Each patient enrolled in the study, underwent a morning fasting blood sample utilising red-stoppered Vacutainers (for the determination of serum protein electrophoresis, creatinine, main parameters of calcium-phosphorus metabolism and markers of skeletal turnover) and one purple-stoppered Vacutainer for haemachrome measurement. Routine blood tests were carried out the same day, the remainder of the blood samples were immediately centrifuged, separated in aliquots and frozen at −80°C until assayed. The assays were completed within 3 months from the time the blood samples were taken. Haematological parameters were determined using the haematology analyser from Bayer (ADVIA 120; Diamond Diagnostics, Holliston, MA, USA). Serum protein electrophoresis and immunofixation were evaluated by the Hydrasys system (Sebia, Italy). Serum levels of total calcium (Ca), phosphorus (P), magnesium (Mg) and creatinine (Cr) were measured by means of a multi-channel analyser (Technicon Autoanalyzer RA 500, Tarrytown, NY, USA). Ionised calcium was measured, immediately after sampling, by use of an ion-specific electrode (Nova 8, ion selective electrode; Nova Biochemical, Walthman, MA, USA), as previously described (Minisola et al, 1993). Creatinine clearance (CrCl) was estimated by the formula (Cockcroft & Gault, 1976), taking into consideration serum creatinine (μmol/l), age (years), body weight (kg) and gender; estimated creatinine clearance = (140 − age) × weight × K/serum creatinine; the constant K being equal to 1·04 for women. Serum levels of calcidiol [25(OH)D] were determined by radio-immunoassay (DiaSorin Inc., Stillwater, MN, USA) as previously described (Hollis et al, 1993), which had intra- and inter-assay coefficients of variation of 8·1 and 10·2% respectively. Circulating parathyroid hormone (PTH) levels were determined by an immunoradiometric assay, which measured serum hormone levels by using two affinity-purified polyclonal antibodies, one specific for the aminoterminal 1–34 portion of the PTH molecule and the second specific for the 39–84 sequence of the hormone (N-tact PTHSP, DiaSorin Inc., Stillwater, MN, USA). Intra- and inter-assay coefficients of variation, in our laboratory, were less than 3·0 and 5·5% (Pepe et al, 2005). Serum levels of C-terminal telopeptide of collagen type I (βCTX) were measured by enzyme-linked immunosorbent assay (ELISA) (Serum CrossLaps ELISA, Nordic Bioscience Diagnostics A/S Herlev, Denmark) (Rosenquist et al, 1998). Intra- and inter-assay coefficients of variation, in our laboratory, were less than 5·1 and 5·4% respectively. Bone isoenzyme of alkaline phosphatase (BALP) was determined with an immunoenzymatic assay (Metra BAP EIA Kit, Quidel Corporation, San Diego, CA, USA) (Gomez et al, 1995); intra- and inter-assay coefficients of variation were less than 5·6 and 7·8% respectively. Serum osteocalcin (BGP) was determined with an immunoradiometric assay (BGP; N-tact Osteo SP, DiaSorin, Stillwater, MN, USA) (Minisola et al, 1999); intra- and inter-assay coefficients of variation were less than 4·5 and 9·5% respectively. Bone sialoprotein (BSP) was determined using a radioimmunoassay (Immunodiagnostik AG, Bensheim, Germany) described elsewhere (Karmatschek et al, 1997). Intra- and inter-assay coefficients of variation were less than 6% and 9% respectively. Serum concentration of OPG and of the soluble receptor activator of nuclear factor kappa B ligand (RANKL) were both measured by ELISA (Biomedica, Vienna, Austria); the ratio of these two parameters was obtained for all patients. Intra- and inter-assay coefficients of variation, in our laboratory, were less than 10 for OPG and 5% and 9% respectively for RANKL. Each patient and control subject underwent standardised lateral radiographs of the thoracic and lumbar spine, centred at T8 and L3, respectively, at a film focus distance of 105 cm. After visual inspection of these radiographs by two independent experienced observers, vertebral deformity was defined when anterior, middle or posterior height loss was more than 20% with respect to the adjacent vertebra, according to Genant's method (Genant et al, 1993). Bone mineral density (BMD) of the lumbar spine (L1–L4) in the anterior–posterior projection and of the hip (femoral neck and total hip) was measured in each patient with dual energy X-ray absorptiometry (Hologic QDR 4500, Hologic Inc., Waltham, MA, USA). The precision error of lumbar spine and total hip measurement was 1·3 and 1·7% respectively.