Global fecal metabolome analysis due to different diets

In order to get an overview how the fecal metabolome was impacted by the baseline, HRS and LRS diet and how they interacted with each other, the fecal metabolite profiles of all participants of all diet periods were analyzed through PCA. Both ionization modes were considered separately, including metabolomic data analyzed in (-) FT-ICR-MS (Figure 2.4-1 A) and (+) FT-ICR-MS (Figure 2.4-1 B). The scores scatter plot of the (-) FT-ICR-MS mode data revealed a slight separation between the HRS diet and the baseline diet, as well as between the HRS and LRS diet. On the contrary, the LRS diet and the baseline diet seemed to have a similar pattern and appeared to be mainly clustered together in the scores scatter plot. In the (+) FT-ICR-MS mode, the scores scatter plot revealed a slight separation between the baseline diet and both RS diets, whereas the HRS diet and the LRS diet did not show a separation at all.

Figure 2.4-1: Over view of the fecal metabolome visualized in unsupervised PCA scores scatter plots.

Comparison of different groups through PCA (UV scaling) of participants of the baseline (blue), HRS (red) and LRS (green) diet with respect to different amounts of dietary starch, analyzed in (-) FT-ICR-MS (A) and (+) FT-ICR-MS (B) mode. All participants of all of dietary stages, including both time points of the dietary starch intake are illustrated.

Afterwards, the fecal samples of participants at different dietary stages and different time points were investigated, wherefore several interrogations were of interest: 1. The impact of varying amounts of RS.

2. The order of RS consumption and 3. The time effect of dietary starch intake. Therefore, the

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participants were divided into 5 main groups, namely the baseline diet (G1), the RS groups at day 28 with HRS diet (G2) and LRS diet (G3), as well as the RS groups at day 56, consuming LRS (G4) and HRS (G5) diet. The baseline (G1) was compared to HRS (G2) or LRS (G3) to investigate the impact of varying amount of RS, after the baseline diet, where no RS was given and individuals were fed the same diet. Further, the HRS diet at day 28 (G2) was compared to the LRS diet at day 28 (G3) to investigate the impact of HRS or LRS at the same time point, as given in the models comparing G4 and G5 at day 56, too. There, it will be investigated if the time metabolite profile comparing the HRS and LRS diet differs between day 28 and day 56. Therefore, the individuals were fed again with the baseline diet for 7 days, followed by the second diet period at day 56 to create a comparable base between the starting point of the study and the second diet period. Another interrogation was, if the fecal metabolite profiles of the same diet at different time points were similar, wherefore the metabolite profiles between both HRS diets (G2 and G5), as well as both LRS diets (G3 and G4) were compared.

To investigate this, the fecal metabolome measured either in (-) FT-ICR-MS or (+) FT-ICR-MS mode was investigated through PCA. The scores scatter plots of the fecal metabolome measured in (-) FT-ICR-MS mode (Figure 2.4-2) showed a clear separation between baseline diet (G1) and HRS diet at day 28 (G2), but no separation could be achieved comparing samples of the baseline diet and LRS diet at day 28 (G3). Further, a separation between HRS (G2) and LRS (G3) at day 28, between HRS (G4) and HRS (G5) at day 56 as well as in the merged HRS (G2, G5) and merged LRS (G3, G4) diet groups could be observed in the scores scatter plots.

Figure 2.4-2: Unsupervised PCA scores scatter plots comparing the fecal metabolome

Comparison of different groups through PCA (UV scaling) of participants of the baseline (blue), HRS (red) and LRS (green) diet with respect to different time points, analyzed in (-) FT-ICR-MS mode. Top left: Baseline versus HRS (Day 28). Top right: Baseline versus LRS (Day 28). Middle left: HRS (Day 28) versus LRS (Day 28). Middle right:

HRS (Day 56) versus LRS (Day 56). Bottom: HRS versus LRS, merged time points.

In (-) FT-ICR-MS mode, the metabolite profile through either high or low amount of RS compared to the baseline diet was changing extremely through the HRS diet, but relatively sparse through LRS diet, implying that the amount of RS played a crucial role on the response of the fecal metabolome.

Therefore, the metabolite profile comparing the HRS and LRS diet – in both time points - was altered as well, whereas more significant features could be assigned to the HRS diet.

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Principal Component Analysis comparing the fecal metabolome through dietary starch intake, analyzed in (-) FT-ICR-MS

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In general, the metabolite profile of the HRS diet at day 28 was comparable to the one of the HRS diet at day 56. However, it turned out that the metabolites significantly altered at day 28 of HRS diet were also present in the samples of the HRS diet at day 56, but showed comparably low intensity levels if LRS was consumed as diet 1 (day 28) followed by the HRS diet as diet 2 (day 56). This leads to the analysis, if the fecal metabolite profile changed between HRS at day 28 (G2) and LRS at day 56 (G4) of the same individuals, as well as between LRS at day 28 (G3) and HRS at day 56 (G5) of the same individuals. It was detected, that if HRS diet was consumed first, followed by the LRS diet, changes were more dominant (Figure 2.4-3 A), than when LRS diet was consumed first and followed by the HRS diet (Figure 2.4-3 B). Nevertheless, the significant metabolites between HRS and LRS at both time points were comparable, though with different intensity levels.

Figure 2.4-3: Unsupervised PCA scores scatter plots comparing the fecal metabolome due to different order.

Comparison of different groups through PCA (UV scaling) of participants of the HRS (red) and LRS (green) diet with respect to different order of low and high RS intake of the same individuals, analyzed in (-) FT-ICR-MS mode.

A: HRS of Day 28 versus LRS (Day 56). B: LRS (Day 28) versus HRS (Day 56).

In (+) FT-ICR-MS mode, not only baseline diet and HRS, but also baseline and LRS diet showed a separation in the scores scatter plot (Figure 2.4-4). Here, it was remarkable that the separation was driven by baseline characteristic metabolites, wherefore the comparison between HRS and LRS diets showed no separation at all. In (+) FT-ICR-MS mode, the separations in the scores scatter plots of the PCA were predominantly driven by metabolites discriminative between the baseline diet and the RS diets. Differences in the metabolite profiles between the HRS or LRS diets at the same time points, and

Principal Component Analysis evaluating the impact on the fecal metabolome by different order of the RS consumption, analyzed in (-) FT-ICR-MS

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between day 28 and day 56 of the HRS and LRS diets were rather sparse, compared to the significant changes between HRS and LRS observed in the (-) FT-ICR-MS analyses.

Figure 2.4-4: Unsupervised PCA scores scatter plots comparing the fecal metabolome.

Comparison of different groups through PCA (UV scaling) of participants of the baseline (blue), HRS (red) and LRS (green) diet with respect to different time points, analyzed in (+) FT-ICR-MS mode. Top left: Baseline versus HRS (DIET 1). Top right: Baseline versus LRS (DIET 1). Bottom: HRS versus LRS, merged time points.

There were no differences in the metabolite composition between both HRS diets at two different time points. Also, between both LRS diets at different time points no differences could be observed.

Additionally, comparing the two RS groups by merging both HRS and both LRS diets, no changes could be observed. In 2015, Ordiz et al. investigated the impact of RS type 2 on the fecal metabolite profile of Malawi children suffering from intestinal inflammation (Ordiz et al. 2015). They did not see that RS reduced the inflammation, but they also detected different metabolite profiles between RS-rich diet and their habitual diet, predominantly caused by small organic metabolites increased by RS consumption.

Also Lu et al. observed different metabolite profiles in fecal water samples of growing pigs after the consumption of a low or high RS diet (Lu et al. 2016). These findings agreed with our result that RS altered the metabolite profile differently than the baseline diet and a diet low in RS.

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Principal Component Analysis comparing the fecal metabolome through dietary starch intake, analyzed in (+) FT-ICR-MS

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