Challenges for measuring oxytocin: The blind men and the elephant?
Introduction
Since its discovery more than a century ago, oxytocin has become one of the most intensively studied molecules in behavioral biology. Once thought of primarily as a female reproductive hormone, oxytocin is now recognized for having diverse roles in the mammalian nervous system, and is implicated in processes ranging from social monogamy and theory of mind to regulation of appetite, the immune and autonomic systems, and bone mass and cardiomyocyte differentiation (Blackburn et al., 1992; Carter, 2014; Carter and Perkeybile, 2018; Feldman et al., 2007; Li et al., 2017; MacKinnon et al., 2018; Paquin et al., 2002; Peltola et al., 2018; Tamma et al., 2009; Wai et al., 2018). In the last five years, Psychoneuroendocrinology has published more than 500 articles with oxytocin in the title, with many of these articles including measures of endogenous oxytocin concentrations. Despite this longstanding interest, methods of measuring endogenous oxytocin are still in active development, and questions about the validity of various techniques have spurred controversy and confusion (Carter et al., 2007; Carter, 2014; Jurek and Neumann, 2018; Leng and Sabatier, 2016; McCullough et al., 2013).
Specifically, two of the most common approaches for plasma sample preparation (use of extracted or non-extracted samples; see below for details) lead to entirely uncorrelated results (Leng and Sabatier, 2016); even when using the same protocols, different commercially available assays can produce markedly divergent measurements (Lefevre et al., 2017; MacLean et al., 2018). Questions about endogenous oxytocin concentrations have been further complicated by recent findings that the vast majority of endogenous oxytocin may be bound to other molecules in plasma and serum. In many cases oxytocin may evade detection unless these bonds are broken prior to measurement (Brandtzaeg et al., 2016; Martin and Carter, 2013). Although efforts to develop reliable measures / confirm the validity of bound oxytocin have proven challenging (Franke et al., 2019), multiple techniques have been developed in recent years, with utility for both immunoassay and mass spectrometry applications (Brandtzaeg et al., 2016; Liu et al., 2019). The widely varying oxytocin concentrations detected by different approaches to sample preparation and measurement – and lack of correlation between these techniques – has led to the suggestion that particular approaches may be ‘no more than a random number generator’ (Leng and Sabatier, 2016). Reflecting on the poor agreement between various approaches, others have suggested that it is urgent that we adopt a single approach to be implemented as the standard in the field (McCullough et al., 2013). Although we agree that progress will depend on a better understanding of why different methods produce such different results, we suggest that it is premature to accept any single approach as a gold standard, since discrepancies between methods are not necessarily an indicator that some methods are valid whereas others are not. Instead, we suggest that the current challenges in the measurement of oxytocin may be analogous to the parable of the blind men and the elephant.
In this parable, a group of blind men conceptualize what an elephant is like by touching its body. Each man touches a different part of the elephant, and afterward recounts his perspective. Of course, all of the men find themselves in utter disagreement about what an elephant is like, having been exposed to only partial information about a complex system. We propose that the current diversity of approaches to measuring oxytocin may be analogous to the blind men in this parable. Varying approaches to sample preparation and measurement of oxytocin may yield different and often conflicting information due to differential sensitivity to diverse conformational states of the oxytocin molecule. We propose that this phenomenon arises because oxytocin, like an elephant, is biologically complex.
Section snippets
Characteristics of the oxytocin molecule
Oxytocin measured in plasma is reported to have a short half-life, ranging from ˜1-5 min, and in vivo, is rapidly degraded by diverse peptidases in the passage of blood through the liver and kidneys (Chard et al., 1970; Morin et al., 2008; Rydén and Sjöholm, 1969). But perhaps more importantly, oxytocin also strongly binds to other molecules (including itself), which are common in biological matrices (Avanti et al., 2013, 2012; Brandtzaeg et al., 2016; Liu et al., 2019; Yamamoto et al., 2019).
Measuring oxytocin
The most common approaches to measuring oxytocin involve immunoassay or mass spectrometry. These techniques detect analytes in fundamentally different ways, and as a result, are characterized by different strengths and limitations (Table 1). Immunoassays rely on binding between oxytocin and an antibody (Fig. 1). Oxytocin antibodies vary in the epitopes they recognize, which can lead to variable results between immunoassays (Lefevre et al., 2017). Additionally, because detection is based on
Sample preparation
Beyond the specific detection platform, pre-analytical steps have the potential to dramatically influence what states of oxytocin are available for detection. Perhaps the most influential preanalytical step is sample extraction. In short, extraction refers to a range of approaches designed to eliminate interfering substances from a complex matrix, while retaining, and often concentrating the target analyte. The simplest oxytocin extractions involve protein precipitation, in which a solvent is
Implications
The issues outlined above reveal a multitude of factors which can influence oxytocin measurement, beginning at the preanalytical stages, and extending through the process of quantitation. Preanalytical steps may discard some of the most abundant forms of oxytocin, fail to satisfactorily recover the analyte, or degrade the molecule through heat and/or oxidation. Quantitation by immunoassay will depend on whether particular epitopes remain available for binding, which may depend on whether
Conflicts of interest
The authors have no conflicts of interest to report.
Acknowledgements
Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Numbers R21HD095217 and P01HD07575. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We are also grateful to Fetzer Institute, Kalamazoo, Michigan, for support of this work. SRW is supported by the Research Council
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