NMR spectroscopy enables simultaneous quantification of carbohydrates for diagnosis of intestinal and gastric permeability

Sarah Stryeck,Angela Horvath,Vanessa Stadlbauer,Bettina Leber,Tobias Madl

doi:10.1038/s41598-018-33104-8

Abstract

Increased intestinal or gastric permeability is one of the major hallmarks of liver cirrhosis. The current gold standard for diagnosis of aberrant gut permeability due to disease is the triple-sugar test, where carbohydrates are orally administered and urinary excretion is measured. Hereby, elevated lactulose levels indicate intestinal permeability, whereas increased sucrose levels reveal gastric permeability. However, reliable detection and quantification of these sugars in a complex biological fluid still remains challenging due to interfering substances. Here we used Nuclear Magnetic Resonance (NMR) spectroscopy with a simple and fast protocol, without any additional sample extraction steps, for straight-forward simultaneous quantification of sugars in urine in order to detect increased intestinal and gastric permeability. Collected urine samples were diluted in buffer and one- and two-dimensional proton spectra were recorded in order to reveal carbohydrate concentrations in individual urine samples containing mannitol, sucrose and/or lactulose. Overall, this article presents a fast and robust method for simultaneous quantification of different sugars down to low micro-molar concentrations for research studies and can be further extended for clinical studies with automation of the quantification process.

Highlights

Liver cirrhosis is a major global health burden with a continuously rising incidence
Sample preparation steps are not required in Nuclear Magnetic Resonance (NMR) spectroscopy-based measurements; hereby urine samples can be measured directly
Most metabolites, including mannitol, sucrose and lactulose, comprise several chemically different protons leading to complex spectral patterns, signal overlap, and this complicates reliable identification and quantification of metabolites (Fig. 1A and B; for full 1H NMR spectra see Fig. S1A and B)

Summary

Introduction

Liver cirrhosis is a major global health burden with a continuously rising incidence. For the ‘triple-sugar test’ elevated lactulose or sucrose levels in urine are a common readout for increased intestinal or gastric permeability, respectively, due to a huge increase in paracellular transport of metabolites into the systemic circulation and subsequent urinary excretion. A pathologically increased intestinal permeability index (lactulose/mannitol ratio >0.07) would presume even higher concentrations of carbohydrates, which require only short measurement times of less than 10 minutes[3,4]. Our study presents a rapid, robust, and non-invasive NMR-based ‘triple-sugar test’ which enables simultaneous quantification of sugars in human urine using NMR spectroscopy in order to determine gastric and/or intestinal permeability in samples for research studies, and for larger sample sizes in clinical applications. In addition to sugar quantification, untargeted urinary metabolite profiles are obtained for free without the need for any additional measurement time

Methods

Results

Conclusion