Abstract
BackgroundUrine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of data normalization procedures on principal component analysis (PCA) and quantitative analysis of NMR-based metabonomics data were investigated. Additionally, the effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses.ResultsThe data normalization factor was shown to have a great impact on the statistical and quantitative results indicating the need to carefully consider how to best normalize the data within a particular study and when comparing different studies. PCA applied to the data obtained from both NMR and UPLC/MS platforms reveals similar age-related differences. NMR indicated many metabolites associated with the Krebs cycle decrease while citrate and 2-oxoglutarate, also associated with the Krebs cycle, increase in older rats.ConclusionThis study compared four different normalization methods for the NMR-based metabonomics spectra from an age-related study. It was shown that each method of normalization has a great effect on both the statistical and quantitative analyses. Each normalization method resulted in altered relative positions of significant PCA loadings for each sample spectra but it did not alter which chemical shifts had the highest loadings. The greater the normalization factor was related to age, the greater the separation between age groups was observed in subsequent PCA analyses. The normalization factor that showed the least age dependence was total NMR intensity, which was consistent with UPLC/MS data. Normalization by total intensity attempts to make corrections due to dietary and water intake of the individual animal, which is especially useful in metabonomics evaluations of urine. Additionally, metabonomics evaluations of age-related effects showed decreased concentrations of many Krebs cycle intermediates along with increased levels of oxidized antioxidants in urine of older rats, which is consistent with current theories on aging and its association with diminishing mitochondrial function and increasing levels of reactive oxygen species. Analysis of urine by both NMR and UPLC/MS provides a comprehensive and complementary means of examining metabolic events in aging rats.
Highlights
Urine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and Ultra Performance Liquid Chromatography (UPLC)/MS
The principal component analysis (PCA) plot presented in Additional File 1 was generated from integral bins for each spectrum normalized to the total spectral intensity of the individual spectra
There is a clear separation in the data based upon age with the 25 and 40 day old groups separating along principle component (PC) 1 and the 80 day old group separating from the 25 and 40 day old groups along PC2
Summary
Urine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses. Metabonomics has been widely used to investigate new biomarkers of disease at different stages and drug-induced toxicities using analytical techniques including NMR [1,2,3] and HPLC/MS [4,5,6,7]. It is important to note that the metabolite profile in animal urine can be affected by various environmental factors that include gender, age, species, diet, water intake, and gut microflora [8,9,10]. As a result, choosing an inappropriate control population and data normalization factors for a metabonomics study may reveal biased biomarkers of disease or drug toxicity. It is necessary to first study the age-related changes in the urinary metabolites of control animals in order to facilitate drug toxicity evaluation using metabonomics techniques
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