2 BMD have been noted prior to return of menses [10] with an increase in suppressed bone formation and a fall in bone resorption. Osteocalcin and N-telopeptide (NTX), es- tablished biochemical markers of bone formation and resorption, respectively, have been shown to appropriately increase and decrease, respectively, with weight recovery [10] accompanied with a 3-4% increase in BMD in as little as 4 months. In this longitudinal study in which we examine women with AN before and after weight normalization and compare them with healthy female control subjects, we propose that a substantial amount of the unexplained increase in REE during refeeding of AN patients is channeled towards BMD recovery such that signiﬁcant bone rebuilding is required before energy stores become available for restora- tion of gonadal function. 2. Materials and Methods 2.1. Subjects. We studied 37 patients aged 23.4 ± 4.8 (range 18–36 years) with AN and 16 healthy control women aged 25.1 ± 4.7 (range 18–35 years). Patients were receiving inpa- tient treatment on the Eating Disorders Research Unit at the New York State Psychiatric Institute, Columbia University Medical Center (NYSPI, CUMC). All met criteria for AN from the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders. Subjects were recruited, screened, and subjected to exclusion criteria as previously described [10]. The 16 healthy control women were recruited from the New York City area and the Columbia University campus by public advertisements. All were healthy, eumenorrheic, and matched with patients by age and percentage of ideal body weight (IBW) as previously described [10]. None of the control women had a history of an eating disorder or psychi- atric or medical illness. Potential control subjects receiving hormonal or other medications known to aﬀect reproductive function or bone metabolism were excluded. All of the control women exercised less than 3 h/wk. All subjects provided written informed consent. The study protocol was approved by the IRB of the NYSPI, CUMC, and St. Luke’s-Roosevelt Hospital Center. Proce- dures were followed according to approved ethical guide- lines. 2.2. Bone Density, Body Fat, Fat-Free Mass Index (FFMI), and Fat-Free Mass (FFM). Spine, pelvis, leg, and total BMDs were determined using dual energy X-ray absorptiometry (DEXA) from a DPX scanner (Lunar Corp., Madison, WI) using version 3.6 software. The reports from the DPX-L scanner (GE Systems, Madison, WI) were analyzed with the use of version 3.6 software and were used to determine regional BMD of the hip and spine, total body bone mineral content, and total percentage of body fat. The CVs for BMD measurements ranged from 0.5% to 1.0% [11]. When measured by DXA, percentage of body fat is independent of BMD because this value is measured directly by recognized standard means in fat depots at sites where bone is not present [12]. FFM is calculated by the following: fat mass = weight × fat% and FFM = weight − fat mass. FFM is also Journal of Osteoporosis known as lean body mass [13]. FFMI is calculated as FFM/ height2 [14]. 2.3. REE. Each subject’s REE was measured in a respiratory chamber-indirect calorimeter. The chamber was equipped with a high-precision gas (oxygen and carbon dioxide) exchange measurement system. A linear state space model converts gas exchange measurements to energy expenditure estimates. REE was measured early in the morning, with subjects in a fasted state for 12–15 h before the experiment. Subjects remained in the chamber for 1 h, and REE calcula- tions were based on the average of 3 10-minute readings after 30 minutes had elapsed [15]. 2.4. Biochemical Analyses. Serum osteocalcin was measured using a human immunoradiometric assay (Immunotopics International, San Clemente, CA) with a sensitivity of 0.5 ng/ mL and an interassay CV of 5.5–6.7%. Urine NTX was measured with the use of an enzyme-linked immunoassay (Ostex International Inc, Seattle, WA) with a detection limit of 20 nmol bone collagen equivalent and an interassay CV of 4.1%. Assays for estradiol, FSH, LH, PRL, testosterone, DHEAS, T3 and T4 (both total and free), TSH, and cortisol were performed as previously described [10]. IGF-1 was assessed by RIA after alcohol extraction with an intra-assay coeﬃcient of variation of 2.4–3.0% (Nichols Institute Diag- nostics, San Juan Capistrano, CA). Leptin and total ghrelin levels were measured using commercial ELISA kits (Diagnos- tic Systems, Webster, TX). Assay sensitivity was 0.1 ng/mL for each. Both hormones were measured in nonfasting subjects. For leptin, the intra-assay coeﬃcient of variation was 3.6%, and the interassay coeﬃcient of variation was 4.9%. For ghrelin, the intra-assay coeﬃcient of variation was 4.9%, and the interassay coeﬃcient of variation was 5.5%, based on ﬁve assays. Blood and urine samples from patients were obtained at the initiation of hospitalization and at maintenance of weight gain to 90% IBW for ≥2 weeks except for one patient who reached 79% IBW. Venous samples from controls and menstruating subjects were obtained during the follicular phase of the menstrual cycle (days 3–10) as determined by a take-home ovulation test kit to conﬁrm ovulatory cycles. 2.5. Treatment. Treatment for patients with AN followed a predominantly behavioral approach at the NYSPI aimed at normalizing weight and eating. Target weight was a mini- mum of 90% IBW based on 1959 Metropolitan Life Actuarial Tables. All but one patient reached a minimum of 90% IBW; that patient remained amenorrheic at 79% IBW. Median number of days of admission was 66. On admission, patients were fed a standard hospital diet of 1800 kcal (≈55% of energy from carbohydrates, 15% of energy from protein, and 30% of energy from fat), given as 3 meals/d and a snack. Patients were observed to eat 100% of the food prescribed and for 1 h afterwards. If patients did not gain weight, calo- ries were increased in 400-kcal increments in food or liquid nutritional supplement (Ensure Plus; Abbott Laboratories, Abbott Park, IL). After a 1-2 wk medical stabilization period, patients began the active weight-gain treatment phase that