2003 Progress Report: Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core

EPA Grant Number: R829388C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R829388
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism
Center Director: Pessah, Isaac N.
Title: Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
Investigators: Hammock, Bruce , German, Bruce , Lango, Jozsef , Watkins, Steve
Current Investigators: Hammock, Bruce , Dettmer, Katja , German, Bruce , Green, Peter , Lango, Jozsef
Institution: University of California - Davis
EPA Project Officer: Hahn, Intaek
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2002 through September 29, 2003
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Health , Health Effects , Children's Health

Objective:

The two major goals of this core are to develop strategies to profile xenobiotics of concern to childhood neurodevelopment in biological fluids and provide support in metabolomics. The long-term approach is to establish a horizontally integrated database from the genome through the autistic phenotype to aid in developing and testing hypotheses regarding the disorder. Thus the core provides established analytical support for xenobiotic and proteomic profiling, and is currently developing tools to study the metabolome.

Our specific aims are: I.) Determine xenobiotics in serum, urine and food; II.) Determine levels of nutritional and structural lipids in serum samples; III.) Provide general analytical support for the project including the development of methods for possible biomarkers that are considered important.

Progress Summary:

We provide walk up service in a variety of analytical areas in either running samples for scientists in the project, providing equipment and training to run the samples or consulting on method development. Our work has focused in several areas. The first area is development of infrastructure in the analytical core to support the project as a whole. The laboratory provides GLC-MS support and both LC-triple quadrapole and LC-time of flight mass spectrometers. We are capable of both peptide and small molecule analysis, but generally emphasize small molecules. During the last year we have added a robotic unit for processing sample in an automated fashion in a 96 well array. We have added 96 well sampling capability to the LC-MS instruments. In addition we have acquired an infrared spectrometer to support routine analysis and have on order a microwave extraction unit to evaluate for more efficient sample extraction. Two new 96 and 384 well ELISA readers are available to all members of the project for immunoassay work. Freezers have been installed which can be indexed with project software for sample tracking.

The philosophy is to have a variety of analytical methods to look at xenobiotics in tissues as well as natural biomarkers. In some cases where there is a clear hypothesis of exposure and relation to autism, we will use high throughput immunoassays. To this end Shirley Gee has obtained antibodies to PCBs and is establishing the assay for high throughput serum analysis. We are collaborating with Dr. Mike Denison of this project to compare an aryl hydrocarbon hydroxylase based reporter assay with an immunoassay for tetrachlorodioxin and related compounds. We are just starting to evaluate these two technologies in a large comparison of bioassay and GLC-MS analysis for dioxin. Hopefully one or more of these technologies will be available in high throughput mode for analysis of serum samples from autistic children. Dr. Takaho Watanabe in the laboratory is exploring the use of LC-MS technology to dramatically improve the sensitivity of the detection of pharmaceuticals in serum. Typically 5 or 10 ml of serum are used in these analyses. He now is running pharmacokinetics of low abundance drugs using less than 5 ul of serum. This 1000x improvement in sensitivity means that rather than subjecting children to the trauma of removing blood with syringe, we can use finger pricks. This work provided the preliminary data for a seed project funded through the Center under the direction of Dr. Robert Hendren. The project will test the hypothesis that the differential response of children with autism spectrum disorders to risperidone and sertraline is do in part to pharmacokinetic properties. This project is in collaboration with Dr. Alan Buckpitt of the veterinary school.

One of the major goals of this core is to provide support in metabolomics. The philosophy of our long-term approach is shown in the figure below. We want to drive toward a horizontally integrated database from the genome through the autistic phenotype to aid in developing and testing hypotheses regarding the disease. We provide analytical support for proteomics but we are developing tools to study the metabolome. This high throughput metabolite profiling is taking several forms.

First, we are attempting to cast a wide net and use the electrospray time of flight mass spectrometer to look in a semi quantitative way at a large variety of metabolites in urine and serum of autistic children. The last year has been spent optimizing extraction and concentration protocols. Data have been generated on both GLC –MS and LC-MS analysis by Dr. Jozsef Lango and we now are looking at how to analyze these data with the analytical core. This broad screen approach has the advantages of not being limited by hypotheses and looking at many analytes at one time. It is limited by the data being only semi-quantitative, platform dependent, and of course the many variables in the samples including sex, nutrition, age and many other factors. We hope to establish collaboration to use two other technologies for broad profiling of samples. One of these approaches is nuclear magnetic resonance of the serum samples and the other is Fourier transform mass spectrometry. In theory this MS technique should give a less biased view of a wide spectrum of metabolites and facilitate more accurate quantitation in the absence of standards. These advantages are off set by the high cost of instrumentation. Second, we are defining several metabolite classes and developing highly sensitive, accurate and precise methods for analyzing the compounds in this class. This approach has the obvious advantages of giving a much higher quality of data and in providing amounts of data that are simpler to analyze. However, it is certain to miss important classes of metabolites. We are following several areas.

One area is the analysis of regulatory lipids. Here we are testing the hypothesis that oxidized lipid levels in urine and serum differ in autistic children. As described below this method may be particularly applicable to the project in collaboration with Dr. Robin Hansen on the effects of gluten free diets on health of autistic children. Oxidized lipids derived from the arachidonic acid and linoleic acid cascades are recognized as mediators of inflammatory and proliferative responses. This might be important in the case of ASD since children with autism often manifest mild to moderate degrees of gastrointestinal inflammation. During the last year we transferred our GLC-MS method for these metabolites to LC-MS/MS (Newman et al, 2002) and recently our method has been expanded to include cyclooxygenase and lipogenase metabolites (Ogingwa et al, submitted). This technology for metabolic profiling of oxidized lipids in urine samples can be used to evaluate the potential activation of biochemical pathways of lipid mediated inflammation. A wide spectrum of oxidized lipids originating from different branches of the arachidonic acid and linoleic acid cascades will be analyzed in urine samples using HPLC-Tandem MS. The figure is a schematic representation of the arachidonic acid cascade with metabolic enzymes indicated in italics. Metabolites are shown in bold, with unstable intermediates in white boxes and future analytical targets in grey boxes. The expansion of this method to include LOX and COX metabolites of arachidonic acid, along with analogous linoleate-derived oxylipids, represent significant progress in developing a metabolite profile of regulatory lipids.

graph

A second metabolite class involves compounds involved i tryptophan metabolism. Dysfunctions of trytophan metabolism have been reported in association with ASD. Specifically, the tryptophan-derived neurotransmitter serotonin is elevated in platelets and indolyl-3-acryloylglycine (IAG) is present in abnormally high quantities in the urine of autistic persons. An unusual metabolite of tryptophan, indole-3- acrylic acid, has been proposed as the precursor for IAG formation. To provide thorough coverage of the tryptophan metabolism, over 18 urinary metabolites within the serotonin, kynurenine, nicotinamide, indole pyruvate, and indole pathways which hold information about critical metabolic branch points will be analyzed using online solid phase extraction-HPLC-Tandem MS. The method validation is almost completed.

The preliminary analysis of urine from an autistic child and a control subject suggests significant differences regarding indole-3- acrylic acid. However, the analysis of a larger sample set is necessary to verify this hypothesis.

Methods are also being developed for general oxidative stress and for vitamin D metabolites. Additional metabolite classes will be targeted as suggested by collaborators in the program. One example of this approach is that a joint project between Dr. Robin Hansen and Katja Dettmer of the analytical core has been funded to look at gluten degradation products that are thought to be biologically active in autistic children.

Future Activities:

Finally we are working on making standard clinical assays more sensitive so that they can be run with a high degree of accuracy, precision, linearity and sensitivity on very small samples of blood or saliva. This general technology will expand our ability to obtain reliable analytical data on autistic children using less invasive procedures.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other subproject views: All 20 publications 19 publications in selected types All 18 journal articles
Other center views: All 146 publications 134 publications in selected types All 133 journal articles
Type Citation Sub Project Document Sources
Journal Article Fang X, Weintraub NL, Oltman CL, Stoll LL, Kaduce TL, Harmon S, Dellsperger KC, Morisseau C, Hammock BD, Spector AA. Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide. American Journal of Physiology-Heart and Circulatory Physiology 2002;283(6):H2306-H2314. R829388 (2006)
R829388 (Final)
R829388C001 (2003)
R829388C001 (2005)
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  • Journal Article Newman JW, Watanabe T, Hammock BD. The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC-MS/MS. Journal of Lipid Research 2002;43(9):1563-1578. R829388 (2006)
    R829388 (Final)
    R829388C001 (2003)
    R829388C001 (2005)
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  • Journal Article Newman JW, Morisseau C, Harris TR, Hammock BD. The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity. Proceedings of the National Academy of Sciences of the United States of America 2003;100(4):1558-1563. R829388 (2006)
    R829388 (Final)
    R829388C001 (2003)
    R829388C001 (2005)
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  • Supplemental Keywords:

    Autism, metabolomics, lipids, proteomics, xenobiotics,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Toxicology, Health Risk Assessment, Chemistry, Epidemiology, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, Physical Processes, Children's Health, genetic susceptability, Biology, Risk Assessment, chemical exposure, neurotoxic, xenobiotics, biomarkers, gene-environment interaction, neurodevelopment, pesticides, exposure, halogenated aromatics, children, neurobehavioral, neurodevelopmental, neurotoxicity, etiology, susceptibility, human exposure, neurobehavioral effects, autism, biological markers, mechanisms, exposure assessment, neurological development, biomarker, synergistic interactions, mercurials

    Progress and Final Reports:

    Original Abstract
  • Final

  • Main Center Abstract and Reports:

    R829388    CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829388C001 Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
    R829388C002 Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
    R829388C003 Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
    R829388C004 Environmental Factors in the Etiology of Autism; Childhood Autism Risks from Genetics and the Environment (The CHARGE Study)
    R829388C005 Environmental Factors in the Etiology of Autism; Animal Models of Autism
    R829388C006 Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism