Norman M. Ratcliffe

Fecal Microbiome and Volatile Organic Compound Metabolome in Obese Humans With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS The histopathology of nonalcoholic fatty liver disease (NAFLD) is similar to that of alcoholic liver disease. Colonic bacteria are a source of many metabolic products, including ethanol and other volatile organic compounds (VOC) that may have toxic effects on the human host after intestinal absorption and delivery to the liver via the portal vein. Recent data suggest that the composition of the gut microbiota in obese human beings is different from that of healthy-weight individuals. The aim of this study was to compare the colonic microbiome and VOC metabolome of obese NAFLD patients (n = 30) with healthy controls (n = 30). METHODS Multitag pyrosequencing was used to characterize the fecal microbiota. Fecal VOC profiles were measured by gas chromatography-mass spectrometry. RESULTS There were statistically significant differences in liver biochemistry and metabolic parameters in NAFLD. Deep sequencing of the fecal microbiome revealed over-representation of Lactobacillus species and selected members of phylum Firmicutes (Lachnospiraceae; genera, Dorea, Robinsoniella, and Roseburia) in NAFLD patients, which was statistically significant. One member of phylum Firmicutes was under-represented significantly in the fecal microbiome of NAFLD patients (Ruminococcaceae; genus, Oscillibacter). Fecal VOC profiles of the 2 patient groups were different, with a significant increase in fecal ester compounds observed in NAFLD patients. CONCLUSIONS A significant increase in fecal ester VOC is associated with compositional shifts in the microbiome of obese NAFLD patients. These novel bacterial metabolomic and metagenomic factors are implicated in the etiology and complications of obesity.

Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease.

Little is known about the volatile organic compounds (VOCs) in feces and their potential health consequences. Patients and healthcare professionals have observed that feces often smell abnormal during gastrointestinal disease. The aim of this work was to define the volatiles emitted from the feces of healthy donors and patients with gastrointestinal disease. Our hypotheses were that i) VOCs would be shared in health;ii) VOCs would be constant in individuals; and iii) specific changes in VOCs would occur in disease. Volatile emissions in health were defined in a cohort and a longitudinal study. Subsequently, the pattern of volatiles found in the cohort study were compared to that found from patients with ulcerative colitis, Campylobacter jejuni, and Clostridium difficile. Volatiles from feces were collected by solid‐phase microextraction and analyzed by gas chromatography/mass spectrometry. In the cohort study, 297 volatiles were identified. In all samples, ethanoic, butanoic, pentanoic acids, benzaldehyde, ethanal, carbon disulfide, dimethyldisulfide, acetone, 2‐butanone, 2, 3‐butanedione, 6‐Methyl‐5‐hep‐ten‐2‐one, indole, and 4‐Methylphenol were found. Forty‐four compounds were shared by 80% of subjects. In the longitudinal study, 292 volatiles were identified, with some inter and intra subject variations in VOC concentrations with time. When compared to healthy donors, volatile patterns from feces of patients with ulcerative colitis, C. difficile, and C. jejuni were each significantly different. These findings could lead the way to the development of a rapid diagnostic device based on VOC detection.—Garner C. E., Smith, S., de Lacy Costello B., White, P., Spencer, R., Probert, C. S. J., Ratcliffe N. M. Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease. FASEB J. 21, 1675–1688 (2007)

Highly sensitive mixed oxide sensors for the detection of ethanol

Sensors consisting of mixtures of tin dioxide and zinc oxide powders in a range of proportions were constructed. Each mixture was applied to an electrode-bearing alumina substrate either as a paste, or by screen printing. The responses of these sensors, and of three commercially-available Figaro sensors, to ethanol vapour in the 1–1000 parts-per-billion (ppb) range were measured. At 100 ppb of ethanol vapour, the most sensitive paste sensor (25% SnO2/75% ZnO) exhibited a response that was more than twice that of the screen-printed sensors, and almost 60 times greater than that of the most sensitive Figaro sensor (TGS822).

An investigation of fecal volatile organic metabolites in irritable bowel syndrome.

Diagnosing irritable bowel syndrome (IBS) can be a challenge; many clinicians resort to invasive investigations in order to rule out other diseases and reassure their patients. Volatile organic metabolites (VOMs) are emitted from feces; understanding changes in the patterns of these VOMs could aid our understanding of the etiology of the disease and the development of biomarkers, which can assist in the diagnosis of IBS. We report the first comprehensive study of the fecal VOMs patterns in patients with diarrhea-predominant IBS (IBS-D), active Crohns disease (CD), ulcerative colitis (UC) and healthy controls. 30 patients with IBS-D, 62 with CD, 48 with UC and 109 healthy controls were studied. Diagnosis of IBS-D was made using the Manning criteria and all patients with CD and UC met endoscopic, histologic and/or radiologic criteria. Fecal VOMs were extracted by solid phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). 240 VOMs were identified. Univariate analysis showed that esters of short chain fatty acids, cyclohexanecarboxylic acid and its ester derivatives were associated with IBS-D (p<0.05), while aldehydes were more abundant in IBD (p<0.05). A predictive model, developed by multivariate analysis, separated IBS-D from active CD, UC and healthy controls with a sensitivity of 94%, 96% and 90%; and a specificity of 82%, 80% and 80% respectively (p<0.05). The understanding of the derivation of these VOMs may cast light on the etiology of IBS-D and IBD. These data show that fecal VOMs analyses could contribute to the diagnosis of IBS-D, for which there is no laboratory test, as well as IBD.

The importance of methane breath testing: a review

Sugar malabsorption in the bowel can lead to bloating, cramps, diarrhea and other symptoms of irritable bowel syndrome as well as affecting absorption of other nutrients. The hydrogen breath test is now a well established noninvasive test for assessing malabsorption of sugars in the small intestine. However, there are patients who can suffer from the same spectrum of malabsorption issues but who produce little or no hydrogen, instead producing relatively large amounts of methane. These patients will avoid detection with the traditional breath test for malabsorption based on hydrogen detection. Likewise the hydrogen breath test is an established method for small intestinal bacterial overgrowth (SIBO) diagnoses. Therefore, a number of false negatives would be expected for patients who solely produce methane. Usually patients produce either hydrogen or methane, and only rarely there are significant co-producers, as typically the methane is produced at the expense of hydrogen by microbial conversion of carbon dioxide. Various studies show that methanogens occur in about a third of all adult humans; therefore, there is significant potential for malabsorbers to remain undiagnosed if a simple hydrogen breath test is used. As an example, the hydrogen-based lactose malabsorption test is considered to result in about 5-15% false negatives mainly due to methane production. Until recently methane measurements were more in the domain of research laboratories, unlike hydrogen analyses which can now be undertaken at a relatively low cost mainly due to the invention of reliable electrochemical hydrogen sensors. More recently, simpler lower cost instrumentation has become commercially available which can directly measure both hydrogen and methane simultaneously on human breath. This makes more widespread clinical testing a realistic possibility. The production of small amounts of hydrogen and/or methane does not normally produce symptoms, whereas the production of higher levels can lead to a wide range of symptoms ranging from functional disorders of the bowel to low level depression. It is possible that excess methane levels may have more health consequences than excess hydrogen levels. This review describes the health consequences of methane production in humans and animals including a summary of the state of the art in detection methods. In conclusion, the combined measurement of hydrogen and methane should offer considerable improvement in the diagnosis of malabsorption syndromes and SIBO when compared with a single hydrogen breath test.

A novel method for rapidly diagnosing the causes of diarrhoea

Background: The microbiological diagnosis of infectious diarrhoea may take several days using conventional techniques. In order to determine whether flatus can be used to make a rapid diagnosis, the volatile organic compounds associated with diarrhoea were analysed. Methods: Stool samples were collected from 35 patients with infectious diarrhoea and from six healthy controls. Gaseous compounds were extracted from a headspace using solid phase microextraction and analysed using gas chromatography and mass spectroscopy. Results: Characteristic patterns of volatile gases were found for the main causes of infectious diarrhoea in hospitals. Furan species without indoles indicated Clostridium difficile, ethyl dodecanoate indicated rotavirus, ammonia without ethyl dodecanoate suggested other enteric viruses, and the absence of hydrocarbons and terpenes indicated Campylobacter infection. Conclusion: These results could be the basis of rapid near patient diagnosis of infectious diarrhoea.

Novel composite organic–inorganic semiconductor sensors for the quantitative detection of target organic vapours

Composites of tin dioxide (an n-type semiconductor) and derivatives of the conducting polymer polypyrrole (a p-type semiconductor) gave reversible changes in electrical resistance at room temperature when exposed to a range of organic vapours. The optimum amount of polymer giving highest sensitivity was found by experiment to be 2.5% by mass for the polypyrrole chloride-tin dioxide composite. Composites containing 2.5% polymer by mass but differing in polymer derivative, were fabricated and exposed to low concentrations of ethanol, methanol, acetone, methyl acetate and ethyl acetate. All were found to give significant and reversible decreases in electrical resistance. Direct comparison with sensors constructed solely of tin dioxide or polypyrrole at room temperature showed the composites to be more sensitive. The gas sensitivity of the composite materials depended on the type of polymer derivative incorporated and the dopant anion associated with the polymer. The composites were simple to fabricate and gave differing response profiles to a range of organic vapours.

Analysis of faecal volatile organic compounds in preterm infants who develop necrotising enterocolitis: a pilot study.

Objective: To determine differences in the profiles of volatile organic compounds (VOCs) in faecal samples from preterm infants who develop necrotising enterocolitis (NEC) compared with non-NEC controls. Materials and Methods: Daily faecal samples from preterm infants were collected prospectively during an 8-month period from a level 3 regional neonatal intensive care unit. Six infants subsequently developed NEC and were matched with 7 non-NEC infants. Solid-phase microextraction and gas chromatography and mass spectrometry were used to extract and identify the VOCs from the headspace above the faecal samples taken before the onset of NEC and after the disease was diagnosed. Faecal samples at similar ages were also studied from the control infants. Results: Two hundred twenty-four different VOCs were extracted from 65 samples. Volatile organic compounds increased in number with age for non-NEC infants. In the days before and after the diagnosis of NEC a reduction in the number of VOCs extracted was observed. In addition, 4 specific esters present in controls—2-ethylhexyl acetic ester, decanoic acid ethyl ester, dodecanoic acid ethyl ester, and hexadecanoic acid ethyl ester—were consistently absent from all faecal samples in those infants who developed NEC in the 4 days before the onset of the disease. Conclusion: This pilot study shows that VOC extraction from faeces may be used to identify infants that are at risk of developing NEC.

A comparative study of the analysis of human urine headspace using gas chromatography–mass spectrometry

First-void urine samples were obtained from 24 elderly, asymptomatic men (median age 62.9 years). The headspace above pH adjusted urine samples were extracted using a carboxen/polydimethylsiloxane solid phase micro-extraction fibre and the volatile organic compounds analysed by gas chromatography/mass spectrometry. A total of 147 compounds were identified in the headspace of urine. The acidified samples recorded a total of 92 compounds, 27 of which were ubiquitous, basified samples 70 compounds, with 12 ubiquitous and unmodified pH samples 49, with 6 ubiquitous. Five compounds were ubiquitous irrespective of pH: acetone, methylene chloride, 4-heptanone, 2-pentanone and 2-butanone. A comparative analysis of unfrozen and frozen-thawed urine (stored at room temperature for 0, 1 and 8 h) showed that samples retained the same number of compounds although variations in the peak areas for some compounds were observed.

Experimental reaction–diffusion pre-processor for shape recognition☆

Abstract We have produced an experimental implementation of a massively-parallel reaction–diffusion processor which performs one of the most essential parts of shape recognition—construction of a skeleton. A skeleton is a unique, stable and invariant representation of a shape, therefore computation of the skeleton is an essential tool of computer vision. Skeleton computation is a typical ‘natural’ spatial problem that can be solved with the use of biological, chemical or physical phenomena. One possible approach—a reaction–diffusion based computation—is explored in this Letter. A contour is represented by a concentration profile of one reagent, a planar substrate is mixed with another reagent. The reagent, representing the original contour diffuses to form a coloured phase in a reaction with the substrate-reagent. However, at sites where two diffusion wave fronts meet no coloured phase is formed and the substrate retains its uncoloured state. These loci of the computation space represent a skeleton of the given contour. In the Letter we only describe a laboratory prototype of a reaction–diffusion processor that computes a skeleton, no further tasks of image processing are undertaken, one could say we have designed an unconventional chemical pre-processor for shape recognition.