The Approach to Sample Acquisition and Its Impact on the Derived Human Fecal Microbiome and VOC Metabolome

<div><p>Recent studies have illustrated the importance of the microbiota in maintaining a healthy state, as well as promoting disease states. The intestinal microbiota exerts its effects primarily through its metabolites, and metabolomics investigations have begun to evaluate the diagnostic and health implications of volatile organic compounds (VOCs) isolated from human feces, enabled by specialized sampling methods such as headspace solid-phase microextraction (hSPME). The approach to stool sample collection is an important consideration that could potentially introduce bias and affect the outcome of a fecal metagenomic and metabolomic investigation. To address this concern, a comparison of endoscopically collected (<i>in vivo</i>) and home collected (<i>ex vivo</i>) fecal samples was performed, revealing slight variability in the derived microbiomes. In contrast, the VOC metabolomes differ widely between the home collected and endoscopy collected samples. Additionally, as the VOC extraction profile is hyperbolic, with short extraction durations more vulnerable to variation than extractions continued to equilibrium, a second goal of our investigation was to ascertain if hSPME-based fecal metabolomics studies might be biased by the extraction duration employed. As anticipated, prolonged extraction (18 hours) results in the identification of considerably more metabolites than short (20 minute) extractions. A comparison of the metabolomes reveals several analytes deemed unique to a cohort with the 20 minute extraction, but found common to both cohorts when the VOC extraction was performed for 18 hours. Moreover, numerous analytes perceived to have significant fold change with a 20 minute extraction were found insignificant in fold change with the prolonged extraction, underscoring the potential for bias associated with a 20 minute hSPME.</p> </div

Alcohol Induced Alterations to the Human Fecal VOC Metabolome

<div><p>Studies have shown that excessive alcohol consumption impacts the intestinal microbiota composition, causing disruption of homeostasis (dysbiosis). However, this observed change is not indicative of the dysbiotic intestinal microbiota function that could result in the production of injurious and toxic products. Thus, knowledge of the effects of alcohol on the intestinal microbiota function and their metabolites is warranted, in order to better understand the role of the intestinal microbiota in alcohol associated organ failure. Here, we report the results of a differential metabolomic analysis comparing volatile organic compounds (VOC) detected in the stool of alcoholics and non-alcoholic healthy controls. We performed the analysis with fecal samples collected after passage as well as with samples collected directly from the sigmoid lumen. Regardless of the approach to fecal collection, we found a stool VOC metabolomic signature in alcoholics that is different from healthy controls. The most notable metabolite alterations in the alcoholic samples include: (1) an elevation in the oxidative stress biomarker tetradecane; (2) a decrease in five fatty alcohols with anti-oxidant property; (3) a decrease in the short chain fatty acids propionate and isobutyrate, important in maintaining intestinal epithelial cell health and barrier integrity; (4) a decrease in alcohol consumption natural suppressant caryophyllene; (5) a decrease in natural product and hepatic steatosis attenuator camphene; and (6) decreased dimethyl disulfide and dimethyl trisulfide, microbial products of decomposition. Our results showed that intestinal microbiota function is altered in alcoholics which might promote alcohol associated pathologies.</p></div

Comparison of SCFA abundance among the cohorts.

<p>Box plots are presented, depicted as described in Fig.8. A) Among the endoscopy collected samples, while most of the SCFAs are comparable in abundance within the healthy and alcoholic cohorts, propanoic acid demonstrates a statistically significant reduction in median abundance in the alcoholic cohort relative to the healthy cohort (<i>p</i> = 0.03, fold change = 2.32). B) In the home collected samples, butyrate has a 1.6 fold reduction in median abundance, while the remaining SCFAs are very similar between the healthy and alcoholic fecal samples. See text for further discussion.</p

Fold change analysis of the metabolite abundance between the healthy and alcoholic fecal samples.

<p>The fold change (FC) is calculated as the log transformation of the ratio between the mean metabolite abundance in the alcoholic cohort relative to the healthy cohort. The analysis was performed with both the endoscopy (A) and home passage collected (B) metabolomes. A log₂(FC) greater than 1.5 is deemed significant (equivalent to a threefold or greater change in metabolite abundance). A fold change analysis comparing median analyte values produces similar results (not shown).</p

Metabolites with a statistically significant difference in abundance between the healthy and alcoholic cohorts (<i>p</i><0.05; <i>p</i>

<p>Metabolites with a statistically significant difference in abundance between the healthy and alcoholic cohorts (<i>p</i><0.05; <i>p</i></p

Metabolite correlation network of the endoscopy collected (A) and home collected (B) fecal VOC metabolomes.

<p>Pearson’s correlation coefficients were calculated for metabolites present in 80% or greater of the total fecal samples. A Pearson correlation value greater than 0.95 is depicted as a green line between metabolites (negative correlations are not shown, as correlation values less than-0.95 were not obtained). To facilitate comparison of the networks, metabolites are numerically represented and their placement around the circumference of each network is fixed among the paired plots. Regardless of the fecal collection method used, the fecal samples from the alcoholic participants have a notably different correlation network than that seen in the fecal samples from non-alcoholics. This difference is even more apparent in correlation networks derived using metabolites present in ≥21% of all fecal samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119362#pone.0119362.s002" target="_blank">S2 Fig</a>.).</p

Comparison of protein putrefaction products among the cohorts.

<p>Box plots depicting relative metabolite abundance are shown, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119362#pone.0119362.g008" target="_blank">Fig. 8</a>. A) Among the endoscopy collected samples, with the exception of methyl indole, the metabolites are comparable in abundance within the healthy and alcoholic cohorts. Note however, the difference observed with methyl indole is not statistically significant (<i>p</i> = 0.519). B) In the home collected samples, metabolite abundance is very similar between the healthy and alcoholic fecal samples.</p

Cohort metabolite composition and abundance.

<p>The pooled analytes present in each cohort were distributed among the indicated chemical classes and then tallied. A and B) The graphs indicate the total number of analytes in each class for the 20 minute hSPME (A) or the 18 hour hSPME (B) metabolomes. The sum of the standard deviation across the chemical classes is 134 for the 20 minute data and 108 for the 18 hour data, emphasizing the greater similarity between the cohorts in the latter. C and D) The relative abundance (peak height) of the analytes present in each cohort were distributed among the indicated chemical classes and then summed. The graphs indicate the relative abundance of each class for the 20 minute hSPME (C) or the 18 hour hSPME (D) metabolomes. Although the cohort similarity is apparent regardless of extraction duration, the sum of the coefficient of variation across the chemical classes totals 3.5 for the 20 minute data and 1.1 for the 18 hour data, highlighting the greater similarity between the cohorts in the latter.</p

Fold change analysis of the metabolite abundance between the endoscopy and home passage collected samples.

<p>The fold change (FC) is calculated as the log transformation of the ratio between the mean metabolite abundance in the endoscopy cohort relative to the home passage collected cohort. The analysis was performed with both the 20 minute (A) and 18 hour (B) metabolomes. </p

Heat map showing the unsupervised hierarchical clustering of the fecal samples according to the similarity of metabolome composition.

<p>The endoscopy collected fecal metabolomes are compared in (A) while the home collected fecal metabolomes are compared in (B). The samples are arranged in rows, the metabolites in columns, and shades of red represent elevation of a metabolite while shades of green represent decrease of a metabolite, relative to the median metabolite levels (see color scale). In the dendrograms, the clustering clearly differentiates the alcoholic and healthy fecal samples.</p