Elsevier

Journal of Hepatology

Volume 58, Issue 3, March 2013, Pages 543-548
Journal of Hepatology

Research Article
Non-alcoholic steatohepatitis: A non-invasive diagnosis by analysis of exhaled breath

https://doi.org/10.1016/j.jhep.2012.10.030Get rights and content

Background & Aims

Histological evaluation of a liver biopsy is the current gold standard to diagnose non-alcoholic steatohepatitis (NASH), but the procedure to obtain biopsies is associated with morbidity and high costs. Hence, only subjects at high risk are biopsied, leading to underestimation of NASH prevalence, and undertreatment. Since analysis of volatile organic compounds in breath has been shown to accurately identify subjects with other chronic inflammatory diseases, we investigated its potential as a non-invasive tool to diagnose NASH.

Methods

Wedge-shaped liver biopsies from 65 subjects (BMI 24.8–64.3 kg/m2) were obtained during surgery and histologically evaluated. The profile of volatile organic compounds in pre-operative breath samples was analyzed by gas chromatography–mass spectrometry and related to liver histology scores and plasma parameters of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Results

Three exhaled compounds were sufficient to distinguish subjects with (n = 39) and without NASH (n = 26), with an area under the ROC curve of 0.77. The negative and positive predictive values were 82% and 81%. In contrast, elevated ALT levels or increased AST/ALT ratios both showed negative predictive values of 43%, and positive predictive values of 88% and 70%, respectively. The breath test reduced the hypothetical percentage of undiagnosed NASH patients from 67–79% to 10%, and of misdiagnosed subjects from 49–51% to 18%.

Conclusions

Analysis of volatile organic compounds in exhaled air is a promising method to indicate NASH presence and absence. In comparison to plasma transaminase levels, the breath test significantly reduced the percentage of missed NASH patients and the number of unnecessarily biopsied subjects.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide, affecting one in three adults, and one in ten adolescents in the USA [1], [2]. NAFLD is present in the majority of patients with metabolic risk factors such as obesity and type 2 diabetes mellitus (T2DM) [1]. While steatosis, the early stage of NAFLD, is considered to be benign and reversible, progression towards more advanced stages often occurs. These advanced stages, referred to as non-alcoholic steatohepatitis (NASH), are characterized by inflammation [3], [4]. Importantly, NASH is in turn associated with the development of hepatic fibrosis, cirrhosis, hepatocellular carcinoma, and an increased risk of liver failure and liver-related mortality [3], [4]. It is therefore clinically relevant to differentiate between patients with sole hepatic steatosis and those suffering from NASH, at an early stage.

Currently, a liver biopsy remains necessary to accurately diagnose NASH and to assess its severity [5], [6]. However, the procedure to obtain a liver biopsy is invasive and associated with considerable discomfort, costs, and morbidity; significant complications are encountered in 0.5% of cases [7], [8]. In order to optimize the risk-benefit ratio, it is advocated to obtain a needle biopsy from all obese patients with clinical risk factors, and a per-operative biopsy from all morbidly obese patients undergoing abdominal surgery [9]. Apart from obesity, acknowledged risk factors are elevated plasma levels of alanine aminotransferase (ALT), an elevated ratio of aspartate aminotransferase (AST) to ALT (AST/ALT ratio), insulin resistance, hypertension, sleep apnea, and increased plasma levels of triglycerides [10]. However, performing liver biopsy procedures based upon these risk factors leads to a selection bias in clinical practice, especially since mainly the presence of obesity and plasma levels of aminotransferases are taken into account. On the one hand, these plasma levels are often maintained within the normal range despite advanced disease [11], resulting in an underestimation of NASH prevalence, as well as undertreatment. On the other hand, if the indication to obtain a liver biopsy is based upon elevated aminotransferase levels, a large proportion of biopsies are obtained from subjects who do not suffer from NASH, since elevated AST and/or ALT levels are not specific for the presence of this liver disease.

In view of (1) the clinical relevance of NASH, (2) the difficulties of selecting the appropriate population to biopsy, and (3) the biopsy-related burden, a less invasive method to identify patients with NASH is urgently required. Such a method could be the analysis of volatile organic compounds (VOC) in exhaled breath. VOC are considered as markers of oxidative stress and can indicate the presence of reactive oxygen species that derive, for example, from peroxidation of polyunsaturated fatty acids [12]. Components in exhaled air have been previously shown to reflect the presence of inflammatory diseases affecting the airways [13], [14] and liver [15], [16], [17]. Hence, analysis of VOC in exhaled air may be useful for predicting NASH presence. In this pilot study, we found that subjects with NASH can be accurately distinguished from those without NASH based upon analysis of VOC in exhaled breath.

Section snippets

Study design

Sixty-five subjects were included consecutively between October 2007 and May 2011, before they underwent laparoscopic abdominal surgery; either cholecystectomy or primary bariatric surgery. Subjects ranged from overweight to severely obese with a BMI range of 24.8–64.3 kg/m2. The laparoscopic abdominal surgery was performed either at the Maastricht University Medical Centre or the Atrium Medical Centre Parkstad by the same surgeon (JWG). Exclusion criteria were acute, recent, and chronic

Population characteristics

The body mass index (BMI) ranged from 24.8 to 64.3 kg/m2 (mean 43.7 kg/m2), population characteristics are summarized in Table 1. NASH was diagnosed in 39 subjects (60%). The average plasma ALT and AST levels were higher in subjects with NASH compared to subjects without NASH (Table 1). Importantly, parameters such as gender, age, BMI, and HbA1c did not differ significantly. The 39 NASH patients were scored according to the scoring systems of Brunt and Kleiner; most subjects suffered from

Discussion

In order to find a less invasive method to diagnose NASH, VOC in exhaled breath were examined in relation to the evaluation of wedge-shaped liver biopsies in a cohort of 65 overweight and obese subjects. The feasibility to predict the presence of NASH based upon three components in exhaled breath (n-tridecane, 3-methyl-butanonitrile, and 1-propanol) was found to be high compared to plasma and clinical parameters. Moreover, by means of these three exhaled breath components, almost all of the

Financial support

This research was supported by the Senter Novem Innovation Oriented Research Program on Genomics, grant IGE05012 and a Transnational University Limburg (TUL) grant.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

The authors thank Yanti Slaats for her contribution to the sample collection at the start of this study and Jelena Arsenijevic for her assistance in the statistical analyses.

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