Conventional tools for measuring dietary exposure have well recognized limitations. Measurement of food-derived metabolites in biofluids provides an alternative approach and our aim was to develop an experimental protocol which ensures that extraneous variability does not obscure metabolic signals from ingested foods. Healthy adults consumed a standardized meal in the evening before each test day and collected pooled overnight urine. On each test day of three different studies, urine was collected in the fasted state and at different time points after consumption of a standardized breakfast. Metabolite fingerprinting of samples using Flow Infusion Electrospray-Ionization Mass Spectrometry followed by multivariate data analysis showed strong discrimination between overnight, fasting and postprandial samples, in each study separately and when data from the three studies were pooled. Such differences were robust and highly reproducible within individuals on separate occasions. Urine volume was an efficient data normalization factor for metabolite fingerprinting data. Postprandial urines had a stable chemical composition over a period of 2–4 h after eating a standardized breakfast, suggesting that there is a flexible time window for urine collection. Fasting urine samples provided a stable baseline for universal comparisons with postprandial samples. A dietary exposure biomarker discovery protocol was validated by demonstrating that top-ranked signals discriminating between fasting and 2–4 h postprandial urine samples could be linked to metabolites abundant in some components of the standardized breakfast. We conclude that the protocol developed will have value in the search for biomarker leads of dietary exposure.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0289-0) contains supplementary material, which is available to authorized users.
We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.
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