Fatty acids and derivatives altered in breastfed and formula-fed infants

The fecal samples of the exclusively fed infants, which previously were evaluated by month revealed several mass signals assigned as FAs to be altered between breastfed and formula-fed infants over time. All detected FAs, including the saturated, medium-chain fatty acids (MCFAs) dodecanoic acid

(C12:0, Figure 3.4-13 A) and LCFA tetradecanoic acid (C14:0, Figure 3.4-13 B, MS/MS spectrum in Figure 5.2-4 A), were significantly increased in the breastfed infants (Bazanella et al. 2017). C12:0 and C14:0 were significantly increased in the breastfed infants up to month 5 (Figure 3.4-13 A, B). On the contrary, Chow et al. detected dodecanoic acid and tetradecanoic acid to be significantly higher in formula-fed infants (Chow et al. 2014).

Figure 3.4-13: Saturated fatty acids increased in breast fed infants up to month 5.

Boxplots of saturated fatty acids significantly increased in breast fed infants (blue) in month 1 (green), month 3 (blue) and month 5 (yellow). Significance was tested by post hoc Kruskal-Nemenyi test; A: Dodecanoic acid, B:

Tetradecanoic acid, A: Month 1 ‡ value = 2.00E-03 (B vs. F-) and value = 1.40E-03 (B vs. F+); Month 3 ‡ p-value = 9.20E-04 (B vs. F-) and p-p-value = 1.92E-03 (B vs. F+); Month 5 ‡ p-p-value = 1.20E-02 (B vs. F-). B: Month 1 ‡ p-value = 2.95E-02 (B vs. F-) and p-value = 9.40E-03 (B vs. F+); Month 3 ‡ p-value = 5.60E-03 (B vs. F+);

Month 5 ‡ p-value = 7.00E-03 (B vs. F-) and p-value = 2.90E-02 (B vs. F+). Further details are listed in Table 6.2-15

The unsaturated LCFA hexadecenoic acid (C16:1, Figure 3.4-14 A), and LCPUFAs, such as eicosatetraenoic acid (ETA, C20:4, Figure 3.4-14 B, MS/MS spectrum in Figure 5.2-4 B) and icosapentaenoic acid (EPA, C20:5, Figure 3.4-14 C) were significantly higher in breastfed infants.

Further details are given in Table 6.2-15. The experimental MS/MS spectra were compared manually against the METLIN database within an error of 0.05 Da (Smith et al. 2005). Further, experimental (-)-ToF-MS/MS spectra were compared against mass spectra acquired by Walker et al. (Walker et al.

2014).

It was revealed that C16:1, C20:4 and C20:5 were significantly higher up to month 7. On the contrary, in month 7 only C20:4 appeared to be significantly higher by breastfeeding. Eicosatetraenoic acid (C20:4) was another ingredient in the different formula feds; however, the intensity of eicosatetraenoic acid was higher in the breastfed infants than in those fed with formula. It was observed that in month 1 until month 5 the intensity level of hexadecenoic acid in the probiotics formula group leads to an

breastfed F- Placebo formula F+ Interventional formula

approximation towards the breastfed infants, which pattern was not prevalent any more in month 7 and almost disappeared.

Additionally, it was remarkable that inter-individual differences in the breastfed group were much higher than in both formula groups. Concerning this, it is well known, that the fatty acids in human milk are strongly influenced by maternal diet, which has consequences to the fecal metabolome of infants.

Especially, LCPUFAs are said to be strongly related to maternal diet (Ballard and Morrow 2013). The high inter-individual changes in the breastfed might imply this message by Ballard et al.. However, the metabolic processes for biosynthesis, metabolism of the variety of fatty acids and their excretion are multiple, whereas about their presence in the fecal samples of breastfed and formula-fed infants no distinct conclusions can be drawn.

Figure 3.4-14: Unsaturated long-chain fatty acids significantly increased in breastfed infants.

Boxplots of monounsaturated fatty acids, such as (A) hexadecenoic acid and long-chain polyunsaturated fatty acids, such as (B) eicosatetraenoic acid and (C) icosapentaenoic acid significantly increased in breast fed infants (blue) compared to interventional formula-fed (green) and placebo formula-fed infants (red) in month 1 (green), month 3 (blue), month 5 (yellow) and month 7 (light blue). A: Month 1 ‡ p-value = 1.50E-03 (B vs. F-) and p-value

= 5.28E-02 (B vs. F+); Month 3 ‡ value = 1.30E-03 (B vs. F-); Month 5 ‡ value = 1.20E-02 (B vs. F-) and * p-value = 2.30E-02 (F- vs. F+). B: Month 1 ‡ p-p-value = 5.10E-02 (B vs. F-) and p-p-value = 2.10E-02 (B vs. F+); Month 3 ‡ p-value = 3.10E-03 (B vs. F+); Month 5 ‡ p-value = 9.60E-03 (B vs. F-) and p-value = 2.66E-02 (B vs. F+);

Month 7 ‡ p-value = 2.30E-02 (B vs. F-) and p-value = 2.30E-02 (B vs. F+). C: Month 1 ‡ p-value = 5.90E-03 (B vs. F-) and p-value = 3.08E-02 (B vs. F+); Month 3 ‡ p-value = 8.90E-03 (B vs. F-) and p-value = 2.70E-03 (B vs.

F+); Month 5 ‡ p-value = 1.30E-02 (B vs. F-). Level of significance was tested by post hoc Kruskal-Nemenyi test.

Further details are listed in Table 6.2-15.

Additionally, the medium-chain dicarboxylic dodecanedioic acid (Figure 3.4-15 A, MS/MS spectrum in Figure 5.2-5 B), also known as traumatic acid, as well as dihydroxyoleic acid (Figure 3.4-15 B, MS/MS

0

breastfed F- Placebo formula F+ Interventional formula

spectrum in Figure 5.2-5 A) were significantly increased in the formula-fed infants. It was noteworthy that in 99% of the breastfed infants, dodecanedioic acid was not present at all in the first year of life.

Dodecanedioic acid and dihydroxyoleic acid were not significantly changed between infants fed with placebo formula or interventional formula, but also responded differently in the two formulas as seen in varying intensity levels of the respective feeding group. However, probiotics in formula did not bring the patterns of dodecenedioic acid and dihydroxyoleic acid closer to the breastfed one, as seen in fatty acid profiles of hexadecenoic acid and LCPUFA.

Figure 3.4-15: Dodecenedioic acid (A) and dihydroxyoleic acid (B) increased in formula-fed infants.

Boxplots of dodecenedioic acid (A) and dihydroxyoleic acid (B) significantly increased in breast fed infants (blue) compared to placebo formula-fed (red) and interventional formula-fed (green) in month 1 (green), month 3 (blue), month 5 (yellow), month 7 (light blue), month 9 (purple) and month 12 (red). A: Month 1 # p-value = 7.00E-03 (B vs. F-); Month 3 # p-value = 1.30E-02 (B vs. F-) and * p-value =2.00E-03 (B vs. F+); Month 5 # p-value =

Significance was tested with the post hoc Kruskal-Nemenyi test; further details are listed in Table 6.2-16.

Further, hydroxyphenyllactic acid (Figure 3.4-16, MS/MS spectrum in Figure 5.2-4 C) was increased in the fecal samples of breastfed infants over time. Although the fecal hydroxyphenyllactic acid was not significantly changed between infants fed with the placebo formula or the interventional formula, here, either, different intensity levels were observed between the two formula-fed infants (Figure 3.4-16).

Intensity x10⁴

breastfed F- Placebo formula F+ Interventional formula

A

B

Figure 3.4-16: Hydroxyphenyllactic acid increased in breastfed infants over time.

Boxplots of hydroxyphenyllactic acid significantly increased in breast fed infants (blue) compared to placebo formula-fed (red) and interventional formula-fed (green) in month 1 (green), month 3 (blue), month 5 (yellow), month 7 (light blue), month 9 (purple) and month 12 (red). Month 3 # p-value = 5.80E-05 (B vs. F-) and * p-value = 5.40E-03 (B vs. F+); Month 5 # p-value = 2.10E-04 (B vs. F-) and * p-value = 2.42E-02 (B vs. F+); Month 7 # p-value = 2.50E-03 ( B vs. F-) and * p-value = 2.26E-02 (B vs. F+); Month 12 # p-value = 1.70E-03 (B vs. F+). Significance was tested with the post hoc Kruskal-Nemenyi test; further details are listed in Table 6.2-16.

Hydroxyphenyllactic acid belongs to the class of phenylpropanoic acids and is a tyrosine metabolite that derives from microbial breakdown of undigested proteins. (Chow et al. 2014). Beloborodova et al.

detected that phenyllactic and p-hydroxyphenyllactic acids are produced by bifidobacteria and lactobacilli in vitro (Beloborodova et al. 2012). Breast milk is a source of bifidobacteria and lactobacilli species, which are known to be transferred to the infants by breastfeeding (Martin et al. 2003, Martin et al. 2012, Soto et al. 2014). Including the findings from Beloborodova et al., higher levels of hydroxyphenyllactic acid in breastfed infants were reasonable. Concerning this, in the formula-fed infants the intensity of hydroxyphenyllactic acid was comparatively small, even though the interventional formula was substituted with bifidobacteria strains. Although hydroxyphenyllactic acid did not show differences between probiotic formula and the placebo one in month 1, the differences developed over time and got more dominant in the following months. It was observed, that the patterns of hydroxyphenyllactic acid in the probiotics group showed a little approximation towards the breastfed group until month 9. Breastfeeding is supposed to improve infants’ health and lowers the development of several diseases, such as otitis media, necrotizing enterocolitis, inflammatory bowel disease, diabetes or allergic diseases (Soto et al. 2014). Our results indicated similar effects of breast milk and probiotic formula and strengthened the use of probiotics to improve infants’ health.

Intensity x10⁵

breastfed F- Placebo formula F+ Interventional formula