Fetuin a

Plasma fetuin-A (R&D system, Minnesota, USA), proinsulin (Mercodia, Uppsala, Sweden), and IGF-II (R&D system, MI, USA) were measured by enzyme-linked immunosorbent assay (ELISA) kits, and the absorbance was determined using a microplate spectrophotometer (Beckman CX7, USA). Serum insulin and insulin-like growth factor 1 (IGF-I) concentrations were detected by automated chemiluminescent assays (ADVIA Centaur and Immulite 2000, SIEMENS, Germany). The minimal detectable concentrations were 0.62 ng/ml for fetuin-A, 3.5 pmol/L for insulin, 1.7 pmol/L for proinsulin, 25 ng/ml for IGF-I and 1.88 pg/ml for IGF-II, respectively. The intra-assay and inter-assay coefficients of variation were in the ranges of 0.5–8.3% for fetuin-A, 2.0–6.5% for insulin and IGF-I, 0.34–5.0% for proinsulin, and 2.4–9.3% for IGF-II, respectively. We measured not only insulin but also proinsulin because both are positively associated with birth weight (23 (link)).

Venous blood samples were collected from the antecubital vein between 8 and 10 a.m. after an overnight fast before surgery, as well as at one week, three and 12 months after surgery. Blood samples were immediately transferred to a tube containing aprotinin (500 units/mL) (Lee et al., 2011a (link)). After centrifugation at 300× g, plasma was separated, dispensed into polypropylene tubes in aliquots, and stored at −20 °C until analysis. Enzyme immuno-assays for plasma fetuin-A (R&D Systems, Minneapolis, MN, USA) and LECT-2 (Medical & Biological Laboratories CO., LTD., Nagoya, Japan), were carried out in a single batch run and in a blinded fashion, as described in our previous study (Lee et al., 2011a (link)).

One investigator (unblinded to treatment) measured weight and height (Harpenden Stadiometer) and scored hirsutism (Ferriman-Gallwey). Systolic and diastolic blood pressures were recorded after a 5-minute rest with the girl supine, using an electronic sphygmomanometer (767 series, Welch Allyn, Spain).
Endocrine-metabolic assessments were performed in the early morning, in the follicular phase (days 3–7) of the cycle or after 2 months of amenorrhea, as described [5 (link)]. Briefly, circulating insulin and SHBG were assayed by immunochemiluminescence (IMMULITE 2000, Diagnostic Products, Los Angeles, CA). HOMA-insulin resistance (HOMA-IR) was calculated as [fasting insulin in mU/L] × [fasting glucose in mg/dL]/405. Serum C-reactive protein (CRP) was analyzed by immunochemiluminescence (ARCHITECT i2000SR, Abbott Diagnostics, Abbot Park, IL, USA); intra- and interassay coefficients of variation (CVs) were <10%. HMW adiponectin was assessed by ELISA (R&D Systems, Minneapolis, MN, USA); intra- and interassay CVs were <9%. Circulating fetuin-A was assessed with a specific ELISA (fetuin-A, R&D systems, Minneapolis, MN, USA); the intra- and interassay CVs were 4.2% and 7.4%, respectively.

Serum levels of FGF23 (full length, KAINOS Lab Inc., Tokyo, Japan), RANKL (R&D, systems, Inc., Minneapolis, MN), OPG (R&D) CTx (Immunodiagnostic Systems Limited, Boldon, UK), urinary Fetuin A (R&D) and α-Klotho (Immuno-Biological Laboratories Co, Ltd, Gunma, Japan) were measured by using an ELISA kit based on the manufacturers’ instructions.

Plasma samples were immediately centrifuged at 3000× g for 15 min at 4 °C (U-32012 Centrifuge, Boeco^®, Hamburg, Germany) upon collection and stored at −80 °C. Plasma Fetuin-A and adiponectin concentrations were determined by using enzyme-linked immunosorbent assays (Fetuin-A, Human Fetuin-A Immunoassay, R&D Systems^®, Minneapolis, MN, USA; Adiponectin, Human Total Adiponectin/Acrp30 Immunoassay, R&D Systems^®, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. The manufactural dilution factor was used while each sample ran in duplicate. The average coefficients of variation for plasma intra-assay (inter-assay) precision were 4.3% (8.0%) and 3.5% (6.5%) for Fetuin-A and adiponectin, respectively. The FAR value was derived from fasting Fetuin-A (ng/mL) divided by adiponectin (ng/mL).