negative correlations with age. The younger children demonstrated significant improvements on behavioral rating scales compared to the older children. In a younger pair of identical twins, one twin showed significantly greater improvements in 4 out of 5 markers of oxidative stress, which corresponded with better overall behavioral rating scale scores than the other twin. CONCLUSIONS: Younger children who slept in the cleanroom altered elemental levels, decreased immune dysregulation, and improved behavioral rating scales, suggesting that their detoxification metabolism was briefly enhanced. The older children displayed a worsening in behavioral rating scale performance, which may have been caused by the mobilization of toxins from their tissues. The interpretation of this exploratory study is limited by lack of a control group and small sample size. The changes in physiology and behavior noted suggest that performance of larger, prospective controlled studies of exposure to nighttime or 24 hour cleanroom conditions for longer time periods may be useful for understanding detoxification in children with autism. TRIAL REGISTRATION: Clinical Trial Registration Number NCT02195401 (Obtained July 18, 2014).

Blood inorganic mercury is directly associated with glucose levels in the human population and may be linked to processed food intake.

BACKGROUND: The goals of the study were (1) to determine the impact of inorganic mercury exposure on glucose homeostasis; and (2) to evaluate the effectiveness of two community-based interventions in promoting dietary changes among American Indian college students to reduce risk factors for Type-2 Diabetes including fasting glucose, insulin, and mercury levels, weight, and body mass index. METHODS: To accomplish goal one, the National Health and Nutrition Examination Survey (NHANES) dataset was analyzed using a previously published method to determine if there is a relationship between inorganic blood mercury and fasting glucose. To accomplish goal two, ten college students were recruited and randomly assigned to a group receiving the online macroepigenetics nutrition course and the support group for eliminating corn sweeteners. Participants in both groups were assessed for diet patterns, weight, body mass index (BMI), fasting glucose, insulin, and mercury levels. The interventions were implemented over a 10-week period. RESULTS: Analysis of the NHANES data (n=16,232) determined a direct relationship between inorganic mercury in blood and fasting glucose levels (p<0.001). The participants who took the online macroepigenetics nutrition intervention course significantly improved their diets (p<0.01), and fasting blood glucose levels (p<0.01) while having lower levels of inorganic mercury in their blood compared to the subjects in the group who eliminated corn sweeteners from their diet and participated in the support group. The trend in lower blood inorganic mercury was strong with p=0.052. The participants in the support group who eliminated corn sweeteners from their diet achieved significant weight loss (p<0.01) and reduced their body mass index (p<0.01). CONCLUSION: Total blood mercury levels may be influenced by dietary intake of highly processed foods and lower inorganic mercury levels are associated with lower fasting glucose levels. Alternative community-based interventions emphasizing the role food ingredients and toxic substances play in gene modulation and the development of diseases can result in significant dietary improvements and reductions in risk factors associated with type-2 diabetes. A healthier diet can be promoted among community members using a novel online nutrition course. Consumption of corn sweeteners may be a risk factor in the development of obesity.

Molecular speciated isotope dilution mass spectrometric methods for accurate, reproducible and direct quantification of reduced, oxidized and total glutathione in biological samples.

Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.

An accurate and transferable protocol for reproducible quantification of organic pollutants in human serum using direct isotope dilution mass spectrometry.

A robust method has been developed for easy transfer between analytical laboratories to obtain highly accurate and reproducible quantification of persistent organic pollutants (POPs) in micro-volumes of serum. This method is suited for analysts researching the impact of environmental exposure on human health. When performed by highly trained analysts, existing methods can produce high quality data; however, complex sample preparation steps often cannot be consistently replicated by laboratories, leading to variance in extraction recovery and quantitation. By combining stir-bar sorptive extraction (SBSE) with direct isotope dilution (D-ID) mass spectrometry quantification, a new analytical method was developed. The D-ID quantification significantly improved accuracy, corrected sample-to-sample irreproducibility, and reduced sample preparation time. Independent production of statistically identical data then confirmed transfer of the validated operating protocol to an off-site laboratory with different instrument models. SBSE performance was compared with industry-accepted extraction techniques. D-ID quantification was compared with peer-reviewed relative isotopic response factor (RF) quantification methods. Holding other variables constant, D-ID improved accuracy by 250% and precision by 300% compared with RF; SBSE improved accuracy by 37% compared to industry-accepted extraction methods. Limits of quantification of the analytes ranged from 60 pg g(-1) to 1 µg g(-1). Protocol transfer exhibited <7% mean between-laboratory error and <2% mean within-laboratory RSD. These results indicate that a transferable method has been developed for academic, government, commercial, and clinical laboratories seeking to maximize throughput and improve quantitative validity. This validated method was applied in a recent clinical study to assess non-communicable disease in children in Pennsylvania, USA.

Method development for the redox speciation analysis of iron by ion chromatography-inductively coupled plasma mass spectrometry and carryover assessment using isotopically labeled analyte analogues.

An ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) method was developed for the redox speciation analysis of iron (Fe) based on in-column complexation of Fe(2+) and Fe(3+) by dipicolinic acid (DPA). The effects of column type, mobile phase composition and molecular ion interference were studied in the method optimization. The carryover of the target species in the IC-ICP-MS method was uniquely and effectively evaluated using isotopically enriched analogues of the analytes ((54)Fe(2+) and (57)Fe(3+)). Standard solutions of the enriched standards were injected into the system following analysis of a sample, and the ratios of the isotopes of iron in the enriched standards were calculated based on the chromatographic peak areas. The concentrations of the analytes carried over from the sample to the enriched standards were determined using the quantitative relationship in isotope dilution mass spectrometry (IDMS). In contrast to the routine way of evaluating carryover effect by injecting a blank solution after sample analysis, the use of isotopically enriched standards identified significant analyte carryover in the present method. Extensive experiments were carried out to systematically identify the source of the carryover and to eliminate the problem; the separation column was found to be the exclusive source. More than 95% of the analyte carryover was eliminated by reducing the length of the column. The detection limit of the IC-ICP-MS method (MDL) for the iron species was 2ngg(-1). The method was used to determine Fe(2+) and Fe(3+) in synthetic aqueous standard solutions and a beverage sample.

Measurement of mercury species in whole blood using speciated isotope dilution methodology integrated with microwave-enhanced solubilization and spike equilibration, headspace-solid-phase microextraction, and GC-ICP-MS analysis.

A biomonitoring method was developed for the determination of inorganic-, methyl-, and ethylmercury (Hg(2+), CH3Hg(+), and C2H5Hg(+), respectively) in whole blood by triple-spiking speciated isotope dilution mass spectrometry (SIDMS) using headspace (HS) solid-phase microextraction (SPME) in combination with gas chromatographic (GC) separation and inductively coupled plasma mass spectrometric (ICP-MS) detection. After spiking the blood sample with isotopically enriched analogues of the analytes ((199)Hg(2+), CH3(200)Hg(+) and C2H5(201)Hg(+)), the endogenous Hg species were solubilized in 2.0 mol L(-1) HNO3 and equilibrated with the spikes using a microwave-enhanced protocol. The microwaved sample was treated with a 1% (w/v) aqueous solution of sodium tetrapropylborate (buffered to pH 5.2), and the propylated Hg species were sampled in the HS using a Carboxen/polydimethylsiloxane-coated SPME fiber. The extracted species were thermally desorbed from the fiber in the GC injection port and determined by GC-ICP-MS. The analytes were quantified, with simultaneous correction for their method-induced transformation, on the basis of the mathematical relationship in triple-spiking SIDMS. The method was validated using a bovine blood standard reference material (SRM 966, Level 2). Analysis of human blood samples demonstrated the accuracy and reproducibility of the method, which can detect the Hg species down to 30 pg g(-1) in blood. The validity of the analytical results found for the blood samples was demonstrated using mass balance by comparing the sum of the concentrations of the individual Hg species with the total Hg in the corresponding samples; the latter was determined by isotope dilution mass spectrometry (IDMS) after decomposing the blood using EPA Method 3052 with single-spiking.

Speciation analysis of arsenic in prenatal and children's dietary supplements using microwave-enhanced extraction and ion chromatography-inductively coupled plasma mass spectrometry.

A study was conducted to develop a microwave-enhanced extraction method for the determination of arsenic species in prenatal and children's dietary supplements prepared from plant materials. The method was optimized by evaluating the efficiency of various solutions previously used to extract arsenic from the types of plant materials used in the dietary supplement formulations. A multivitamin standard reference material (NIST SRM 3280) and a prenatal supplement sample were analyzed in the method optimization. The identified optimum conditions were 0.25 g of sample, 5 mL of 0.3 mol L(-1) orthophosphoric acid (H3PO4) and microwave heating at 90 °C for 30 min. The extracted arsenic was speciated by cation exchange ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The method detection limit (MDL) for the arsenic species was in the range 2-8 ng g(-1). Ten widely consumed prenatal and children's dietary supplements were analyzed using the optimized protocol. The supplements were found to have total arsenic in the concentration range 59-531 ng g(-1). The extraction procedure recovered 61-92% of the arsenic from the supplements. All the supplementary products were found to contain arsenite (As(3+)) and dimethylarsinic acid (DMA). Arsenate (As(5+)) was found in two of the supplements, and an unknown specie of arsenic was detected in one product. The results of the analysis were validated using mass balance by comparing the sum of the extracted and non-extracted arsenic with the total concentration of the element in the corresponding samples.

Determination of chromium species in dietary supplements using speciated isotope dilution mass spectrometry with mass balance.

In order to determine the health impact of chromium in dietary supplements, the Cr(III) and Cr(VI) must be independently measured and verified with mass balance (sum of both species equaling independent measurements of total chromium), as both may be present in finished products. Because Cr(III) is stable in acidic conditions and Cr(VI) in alkaline conditions, interconversions between species may occur in complex matrices and during analytical extraction, increasing the difficulty of quantification. A study was conducted to determine Cr(VI) and Cr(III) in dietary supplements. EPA Method 3060A extraction protocol was performed to extract Cr(VI), and EPA Method 3052 was performed on the extracted residue to digest the remaining Cr(III). Speciated isotope dilution mass spectrometry (SIDMS), as described in the EPA Method 6800 (update V), was implemented with ion-exchange chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS). Method 6800 uniquely enables tracking and correcting for the bidirectional chromium interspecies conversions that occur during extraction and sample handling prior to instrumental analysis. Mass balance results indicated that the off-the-shelf dietary supplements analyzed during this study contained hexavalent chromium ranging from

Improved sample preparation of glyphosate and methylphosphonic acid by EPA method 6800A and time-of-flight mass spectrometry using novel solid-phase extraction.

The employment of chemical weapons by rogue states and/or terrorist organizations is an ongoing concern in the United States. The quantitative analysis of nerve agents must be rapid and reliable for use in the private and public sectors. Current methods describe a tedious and time-consuming derivatization for gas chromatography-mass spectrometry and liquid chromatography in tandem with mass spectrometry. Two solid-phase extraction (SPE) techniques for the analysis of glyphosate and methylphosphonic acid are described with the utilization of isotopically enriched analytes for quantitation via atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry (APCI-Q-TOF-MS) that does not require derivatization. Solid-phase extraction-isotope dilution mass spectrometry (SPE-IDMS) involves pre-equilibration of a naturally occurring sample with an isotopically enriched standard. The second extraction method, i-Spike, involves loading an isotopically enriched standard onto the SPE column before the naturally occurring sample. The sample and the spike are then co-eluted from the column enabling precise and accurate quantitation via IDMS. The SPE methods in conjunction with IDMS eliminate concerns of incomplete elution, matrix and sorbent effects, and MS drift. For accurate quantitation with IDMS, the isotopic contribution of all atoms in the target molecule must be statistically taken into account. This paper describes two newly developed sample preparation techniques for the analysis of nerve agent surrogates in drinking water as well as statistical probability analysis for proper molecular IDMS. The methods described in this paper demonstrate accurate molecular IDMS using APCI-Q-TOF-MS with limits of quantitation as low as 0.400 mg/kg for glyphosate and 0.031 mg/kg for methylphosphonic acid.

α-Galactosidase A expressed in the salivary glands partially corrects organ biochemical deficits in the fabry mouse through endocrine trafficking.

Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA -/0 or -/- mice received an adenoviral vector (2 × 10(10) or 1 × 10(9) viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/µg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.

Simultaneous extraction of arsenic and selenium species from rice products by microwave-assisted enzymatic extraction and analysis by ion chromatography-inductively coupled plasma-mass spectrometry.

A microwave-assisted enzymatic extraction (MAEE) method was developed for the simultaneous extraction of arsenic (As) and selenium (Se) species in rice products. The total arsenic and selenium content in the enzymatic extracts were determined by inductively coupled plasma mass spectrometry (ICP-MS), while the speciation analysis was performed by ion chromatography coupled to inductively coupled plasma-mass spectrometry (IC-ICP-MS). The main factors affecting the enzymatic extraction process were evaluated in NIST SRM-1568a rice flour. The optimum extraction conditions were 500 mg of sample, 50 mg of protease XIV, and 25 mg of alpha-amylase in aqueous medium during 40 min at 37 degrees C. The extraction recoveries of total As and Se reached 100 +/- 3 and 80 +/- 4%, respectively. The species stability study during the MAEE process did not show transformation of the target species in rice products. The results of As speciation obtained for SRM-1568a were in agreement with previous studies of As speciation performed on the same reference material. The proposed method was applied to the determination of As and Se species in rice and rice-based cereals. Arsenite [As(III)], arsenate [As(V)], dimethylarsinic acid (DMA), and selenomethionine (SeMet) were the predominant species identified in rice products.

The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders.

The frequency of zinc deficiency, copper toxicity and low zinc/copper in children with autism spectrum disorders (ASDs) may indicate decrement in metallothionein system functioning. A retrospective review of plasma zinc, serum copper and zinc/copper was performed on data from 230 children with autistic disorder, pervasive developmental disorder-NOS and Asperger's syndrome. The entire cohort's mean zinc level was 77.2 microg dl(-1), mean copper level was 131.5 microg dl(-1), and mean Zn/Cu was 0.608, which was below the 0.7 cut-off of the lowest 2.5% of healthy children. The plasma zinc/serum copper ratio may be a biomarker of heavy metal, particularly mercury, toxicity in children with ASDs.

Simultaneous determination of arsenic and selenium species in fish tissues using microwave-assisted enzymatic extraction and ion chromatography-inductively coupled plasma mass spectrometry.

A microwave-assisted enzymatic extraction (MAEE) method was developed for the simultaneous extraction of arsenic (As) and selenium (Se) species in fish tissues. The extraction efficiency of total As and Se and the stability of As and Se species were evaluated by analyzing DOLT-3 (dogfish liver). Enzymatic extraction using pronase E/lipase mixture assisted by microwave energy was found to give satisfactory extraction recoveries for As and Se without promoting interspecies conversion. The optimum extraction conditions were found to be 0.2 g of sample, 20 mg pronase E and 5mg lipase in 10 mL of 50 mM phosphate buffer, pH 7.25 at 37 degrees C. The total extraction time was 30 min. The speciation analysis was performed by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The accuracy of the developed extraction procedure was verified by analyzing two reference materials, DOLT-3 and BCR-627. The extraction recoveries in those reference materials ranged between 82 and 94% for As and 57 and 97% for Se. The accuracy of arsenic species measurement was tested by the analysis of BCR 627. The proposed method was applied to determine As and Se species in fish tissues purchased from a local fish market. Arsenobetaine (AsB) and selenomethionine (SeMet) were the major species detected in fish tissues. In the analyzed fish extracts, the sum of As species detected was in good agreement with the total As extracted. However, for Se, the sum of its species was lower than the total Se extracted, revealing the presence of Se-containing peptides or proteins.