Neuropeptide y

Simultaneous profiling of multiple cytokines in addition to a set of brain-derived proteins (endocrine, neuropeptide) was performed in saliva samples in the cytokine reference laboratory located in the University of Minnesota Department of Pediatrics using a commercially available 22-plex Human Cytokine Array Panel (LUH000, R&D Systems, Minneapolis, MN, USA). Salivary levels of cytokines/chemokines, agouti-related peptide (AgRP), and prolactin were determined by multiplex method on the Luminex platform (Austin, TX, USA) with Bioplex software (BioRad, Hercules, CA, USA) using human-specific bead sets from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assays (ELISAs) were used to determine levels of cortisol (Alpco Diagnostics), dynorphin A (Phoenix Pharmaceuticals), neuropeptide Y (RayBiotech), somatostatin (BACHEM—Peninsula Labs), and NGF (Promega). Values were interpolated from standard curves of the relevant recombinant human proteins set up on each plate. Dilution series and standard curves were run for all samples; all assays were performed in duplicate.

The following reagents were purchased from the indicated sources: phosphate-buffered saline, perchloric acid, potassium hydroxide (KOH), NaOD, DCl, and D₂O (Sigma-Aldrich, St. Louis, MO, USA), cortisol (Alpco Diagnostics, Salem, NH, USA), dynorphin A (Phoenix Pharmaceuticals, Burlingame, CA, USA), neuropeptide Y (RayBiotech, Norcross, GA, USA), somatostatin (BACHEM—Peninsula Labs, San Carlos, CA, USA), and nerve growth factor (NGF; Promega, Madison, WI, USA).

Plasma biomarkers were assayed by enzyme-linked immunosorbent assay. Kits were purchased from the following sources: neuropeptide Y (NPY), RANTES (Regulated on activation, normal T-cell-expressed and secreted), and E-selectin from Ray Biotech (Norcross, GA); serotonin from Enzo Life Sciences Inc (Farmingdale, NY); CS846 (aggrecan chondroitin sulfate 846) from IBEX Pharmaceuticals Inc (Montreal, Quebec, Canada); and CTX-II (C-terminal telopeptide of type II collagen) from Immunodiagnosticsystems, IDS Inc (Gaiersburgh, MD). All samples were assayed in duplicate and averaged following the manufactures’ instructions and reported as concentration (ng/ml).