Análisis de diferentes escenarios económicos

4.2.1 Participants

Ethical clearance was granted from the Loughborough University Ethics Approvals (Human Participants) Sub-Committee (reference R16-P074) and all participants provided written informed consent. Adults (n=88, 18+ years) were recruited from the local community in the Loughborough Town area. Participants were free of blood borne viruses; there were no other exclusion criteria.

4.2.2 Study visit

Participants came to the National Centre for Sport and Exercise Medicine at Loughborough University for one visit. After obtaining written consent, anthropometric measurements of height and weight were recorded; two participants refused measurement of height and weight and therefore were not included in analysis requiring these variables. A finger prick capillary blood sample was collected into three 300μL EDTA-coated microvette tubes (Microvette® CB 300 µl, K2 EDTA, Sarstedt, Germany) for blood cell count and DNA extraction. Four dried blood spots (DBS) were collected for fatty acid analysis.

4.2.3 Fatty acid measurements

DBSs were collected on Whatman cards (903 Protein saver, Sigma-Aldrich) pre- treated with butylated hydroxytoluene (supplied by the Loughborough University Chemistry department) to stabilise the blood fatty acids for up to eight weeks at room temperature (Metherel et al., 2013). Each DBS was punched from the card, weighed and frozen at -80o_{C within eight weeks of collection. Gas chromatography mass}

spectrometry (GC-MS), carried out by Boakye Gyimah in the Loughborough University Chemistry department, was used to analyse the fatty acid composition of the DBS. Methyl esters were prepared using the method from Ichihara and Fukubayashi, (2010). Briefly, one of the 30mm2 _{punched samples was derivatised by acetyl chloride}

(99%, Sigma Aldrich) and methanol (HPLC grade, Fisher Scientific) at 70o_{C for 60}

minutes to produce fatty acid methyl esters (FAMEs) and reconstituted in 100µl hexane (HPLC grade, Fisher Scientific) containing 10µg/ml of internal standard

(methyl heptadecanoate, analytical standard 51633-1G, Sigma Aldrich). The sample was analysed by GC-MS, FAMEs were characterized by electron ionization in positive ion full scan mode. Fatty acid quantities were determined based on the relative abundance to the internal standard. Fatty acids are presented as their percentages of total fatty acids (%TFA.).

4.2.4 DNA extraction and bisulphite conversion

One aliquot of the capillary blood was used for a cell count (Beckman Coulter Counter, USA). Aliquots of capillary blood were frozen at -80o_{C until analysis. DNA was}

extracted from the capillary blood and bisulphite-converted using EpiTectTM _Fast

LyseAll Bisulphite conversion kit (Qiagen, Germany) using the manufacture’s protocol. Briefly, the blood sample was lysed, and proteins denatured, the resulting pellet was re-suspended in PBS and added directly into the bisulphite reaction. The DNA was sodium bisulphite-treated and subjected to two cycles of denaturing at 95o_{C for 5}

minutes and incubation 60o_{C for 20 minutes. Bisulphite-converted DNA was}

desulfonated and purified using MinElute spin columns provided. Successful bisulphite conversion was assessed using a bisulphite-converted DNA-specific dispensation during the pyrosequencing, as displayed on a pyrogram in Figure 4-1 highlighted in orange.

Figure 4-1 Example pyrogram for TNF exon 1 assay covering Cytosine-Guanine dinucleotides (CpG) sites +197, +202, +214 and +222 from the transcription start site, highlighted grey. Dispensation 15, highlighted in orange, served as a bisulphite conversion control.

4.2.5 DNA methylation analysis

The methylation of four CpG sites within Exon 1 of the TNF were measured using bisulphite pyrosequencing (PyroMark® _{Q48 Autoprep System, Qiagen, Germany).}

Primers, designed in Chapter 3, are provided in Table 4-1. The extracted bisulphite- converted DNA was amplified using the PyroMark® _{PCR kit (Qiagen, Germany) in a}

Veriti® _{thermocycler (Applied Biosystems Inc., USA). PCR product was used for CpG}

quantification with the PyroMark® _{Q48 Autoprep (Qiagen, Germany) using Advanced}

CpG Reagents (Qiagen, Germany) in accordance with the manufacturers protocol. The assay covered the methylation sites +197, +202, +214 and +222 bp from the transcription start site of TNF. The percentage methylation of the four CpG sites was calculated within the software (PyroMark® _{Q48 Autoprep 2.4.1 Software, Qiagen,}

Germany) and the methylation percentages were exported for further analysis.

Table 4-1: Pyrosequencing primers for TNF (MIM 191160) exon 1. The assay covers four methylation sites +197, +202, +214 and +222 base pairs from the transcription start site.

Pyrosequencing Primer Sequence Forward PCR

Primer 5’-GGAAAGGATATTATGAGTATTGAAAGTATG-3’

Reverse PCR Primer 5’-biotin-CTAAAACCCCCCTATCTTCTTAAA-3’ Sequencing Primer 5’-ATTATGAGTATTGAAAGTATGAT-3’

4.2.6 Statistical Analysis

All statistical analysis was performed using IBM SPSS statistics software (SPSS version 23). The data distribution was assessed for normality by Shapiro-Wilk's test (p > 0.05). Group differences between sex were assessed using an independent sample t-test for normally distributed data and a Man Whitney-U test where appropriate. Differences between sex for categorical data were assessed using chi-square. A key consideration in DNA methylation studies is the composition of the white blood cells (WBCs) from which the DNA is extracted. The DNA methylation values are therefore presented raw and adjusted for the cell heterogeneity of the capillary blood. The method presented by Jones et al., (2015) was used to adjust DNA methylation values to account for the white blood cell composition. In brief, the adjusted figure is a sum of the mean methylation for the site and the unstandardised residual from a linear regression between DNA methylation (dependent variable) and the individual white blood cell counts (independent variables).

Spearman’s correlation analysis was used to assess the relationship between the methylation (raw and adjusted) percentage of the four sites studied and the fatty acids identified within the blood. A p-value lower than 0.05 was considered significant. Using an expected b = 0.2 and r = 0.3, sample size required was 85.