Walsh Medical Media

Heading : Methods

Participants

A case–control study was carried out on 25 adolescent girls diagnosed with AN according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV) [<a href="#7" title="7">7</a>]. Patients were recruited from the Outpatient Child Psychiatry Clinic, Institute of Psychiatry and Pediatric Psychiatry Clinic, Children’s Hospital, Faculty of Medicine, Ain Shams University (Cairo, Egypt), during March 2010-October 2013.

Of the 25 patients, 22 were recruited from the Outpatient Child Psychiatry Clinic (of whom four patients were referred from the Gastroenterology department with the complaint of persistent vomiting and three patients were referred from the Gynecology department for amenorrhea) and three patients from the Pediatric Psychiatry Clinic.

Patients with AN were studied in comparison with 30 age - and pubertal stage- matched girls who served as controls. None of the controls had clinical findings indicative of neuropsychiatric manifestations or endocrinal disorders. Exclusion criteria included treatment with estrogen, vitamin D, or calcium before the study or taking medications that might influence the HPA axis. Parents of the patients studied signed an informed written consent for participation in the study. This study was approved by the Bioethical Research Committee, Faculty of Medicine, Ain Shams University Hospitals (Cairo, Egypt).

Procedures

The studied adolescents were subjected to the following:

(1) Complete assessment of medical history from the patients or their caregivers, with a focus on developmental history, symptoms of malabsorption, for example, abdominal distention, diarrhea with pale foul-smelling, bulky stools, vomiting, poor weight gain, and growth retardation.

(2) Complete clinical examination to exclude other causes of weight loss, for example, malabsorption.

(3) Neuropsychiatric assessment (for patients only)

(a) Patients were diagnosed with AN according to the DSM-IV criteria [<a href="#7" title="7">7</a>]. Twenty one patients had restricting AN and the remaining four had binge eating/purging AN.

(4) Auxological measurements:

(a) Body weight (kg) was measured on a digital electronic scale. The scale was set to zero before the girl was placed on the scale and was checked weekly using known calibration weights. Weight measurements were taken with the girl wearing little outer clothing and no shoes and then expressed as weight for height standard deviation score (SDS).

(b) Height (cm) was measured without shoes using a wall-mounted Harpenden stadiometer and then expressed as height for age SDS.

(c) BMI is defined as weight (kg)/height (m2). SDSs of height for age and weight for height were calculated according to the norms of Tanner et al. [<a href="#8" title="8">8</a>] and that of BMI were calculated according to the norms of Cole et al. [<a href="#9" title="9">9</a>].

(5) BA assessment: BA was determined in all patients with AN and controls by plain radiograph of the left hand and wrist using the method of Greulich and Pyle [<a href="#10" title="10">10</a>].

(6) Bone density and body composition: areal bone mineral density (BMD, g/cm2) was measured at the whole body, the posteroanterior lumbar spine (L1–L4), and the left femoral neck, and the whole body composition parameters [whole-body bone mineral content (BMC) (g), lean body mass (g), whole-body fat (g), and percentage body fat] were also determined for each individual at the Institute of Postgraduate Childhood Researches, Ain Shams University. All the parameters mentioned above were determined usingthe Hologic QDR Discovery DEXA fan-beam scanner (software v. 12.3, Hologic, Inc. Company, Bedford, USA; fast-array mode). Calibration stability was monitored using two site-specific phantoms (hologic anthropomorphic spine and whole body phantoms). The precision errors for BMD and BMC are less than 1% for the spine phantom and less than 2.5% for the whole-body phantom. This software release has special features for pediatric and adolescent scans. The spine, hip, and whole-body analyses used an automatic low bone density detection algorithm that increases the sensitivity of finding low-density bone. For hip and spine scans, two bone detection thresholds were applied to all scans [<a href="#11" title="11">11</a>].

Precautions taken before DEXA scanning were as follows:

(1) Artifacts including enteric tubes, metallic objects, and jewelry were excluded from the image [<a href="#12" title="12">12</a>].

(2) Sedatives were administered to some girls in order to ensure their stability throughout the scanning time, especially during the whole-body scan (which takes longer) [<a href="#13" title="13">13</a>].

Laboratory investigations

(a) Serum IGF-1 and GH levels were analyzed using commercial reagents (Incstar Corporation, Stillwater, Minnesota, USA) after extraction of the plasma samples with acid ethanol. Serum basal growth hormone (GH) was determined using commercial reagents (Pharmacia Diagnostics, Uppsala, Sweden) by a solid-phase, enzyme-labeled chemiluminescent immunometric assay (by the Immulite 2000 Analyzer; Siemens, Cruinn Diagnostics Limited, Dublin, Ireland).

(b) Serum total ionized calcium concentration was determined by a spectrophotometer using a Hitachi 917 autoanalyzer (Hoffmann-La Roche Limited, Basel, Switzerland) and Roch reagents. A calcium-binding dye, orthocresolphthalein complex one, was used, which changes its color on binding to calcium. The intensity of the color formed is directly proportional to the concentration of calcium in the sample, and the absorbance of the color formed was determined at 650 nm. A reference range of 8.5-10.5 mg/dl was used [<a href="#14" title="14">14</a>].

(c) Serum inorganic phosphorus levels were determined using a Hitachi 917 autoanalyzer and Roche reagents. This method is based on the reaction of phosphate ions with ammonium molybdate to form a phosphomolybdate complex, which is colorless and is determined directly by UV absorbance at 340 nm. A reference range of 3.5-5.5 mg/dl was used [<a href="#15" title="15">15</a>].

(d) Serum levels of 25(OH)D were assessed by an enzyme-linked immunosorbent assay (ELISA) using a competitive protein-binding assay kit for the measurement of 25(OH)D, which is based on the competition of 25(OH)D present in the sample with 25(OH) vitamin tracer, for the binding pocket of vitamin D protein (VDBP, Gc globulin). Serum levels of 25(OH) D less than 30 and 10 ng/ml were defined as vitamin D insufficiency and vitamin D deficiency, respectively, whereas levels more than 30 ng/ml were defined as vitamin D sufficiency [<a href="#16" title="16">16</a>].

(e) Serum osteocalcin (OC) level as a bone formation marker was determined using an ELISA. Osteocalcin ELISA is based on the application of two highly specific monoclonal antibodies (M-abs) against human osteocalcin. An antibody recognizing the mid-region (amino acids 20–29) was used as the capture antibody and a peroxidase-conjugated antibody recognizing intact osteocalcin (amino acid 1–49) was used for detection; the N-terminal-mid fragment (amino acids 1–43) was also detected. A reference range of 0-100 ug/l was used [<a href="#17" title="17">17</a>].

Insulin –like Growth Factor 1 in Adolescent Girls with Anorexia Nervosa: Relation to Bone Formation Parameters

Heading : Methods