Assay Procedures
Blood samples for oxytocin assay were collected from the antecubital vein into pre-chilled 5 ml EDTA tubes with 250 KIU of apoprotinin, and refrigerated until processing. Plasma was isolated by centrifugation at 1800 g, 15 minutes, 4°C, and stored in aliquots at −70°C. Oxytocin immunoreactivity levels were quantified in duplicates using a commercial oxytocin ELISA kit (Enzo Life Sciences, NY, USA, formerly Assays Designs, MI, USA), as recommended in previous publications [11]. Thawed samples on ice were diluted 1∶2 times in assay buffer and assayed according to manufacturer's instructions. The oxytocin assay had a sensitivity of 11.7 pg/ml, and inter- and intra-assay coefficient of variations below 15%.
Currently there are differences in opinions surrounding the measurement of oxytocin and particularly concerning the requirement of sample extraction. The commercially available oxytocin EIA kit from Enzo Life (formerly Assay Designs), which has been validated by for linearity, cross reactivity, matrix effects, accuracy, precision and recovery [26], was used in the current study. The experience of some investigators suggests that extraction of the samples leads to significant loss of measureable oxytocin. Importantly, the oxytocin data from non-extracted samples makes biological sense as compared to those from extracted samples, which often gave rise to non-detectable levels of oxytocin. Szeto et al., 2011 confirmed these technical findings; as much as two-three fold of the authentic oxytocin was removed by extraction, and 5% of the extracted samples had non-detectable oxytocin levels [21]. Moreover, it would not be appropriate to measure extremely low levels of target analyte, viz., following extraction in the case of oxytocin, using commercial immunoassays that are insufficiently sensitive, which gives rise to erroneous results. We are also not keen to extract the samples using the solid-phase extraction method, as the procedure requires large volume of samples, and often gives rise to low recovery of analytes, high variability in results and incomplete removal of interferences. Hence, we chose not to extract. During our assay runs, we performed 1∶2 dilutions on the unextracted samples so that the measured oxytocin concentrations fall within the “measurable” portion of the standard curve. Pre-dilution of samples is also a common technical approach used to reduce assay interference due to sample matrix.
Taking into consideration the labile nature of oxytocin in biological matrix [27], we followed strict protocol during sample collection to limit its enzymatic breakdown. All blood samples were collected into pre-chilled EDTA tubes containing protease inhibitor. Processing of samples was performed at 4 deg C. During assay, thawed samples were kept on ice. In Szeto et al., human EDTA plasma/serum were obtained and stored at −80 deg C until assay. There was no mention of proper sample collection and hence, the stability of oxytocin in their plasma samples needs to be examined, which may offer one possible explanation of the degraded oxytocin products found in their samples.
The concentrations of oxytocin in unextracted blood samples are 100-fold more than in extracted samples [21]. Martin-Protean, a biotechnology company that specialises in protein analysis, reported oxytocin values to be higher (at levels of 1000 pg/ml) using novel isolation methods and mass spectrometry, and proposed a new model of explanation that incorporates oxytocin carrier protein neurophysin 1 [28]. In their website (http://martin-protean.com/), “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”.
Szeto et al. did perform stability tests using tritiated oxytocin added to plasma, and found that oxytocin is stable under different temperatures and after multiple freeze/thaw cycles. On the other hand, they concluded that plasma oxytocin has a short half life of 3–6 minutes and rapidly degrade into products that are more stable underscoring the careful handling procedure employed in our study. While Szeto et al. reported a lack of correlation between oxytocin levels in extracted versus non-extracted plasma samples (r = 0.09), another study reported high correlation (r = 0.89) [29].
Szeto et al. concluded that degradation products of oxytocin are likely to contribute to the measured levels of oxytocin in unextracted samples. However, it is doubtful that these are degraded products of oxytocin as they have molecular masses more than that of oxytocin. Based on their chromatography results, we believe there is a strong possibility that the assay is measuring immunoreactive oxytocin that comprises authentic oxytocin as well as oxytocin prohormones (OX-T) [30] or other forms of oxytocin in the unextracted samples. Recently, a novel form of oxytocin has been described in multiple species of squirrel monkeys, with a substitution of a leucine to a proline in amino acid position 8 [31]. The variant forms of oxytocin cannot be disregarded and appear to be biologically significant, as many other researchers have used the same kit to measure oxytocin in unextracted samples and found a myriad of associations with relevant physiological outcomes.