# The Stool Volatile Metabolome of Pre-Term Babies
## 2 Results
### Metabolomic Profile of Stool Samples from Pre-Term Babies  
There were 36 volatile organic compounds (VOCs) present in at least 25% of samples (Table 2, Appendix A Table A1). The three short chain fatty acids, acetic acid, propionic acid and butanoic acid, were present in 91%, 53% and 42% of samples, respectively. Aldehydes and alcohols were the largest groups with 6 compounds in each group. We then investigated the impact of the infants' postnatal age (all samples were included, \(n=152\)). A mixed effect regression model of VOC number per patient and postnatal age (days) showed a significant (\(p\)-value < 0.0001) increase in VOCs during time of 1.0126 compound per day (95% Wald confidence interval 1.009, 1.016). Subsequently, samples were grouped by age (Table 3). The number of VOCs was limited in the first 5 days of life (Table 3) (Figure 1). Anova analysis (f-ratio = 16.55624, \(p<0.00001\), post hoc HSD) showed that the number of VOCs was significantly different among the groups: R1 < R2**, R3***, R4*** and R2 < R3*, R4***. R3 and R4 not significantly different. (* 0.05, *** < 0.01, *** < 0.001, *** < 0.0001). \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Median** & **Range** \\ \hline Gestational age (weeks) & 29 & 23–31 + 6 d \\ Birthweight (g) & 1095 & 585–1820 \\ Samples per donor & 3 & 2–6 \\ \hline d: days. & \\ \end{tabular}
\end{table} 
Table 1: Summary of basic demographic features and sampling from 51 preterm babies. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{**Short Chain Fatty Acids**} & **Branched Chain Fatty Acids** & **Methylated Aldehydes** & **Esters** \\ \hline Acetic acid & 2-methylbutanoic acid & Isovaleraldehyde & Ethyl acetate \\ Propionic acid & Isovaleric acid & 2-methylbutyraldehyde & Proply acetate \\ Butanoic acid & & Isobutylraldehyde & Ethyl propionate \\  & & Propyl propionate \\ \hline
**Aldehydes** & **Alcohols** & **Ketones/Diketones** & **Others** \\ \hline Hexanal & Ethanol & 2-heptanone & 2-ethylfuran \\ Heptanal & Propanol & 4-heptanone & 2-pentylfuran \\ Octanal & 1-pentanol & 6-methyl-5-hepten-2-one & D-limonene \\ Nonanal & 1-hexanol & Acetion & Methoxy-phenyl-oxime \\ Benzaldehyde & 1-octer-3-ol & 2,3-butanedione & 1,4-xylene \\ Phenylacetaldehyde & 2-ethylhexanol & & Ethylbenzene \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Summary of 36 volatile organic compounds (VOCs) found in at least 25% of samples. Scrutiny of the data showed that some VOCs were present in the majority of babies in each age group. Others started to appear in the second or third groups. Nine VOCs were present in >66% of samples in first group (1-5 days): 4 were aldehydes--hexanal (100%), heptanal (67%), octanal (78%) and nonanal (67%); 2 others were methylated aldehydes, 2-methylbutylraldehyde (67%) and isovaleraldehyde (89%); the remainder were acetic acid (89%), 2-pentylfuran (72%), and 1-octen-3-ol (67%). These 9 VOCs remained common in the later samples. The second group (6-10 days) had 4 further VOCs that were found on >60% of samples: these were 2-methylbutanoic acid (61%), isovaleric acid (70%), 2,3-butanedione (64%) and 6-methyl-5-hepten-2-one (59%). While acetic acid was common in all 4 groups, propionic acid (range 38-64%) and butanoic acid (11-62%) were not. In Figure 2, we focus on a selection of compounds, showing that some of these, specifically, aldehydes and acetic acid, were present since birth and others (acids, esters, ketones and alcohol), increased after day 5. Figure 1: Scatterplot and boxplots to show the number of volatile organic compounds (VOCs) in each of the age groups. Each dot represents a sample (all samples were included, n = 152). (R1 = up to 5 (n = 18), R2 = 6–10 (n = 44), R3 = 11–20 (n = 56), R4 = 21–70 days (n = 34).