Funciones por Cargo Laboral

Before any samples taken for this project can be analysed and interpreted with confidence, a reliable and accurate assay for oxytocin must be in place. Studies looking to investigate peripheral levels of the hormone have existed since the 1950s (Ginsburg and Smith, 1959) and have become popular in the 21stcentury. Oxytocin has been assayed in various substrates including saliva (Carteret al.2007, Feldmanet al.2010a and.2010b, Weismanet al.2012a and 2012b), urine (Nagasawaet al.2009, Moscovice and Ziegler 2012, Crockfordet al.2013), cerebro-spinal fluid (Devarajan and Rusak, 2004, Martínez-Lorenzanaet al.2008), and milk (Leakeet al.1981, Prakashet al.2009). However, the most common medium used for oxytocin detection is plasma (of 50 papers reviewed using one or multiple substrates, n=39 plasma, n=9 saliva, n=8 urine, n=6 cerebro-spinal fluid, n=5 serum and n=2 milk). While no study has detected the hormone in a pinniped species before, oxytocin is a widely conserved hormone across mammals (Gimpl and Fahrenholz 2001) and prior studies have

successfully analysed plasma oxytocin levels in humans (Dawoodet al.1981, van der Postet al.1997, Stratheanet al.2009), sheep (Sheldrick and Flint 1981, Da Costaet al.1996), rodents (Devarajan and Rusak 2004, Carteret al.2007, Martinez-Lorenzana et al.2008) dogs (Odendaal and Meintjes 2003) and primates (Amicoet al.1990, Maestripieriet al.2009, Moscovice and Ziegler 2012, Crockfordet al.2013). Developing a protocol for detecting pinniped oxytocin using existing analysis kits should therefore be possible, despite peculiarities in phocid plasma (Hall 1998) that may interfere with the assays.

There are a variety of techniques that can be used to analyse oxytocin levels in mammals, including RIA (Stratheanet al.2009) ELISA (Martinez-Lorenzanaet al. 2008) high performance liquid chromatography (HPLC) (Dudkiewicz-Wilczynskaet al.2000) and other experimental quantification methods such as surface-enhanced laser desorption–ionization time-of-flight mass spectrometry (SELDI-TOF MS) (Cool and DeBrosse 2003). In this chapter commercially available assay kits will be tested against each other to determine the most sensitive and accurate way to analyse samples for all studies that follow in this thesis.

Chapter 3: Measuring Oxytocin

Despite the increasing number of studies using plasma to investigate oxytocin, there is still contention about the appropriate way to collect and analyse samples accurately, and there has been a call for new research into reliable, accurate methodologies for investigators to utilise in future research (McCulloughet al.2013). Such contention in the literature makes selection of the most appropriate protocol difficult. The majority of studies published use an ELISA kit manufactured by Assay Designs Inc (Ann Arbor, MI, USA). However, despite this manufacturer providing a protocol for collection and preparation prior to analysis, there is huge variation between studies in the methods researchers actually employ (Table 3-1.).

Table 3-1. Variety of analysis protocols in 39 different published studies on oxytocin concentrations in blood.

Extracted Plasma Vacutainer Choice Inhibitor Use

Yes 13 EDTA 12 Yes 7

No 17 Heparin 10 No 7

Not Specified/Serum 9 Not Specified 17 Not Specified 25

In addition to the varying analysis protocols, there is disagreement in the literature over whether oxytocin concentrations measured in raw plasma are correlated with those in extracted samples, and how the two are related. Currently some studies rely on the hypothesis that there is a direct relationship between oxytocin concentrations determined in extracted and raw samples (Hogeet al., 2012). Michopouloset al. (2011) provide data showing a correlation between oxytocin levels in raw and

extracted serum samples and Robinson (1980) developed an assay protocol utilising a RIA to accurately detect oxytocin in raw plasma samples. However, two papers to date have directly tested the benefits of extracting plasma samples prior to analysis, with both recommending the use of solid phase extraction with C18 Sep-Pak columns (Bachem, San Carlos, CA) as the best method to gain accurate detection levels for oxytocin in plasma (Szetoet al.2011, Cool and DeBrosse 2003). Szetoet al.(2011) go on to report no correlation between oxytocin concentrations measured in raw plasma and those in extracted plasma, and call for further work to be carried out in order to develop reliable methods for oxytocin analysis. In this chapter, we use a longitudinal sample set to investigate the effect of extraction, vacutainer type, freezer

temperature and use of inhibitor on oxytocin concentrations detected in plasma samples.

In addition to developing an accurate assay for this hormone specifically in phocid seals, it is vital to ensure that abnormal concentrations of oxytocin are not being generated during the capture and sampling procedure. The relationship between oxytocin and cortisol is not fully understood, but manipulation studies injecting either of the hormones have found that oxytocin plays a role in the regulation of stress responses (Devarajan and Rusak 2004). Oxytocin infusions prior to a stressor was found to lower the cortisol response in sheep (Cook 1997) and humans (Ditzenet al. 2009), oxytocin injections consistently decreases corticotrope function levels in humans (Legroset al.1984 and 1987) and cortisol injections cause increases in plasma oxytocin levels (Topset al.2012). Additionally, oxytocin is documented to be released during extreme stressors such as restraint over an extended time period in rodents (Grippoet al.2009, Pournajafi and Carter, unpublished observations) and is hypothesised to occur during procedures such as injections (Devarajan and Rusak 2004). Therefore any attempt to study basal levels of this hormone in seals, which must be manually or chemically restrained during the sampling procedure, must be accompanied by investigation of whether oxytocin is released when sampling occurs.

The objectives of this chapter were:

1. To test the accuracy and suitability of the three commercially available oxytocin assay kits for use analysing phocid plasma samples.

2. To investigate the effect of extraction, vacutainer choice, storage temperature and the use of an inhibitor on the concentration of oxytocin detected in plasma samples.

3. To explore the relationship between raw and extracted oxytocin

concentrations, and determine whether one can be used to accurately predict the other.

4. To quantify any elevation in oxytocin that may occur from rising cortisol levels due to capture and handling stress.

Chapter 3: Measuring Oxytocin