In order to determine the concentration of trace minerals, analyses were performed using inductively coupled plasma mass spectrometry (ICP-MS) following the method described by Maynar et al. [24 (link)].
The decomposition of the organic matrix was achieved by heating it for 10 h at 90 °C after adding 0.8 mL HNO3 and 0.4 mL H2O2 to 2 mL of plasma samples. The samples were then dried at 200 °C on a hot plate. Sample reconstitution was carried out by adding 0.5 mL of nitric acid, 10 μL of indium (In) (10 mg/L) as an internal standard, and ultrapure water to complete 10 mL. Digested solutions were analyzed with an ICP-MS Nexion model 300D (PerkinElmer, Inc., Shelton, CT, USA). Three replicates were analyzed per sample. The values of the standard materials of each element (10 μg/L) used for quality controls were in accordance with intra and inter-assay coefficient variations of less than 5%.
The hormone quantification was conducted using enzyme-linked immunosorbent assay (ELISA) with an ER-500 (Sinnowa, Germany), using the commercial tests for insulin, cortisol, testosterone, and luteinizing hormone from CisRadioquímica, SA (Madrid, Spain). Hormonal analyses were performed in duplicate by the same technician. Coefficients of variation (between and within) were less than 10% for all biochemical analyses.
The decomposition of the organic matrix was achieved by heating it for 10 h at 90 °C after adding 0.8 mL HNO3 and 0.4 mL H2O2 to 2 mL of plasma samples. The samples were then dried at 200 °C on a hot plate. Sample reconstitution was carried out by adding 0.5 mL of nitric acid, 10 μL of indium (In) (10 mg/L) as an internal standard, and ultrapure water to complete 10 mL. Digested solutions were analyzed with an ICP-MS Nexion model 300D (PerkinElmer, Inc., Shelton, CT, USA). Three replicates were analyzed per sample. The values of the standard materials of each element (10 μg/L) used for quality controls were in accordance with intra and inter-assay coefficient variations of less than 5%.
The hormone quantification was conducted using enzyme-linked immunosorbent assay (ELISA) with an ER-500 (Sinnowa, Germany), using the commercial tests for insulin, cortisol, testosterone, and luteinizing hormone from CisRadioquímica, SA (Madrid, Spain). Hormonal analyses were performed in duplicate by the same technician. Coefficients of variation (between and within) were less than 10% for all biochemical analyses.
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