Network premiere for fumarase-insufficient cells

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  • Published: Jan 16, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: Network premiere for fumarase-insufficient cells

Fumarase insufficiency requires further study

Fumarase is a vital enzyme in many living organisms, including humans, converting fumarate to malate. Fumarase deficiency is a rare genetic disorder associated with encephalopathy and serious mental retardation. However, there is also interest concerning the possible effects of a less severe decrease in the amount or effectiveness of fumarase (fumarase insufficiency), where enough of the enzyme is present for normal mental development. Previous work had shown that Dahl salt-sensitive rats (a strain of rat prone to hypertension when given high levels of salt) had a different amino sequence in their fumarase to salt-insensitive rats, resulting in their fumarase not functioning as well as normal. This observation raised the possibility that some cases of human hypertension might be linked to fumarase insufficiency.

The Xi’an researchers looked at metabolites from human umbilical vein endothelial cells (HUVECs) that had been modified using interfering RNA to give fumarase insufficiency. They used network methods to compare the metabolites from the modified cells (the ‘metabolome’) to those from HUVECs treated with ‘scrambled’ RNA (negative control).

GC-MS and network methods used to compare metabolites

Intracellular metabolites were isolated from cell pellets using a mixture of chloroform, methanol and water (2:5:2 v/v/v) with ultrasound. Deuterated myristic acid (d27) was added as an internal standard for a GC retention time lock. Centrifugation and lyophilisation gave a crude product that was then derivatised with methoxyamine hydrochloride and pyridine to convert carbonyl groups to oximes. Further derivatisation (silylation) was carried out with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and 1% trimethylchlorosilane (TMCS).

GC employed an Agilent 7890A instrument fitted with a J&W DB-5 column. The temperature was ramped from 60 to 325 °C. Mass spectrometry was carried out with an Agilent 5975C Inert MSD (mass selective detector) operating in full scan mode using electron ionisation (EI). Peak annotation was carried out using NIST’s AMDIS programme, while compound identification used the Agilent Fiehn GC-MS Metabolomics RTL library. The data, consisting of 62 varied compounds, were transferred into Agilent’s Mass Profiler Professional (MPP) software for further analysis.

The data were imported into MATLAB 2009A where differences between the two groups of metabolites were identified using a Mann–Whitney–Wilcoxon test, giving 24 metabolites with significantly different concentrations (eight amino acids were significantly lower in the fumarase-insufficiency group). Pathway analysis was carried out on those metabolites that significantly differed between the two groups using MetaboAnalyst 3.0 software.

A correlation network was then constructed based on Pearson correlation coefficients, with a large number of nodes and connections. Visualisation of the network employed R version 3.2.3 software from Revolution Analytics. Five metabolites, fumaric acid, glycine, aspartic acid, malic acid and glutamic acid, were given as ‘hub’ metabolites using the PageRank algorithm (i.e. they were particularly important in the network). These five compounds were also significantly different in the two groups of HUVAC cells. The results confirm that fumarase insufficiency can lead to irregularities in the amino acid profile.

It was also shown that a decrease in fumarase activity in the HUVACs led to an increase in the activity of two other enzymes, glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT). Such raised activity would be expected to reduce alanine and increase pyruvate, as was seen in practice in the metabolite results from the GC-MS.

Fumarase insufficiency linked to amino acid changes

The use of GC-MS and network analysis clearly showed that fumarase insufficiency can be linked to a changed amino acid profile in isolated cells. Much more work is needed to establish whether or not there is any link between fumarase insufficiency and cases of hypertension.

Related Links

Biomedical Chromatography, Early View paper. Hou et al. Combined metabolomic and correlation networks analyses reveal fumarase insufficiency altered amino acids metabolism.

Drug Discovery Today, 2014, 19, 171-182. Kell et al. Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery.

Wikipedia, Network Theory

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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