1. Issues in the Measurement of Oxytocin Armando J. Mendez Department of Medicine Division of Endocrinology, Diabetes And Metabolism Miller School of Medicine University of Miami Miami, Florida

2. Measurement of Oxytocin Increasing interest in the role of oxytocin in behavior and physiology necessitates a reliable method for quantitation of oxytocin levels. Increasing interest in the role of oxytocin in behavior and physiology necessitates a reliable method for quantitation of oxytocin levels. The purpose of this study was to compare commercially available methods for oxytocin measurement and to evaluate the need for sample extraction prior to analysis. The purpose of this study was to compare commercially available methods for oxytocin measurement and to evaluate the need for sample extraction prior to analysis.

3. Variability in Reported Plasma Oxytocin Levels Measured Using Different Methods Concentration (pg/ml) MethodExtractionReference ~2RIA (In-house)Y Amico et al. (1981) ~3RIA (In-house)Y Lee et al. (2003) 0.1-5.0 RIA (Phoenix Peptides) Y Marazzitiv et al (2008) 0-3 RIA (Phoenix Peptides) Y Modahl et al. (1998), ~20 RIA (Phoenix Peptides) N Mennella (2006) ~200-450 EIA (Assay Designs) N Zak et al. (2005) ~250-400EIA (Assay Designs) N Gordon et al. (2008)

4. Methods Oxytocin Immunoassays tested: Enzyme Linked Immunoassay (ELISA; Assay Designs) Radioimmunoassay (RIA; Phoenix Peptides) Plasma Extraction: Solid phase extraction using C18 columns and 1 ml of sample. Extraction efficiency = 95 ± 5 % Provides a 10-fold concentration of sample prior to analysis.

5. Oxytocin Levels in Extracted vs. Unextracted Plasma by Radioimmunoassay

6. Oxytocin Levels in Extracted vs. Unextracted Plasma by ELISA

7. Gel Filtration Chromatography to Evaluate Oxytocin Immunoreactivity (ELISA) by Molecular Size in Plasma Before and After Extraction Fraction Size

8. Comparison of Oxytocin Assays

9. Oxytocin Immunoreactivity of Plasma extracts after HPLC fractionation Immunoreactive Oxytocin (% of Total)

10. Summary Plasma values are 100-fold higher in unextracted plasma compared to extracted samples by ELISA, and are uncorrelated to extracted samples. The commercially available radioimmunoassay lacks sensitivity to measure basal plasma oxytocin, resulting in values below the level of detection. There are multiple immunoreactive species in plasma detected by the ELISA assay that may account for higher levels in unextracted plasma Using the ELISA, HPLC analysis of plasma extracts demonstrated a peaks of immunoreactivity coincident with oxytocin, and a second unidentified peak. The oxytocin peak accounted for less than 30% of the total immunoreactivity.

11. Conclusion There is a need for a widely available, reliable and valid methods to measure plasma oxytocin.