San Diego State University postdoctoral researcher Katrine Whiteson, Ph. D. has found that a molecule previously linked to lung injuries in factory workers producing microwave popcorn might also play an important role in microbial lung infections suffered by people with cystic fibrosis (CF). The molecule, known as 2,3-butanedione, which tastes “buttery” and is the main ingredient in microwave popcorn flavoring, is known by beer brewers as diacetyl, and also found in yogurt and wine, can be detected in higher concentrations in CF patients than in healthy ones.
Dr. Whiteson and her colleagues recently published their findings in the ISME Journal in a paper entitled “Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation,” (9 January 2014; doi: 10.1038/ismej.2013.229), co-authored by Dr. Whiteson, Yan Wei Lim, Robert Schmieder, and Forest Rohwer — all of San Diego State U.; Simone Meinardi and Donald R. Blake of the Department of Chemistry, University of California, Irvine, CA; Heather Maughan of the Ronin Institute, Montclair, NJ; and Douglas Conrad of the Department of Medicine, University of California, San Diego, La Jolla, CA.
The scientists note that cystic fibrosis patients’ airways are chronically colonized by patient-specific polymicrobial communities, and conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. They observe that recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production.
In order to examine bacterial fermentation in the respiratory tract, the researchers measured breath gas metabolites, then sequenced several metagenomes from CF and non-CF volunteers. They found that 2,3-butanedione was produced in nearly all respiratory tracts, and breath concentrations of the molecule varied between CF patients at the same time point, with some patients having high enough levels of 2,3-butanedione to irreversibly damage lung tissue.
They note that antibiotic therapy likely dictates activities of 2,3-butanedione-producing microbes, suggesting a need for further study with larger sample size. Sputum microbiomes were observed to be dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, revealing potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were found to be disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa.
The coauthors propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification, and hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.
In an interview this month with pharmaphorum.com, Dr. Whiteson elaborates on her research team’s discovery of 2,3-butanedione’s suspected role in exacerbating Cystic Fibrosis flare ups, noting that factory workers exposed to high concentrations of this molecule have had their lungs destroyed by inflammation and scarring (bronchiolitis Obliterans), and several have even had needed lung transplants. She explains that the 2,3-butanedione molecule is produced by microbes resident in most people’s mouths, including Streptococcus spp.
In a news release, San Diego State’s Michael Price notes that CF patients experience day-to-day persistent coughing and increased mucus production, punctuated by periodic flare-ups of these symptoms, known as “exacerbations,” and that health experts believe most permanent scarring and damage to lung tissue in CF patients, occurs during these exacerbations. However, if CF patients had some warning of oncoming or imminent exacerbations, they could respond with earlier or more specific treatment, potentially warding off some of the damaging effects. However, currently there is no proven way to detect when someone’s about to have an exacerbation.
Price cites Dr. Whiteson observing that when CF patients visit the doctor, they inhale vaporized salt to induce deep coughing, producing a mucus sample, and that when clinical labs culture the microbes in these samples, they usually don’t find big differences between the microbes that grow when CF patients are feeling healthy and when they are feeling sick. This leaves it up to doctors to choose antibiotic therapies based on trial and error, so the operative question that has plagued researchers is: “What’s different about the CF lung during these exacerbations?”
In their study, Dr. Whiteson and her research colleagues measured the breath gases produced by both CF patients and healthy volunteers, and as noted, in analyzing those gases, they found elevated levels of 2,3-butanedione /diacetyl in the CF patients’ lungs.
Dr. Whiteson and her team theorize that various species of the oral microbe Streptococcus produce the diacetyl via a fermentation process, and that the molecule can also activate harmful effects of other bacteria common in the lungs of CF patients. For example, when the bacteria P. aeruginosa come into contact with diacetyl, it causes the bacteria to produce toxic compounds which may be partially responsible for CF’s characteristic lung damage.
Price says Dr. Whiteson and her colleagues envision a new technology based on their recent work that would function something like a breathalyzer does for alcohol, to detect the presence of diacetyl. By regularly monitoring the presence of the molecule in their breath, CF patients might get an early detection signal indicating that an exacerbation is imminent, and then take antibiotics to prevent it from occurring. That won’t be the final weapon in the battle against cystic fibrosis, which is a complex disease, and the damage caused by 2,3-butanedione /diacetyl is likely only one part of its profile. But it’s a good step, she affirms.
Dr. Whiteson is currently working with Silicon Valley biotech company, Metabolomx to develop a microchip capable of detecting 2,3-butanedione /diacetyl or other indicators of imminent CF exacerbations.
Early Detection – The Emerging Field Of Metabolomics
Metabolomx’s researchers note that disease diagnosis has largely been conducted by blood and urine assays of large biomolecules, often proteins or nucleic acid. Moving beyond genomics and proteomics, the emerging field of metabolomics directly analyzes the activity of the metabolic pathways themselves by measurement of small molecule metabolites. These small molecule metabolites are volatile organic compounds (VOC’s) that appear in alveolar blood and partition into the gas phase in exhaled breath, producing a metabolic signature of disease in breath that can distinguished from that of healthy subjects.
Human breath contains hundreds of VOCs produced both endogenously and from external environmental sources, so in order to detect the few metabolite biomarkers of disease against a background of hundreds of other VOCs, a breath analysis system must be highly dimensional, able to distinguish the signature pattern of diverse VOC biomarkers in a diverse chemical background. The metabolite biomarkers are often important at very low part per billion (ppb) concentrations. Therefore it is critical for the breath analysis system sensor to be very sensitive, able to detect diverse VOC biomarkers often in low single digit ppb concentration. The Metabolomx sensor has the high dimensionality and sensitivity to capture the chemical signature pattern of the complex mixture of VOC’s present in breath.
At the core of the Metabolomx breath analysis system is a novel, proprietary colorimetric sensor array (CSA) — a matrix of colored chemical indicators of diverse reactivities embedded in a nanoporous sol-gel matrix. Each indicator has distinct chemical reactivity with volatile species and changes color differently upon exposure to analytes. The resulting pattern of color changes comprises a high-dimensional chemical signature pattern. The image below demonstrates the patterns produced by 10 distinct bacteria, all 10 of which Metabolomx says were identified accurately (98.8%) in 50 blind trials. Note that even different strains of the same species cause distinct patterns.
The sensor is constructed on a simple plastic or paper-like media, is inexpensive and disposable. Cost and performance easily match the requirements for a one time use medical exam. The CSA exam card used in the Metabolomx breath analysis instrument is printed on a plastic substrate, contained in a tube, through which breath can be passed. The exam card is disposable; one is used with each patient exam.
Prior devices may have had many elements but produced sensor signals with only a few material principal components. The CSA is highly dimensional, each of the array of over 100 reactive indicators are highly diverse chemically, a very wide range of volatile organic compounds (VOCs). The colorimetric sensor array has proven very well suited to identifying the fingerprint of highly complex mixtures. The ability to identify the signature of complex mixtures proves crucial for the assessment of disease from breath, as disease processes such as lung cancer tumor metabolism or bacterial metabolism each produce a signature pattern composed of dozens of out-gassed volatile organic compounds (VOCs), the entire pattern being the recognizable signature. A recent breakthrough material and new classes of sensors have improved sensitivity and significantly increased dimensionality. The most recent sensor includes over 100 indicators, older versions were limited to 36.
The Metabolomx breath analysis instrument breath analysis instrument (BAI) captures a precise quantity of breath, in a specific portion of the breath cycle and controls the exposure of a CSA exam card to the captured breath at a precise flow rate and volume. The specific portion of the breath cycle to be sampled depends on the condition to be diagnosed. For example, lung cancer biomarkers are best identified from the deep lung alveolar portion of breath and upper respiratory infections biomarkers are best identified from the early portion of an exhale.
The breath analysis instrument is designed to be mounted on a medical mobile computing cart or on a desktop, and has three major components: an analysis module, a computing platform with display and breath collection apparatus. Exams are easily performed with a few clicks and minimum training, and takes only a few minutes. Metabolomx says many patients entertain themselves with reading or browsing the Internet on a touchpad device.
San Diego State University
The ISME Journal
Katrine Whitesun – Linked-In