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Supplement

Characterization of Diet-Dependent Metabolic Serotypes: Analytical and Biological Variability Issues in Rats^{1 ,2}

Karen E. Vigneau-Callahan^*, Alexander I. Shestopalov, Paul E. Milbury^*,**, Wayne R. Matson^* and Bruce S. Kristal^,3

^* ESA, Inc., Chelmsford, MA 01824^; Dementia Research Service, Burke Medical Research Institute, White Plains, NY 10605; ^** Antioxidants Research Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 and Departments of Biochemistry and Neuroscience, Cornell University Medical College, New York, NY 10021

³To whom correspondence should be addressed. E-mail: bkristal{at}burke.org

This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.

KEY WORDS: • dietary restriction • blood • high performance liquid chromatography • diet • coulometric array

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