Psychosom Med. 2011 Jun;73(5):393-400. doi: 10.1097/PSY.0b013e31821df0c2. Epub 2011 Jun 2.
Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin.
Szeto A1, McCabe PM, Nation DA, Tabak BA, Rossetti MA, McCullough ME, Schneiderman N, Mendez AJ.
Behavioral Medicine Research Center and Department of Psychology, University of Miami, Coral Gables, Florida, USA.
There is increased interest in measuring peripheral oxytocin levels to better understand the role of this peptide in mammalian behavior, physiology, and disease. The purpose of this study was to compare methods for plasma oxytocin measurement using a commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA), to evaluate the need for sample extraction, and to assess the immunospecificity of the assays.
Oxytocin was measured in extracted and unextracted human plasma samples (n = 39). Oxytocin and its degradation products were separated by high-performance liquid chromatography and gel filtration chromatography and then assayed by EIA or RIA to identify oxytocin immunoreactive peaks.
Without extraction, plasma measured by EIA was more than 100-fold higher than in extracted plasma, and the correlation between oxytocin levels in extracted and unextracted plasma was minimal (Spearman ρ = -0.10, p = .54). Using the RIA, most samples (>90%) were below the level of detection with or without extraction. After chromatographic fractionation of sample extracts, multiple immunoreactive products were found to be present in addition to oxytocin, which casts doubts on the specificity of the assays.
Changes in oxytocin levels have been reported in social and behavioral challenge studies. This study indicates that sample extraction is necessary to obtain valid assay results. Changes in oxytocin degradation products are likely to contribute to the previously observed responses in circulating oxytocin levels to behavioral and social challenge. There is a critical need for valid and reliable methods to measure oxytocin in biologic samples.
Comparison of oxytocin levels in plasma (n = 39) with and without extraction. One milliliter of plasma was extracted using 200 mg C18 Sep-Pak columns as described in the Methods. Oxytocin levels in plasma (diluted 1:4 before assay) and the corresponding extract were assayed by the EIA method, and the results are expressed as pg/mL of the original plasma volume. Five of 39 samples had values below the limit of detection and were assigned a value of 0.1 pg/mL for the statistical analysis. Correlation was determined using the Pearson correlation coefficient.
Oxytocin immunoreactivity in plasma and plasma extracts after gel filtration chromatography. Plasma (400 μL, A and C) or extracts of the same samples (B and D) were applied to a Superdex 75 10/300 GL column and eluted with 5-mM ammonium acetate buffer (pH 7.8) at a flow rate of 1.0 mL/min, and 1.5-mL fractions were collected. After collection, the fractions were lyophilized then reconstituted and assayed for oxytocin immunoreactivity by EIA. Samples 1 and 2 had plasma oxytocin levels of 442 and 281 pg/mL, respectively, and after extraction, 15.2 and 6.1 pg/mL, respectively. Elution of MW markers and of oxytocin is indicated above A and C.
Oxytocin immunoreactivity profile in plasma extracts after high-pressure liquid chromatography fractionation. Plasma extracts of different samples were performed as described in the Methods. After collection, the fractions were lyophilized then reconstituted and assayed for oxytocin immunoreactivity by EIA (A–C) or RIA (D and E). Samples 1, 2, and 3 had plasma oxytocin levels of 15.5, 10.1, and 3.4 pg/mL, respectively. Samples 4 and 5 had plasma oxytocin levels of 13.3 and 4.1 pg/mL, respectively. Arrows indicate elution of the oxytocin standard.