Obesity has reached epidemic proportions in most of the western world. It is strongly associated with nonalcoholic fatty liver disease (NAFLD), a form of chronic liver disease that is now considered the most common serious complication of childhood obesity affecting ∼10% of children 1. NAFLD is the hepatic manifestation of metabolic syndrome (MetS) and should be suspected in all overweight or obese children and adolescents. The spectrum of NAFLD ranges from simple steatosis, to nonalcoholic steatohepatitis, to fibrosis, and eventually cirrhosis and its complications 2. The prognosis of NAFLD in children is not clearly defined; however, in the largest natural history study in children to date, up to 80% of patients with repeat biopsies developed some degree of fibrosis during the follow-up period 3. Histological staging and grading by means of liver biopsies is presently the gold standard for diagnosing NAFLD. Liver biopsy is an invasive procedure associated with significant potential complications especially in children. Unfortunately, there are currently no reliable noninvasive methods to screen for NAFLD. The American Academy of Pediatrics recommends that estimation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) should be performed to screen for NAFLD in obese children. However, it has become clear that in both adults and children, liver enzymes perform poorly for diagnosing NAFLD with up to two-thirds of patients with NAFLD having normal levels of ALT and AST 4. Hepatic ultrasonography (US) is the most commonly used imaging modality for NAFLD screening and we have shown recently that it has good accuracy for estimating hepatic steatosis in children 5. However, US is a relatively expensive and labor intensive screening tool, given that one-third of American children are overweight or obese. In light of the increasing incidence of NAFLD and its implications, it is extremely important to develop an easy and noninvasive tool to detect patients with fatty liver and hepatic injury.
Breath testing is becoming an increasingly important noninvasive diagnostic method that can be used in the evaluation of health and disease states in the gastrointestinal tract and beyond 6,7. Breath testing is a simple and safe alternative to more invasive investigations in children. More recent technological advancements in breath testing and analysis through gas and liquid chromatography and mass spectrometry have made it possible to identify thousands of substances in the breath that correlate with different disease states 7. The aims of the current study were to assess (a) the feasibility of breath testing using selective ion flow tube mass spectrometry (SIFT-MS) in obese children and (b) the ability to identify volatile organic compounds (VOCs) that correlate with the presence of NAFLD in these children.
Study participants and clinical data
The study protocol was approved by the Cleveland Clinic Institutional Review Board.
Overweight and obese children between the ages of 6 and 18 years old were recruited from the Pediatric Preventive Cardiology and Metabolic Clinic at the Cleveland Clinic.
Clinical variables were recorded, which included standard procedures for height, weight, BMI, and waist circumference (evaluated at the highest point of the iliac crest with a standing participant). The MetS in this cohort was defined as having three or more of the following five criteria 8: (a) abdominal obesity, defined as waist circumference of at least 90th percentile for age and sex; (b) low HDL-cholesterol, defined as concentrations less than 40 mg/dl; (c) hypertriglyceridemia, defined as triglyceride level greater than 110 mg/dl; (d) hypertension, defined as systolic or diastolic blood pressure of greater than 90th percentile; and (e) impaired fasting glucose (≥110 mg/dl) or known type 2 diabetes mellitus. A conventional hepatic US was performed by an experienced radiologist blinded to clinical and experimental data to evaluate for the presence of NAFLD.
Exhaled breath collection
All exhaled breath samples were collected after an 8-h fast. Study participants completed a mouth rinse with water before the collection of the breath sample to reduce the contamination from VOCs produced in the mouth. Participants were prompted to exhale normally to release residual air from the lungs and then inhale to total lung capacity through a disposable mouth filter (Fig. 2). The inhaled ambient air was also filtered through an attached N7500-2 acid gas cartridge (North Safety Products, Smithfield, Rhode Island, USA). The filters were used to prevent exposure of the participant to viral and bacterial agents and to eliminate exogenous VOCs from the inhaled air. The participants then proceeded to exhale at a rate of 50 ml/s through the mouth filter until the lungs were emptied. The exhaled breath sample was collected into an attached Mylar bag (Convertidora Industrial, Jalisco, Mexico), capped, and analyzed within 4 h. Mylar bags were cleaned by flushing with nitrogen between participants.
Selected ion flow tube mass spectrometry
The exhaled breath samples underwent gas analysis using SIFT-MS on a VOICE200 SIFT-MS instrument (Syft Technologies Ltd, Christchurch, New Zealand). The SIFT-MS technology and instrument used in this study have previously been described elsewhere by our group and others 9–11.
Mass scans of the product ions generated in the chemical ionization mass spectrum from each reagent ion (H3O+, NO+, and O2+) were obtained in the mass scanning (MS) mode. MS between 14–200 amu was used to identify significant peaks at product ion masses representing unknown breath volatiles relating to liver cirrhosis. More accurate concentration data was obtained by selected ion monitoring of product ions of 14 preselected compounds: 2-propanol, acetaldehyde, acetone, acrylonitrile, ammonia, benzene, carbon disulfide, dimethyl sulfide, ethanol, hydrogen sulfide, isoprene, pentane, triethylamine, and trimethylamine. These compounds have been previously identified as common constituents of exhaled breath in patients with and without liver cirrhosis 10. The 14 preselected compounds for selected ion monitoring analysis were monitored using the product ions listed in Table 1.
Data are presented as mean±SD, median (25th, 75th percentiles), or N (%).
Univariable analysis was carried out to compare patients with fatty liver versus no fatty liver. Student’s t-tests or the nonparametric Wilcoxon’s rank-sum tests were used to compare continuous variables and Pearson’s χ2 tests were used for categorical factors. In addition, analysis of covariance was used to assess differences while adjusting for possible confounders; the logarithm of each VOC was modeled as the outcome variables. Classification was performed using canonical discriminant analysis of the MS peaks. Following processing of the MS peaks, forward stepwise variable selection was applied and four masses (variables) were selected. Receiver operating characteristics analysis was used to study the role of VOCs in diagnosis of NAFLD and the area under the receiver operating characteristic curve (AUC) was reported. A P value less than 0.05 was considered statistically significant. SAS (version 9.2; The SAS Institute, Cary, North Carolina, USA), JMP (Pro version 9.0; The SAS Institute), and R (version 2.15.1; The R Foundation for Statistical Computing, Vienna, Austria) were used for all analyses.
Sixty patients were included in the study (37 with NAFLD on US and 23 with normal liver). All children were overweight or obese with a BMI of at least 85% for age. The mean age was 14.1±2.8 years, 50% were female, and 60% were White. The youngest patient to complete breath testing successfully was 7 years old. Patients with NAFLD were more likely to be White than controls (70.3 vs. 43.5%) and less likely to be African American (8.1 vs. 47.8%), P=0.003. AST and ALT were significantly higher in patients with NAFLD compared with obese controls, whereas alkaline phosphatase was significantly lower in the NAFLD group (P<0.05 for all) (Table 1). Of note, MetS was more prevalent in those with NAFLD (75 vs. 52.2%); however, this did not reach statistical significance (P=0.071).
Predicting the presence of nonalcoholic fatty liver disease using unique metabolomic breathprint
A comparison of the SIFT-MS results of patients with NAFLD with those with normal liver on US revealed differences in concentration of more than 15 compounds. Stepwise variable selection was performed using the MS ion peak data. Four ion peaks were used to classify patients of a 42-patient training set into those with NAFLD and those without fatty liver, with five patients being misclassified: 22 patients were diagnosed with NAFLD and 20 patients as being without (−2 log likelihood 32.65; Wilks’ λ 0.518 (P<0.001); Fig. 1a). The use of these four ion peaks gave an excellent accuracy for predicting the presence of NAFLD with an AUC of 0.913 (Fig. 1b). The discriminant analysis model was then successfully tested in a 35-patient validation set that included 15 new patients with NAFLD and the same 20 patients without NAFLD in the training set with nine patients that were misclassified and an AUC of 0.763 as shown in Fig. 2.
Volatile organic compound changes in children with nonalcoholic fatty liver disease
After adjusting for race, the presence of MetS and ALT, the concentrations of breath isoprene, acetone, trimethylamine, acetaldehyde, and pentane were significantly higher in the NAFLD group compared with normal liver group (14.7 ppb vs. 8.9 for isoprene; 71.7 vs. 36.9 for acetone; 5.0 vs. 3.2 for trimethylamine; 35.1 vs. 26.0 for acetaldehyde; and 13.3 vs. 8.8 for pentane, P<0.05 for all) (Table 2).
The main findings of this study include the following: (a) breath testing is feasible in overweight and obese children as young as 7 years of age, (b) concentrations of different VOCs were increased in NAFLD patients compared with those with normal livers on US, (c) breath testing is a promising noninvasive diagnostic tool to screen for NAFLD in overweight and obese children.
Pathological conditions such as obesity and NAFLD can lead to the production of new VOCs or a change in the ratio of VOCs that are produced normally 12. Little work has been done in children to assess the usefulness of these VOCs as biomarkers of disease states. SIFT-MS emerged recently as a new technology for detection of breath gases in humans with several important benefits over other types of mass spectrometers. It has the advantage of performing measurements of complex mixtures regardless of the water vapor content in real time 13. Another great advantage of SIFT-MS is its ability to detect and quantify compounds down to very low concentrations for many VOCs. Concentrations as low as parts per trillion (ppt) are common for several VOCs. In addition to this, the use of three precursor ions (H3O+, NO+, O2+) that react with the breath sample allows for isomeric compounds to be distinguished from each other on the basis of their unique reaction products with these precursors.
Isoprene, acetone, trimethylamine, acetaldehyde, and pentane represent novel biomarkers for NAFLD with plausible mechanistic links to disease pathogenesis. Isoprene is a by-product of cholesterol biosynthesis, which may be upregulated in patients with NAFLD 14. Recent studies in adult patients have demonstrated that cholesterol metabolism in NAFLD is characterized by increased synthesis and decreased absorption 15. Further, data suggest that the intestinal microbiota may generate isoprene 16. Acetone is an abundant VOC in human breath that is linked to carbohydrate metabolism and lipolysis. It is synthesized by liver cells by the decarboxylation of excess acetyl-CoA, which is produced from fatty acid β-oxidation 17,18. Dietary phosphatidylcholine is degraded by the intestinal microflora to form a volatile compound, trimethylamine. Trimethylamine is metabolized by the hepatic flavin monooxygenase family of enzymes into trimethylamine N-oxide 19. Acetaldehyde is an intermediate breakdown product of ethanol metabolism in the liver 20. The endogenous production of ethanol by the microbiota may contribute to the pathogenesis of NAFLD 21.
Interestingly, the presence of pentane in exhaled breath is considered a result of lipid peroxidation of polyunsaturated fatty acids in cellular membranes; a process mediated by free radicals and oxidative stress 17, which plays a significant role in NAFLD development.
Our study has several limitations including the fact that our obese children were seen at a large referral tertiary care medical center and the findings may not be generalizable to overweight pediatric patients in the community. The diagnosis of NAFLD was based on the presence of fatty infiltration on hepatic US and not the gold standard of liver biopsy. However, the utility of hepatic US for detection and quantification of hepatic steatosis in children with NAFLD has been established recently 5. We were not able to control factors that may influence VOC concentrations such as diet. Most importantly, this study was cross-sectional and the VOCs were determined at a single-time point.
Our data provide evidence of significant changes of VOCs in the breath of obese children with NAFLD compared with those with normal livers. Breath testing may become a useful screening and diagnostic tool for this common condition. Further, elucidating different pathways responsible for the production of NAFLD-related VOCs may give insights into novel therapeutic approaches for this common disease.
This work was supported by the BRCP 08-049 Third Frontier Program grant from the Ohio Department of Development.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.
biomarker; breath testing; children; nonalcoholic fatty liver disease; volatile organic compounds