Análisis a partir de las técnicas de insumo producto

All biochemical analysis was conducted first hand, and in-house. All samples were thawed at room temperature, and placed on a vortex before analysis. All reagents were prepared to manufacturer’s specifications. All assays composed of standards and quality controls (QC) in duplicate, with samples in singlet. For calculation of concentrations, a standard curve was constructed and results from QCs and samples were calculated from the curve using a computer software package (Multicalc®; PerkinElmer, Wallac OY; Finland). The standard curve was calculated by plotting the mean absorbance for each standard on the linear y-axis

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against the concentrations on a logarithmic x-axis, a line of best fit was applied to the curve, and sample concentrations determined from the mean absorbance for the standard curve: whereby percentage absorbance = (B – blank OD)/(Bo – blank OD), B = OD of sample or

standard, and Bo = OD of zero standard (total binding). Concentrations were presented as per

standard convention. Optical density was determined using a plate illuminometer (Microplate illuminometer LB 96P, EG&G Berthold, Germany). The coefficient of variance for all assays conducted was < 10%, as determined from standards on each plate. A summary of the assays used to determine hormones, metabolites and cytokines measured across studies is provided in Appendix I. Concentrations derived as a molarity (g.mol-1) then converted to mass (pg.ml-1) by multiplication of the molecular mass of the peptide by the peptide molarity; the reverse was performed for concentrations derived in molecular mass and typically presented as a molarity (Appendix J).

2.5.1 Insulin

The Invitron insulin assay (Invitron Ltd, Monmouth, UK) was used to analysis serum insulin. The insulin assay is a two-site immunoassay, which employs a solid base phase insulin antibody immobilised on microtitre wells, and a soluble insulin antibody labelled with a chemiluminescent acridinium ester. Labelled insulin antibody is added to each well followed by either a standard, QC or sample. Following an incubation period and a wash, optical density was determined. Concentrations were established in pmol.l-1.

The Invitron assay is 100% cross-reactive with insulin Lispro, Aspart, and Glargine, and 300% cross reactive with Determir (Pennartz et al. 2011). Therefore, all patients treated with insulin Determir were excluded from the analysis of serum insulin. In addition, the insulin assay is also 100% cross reactive with human insulin, however, all patients in this series of studies had long-standing type 1 diabetes and were solely dependent upon exogenous insulin administration, thus, the influence of any residual β-cell function was considered negligible (Wang et al. 2012). When basal dose was maintained (chapters 3 and 4), changes in insulin

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concentrations detected by this assay were considered to be due to the appearance / disappearance of rapid-acting insulin analogues. Conversely, where bolus administration was matched in trials (chapter 5), changes were considered to be due to the appearance / disappearance of insulin Glargine. The coefficient of variance for all insulin assays conducted was 7.4 ± 1.1%, as determined from standards on each plate.

2.5.2 Glucagon

Plasma glucagon was analysed using a competitive enzyme immunoassay (Glucagon EIA RAB0202; Sigma Aldrich, MD, USA). The microplate kit is pre-coated with anti-rabbit secondary antibody. After a blocking step and incubation of the plate with anti-glucagon antibody, both biotinylated glucagon peptide and peptide standard or targeted peptide in samples interacts competitively with the glucagon antibody. Uncompleted, or bound, biotinylated glucagon peptide then interacts with streptavidin-horseradish peroxidase (SA- HRP), which catalyses a colour development reaction. The intensity of colorimetric signal is directly proportional to the amount of biotinylated peptide-SA-HRP complex and inversely proportional to the amount of glucagon peptide in the standard or samples. This is due to the competitive binding to glucagon antibody between biotinylated glucagon peptide and peptides in standard or samples. The coefficient of variance for all glucagon assays conducted was 6.9 ± 2.1%, as determined from standards on each plate.

2.5.3 Catecholamines (Adrenaline and Noradrenaline)

Plasma adrenaline and noradrenaline were analysed using the CatCombi enzyme-linked immunosorbent assay (ELISA) kit (IBL, Europe Ltd). The test consists of 2 stages, an extraction phase and an assay phase. The extraction phase consists of an extraction plate to which standards, QC or samples are added. Deionised water is added to all wells to correct for differences in volume. The extraction plate then undergoes a number of incubations and washes, in which an acetylation reaction occurs producing an “extracted sample”. The samples, standards ad QCs are then ready for assay. Adrenaline and noradrenaline anti-serum

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is added before a further incubation. Optical density was immediately determined. Concentrations were derived as a mass (pg.ml-1) then converted to molarity (nmol-1) by division of the molecular mass of the peptide by the peptide molarity (Appendix J). The coefficient of variance for all insulin assays conducted was 9.1 ± 0.8%, as determined from standards on each plate.

2.5.4 Cortisol

Serum cortisol concentrations were determined using the Parameter™ Cortisol Assay (R&D Systems, Minneapolis, USA). For this assay, serum samples require a 20-fold dilution; therefore, 20μl of the serum sample was mixed with 380μl of calibrator diluent. The assay uses a cortisol conjugate (horseradish peroxidase with red dye and preservatives). To which a primary antibody solution (mouse monoclonal antibody to cortisol in buffer with blue dye with preservatives) is added followed by a series of incubation periods and washes before two colour regents (stabilised hydrogen peroxide and stabilised chromogen (tetramethylbenzidine)) are added. Following a further incubation period optical density is determined. The concentrations read from the standard curve were multiplied by the dilution factor to account for dilution. Concentrations were derived as a mass (pg.ml-1) then converted to molarity (nmol.l-1) by division of the molecular mass of the peptide by the peptide molarity (Appendix J). The coefficient of variance for all insulin assays conducted was 9.0 ± 0.7%, as determined from standards on each plate.

2.5.5 Non-esterified fatty acids (NEFA)

The enzymatic non-esterified fatty acids (NEFA) assay (NEFA, Randox Laboratories, UK) was used in accordance with the Randox Daytona Plus (Randox Daytona Plus, Randox Laboratories, UK) for the determination of NEFA concentrations. The assay uses direct photometry to measure a coloured endpoint from the following reaction:

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