Randomized Clinical Trial
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Jun 28, 2015; 7(12): 1701-1707
Published online Jun 28, 2015. doi: 10.4254/wjh.v7.i12.1701
1H nuclear magnetic resonance spectroscopy-based metabonomic study in patients with cirrhosis and hepatic encephalopathy
Konstantinos John Dabos, John Andrew Parkinson, Ian Howard Sadler, John Nicholas Plevris, Peter Clive Hayes
Konstantinos John Dabos, John Nicholas Plevris, Peter Clive Hayes, Centre of Liver and Digestive Disorders, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, Scotland, United Kingdom
John Andrew Parkinson, Department of Chemistry, University of Strathclyde, Glasgow G1 1XW, United Kingdom
Ian Howard Sadler, Department of Chemistry, University of Edinburgh, Edinburgh EH16 4SA, Scotland, United Kingdom
Author contributions: Plevris JN and Hayes PC conceived the study; Dabos KJ and Plevris JN designed the study; Dabos KJ and Parkinson JA performed the data collection; Sadler IH helped with the data collection; Dabos KJ wrote the manuscript; all authors critically reviewed the manuscript and approved it.
Ethics approval: The study was reviewed and approved by the Lothian Research Ethics Committee Institutional Review Board.
Clinical trial registration: As this trial recruited before 1999 it was not registered with a clinical trials registry.
Informed consent: All study participants or their next of kin provided written informed consent prior to study enrolment.
Conflict-of-interest: The authors do not disclose any conflicts of interest.
Data sharing: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Konstantinos John Dabos, MD, PhD, Centre of Liver and Digestive Disorders, Royal Infirmary of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SA, Scotland, United Kingdom. konstantinos.dabos@nhslothian.scot.nhs.uk
Telephone: +44-131-2421627
Received: March 12, 2015
Peer-review started: March 12, 2015
First decision: April 10, 2015
Revised: May 9, 2015
Accepted: June 4, 2015
Article in press: June 8, 2015
Published online: June 28, 2015
Processing time: 109 Days and 16.7 Hours
Abstract

AIM: To identify plasma metabolites used as biomarkers in order to distinguish cirrhotics from controls and encephalopathics.

METHODS: A clinical study involving stable cirrhotic patients with and without overt hepatic encephalopathy was designed. A control group of healthy volunteers was used. Plasma from those patients was analysed using 1H - nuclear magnetic resonance spectroscopy. We used the Carr Purcell Meiboom Gill sequence to process the sample spectra at ambient probe temperature. We used a gated secondary irradiation field for water signal suppression. Samples were calibrated and referenced using the sodium trimethyl silyl propionate peak at 0.00 ppm. For each sample 128 transients (FID’s) were acquired into 32 K complex data points over a spectral width of 6 KHz. 30 degree pulses were applied with an acquisition time of 4.0 s in order to achieve better resolution, followed by a recovery delay of 12 s, to allow for complete relaxation and recovery of the magnetisation. A metabolic profile was created for stable cirrhotic patients without signs of overt hepatic encephalopathy and encephalopathic patients as well as healthy controls. Stepwise discriminant analysis was then used and discriminant factors were created to differentiate between the three groups.

RESULTS: Eighteen stabled cirrhotic patients, eighteen patients with overt hepatic encephalopathy and seventeen healthy volunteers were recruited. Patients with cirrhosis had significantly impaired ketone body metabolism, urea synthesis and gluconeogenesis. This was demonstrated by higher concentrations of acetoacetate (0.23 ± 0.02 vs 0.05 ± 0.00, P < 0.01), and b-hydroxybutarate (0.58 ± 0.14 vs 0.08 ± 0.00, P < 0.01), lower concentrations of glutamine (0.44 ± 0.08 vs 0.63 ± 0.03, P < 0.05), histidine (0.16 ± 0.01 vs 0.36 ± 0.04, P < 0.01) and arginine (0.08 ± 0.01 vs 0.14 ± 0.02, P < 0.03) and higher concentrations of glutamate (1.36 ± 0.25 vs 0.58 ± 0.04, P < 0.01), lactate (1.53 ± 0.11 vs 0.42 ± 0.05, P < 0.01), pyruvate (0.11 ± 0.02 vs 0.03 ± 0.00, P < 0.01) threonine (0.39 ± 0.02 vs 0.08 ± 0.01, P < 0.01) and aspartate (0.37 ± 0.03 vs 0.03 ± 0.01). A five metabolite signature by stepwise discriminant analysis could separate between controls and cirrhotic patients with an accuracy of 98%. In patients with encephalopathy we observed further derangement of ketone body metabolism, impaired production of glycerol and myoinositol, reversal of Fischer’s ratio and impaired glutamine production as demonstrated by lower b-hydroxybutyrate (0.58 ± 0.14 vs 0.16 ± 0.02, P < 0.0002), higher acetoacetate (0.23 ± 0.02 vs 0.41 ± 0.16, P < 0.05), leucine (0.33 ± 0.02 vs 0.49 ± 0.05, P < 0.005) and isoleucine (0.12 ± 0.02 vs 0.27 ± 0.02, P < 0.0004) and lower glutamine (0.44 ± 0.08 vs 0.36 ± 0.04, P < 0.013), glycerol (0.53 ± 0.03 vs 0.19 ± 0.02, P < 0.000) and myoinositol (0.36 ± 0.04 vs 0.18 ± 0.02, P < 0.010) concentrations. A four metabolite signature by stepwise discriminant analysis could separate between encephalopathic and cirrhotic patients with an accuracy of 87%.

CONCLUSION: Patients with cirrhosis and patients with hepatic encephalopathy exhibit distinct metabolic abnormalities and the use of metabonomics can select biomarkers for these diseases.

Keywords: Ketone bodies; Branch chain amino acids; Glutamine; Glycolysis

Core tip: Few studies have approached the metabolic abnormalities of liver cirrhosis and its complication hepatic encephalopathy. This study provides evidence that in stable cirrhosis key metabolic pathways are impaired and confirms the fact that there is impaired gluconeogensis, impaired ketogensis and ketone bodies break down as well as impaired urea cycle. In encephalopathy there is a reversal in the pattern of branch chain amino acids concentrations towards normal. By using stepwise discriminating analysis we were able to separate with remarkable accuracy metabolic phenotypes of cirrhotic patients from controls and also those who suffered from encephalopathy from those cirrhotics who did not.