Liaison assay

Fasting calcium and phosphorus plasma levels were measured by colorimetric methods using a COBAS 8000 analyzer (Roche Diagnostic, Mannheim, Germany). 25(OH)D levels were calculated by a high-specific chemiluminiscence-immunassay (LIAISON Assay, Diasorin, Dietzenbach, Germany), and PTH levels were determined by a highly specific solid-phase, two-site chemiluminescent enzyme-labeled immunometric assay using an Immulite analyzer (DPC Biermann, Bad Nauheim, Germany).
The criteria for classification of vitamin D status of the United States Endocrine Society were used for categorization. Vitamin D deficiency was then defined when 25(OH)D levels were lower than 20 ng/ml (<50 nmol/L), Vitamin D insufficiency when 25(OH)D levels were between 20 and 29 ng/ml (50–75 nmol/L) and Vitamin D sufficiency when 25(OH)D levels reached or surpassed 30 ng/ml (>75 nmol/L)^{21 (link),22 (link)}. Secondary hyperparathyroidism was considered when PTH serum levels were higher than 65 pg/ml^{2 (link),7 (link)}.

Calcium, phosphorous and alkaline phosphatase plasma levels were measured during fasting by colorimetric methods using a COBAS 8000 analyzer (Roche Diagnostic, Mannheim, Germany). Intra- and interassay coefficients of variation were <5%.
Calcidiol was determined by a high-specific chemiluminiscence-immunassay (LIAISON Assay, Diasorin, Dietzenbach, Germany) with intra- and interassay coefficient of variation of 4.2–9.5% and 7.6–2.1%, respectively, and functional sensitivity of 4.0 ng ml⁻¹. PTH was determined by a highly specific solid-phase, two-site chemiluminescent enzyme-labeled immunometric assay using an Immulite analyzer (DPC Biermann, Bad Nauheim, Germany) with intra- and interassay coefficient of variation of 3.8–6.9% and 3.1–7.2%, respectively, and functional sensitivity of 5.0 pg ml⁻¹.^{18 (link)}Vitamin D deficiency was defined as calcidiol lower than 20 ng ml⁻¹ (<50 nmol l⁻¹). Vitamin D insufficiency is when calcidiol levels fluctuate between 20 and 29 ng ml⁻¹ (50–75 nmol l⁻¹) and vitamin D sufficiency is when calcidiol levels reach or overtake 30 ng ml⁻¹ (>75 nmol l⁻¹).^{19 (link), 20 (link)} Secondary hyperparathyroidism was defined when PTH serum levels exceed 65 pg ml⁻¹.^{12 (link), 21 (link)}

All hormonal determinations (leptin, TSH, FT4, IGF-1, IGFBP3), PL, insulin, FSH, LH, estradiol, ACTH, cortisol, 17-OH-P, DHE-S, androstenedione, T and FT were made after blood sample collection under basal fasting conditions (between 8:00 and 9:00 h after an overnight fast).
Hormonal measurements, when appropriate, were quantified by radio immuno-assay (Immuno Diagnostic System, Bolden, UK), enzyme-linked immunosorbent assay (Immulite analyzer, DPC Biermann, Bad Nauheim, Germany) or highly sensitive chemiluminescence immuno-assays (LIAISON Assay, Diasorin, Dietzenbach, Germany). Homeostasis model-assessment (HOMA-IR) indexes were calculated from fasting glucose and insulin concentrations (glucose levels in mmol × insulin in μUml/L/22.5).

Serum 25(OH) vitamin D levels, the exposure variable of interest, were assayed using Liaison assay (DiaSorin Inc, Stillwater, MN), a direct enzyme immunoassay method. This assay has a correlation of 0.9 with the gold standard Immuno Radio Assay (IRA), with intra-assay coefficient of variation <8% and an inter-assay coefficient of variation of <10%. 70% of vitamin D samples were analyzed in a lab that was associated with our clinic and the tests for reliability of the vitamin D assay is as mentioned above. The remaining 30% samples were assessed in various labs around Scranton and the measures of reliability for these labs could not be obtained.

Blood glucose, insulin, total cholesterol (TC), high (HDL) and low (LDL) density lipoprotein cholesterol, triglycerides (TG), and C-reactive protein (CRP) were measured after overnight fasting in all subjects. Values of TC, LDL, HDL, and TG were considered in the normal range if within the 5th and the 95th percentile [60 (link)]. Oral glucose (1.75 g/Kg) tolerance test (OGTT) was performed in obese subjects recording basal levels of blood glucose and insulin after 120 min.
Calcium, phosphorus and alkaline phosphatase (ALP) concentrations were measured by the nephelometric method. Serum active intact parathyroid hormone (PTH) and 25(OH) vitamin D were measured by immunological tests based on the principle of chemiluminescence using commercial kits (Liaison assay; DiaSorin, Stillwater, Minnesota, USA). Osteocalcin serum concentration was measured by enzyme immunoassay (IBL International GmbH, Hamburg, Germany). LIGHT levels were also assessed (R&D Systems, Minneapolis, MN, USA). Two methods to assess insulin resistance, the homeostasis model assessment (HOMA) and the quantitative insulin sensitivity check index (QUICKI) were evaluated.

Blood samples were collected in the morning BPP, BN, AN. Biochemical parameters considered in this study were: ALT, AST, creatinine, 25-hydroxy vitamin D (Liaison^® Assay, DiaSorin, Italy), total cholesterol, HDL, LDL, triglycerides concentrations.