Freeze-drying: an alternative method for the analysis of volatile organic compounds in the headspace of urine samples using solid phase micro-extraction coupled to gas chromatography - mass spectrometry.

BACKGROUND: Volatile organic compounds (VOCs) can be intermediates of metabolic pathways and their levels in biological samples may provide a better understanding about diseases in addition to potential methods for diagnosis. Headspace analysis of VOCs in urine samples using solid phase micro extraction (SPME) coupled to gas chromatography - mass spectrometry (GC-MS) is one of the most used techniques. However, it generally produces a limited profile of VOCs if applied to fresh urine. Sample preparation methods, such as addition of salt, base or acid, have been developed to improve the headspace-SPME-GC-MS analysis of VOCs in urine samples. These methods result in a richer profile of VOCs, however, they may also add potential contaminants to the urine samples, result in increased variability introduced by manually processing the samples and promote degradation of metabolites due to extreme pH levels. Here, we evaluated if freeze-drying can be considered an alternative sample preparation method for headspace-SPME-GC-MS analysis of urine samples. RESULTS: We collected urine from three volunteers and compared the performances of freeze-drying, addition of acid (HCl), addition of base (NaOH), addition of salt (NaCl), fresh urine and frozen urine when identifying and quantifying metabolites in 4 ml samples. Freeze-drying and addition of acid produced a significantly higher number of VOCs identified than any other method, with freeze-drying covering a slightly higher number of chemical classes, showing an improved repeatability and reducing siloxane impurities. CONCLUSION: In this work we compared the performance of sample preparation methods for the SPME-GC-MS analysis of urine samples. To the best of our knowledge, this is the first study evaluating the potential of freeze-dry as an alternative sample preparation method. Our results indicate that freeze-drying has potential to be used as an alternative method for the SPME-GC-MS analysis of urine samples. Additional studies using internal standard, synthetic urine and calibration curves will allow a more precise quantification of metabolites and additional comparisons between methods.Graphical abstractEnhancing VOC profiling from urine samples.

The use of a gas chromatography-sensor system combined with advanced statistical methods, towards the diagnosis of urological malignancies.

Prostate cancer is one of the most common cancers. Serum prostate-specific antigen (PSA) is used to aid the selection of men undergoing biopsies. Its use remains controversial. We propose a GC-sensor algorithm system for classifying urine samples from patients with urological symptoms. This pilot study includes 155 men presenting to urology clinics, 58 were diagnosed with prostate cancer, 24 with bladder cancer and 73 with haematuria and or poor stream, without cancer. Principal component analysis (PCA) was applied to assess the discrimination achieved, while linear discriminant analysis (LDA) and support vector machine (SVM) were used as statistical models for sample classification. Leave-one-out cross-validation (LOOCV), repeated 10-fold cross-validation (10FoldCV), repeated double cross-validation (DoubleCV) and Monte Carlo permutations were applied to assess performance. Significant separation was found between prostate cancer and control samples, bladder cancer and controls and between bladder and prostate cancer samples. For prostate cancer diagnosis, the GC/SVM system classified samples with 95% sensitivity and 96% specificity after LOOCV. For bladder cancer diagnosis, the SVM reported 96% sensitivity and 100% specificity after LOOCV, while the DoubleCV reported 87% sensitivity and 99% specificity, with SVM showing 78% and 98% sensitivity between prostate and bladder cancer samples. Evaluation of the results of the Monte Carlo permutation of class labels obtained chance-like accuracy values around 50% suggesting the observed results for bladder cancer and prostate cancer detection are not due to over fitting. The results of the pilot study presented here indicate that the GC system is able to successfully identify patterns that allow classification of urine samples from patients with urological cancers. An accurate diagnosis based on urine samples would reduce the number of negative prostate biopsies performed, and the frequency of surveillance cystoscopy for bladder cancer patients. Larger cohort studies are planned to investigate the potential of this system. Future work may lead to non-invasive breath analyses for diagnosing urological conditions.

Identifying and quantifying metabolites by scoring peaks of GC-MS data.

BACKGROUND: Metabolomics is one of most recent omics technologies. It has been applied on fields such as food science, nutrition, drug discovery and systems biology. For this, gas chromatography-mass spectrometry (GC-MS) has been largely applied and many computational tools have been developed to support the analysis of metabolomics data. Among them, AMDIS is perhaps the most used tool for identifying and quantifying metabolites. However, AMDIS generates a high number of false-positives and does not have an interface amenable for high-throughput data analysis. Although additional computational tools have been developed for processing AMDIS results and to perform normalisations and statistical analysis of metabolomics data, there is not yet a single free software or package able to reliably identify and quantify metabolites analysed by GC-MS. RESULTS: Here we introduce a new algorithm, PScore, able to score peaks according to their likelihood of representing metabolites defined in a mass spectral library. We implemented PScore in a R package called MetaBox and evaluated the applicability and potential of MetaBox by comparing its performance against AMDIS results when analysing volatile organic compounds (VOC) from standard mixtures of metabolites and from female and male mice faecal samples. MetaBox reported lower percentages of false positives and false negatives, and was able to report a higher number of potential biomarkers associated to the metabolism of female and male mice. CONCLUSIONS: Identification and quantification of metabolites is among the most critical and time-consuming steps in GC-MS metabolome analysis. Here we present an algorithm implemented in a R package, which allows users to construct flexible pipelines and analyse metabolomics data in a high-throughput manner.

Fecal volatile organic compounds: a novel, cheaper method of diagnosing inflammatory bowel disease?

The investigation of a novel, cheaper method of diagnosing inflammatory bowel disease (IBD) is an area of active research. Recently, investigations into the metabolomic profile of IBD patients and animal models of colitis compared to healthy controls has begun to receive considerable attention and correlations between the fecal volatile organic compound (VOC) metabolome and IBD is merging. Patients and clinicians have often reported a change in odor of feces during relapse of IBD. Therefore, this article will focus specifically on the fecal VOC metabolome and its potential role in identifying a novel diagnostic method for IBD.

One-year investigator-blind randomized multicenter trial comparing Asacol 2.4 g once daily with 800 mg three times daily for maintenance of remission in ulcerative colitis.

BACKGROUND: Mesalazine (Asacol) is still widely prescribed in divided doses for ulcerative colitis (UC), despite evidence that adherence is improved by once-daily (OD) prescribing. We aimed to investigate whether OD Asacol was as effective as three times (TDS) daily dosing, and to evaluate the role of treatment adherence. METHODS: An investigator-blind randomized trial was undertaken comparing OD Asacol (three 800 mg tablets) versus one 800 mg TDS in maintenance of remission of UC over 1 year. The primary endpoint was relapse rate, and noninferiority would be concluded if the lower limit of the two-sided 95% confidence interval (CI) of the difference in proportions relapsing (TDS-OD) exceeded -10%. Adherence was measured by tablet counts and self-reported adherence. A subgroup of patients used a bottle cap that recorded all bottle opening events. RESULTS: In all, 213 patients were randomized. In the intention-to-treat (ITT) population, relapse rates were 31% (95% CI 22%-40%) in the OD and 45% (95% CI 35%-54%) in the TDS group. Primary analysis confirmed the noninferiority of OD dosing. Two of the study populations, ITT and per-protocol (PP), showed potential superiority of OD dosing. All measures of adherence showed that it was significantly better in the OD group. Multivariate analysis, however, showed OD dosing was associated with lower relapse risk independently of adherence. CONCLUSIONS: OD dosing with Asacol 2.4 g is as safe and effective as TDS dosing, and secondary analysis confirmed significantly reduced relapse rates. The benefit, however, was clinically borderline and may relate in part to ease of adherence.

The molecular biology of inflammatory bowel diseases.

IBDs (inflammatory bowel diseases) are a group of diseases affecting the gastrointestinal tract. The diseases are multifactorial and cover genetic aspects: susceptibility genes, innate and adaptive responses to inflammation, and structure and efficacy of the mucosal protective barrier. Animal models of IBD have been developed to gain further knowledge of the disease mechanisms. These topics form an overlapping background to enable an improved understanding of the molecular features of these diseases. A series of articles is presented based on the topics covered at the Biochemical Society Focused Meeting The Molecular Biology of Inflammatory Bowel Diseases.

Role of faecal gas analysis for the diagnosis of IBD.

The diagnosis of IBD (inflammatory bowel disease) is based on the clinical evaluation of symptoms and signs leading to a series of investigations. The investigations used are often unpleasant for patients; they are invasive, costly and potentially dangerous. Patients often report that the odour of flatus, or the gas emitted from faeces, is abnormal during a flare of their IBD. Our group has characterized the VOCs (volatile organic compounds) in the headspace gas emitted from faecal samples from healthy subjects, from patients with infectious diarrhoea and from those with Crohn's disease or ulcerative colitis, both in relapse and remission. Painstaking analysis of gas chromatography-MS data (VOC profiling) has revealed patterns of compounds that are strongly associated with specific infectious diseases and with IBD. These compounds represent a change in the microflora and/or the metabolism of bacteria and/or the epithelium in disease states. These profiles offer a potential for rapid non-invasive assessment of a range of infectious and non-infectious gastrointestinal diseases. The study of VOCs may lead to a better understanding of the pathogenesis of IBD.

Volatile organic compounds as diagnostic biomarkers in gastrointestinal and liver diseases.

The assessment of disease activity in various conditions may be performed using a range of different techniques. These include the use of non-invasive tests, such as acute phase inflammatory markers and simple radiological techniques, to more advanced invasive and complex modalities. Over the past two decades the analysis of volatile organic compounds (VOCs) in biological specimens has attracted a considerable amount of clinical interest. The investigation of VOCs, using a variety of analytical techniques, has shown a significant correlation between the pattern and concentration of VOCs and the occurrence of various diseases. This provides a potentially non-invasive means of diagnosis, monitoring of pathological processes and assessment of pharmacological response. It may be rapid, simple and acceptable to patients. In this paper we review the medical literature and research efforts that have been carried out over the past decades, and try to summarize the clinical implications of VOC analysis of various biological emanations including stool, breath and blood samples and their correlation with gastrointestinal and liver diseases.

An economic evaluation comparing concomitant oral and topical mesalazine versus oral mesalazine alone in mild-to-moderately active ulcerative colitis based on results from randomised controlled trial.

INTRODUCTION: A previous randomised controlled trial has demonstrated that oral plus topical mesalazine enema is more effective than oral mesalazine alone for achieving clinical remission in mild-to-moderately active extensive ulcerative colitis (UC). To evaluate whether this strategy is cost-effective we conducted an economic evaluation comparing 1 g topical mesalazine in combination with 4 g oral mesalazine compared to 4 g mesalazine monotherapy in mild-to-moderately active UC. METHODS: The economic evaluation was based on the ability to achieve remission using changes from baseline in the ulcerative colitis disease activity instrument (UCDAI). A cost-utility analysis was used where the main outcome was quality-adjusted life years to reflect improved quality of life associated with achieving remission compared with active disease. A simulated Markov model with five health states was constructed to model cost and outcome changes over time: (1) active UC; (2) mesalazine-refractory active UC; (3) steroid-refractory active UC; (4) infliximab-responsive active UC; and (5) remission. To reflect parameter uncertainty in the cost-effectiveness analysis probabilistic sensitivity analysis (PSA) was conducted by varying relevant clinical parameters. RESULTS: Average treatment costs required to transition a patient from active UC to remission using oral and topical mesalazine compared with oral alone were £1812 and £2390, respectively. Improved remission rates attributed to oral and topical mesalazine resulted in moderate improvements in quality-adjusted life years (QALYs) compared to oral mesalazine alone. Disaggregation of medical costs indicated that medical consultations and diagnostic costs were similar for both treatment arms. An abbreviated analysis which considered costs up to steroid-refractory patients in subacute UC indicated that combination therapy offered a cost-savings of £285 over 16 weeks of therapy compared with monotherapy. CONCLUSIONS: The results indicate that the addition of 1 g topical mesalazine results in significant cost-savings and moderate quality of life improvements. We have also shown that irrespective of which treatment modality is used in steroid-refractory patients (eg, infliximab, azathioprine, ciclosporine) that topical mesalazine is cost-saving.

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-hepten-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.

Refractory coeliac disease: remission with infliximab and immunomodulators.

Coeliac disease is a T-cell-mediated enteropathy induced by gluten. A minority of patients who fail to respond to a gluten-free diet may require intervention with immunomodulating drugs. We report a case of refractory coeliac disease where remission was induced by the anti-tumour necrosis factor-alpha antibody infliximab and was maintained with prednisolone and azathioprine.

Incidence of inflammatory bowel disease is rising and abdominal tuberculosis is falling in Bangladeshis in East London, United Kingdom.

OBJECTIVES: Ulcerative colitis (UC) and Crohn's disease (CD) were previously thought to be uncommon and abdominal tuberculosis (TB) common in patients from Bangladesh. We have reevaluated their incidence in Bangladeshis resident in East London. METHODS: Bangladeshis resident in Tower Hamlets presenting between 1997 and 2001 were identified from pathology, endoscopy, and medical records. Demographic, clinical, and management details were recorded. Incidences were calculated and compared with those from 1981 to 1989. RESULTS: Sixteen Bangladeshi patients with UC, 19 with CD, and 5 with abdominal TB were identified. Between 1997 and 2001, the age-standardized incidence of UC was 8.2/10(5)/yr (95% CI 2.5-13.9) compared with 2.4 (95% CI 0.8-3.8) in 1981-1989, and that of CD was 7.3/10(5)/yr (95% CI 2.0-12.6) (2.3, 95% CI 0.7-3.7 in 1981-1989). The standardized ratios for the incidences of UC and CD in recent periods compared with previous periods were 2.1 (95% CI 0.9-3.9) and 2.5 (1.2-4.6), respectively. There was a significant increase in the number of Bangladeshis developing CD by age <20 yr between the earlier and more recent periods (p < 0.02). The standardized incidence of abdominal TB was 2.5/10(5)/yr (95% CI 0.2-4.8) in 1997-2001, and 7.4 (95% CI 2.1-12.7) in 1985-1989 (p < 0.05). The standardized ratio for the incidence of TB in the two periods was 0.22 (95% CI 0.07-0.53). CONCLUSIONS: In Bangladeshis in East London, the incidence of IBD has increased and of abdominal TB has fallen over the last decade; CD has become a more likely diagnosis than abdominal TB. Clinicians in the Western world need to be aware of the changing incidences of IBD and abdominal TB in South Asians.