Metabonomics evaluations of age-related changes in the urinary compositions of male Sprague Dawley rats and effects of data normalization methods on statistical and quantitative analysis

Abstract

Background: Urine 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.

Results: The 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.

Conclusion: This 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.

Figure 1

Representative NMR spectra of urine. Overlaid representative NMR spectra of predose 0 hour urine from 25, 40 and 80-day old SD rats.

Figure 2

PCA scores and loadings normalized to total intensity. (A) 2D PCA scores and loadings plots of NMR data normalized to total spectral intensity from urine samples of 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old control SD rats at all timepoints. (B) The PC1 and PC2 loadings for the negative NMR PCA analysis.

Figure 3

PCA scores and loadings normalized to weight. (A) 2D PCA scores and loadings plots of NMR data renormalized by animal weight from urine samples of 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old control SD rats at all timepoints. (B) The PC1 and PC2 loadings for the negative NMR PCA analysis.

Figure 4

PCA scores and loadings normalized to 1/weight. (A) 2D PCA scores and loadings plots of NMR data renormalized by 1/animal weight from urine samples of 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old control SD rats at all timepoints. (B) The PC1 and PC2 loadings for the negative NMR PCA analysis.

Figure 5

PCA scores and loadings normalized to [Creatinine]. (A) 2D PCA scores and loadings plots of NMR data renormalized by the concentration of creatinine in the individual samples from urine samples of 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old control SD rats at all timepoints. (B) The PC1 and PC2 loadings for the negative NMR PCA analysis.

Figure 6

Positive mode UPLC/MS PCA scores and loadings plots. (A) Three dimensional PCA trajectory plots for UPLC/MS data in the positive ionization mode data from 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old SD rats at all time points. (B) The PC2 and PC3 loadings for the positive UPLC/MS PCA analysis.

Figure 7

Negative mode UPLC/MS PCA scores and loadings plots. (A) Three dimensional PCA scores plot obtained from UPLC/MS data acquired from SD-rat urine samples collected from data from 25-day (red circles), 40-day (blue circles), and 80-day (yellow circles) old SD rats at all time points in the negative ionization mode. (B) The PC2 and PC3 loadings for the negative UPLC/MS PCA analysis.