A cohort of 50 Caucasian adolescent and young women with AN, referred to the outpatient Clinic for Osteoporosis of the Department of Internal Medicine at the University Hospital of Siena (Italy) for an evaluation of BMD, were enrolled in the study. Inclusion criteria were: age > 18 years, body mass index (BMI) <18 kg/ m2 and a diagnosis of restrictive AN as defined by the DSM-5 . The control group included 30 normal-weight healthy adolescent and young women with 18<BMI<25 kg/m2. All the controls presented no lifetime history of eating disorders and normal menstrual cycles. All the subjects previously treated with antiosteoporosis drugs, except calcium and vitamin D supplements, and those who were suffering illness (cancer, multiple myeloma, hyperparathyroidism etc.) or were receiving therapies able to influence bone metabolism were excluded. Height and weight were measured in a standardized fashion and BMI was calculated as weight in kilograms divided by the square of height in meters.
In all subjects, fasting venous blood samples were drawn in order to assess serum levels of 25-hydroxyvitamin D (25OHD), parathyroid hormone (PTH), serum calcium, serum phosphate and creatinine. Serum 25OHD was determined by a chemiluminescence immunoassay (LIAISON 25OHD Total Assay, DiaSorin Inc, Stillwater, MN, USA). In our institution, the intra- and inter-assay coefficients of variation were 6.8% and 9.2%, respectively. Serum PTH was assessed by a immunoradiometric assay (Total Intact PTH, Antibodies Lab. Inc.; Santee, CA,USA) and the intra- and inter-assay coefficients of variation were 3.6% and 4.9%, respectively.
Bone Mineral Density Measurement
In all women we measured BMD at the lumbar spine (LS-BMD), at femoral neck (FN-BMD) and total hip (TH- BMD) using a dual-energy X-ray absorptiometry (Discovery W, Hologic, Waltham, MA, USA). All DXA scans were performed according to the standard clinical routine procedures. Osteoporosis and osteopenia were diagnosed according to the World Health Organization (WHO) and ISCD definition ; sex-matched Italian reference data were used for the calculation of T score and Z -score. REMS scans were performed at both femoral sites and lumbar spine by employing a dedicated echographic device (EchoStation, Echolight Spa, Lecce, Italy), equipped with a convex transducer operating at the nominal frequency of 3.5 MHz and used as recommended by the manufacturer. In a REMS investigation, the probe is placed on the abdomen or on the hip in order to visualize of the target bone interface and the operator has to set the appropriate values of scan depth and transducer focus. Subsequently, the software detects the sought bone interfaces in the sequence of acquired frames and identifies the regions of interest for the diagnostic evaluation. The selected measured data are finally synthesized in a patient specific spectrum of the considered bone target, which undergoes an advanced comparison with gender, age, site and BMI matched reference spectral models extracted from a dedicated database. Actually, the spectral modifications introduced by the physical properties of the bone structure that has backscattered the ultrasound signals are identified by the comparison procedure, resulting in a BMD estimation and in the consequent diagnostic classification as healthy, osteopenic or osteoporotic [16, 18]. In Figure 1 showed a schematic representation of the REMS acquisition on femoral neck. Data processing methodologies implemented in the REMS approach were detailed in previous papers [16, 18, 19].
Moreover, in AN subjects the presence of prior low major trauma fractures (hip, vertebrae, wrist, ankle, humerus) was ascertained by self-report and confirmed by an examination of clinical and radiological reports. Three anorexic women were excluded due to inadequate quality of BMD or REMS evaluation; therefore, the statistical analysis was carried out in 47 AN and in 30 healthy women.
An informed written consent was obtained from all participants, and the study was approved by the Institutional Review Board of Siena University Hospital (ID-1234/14). All the data were anonymized before being used for the statistical analysis.
All values were expressed as mean ± SD. The Kolmogorov–Smirnov test was used to verify the normality of the distribution of the outcome variables. Clinical data and initial values of the variables measured in the study groups were compared using Student’s t test and Mann–Whitney U-test as appropriate. The degree of correlation between DXA and REMS values was quantified through Pearson's correlation coefficient. The agreement between DXA- and REMS-based BMD values was assessed through the Bland-Altman method, by measuring the residual standard deviation (RSD).
All tests were performed using the SPSS statistical package for Windows version 16.0 (SPSS Inc., Chicago).