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. 2015 Dec;81(23):8093-7.
doi: 10.1128/AEM.02787-15. Epub 2015 Sep 18.

A rapid and specific method for the detection of indole in complex biological samples

Charles Darkoh  1 , Cynthia Chappell  2 , Christopher Gonzales  2 , Pablo Okhuysen  3
Affiliations

A rapid and specific method for the detection of indole in complex biological samples

Charles Darkoh et al. Appl Environ Microbiol. 2015 Dec.

Abstract

Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota.

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Figures

FIG 1
FIG 1
The hydroxylamine-based indole assay (HIA) does not detect other naturally occurring indole analogs. Indole and indole analogs were tested in the hydroxylamine-based (A, B) and Kovács (C, D) assays. Each point (A, C) or bar (B, D) represents the mean value of four different experiments, each done in triplicate. The results of the HIA (A) and the Kovács assay (C) of naturally occurring (biological) indole (filled circles) and indole analogs (3-methylindole, filled squares; indole sulfate, open circles; indoxyl-3-proprionic acid, open squares) are shown for concentrations of 0 to 300 μM. Error bars indicate the standard deviations among the four replicate experiments. The HIA (B) and the Kovács assay (D) show absorbances of the following biological and nonbiological compounds at a concentration of 300 μM: indole (bar 1), 3-methylindole (bar 2), indoxyl-3-proprionic acid (bar 3), indole sulfate (bar 4), 2-methylindole (bar 5), 1-methylindole (bar 6), 3-indoleacetic acid (bar 7), and indoxyl acetate (bar 8).
FIG 2
FIG 2
Frequency distribution of fecal indole levels (in micromolar) in 53 healthy adults as determined by the hydroxylamine-based assay. Each sample was assayed in duplicate, and mean values are expressed as micromolar concentrations. The percentage of subjects in each category is shown.
FIG 3
FIG 3
Comparative quantitation of indole concentrations in bacterial cultures. Enterotoxigenic E. coli strain H10407 was grown for 24 h, and supernatants were tested using the hydroxylamine-based indole assay (HIA) or the Kovács assay. Data represent mean values ± standard deviations from three separate experiments, each done in triplicate.
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