An untargeted metabolomics method for archived newborn dried blood spots in epidemiologic studies
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For pediatric diseases like childhood leukemia, a short latency period points to in-utero exposures as potentially important risk factors. Untargeted metabolomics of small molecules in archived newborn dried blood spots (DBS) offers an avenue for discovering early-life exposures that contribute to disease risks.
The purpose of this study was to develop a quantitative method for untargeted analysis of archived newborn DBS for use in an epidemiological study (California Childhood Leukemia Study, CCLS).
Using experimental DBS from the blood of an adult volunteer, we optimized extraction of small molecules and integrated measurement of potassium as a proxy for blood hematocrit. We then applied this extraction method to 4.7-mm punches from 106 control DBS samples from the CCLS. Sample extracts were analyzed with liquid chromatography—high resolution mass spectrometry (LC-HRMS) and an untargeted workflow was used to screen for metabolites that discriminate population characteristics such as sex, ethnicity, and birth weight.
Thousands of small molecules were measured in extracts of archived DBS. Normalizing for potassium levels removed variability related to varying hematocrit across DBS punches. Of the roughly 1000 prevalent small molecules that were tested, multivariate linear regression detected significant associations with ethnicity (three metabolites) and birth weight (15 metabolites) after adjusting for multiple testing.
This untargeted workflow can be used for analysis of small molecules in archived DBS to discover novel biomarkers, to provide insights into the initiation and progression of diseases, and to provide guidance for disease prevention.
KeywordsDried blood spots Small molecules LC-HRMS Hematocrit Metabolome
We gratefully acknowledge the assistance of Agilent Technologies (Santa Clara, CA, USA) for the loans of the liquid-chromatography mass-spectrometry instruments used in these analyses. We also thank the families for their participation in the CCLS.
This work was supported by the National Institute for Environmental Health Sciences of the U.S. National Institutes of Health (NIEHS) and the U.S. Environmental Protection Agency through grants to the Center for Integrative Research on Childhood Leukemia and the Environment (NIEHS grants P01 ES018172 and P50ES018172 and USEPA grants RD83451101 and RD83615901), by the California Childhood Leukemia Study (NIEHS grants R01ES009137 and P42ES004705), by NIEHS grant P42ES0470518, and by a post-doctoral fellowship from the Environment and Health Fund, Jerusalem, Israel.
Compliance with ethical standards
Conflict of interest
L. Petrick, W. Edmands, C. Schiffman, H. Grigoryan, K. Perttula, Y. Yano, S. Dudoit, T. Whitehead, C. Metayer and S. Rappaport have no conflict of interest to declare.
The ideas and opinions expressed herein are those of the authors and do not necessarily represent the official views of EPA or NIEHS. Endorsement of any product or service mentioned is not intended nor should it be inferred.
The study was approved by the University of California Committee for the Protection of Human Subjects, the California Health and Human Services Agency Committee for the Protection of Human Subjects, and the institutional review boards of all participating hospitals.
Written informed consent was obtained from the adult volunteer subject and parents of all participating subjects.
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