A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis

Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz 1H Nuclear Magnetic Resonance spectroscopy and Ultra-Performance Liquid Chromatography Mass Spectrometry. A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.

Two extraction blanks samples were also prepared for each group and run parallel with other tissue samples in order to assess if there was any metabolite carryover from previous samples and semi-quantify the presence of impurities and contaminants relevant to the extraction procedure. Samples were loaded onto a bead beater (Precellys 24, Bertin Technologies) and a homogenization cycle, consisting of 40 s shaking at 6500 Hz followed by 5 min cooling on dry ice, was repeated 4 times to maximize dissolution of the powder.
Samples were centrifuged (Eppendorf 5417R) at 17949 x g for 20 min at 4 °C and the supernatant was taken into an Eppendorf tube. Content from each beading tube was then transferred into 5 glass vials with PTFE seal (Fisher, UK) each containing 200 µl. Glass vials containing organic metabolites were dried overnight in a fume hood at room temperature and then stored in -40°C freezer. For aqueous extraction, 1.5 mls of water/ methanol (1:1) was added in each 2ml microtube containing the sample which was then run on bead beater for 2 cycles at 6500 Hz each lasting 40 s. This was followed by Centrifuged (Eppendorf 5417R) at 17949 x g for 20 min at 4 °C. A total of 1.25 mL of supernatant was obtained from each sample and further divided into 5 x 250 µL aliquots. Samples were dried in a speed vacuum for 10 hours at 30 °C and stored at -40 °C pending NMR and UPLC-MS analysis.

Preparation of samples for 1 H-NMR spectroscopic analysis of aqueous extracts
Two aliquots of the dried aqueous extracts were combined after sequential reconstitution in 650 µL sodium phosphate buffer solution (0.2 M, 0.05% of sodium 3-trimethylsilyl-1-[2,2,3,3,-2 H 4 ] propionate (TSP), 70% D 2 O, pH 7.4). The aliquots were vortexed for 1 min, sonicated for 5 min and vortexed for additional 1 min, followed by centrifugation for 30 s at 17945 x g at 4 0 C. The supernatant was transferred to the second aliquot and the reconstitution procedure was repeated. The supernatant (500 uL) was then transferred into an NMR tube with an outer diameter of 5 mm.

Preparation of samples for UPLC-MS analysis of aqueous and organic extracts
Dried aqueous extracts were reconstituted in 200 µL of acetonitrile/water (95:5) for UPLC-MS hydrophilic interaction liquid chromatography (HILIC) analysis. Samples were vortexed for 1 min, sonicated for 5 min, vortexed again for 1 min and then centrifuged at 17949 x g at 4 °C for 8 min. Contents were then transferred into LC-MS grade glass total recovery vials (Waters, USA). A total of 50 µl from each sample was added together to make a quality control (QC) sample.
Samples were vortexed for 1 min, sonicated for 5 min, vortexed again for 1 min and then centrifuged at 17949 x g at 4 °C for 8 min. Following centrifugation at 17949 x g at 4 °C for 8 min, contents were transferred into LC-MS grade glass vials with inserts (Waters, USA). 200 µl of each sample was put inside the 1.8mm LCMS glass vials and the remaining 50 µl of each sample was added together to form the quality control (QC) sample.  Supplementary Table 4.

miRNA analysis
Samples were snap frozen and powdered, and subsequently 1ml of TRIzol was added per 100mg of tissue and left to solubulize at room temperature. 0.2ml of chloroform was added per 100mg of tissue and vortexed for 15 seconds to enhance phase separation. Samples were then centrifuged at 10000G for 10 minutes and the supernatant collected and 0.5 ml of isopropanol added to precipitate the RNA. The samples were centrifuged again at 10000G for 10minutes and the supernatant discarded. The pellet was re-suspended in (1x) sodium dodecyl sulphate buffer and NaOAc was added to 3 M. The RNA samples, which were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). Once RNA quality was assured, 100ng of each of the samples was labelled and hybridised by incubating for 20 hours at 55 o C to a Agilent Human microRNA Microarrays 8x60K v16 (Agilent Technologies), after which the microarrays were washed once with the Agilent Gene Expression Wash Buffer for 5 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 5 min. The fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System which determines feature intensities and allows for the possibility to compare two single intensity profiles in a ratio experiment.