The ADVIA 1650 analyzer (Bayer Diagnostics; Bershire, UK) was used to estimate serum creatinine, calcium, albumin, urine creatinine, and urine calcium levels. The Jaffe reaction and enzymatic reaction of Tanganelli was used to measure urine creatinine and serum creatinine respectively. The Doumas, Watson, and Biggs method using bromcresol green as the binding dye was used to estimate serum albumin. The reaction between calcium and o-cresolphthalein complex one was used to measure serum and urine calcium. Serum intact parathyroid hormone (iPTH) was estimated using a solid-phase, two-site, chemiluminescent enzyme-labeled immunometric assay in the Immulite 2000 analyzer (DPC; Scientific Supplies and Technology; Miami, FL). A double-antibody, sequential, 125I-labeled radioimmunoassay was used to estimate serum estradiol. An ADVIA Centaur immunoassay system (Bayer) was used to estimate serum prolactin, follicular-stimulating hormone (FSH) and leutinizing hormone (LH) levels. These assays were two-site “sandwich” immunoassays. Serum 25 hydroxyvitamin D was measured using DiaSorin 25 hydroxy vitamin D radioimmunoassay (DiaSorin; Stillwater, MN). Rate spectrophotometric method using reagents from Sigma Diagnostics (Columbia, MD) was used in the measurement of serum angiotensin-converting enzyme (ACE) levels. Serum 1,25 dihydroxyvitamin D was estimated by a competitive radioimmunoassay kit manufactured by DiaSorin. The vitamin D metabolites were extracted and purified using a C18 cartridge, followed by radioimmunoassay using a polyclonal antibody that is specific to both 1,25(OH)2D2 and 1,25(OH)2D3.