Xponent v 3

Antipsychotic-induced hyperprolactinemia is a proxy indicator of D₂ receptor occupancy manifesting when D₂ receptor occupancy is greater than 72% (96 (link)). Serum prolactin was assessed as a surrogate for central nervous system D2 receptor binding affinity for haloperidol and clozapine. Prolactin levels were determined from serum samples using the MILLIPLEX MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 (EMD Millipore, Burlington, MA, USA; Cat# HCCBP1MAG58K) containing prolactin coupled with the Luminex LX200 (EMD Millipore, Burlington, MA, USA) platform in a magnetic bead format according to manufacturer’s instructions. All serum samples were assayed in duplicate, and a pooled serum sample was included as a positive control. Concentrations are expressed as ng/ml. Data were analyzed using xPONENT v.3.1 software (EMD Millipore, Burlington, MA, USA). The minimum detectable concentration for prolactin was 30.2 pg/ml. The coefficient of determination for prolactin was 0.9999. The intra-assay precision was 4.9–15% and the inter-assay precision 4.1–16.2%. Intra-assay precision is generated from the mean of the %CV’s from 16 reportable results across two different concentrations of analytes in a single assay. Inter-assay precision is generated from the mean of the %CV’s across two different concentrations of analytes across 10 different assays.

Blood samples were taken at baseline and at the end of the study period after a 12 h overnight fast at the Extraction Unit of the Hospital Universitario La Paz (Madrid, Spain). Samples were collected early in a 5 ml vacutainer tube with EDTA, and they were centrifuged at 4 °C over 7 min at 3500 rpm. Finally, samples were kept at −40 °C until analysis. A biochemical serum lipid profile (TC, HDL and LDL cholesterol, triglycerides, apolipoprotein A1, apolipoprotein B, and free fatty acids), and glucose determinations were performed by an enzymatic-spectrophotometric assay using an Olympus AU 5400 apparatus (Izasa, CA, USA). C-reactive protein (CRP) concentrations were determined using a BNII nephelometer (Siemens Healthcare Diagnostics GmbH, Eschborn, Germany). Tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) were determined using a Luminex ®-100 (Luminex Corporation. Texas City, TX, USA) multianalyte profiling system with commercially available immunoassay panels. Total lipid peroxides in plasma were determined as an indicator of oxidative stress by using the thiobarbituric acid reactive substances (TBARS) method21. The results were expressed as µmol MDAeq/mL. Data were analyzed using the xPONENT v.3.1 software (Merck Millipore, Burlington, VT, USA) and were determined using specific protocols of La Paz University Hospital.