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Martin-Protean has developed an innovative process for measuring oxytocin in plasma that produces significantly higher levels than obtained by other mass spectrometry methods. We also have methods for quantifying oxytocin in saliva and urine.
Guidelines to submit plasma and other fluid samples for oxytocin quantification:
Collection of plasma is reasonably standard, we use the EDTA tubes (please don't use the heparin) and we like the protocol for plasma preparation here. When you have prepared your plasma store it frozen in cryovials (we like -80C) and ship the plasma on dry ice. Methods for collecting saliva are not as standardized, we work with crude saliva frozen. If you want to pellet the cells and other solid debris we recommend adding 2mM EDTA, which inhibits a salvial glutamyl endopeptidase. Urine may be frozen in cryovials and shipped on dry ice. Breastmilk may be frozen in ziplock bags (as any nursing mother knows) and shipped on dry ice. Introduction
Quantification of the oxytocin peptide in complex biological samples is difficult. There are methods for quantifying oxytocin in plasma with polyclonal antibodies. These methods do not agree with each other and both incorrectly quantify non-oxytocin components as oxytocin1. Mass spectrometry is an analytical method that affords the ability to identify and quantify oxytocin, if it can be sufficiently isolated from the sample. A method for quantifying oxytocin by mass spectrometry is published2 and finds plasma levels of oxytocin of 1pg/mL. Martin-Protean developed an inventive method for isolating oxytocin from plasma and saliva without an antibody and quantifying the isolated oxytocin by mass spectrometry. We find oxytocin in plasma at levels of 1000pg/mL, we propose a model to explain the differences in our findings.Analytical
Why mass spectrometry? It is sensitive. It is specific. The identity of the oxytocin is confirmed in every measurement. On our instrument the oxytocin detection limit 0.1fmol. Oxytocin identity is a function of retention time on a reverse phase column, molecular weight, and fragmentation fingerprint. Only if the a peptide with a mass/charge of 1007.7 that elutes at the 13.8 minutes and produces the fragmentation fingerprint (bits of peptide with mass/charge < 1007.7) is it counted as oxytocin. The amount of oxytocin in the sample is determined by the area under the peak. The details of the method are proprietary, two patent applications are filed.Conclusions
Oxytocin is quantified in plasma at levels 10-fold higher than EIA measurements and 1000-fold higher than RIA or previous mass spectrometry measurements. The higher oxytocin levels as well as specific details of the isolation of oxytocin from plasma are consistent with a model where the vast majority of oxytocin in the blood is bound in a way that may inhibit detection in other existing approaches. Original isolations of oxytocin found it bound to a “van Dyke protein”3 that was eventually identified as neurophysin I4. Neurophysin I is found in plasma5. One way to explain the differences in observed levels is to propose different partition coefficients for free oxytocin and oxytocin bound to neurophysin I. Different methods may isolate with different efficiencies the oxytocin in the different partitions. Efforts to quantify oxytocin that capture 0.1% of the oxytocin present would not capture the complete oxytocin story and are likely to be dominated by non-biological variation in experimental procedure.References
Angela Szeto, Philip M. McCabe, Daniel A. Nation, Benjamin A. Tabak, Maria A. Rossetti, Michael E. McCullough, Neil Schneiderman, and Armando J. Mendez (2011). Evaluation of Enzyme Immunoassay and Radioimmunoassay Methods for the Measurement of Plasma Oxytocin. Psychosom Med, 73: 393-400.
Guodong Zhang, Yizhong Zhang, Douglas M. Fast, Zhaosheng Lin, and Rick Steenwyk, (2011). Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay. Analytical Biochemistry, 416(1): 45-52.
Roger Acher, Albert Light and Vincent du Vigneaud (1958) Purification of Oxytocin and Vasopressin by Way of a Protein Complex. J. Biol. Chem. 233(1): 116-120.
Ester Breslow and Lorraine Abrash (1966). The binding of oxytocin and oxtytocin analogues by purified bovine neurophysins. Biochemistry 56: 640-646.
Janet A. Amico, Said M. Seif, and Alan G. Robinson (1981). Oxytocin in Human Plasma: Correlation with Neurophysin and Stimulation with Estrogen. J Clin Endocr & Metab 52(5) 988-993.