Intermediates of the tocopherol metabolism increased in formula fed infants

The statistical evaluation of the metabolite profiles of formula- and breastfed infants, revealed two highly significant mass signals, namely m/z 585.3432 and m/z 591.3879. In order to identify both, MS/MS experiments were performed. Through MS/MS experiments, similar fragmentation patterns were observed in both mass signals, with a characteristic loss of 176.03, which is distinctive for the loss of glucuronide fragments as shown in Figure 3.4-11. Therefore, the MS/MS spectra, as the unconjugated form were compared with the Metlin MS/MS database, which classified the two metabolites as γ-tocotrienol and γ-tocopherol.

Figure 3.4-11: Vitamin E metabolite patterns in breastfed and formula-fed infants.

Experimental (-)-TOF-MS/MS spectra at 40 eV of A: γ-Tocotrienol glucuronide; B: γ-Tocopherol glucuronide and boxplots of the (C) γ-tocotrienol glucuronide and (D) γ-tocopherol glucuronide significantly increased in formula-fed infants over time. C: Month 1: # p-value = 9.78E-08; Month 3: * p-value = 6.14E-08; Month 5: † p-value = 6.32E-07; Month 7: ‡ p-value = 2.65E-06; Month 9: ○ p-value = 0.000554; D: Month 1: # p-value = 2.19E-05; Month 3: * p-value = 1.99E-07; Month 5: † p-value = 3.68E-08; Month 7: ‡ p-value = 6.56E-07; Month 9: ○ p-value = 9.30E-07; Month 12: ¥ p-value = 0.004312 (Mann-Whitney-Test). p-values are always in relation to the same month of the different feedings. Further details are listed in Table 6.2-14.

0

Month 1 Month 3 Month 5 Month 7 Month 9 Month 12

# γ-Tocotrienol glucuronide (m/z 585.3432) γ-Tocopherol glucuronide (m/z 591.3879)

Differentiation between the β- and γ-tocopherol or tocotrienols respectively, having the same monoisotopic mass was enabled by different fragmentation patterns. Also Chow et al. detected higher levels (18.36-fold) of γ-tocopherol, an intermediate of the tocopherol metabolism, in exclusively formula-fed infants compared to breastformula-fed infants. Assuming, that all vitamin E intermediates are glucuronidated and then excreted into feces, the monoisotopic masses of metabolites involved in the biosynthesis of tocopherol/tocotrienol were calculated as [M-H]- adducts and fictively conjugated with glucuronide. In this way, α-tocopherol glucuronide, α-tocotrienol glucuronide and δ-tocopherol glucuronide were also detected as significantly increased in the fecal samples of formula-fed infants.

Figure 3.4-12: Over time patterns of intermediates of the biosynthesis of tocopherols.

Boxplots of potential metabolites involved in the biosynthesis of tocopherol differentiating between breastfed and formula-fed infants of month 1 (green), month 3 (blue), month 5 (yellow), month 7 (light blue), month 9 (purple) and month 12 (red). Significance was tested through the Mann-Whitney test; symbols #, *, †, ‡, °, ¥: p-value < 0.05.

Further details are listed in Table 6.2-14.

Usually infants are born with relatively little amounts of vitamin E, as the placental transfer from the mother to the fetus is limited (Preedy and Watson 2007) and need to be supplied with it via their diet.

Both are intermediates in the tocopherol/tocotorienol biosynthesis (vitamin E) and are essential nutrients, which cannot be synthesized endogenously (Chow et al. 2014) and need to be provided by

α- tocotrienol glucuronide (m/z 599.3565) α-tocopherol glucuronide (m/z 605.4029)

Intensity x 10⁴ Intensity x 10⁴

A B

Month 1 Month 3 Month 5 Month 7 Month 9 Month 12

δ-tocopherol glucuronide (m/z 577.3719)

either breast milk or formula. In this study, the amount of vitamin E was 1.3 mg/100mL in each formula fed to the infants, whether it was pre-formula, follow-up or supplementary formula. As γ-tocotrienol and γ-tocopherol were distinctively classified through MS/MS experiments, their patterns in the formula-fed infants over time will be reviewed for possible relationships between the amount of formula the infants were fed and the intensity of tocotrienol and tocopherol detected in the fecal samples of the formula and the breastfed infants. Considering all months together, Pearson correlation (alpha = 0.05) revealed a significantly positive relationship between the amount of formula fed and the intensity of tocotrienol (r(221) = 0.64, p-corr < 0.001) and tocopherol (r(221) = 0.52, p-corr < 0.001) detected in the fecal samples of formula-fed infants. Evaluating the correlations on a monthly view, a significant positive relationship between the amount of formula and the two metabolites was observed up to month 5. The positive relationship between the amount of formula and tocotrienol was further decreasing up to month 12, whereas for tocopherol the correlations are still present up to month 9, as shown in Table 3.4-3.

The different correlation pattern leads to study the impact of tocotrienol on the tocopherol pattern and vice versa by applying the Pearson correlation, too.

Table 3.4-3: Correlations between tocopherol and tocotrienol and the amount of formula fed.

Correlations between the amount of formula and the metabolites, as well as the correlations among the different metabolites were obtained through Pearson correlation, recording the correlation coefficient (r), and the degrees of freedom (df), as well as p-corr values calculated through regression analysis.

Name [M-H]^- r p-corr df

Relationship between the amount of formula and the intensity levels

γ-tocotrienol glucuronide

Correlations between tocotrienol and tocopherol

Month 1 0.80 1.29E-09 36

It could be assumed that up to month 5, tocotrienol and tocopherol correlated predominantly positively with the amount of formula fed. After month 5 the predominating factor seems to be the coexistence of

the two vitamin intermediates and their significant positive relationship, as well as their dependency on one another, since the positive correlation persists up to month 9.

To further investigate, why formula-fed infants display higher intensities of tocotrienol and tocopherol compared to the relatively low amount in breastfed infant, the study of Haug et al in 1987 possibly provides some more insights (Haug et al. 1987). They analyzed the vitamin E content (α-, β- and γ-tocopherol) in breast milk samples of mothers of preterm and full-term infants. They observed that not only the vitamin E content of preterm and full-term breast milk showed similar concentration patterns, but also stage of lactation (colostrum, transitional and mature) of the breast milk samples played an important role on the vitamin E concentrations. Further, they showed that with the duration of lactation the concentration of vitamin E decreases.

Further studies confirm this finding (Kobayashi et al., Syvaoja et al and Boersma et al. (Kobayashi et al. 1975, Syvaoja et al. 1985, Boersma et al. 1991). The same result was obtained by Martysiak-Zurowska et al. (Martysiak-Martysiak-Zurowska et al. 2013), who analyzed α- and γ-tocopherol in breast milk samples of different stages of lactation (day 2, day 14, day 30 and day 90). They found concentration levels of γ-tocopherol in the breast milk samples from 0.022 mg/100mL – 0.060 mg/100mL and 0.207 mg/100mL – 0.999 mg/mL for α-tocopherol, depending on the stage of lactation. Compared to the vitamin E content in the formula within this study (1.3 mg/100 mL), the concentration levels are much higher than those detected in breast milk in previously performed studies. As previously postulated by Chow et al., this indicated either levels in the diet that are too excessive or that the tocopherols cannot be absorbed completely and are excreted into feces (Chow et al. 2014). It can be assumed that the amount of vitamin E in breast milk samples received by the infants is usually sufficient as the concentration levels in the breastfed infant were 1.65 – 7.65-fold lower than in the formula-fed infants (Table 6.2-14) and the formula-fed infants need to excrete this excess with feces.