N measurementsPatients and methodsSettingThe Acute Comprehensive Urgent Treatment of Eating disorders (ACUTE) center at Denver Health Medical Center is a five-bed, multidisciplinary center that cares for the largest number of critically ill anorexic patients in the country. It serves patients too medically compromised to initiate or continue treatment in a psychiatrically based eating disorder program. Therefore, the ACUTE center is a medical stabilization unit treating the most seriously ill anorexic patients. Although hospitalization is recommended for anorexic patients with a body mass index (BMI) under 14 kg/m 2 , the ACUTE center's patients have a mean BMI of 12.6 kg/m2, making them 4-Bromopicolinaldehyde a uniquely ill patient population. It is worthThe total protein content of serum samples was quantified using BCA protein measurement (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Samples were diluted 1:100 in phosphatebuffered saline (PBS) prior PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/23410069 to incubation and spectrophotometric assessment. Bovine serum albumin (Thermo Scientific) was used to generate a standard curve.Complement hemolytic activity and serum levelsSerum samples were thawed and processed immediately. Repetitive freeze-thaw cycles were avoided to minimize in vitro complement activation. The following commercially available ELISA kits were used strictly according to the manufacturer's protocol: MicroVue CH50 Eq EIA kit (Quidel, San Diego, CA, USA; sample dilution per manufacturer's protocol); C3 fixed complement preceptor ELISA kit (Bachem, San Carlos, CA, USA; 1:20 sample dilution); MicroVue C3a EIA kit (Quidel; 1:Flierl et al. Annals of General Psychiatry 2011, 10:16 http://www.annals-general-psychiatry.com/content/10/1/Page 3 ofsample dilution); human complement component C5a ELISA (R D, Minneapolis, 4 MN, USA; 1:20 sample dilution); MicroVue sC5b-9 EIA kit (Quidel). Obtained concentrations were protein adjusted (concentration/mg total protein) in order to address differences in total protein levels between anorexia nervosa patients and healthy volunteers.Statistical analysisBaseline characteristics of the study participants are described with mean and standard deviation (SD) or percentages. Complement levels are expressed as medians with interquartile ranges because they were nonnormally distributed (Anderson-Darling test). Comparisons between the anorexia nervosa patients and healthy volunteers were analyzed with an unpaired t test, Wilcoxon rank sum test, or Fisher's exact test, as appropriate. Spearman correlation coefficients were used to determine association between BMI and complements. A generalized estimating equation (GEE) analysis was used to determine the relationship between BMI and complement level over time for the anorexia nervosa patients. PROC GENMOD was used since it accounts for the repeated measures 1-(4-Bromo-2-pyridyl)piperazine within a patient, allows missing data, and does not require the response to be normally distributed. Differences were considered significant when P <0.05. All analyses were conducted in SAS v.9.1. (SAS, Cary, NC, USA).body mass index of 13.6 ?1.5 kg/m2 compared with 22.2 ?2.6 kg/m2 in the control group PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11834444 (P <0.001). The initial percentage of ideal body weight in the anorexia group was 64.5 ?7.6 , which was 40 lower than the healthy controls (P <0.001). The serum total protein was 61.9 ?8.2 mg/ml in the anorexia group compared with 73.7 ?9.6 in the control group, P = 0.001. Thus, we adjusted for this baseline difference in protein wh.
