Grantee Research Project Results

2004 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: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
Investigators: Hammock, Bruce , German, Bruce , Lango, Jozsef , Dettmer, Katja
Current Investigators: Hammock, Bruce , German, Bruce , Lango, Jozsef , Dettmer, Katja , Green, Peter
Institution: University of California - Davis
EPA Project Officer: Aja, Hayley
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2003 through September 29, 2004
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Children's Health , Human 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:

During the last year the instrumentation in the laboratory has been expanded by a new LC/MS/MS instrument, a Quattro Premier (Waters/Micromass), which was acquired in collaboration with the College of Agriculture and Environmental Science. It is a tandem quadrupole MS equipped with Z-spray, API interface, electrospray probe and atmospheric pressure photo ionization probe. The mass spectrometer is interfaced with a Shimadzu ASP 10 HPLC system equipped with autosampler, dual wavelength UV-VIS detector, high-pressure mixing chamber and a programmable 10-port valve for column switching applications. We also acquired a microwave extraction unit that will be evaluated for more efficient sample extraction. All instrumentation is linked via a secure local area network interfaced with 9 standalone workstations for data analysis and interpretation. We also added a Dual Xeon 3.06 GHz server (1.0 Terra Byte storage) and a Dual Xeon 2.6 GHz server (720 Giga Byte Storage) to expand our data handling and storage capacity. In order to support work in the field of metabolomics, a major project in the core, we purchased MarkerLynx, a software module for the instrument software MassLynx™ 4.0 (Micromass), specifically designed to process high resolution LC-MS metabolomics data. Multivariate data analysis including hierarchal cluster analysis, principal component analysis and principal component regression can be performed using the newly acquired chemometrics software Pirouette® Version 3.11 (Infometrix).

The Core philosophy is to have a variety of analytical methods to look at xenobiotics in tissues as well as natural biomarkers. Shirley Gee has obtained antibodies to PCBs and is establishing the assay for high throughput serum analysis. We are collaborating with Dr. Mike Denison to compare an aryl hydrocarbon hydroxylase based reporter assay with an immunoassay for tetrachlorodioxin and related compounds. We are evaluating these two technologies in a large comparison of bioassay and GLC-MS analysis for dioxin.

Dr. Takaho Watanabe in the laboratory has developed LC-MS based methods to dramatically improve the sensitivity of the detection of pharmaceuticals in serum. Instead of using 5 or 10 mL of serum, typically drawn for these analyses, he 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 a syringe, we can use finger pricks. Dr. Watanabe has developed a method to analyze risperidone and sertraline in low volume serum samples. This work provided preliminary data for a project under the direction of Dr. Robert Hendren testing the hypothesis that the differential response of children with autism spectrum disorders to risperidone and sertraline is due in part to pharmacokinetic properties.

Dr. Lango has worked with Dr. Pessah to identity hyperreactive cysteines within the ryanodine receptor using mass spectrometry. Redox modulation of proteins and ion channels through critical cysteines is thought to underlie the regulation of many intracellular signaling pathways.

One of the major goals of this core is to provide support in metabolomics. 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. We have developed a method for mass spectrometry based metabolic fingerprinting in biofluids using on-line solid phase extraction-HPLC separation and time of flight mass spectrometry (TOF) with electrospray ionization. The procedure has been applied to urine samples, bronchiolar alveolar lavage fluid (BALF) and cell culture media. In the metabolic fingerprinting approach, we do not intend to identify single analytes initially, but rather to compare fingerprints obtained from study samples and control samples. Instrument raw data is exported for multivariate data analysis using spectral binning routines developed in our lab or the MarkerLynx™ application manager. MarkerLynx™, a software module of the instrument software MassLynx™ 4.0 (Micromass). Multivariate data analysis including hierarchal cluster analysis, principal component analysis (PCA) and principal component regression is initially performed using the chemometrics software Pirouette® Version 3.11 (Infometrix). If unique phenotype clusters are observed in the multivariate analysis, the peaks responsible for clustering will be identified and subjected to structural elucidation. Once structures of key discriminating metabolites are found we can develop highly quantitative analytical methods for these materials. In addition to the LC-TOF-MS approach, we are currently developing procedures based on GC-MS for fingerprinting of less polar organic metabolites that are not readily ionizable in electrospray and have collaborations to examine NMR evaluation of metabolomic profiles.

Second, we are defining several metabolite classes and developing highly sensitive, accurate and precise methods for analyzing the compounds in this class. One class of compounds involves tryptophan metabolites. Dysfunctions of tryptophan 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.

The quantitative analysis of urinary tryptophan metabolites is performed by on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with MS/MS detection. Between 5 and 50 μL of the urine sample is injected on a pre-column, followed by a washing step. Subsequently, the solvent flow through the pre-column is reversed with solvent flow being directed to an analytical reversed-phase HPLC column. We are using an Oasis HLB pre-column and the extraction efficiency of this column for the target analytes has been evaluated in preliminary experiments.

The chromatographic separation is followed by electrospray ionization in both positive and negative mode and multi-reaction monitoring (MRM) mass spectral data are acquired. The assay currently delivers quantitative data for 16 tryptophan metabolites providing information for four major metabolic pathways. Currently, we are working on transferring the method to a new tandem mass spectrometer (Quattro Premier, Micromass) in order to improve detection limits of the method. If the new instrument provides significantly improved performance, all samples will be analyzed with this system.

A widely spread hypothesis used to explain autistic symptoms is the “opioid peptide excess” theory. This hypothesis postulates that excessive amounts of naturally occurring opioid substances detected in urine produce the behavioral aberrations of autism. Milk (casein) and wheat (gluten) proteins are thought to be incompletely broken down in the intestine. The resulting neuroactive peptides, such as casomorphin from casein or gliadinomorphin form gluten, are absorbed by a “leaky gut”, enter the circulation, cross the blood brain barrier and exert an "opioid-like" effect in the brain. We have developed a specific, quantitative method for the analysis of 6 urinary peptides. The opioid peptides gliadinomorphin and β-casomorphin are used as lead exogenous neuropeptides. In addition, the endogenous peptides endomorphin 1, endomorphin 2, deltorphin 1 and deltorphin 2 are quantitatively analyzed. Since gliadinomorphin is not commercially available, we are working with a collaborator in the UC Davis chemistry department who has synthesized gliadinomorphin and deuterated gliadinomorphin as standards for quantification purposes.

Quantitative peptide analysis is performed using on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with MS/MS detection. On-line SPE–HPLC increases method automation and laboratory throughput. Moreover, analyte losses are minimized and the amount of sample required per analysis is reduced, since the entire sample is analyzed. We have optimized the LC-MS parameters and the online extraction method with our standards. The efficiency and reproducibility of this extraction method was analyzed with spiked urine samples. Three urine samples from different individuals were spiked with standard solutions resulting in concentrations of 100, 250 and 428 ng/mL. The samples were analyzed as described. The recovery rates are above 60% for all analytes. In case of gliadinomorphin and β-casomorphin recovery rates between 84 and 100% were achieved. With the exception of deltorphin 2, the reproducibility is good with relative standard deviations less than 30%. Limits of detection are in the range of 100 – 500 pg/mL using an injection volume of 20 μL.

In a preliminary study, 77 first morning urine samples were quantitatively analyzed for urinary peptides. The samples set contained 15 samples from autistic children and 7 control samples as well as 55 unknown samples from a blinded study. All urine samples were age and gender matched samples from autistic and control children. Neuropeptides were not detected above the limit of detection in the studied samples, thus the opioid peptide excess theory was not validated with this small sample set.

A third class of compounds are oxidized lipids derived from the arachidonic acid and linoleic acid cascades. Oxilipins 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. The use of metabolic profiling of oxidized lipids in urine samples can be used to evaluate the potential activation of biochemical pathways of lipid mediated inflammation. We are using a novel LC/MS/MS – based method to quantify lipid mediators (Newman et al., 2002). This method has served as the foundation for a continued expansion of the technique, which now routinely measure ~40 metabolites of arachidonate and linoleate metabolism emanating from COX, LOX and cytochrome P450 dependent metabolism. We are applying this method to a subset of urine samples. If we see significant differences in the oxylipid profiles from autistic and control samples, further evaluation will be performed.

As part of the Analytical Core, Dr. Peter Green in the Department of Civil and Environmental Engineering is measuring Hg and a wide range of other metals -- physiological as well as trace and/or toxic.  Using half of the 0.5mL of whole blood frozen for this purpose, a dilution of 20-fold is performed to bring the total dissolved salts concentration down to the operating range of the ICP-MS (Inductively Coupled Plasma Mass Spectrometer) without compromising sufficient sensitivity needed.  The detection limit is 0.02ng/mL, where indeed a few of the first batches of samples have been measured.  Others are more than 100-fold higher, giving us a rich data set to begin interpreting and comparing with other information.  Even with only preliminary results for the other elements, a simple summary follows.  Common elements such as Mg, Ca, Cu, Zn, As and Se all seem to be quite tightly grouped with little variation among the samples - except for a few either higher or lower.  Less common elements such as Li, V, Cr, Mn, Co, Ni, Rb, Sr, Cs and Ba were all determined and show somewhat greater variation.  Most striking was fairly strong variation of Pb.

Journal Articles:

No journal articles submitted with this report: View all 20 publications for this subproject

Supplemental Keywords:

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

Progress and Final Reports:

Original Abstract

Final

Main Center Abstract and Reports:

R829388    UC Davis Center for Children's Environmental Health and Disease Prevention

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