ارزیابی روش های استخراج کلاژن برای تحلیل ایزوتوپ های پایدار در مطالعات رژیم غذایی Evaluating bone collagen extraction methods for stable isotope analysis in dietary studies

۱٫ Introduction Stable isotope analyses of d13C and d15N measured in bone collagen are routinely used for the reconstruction of ancient diets and subsistence patterns (e.g., Ambrose, 1993; Bocherens et al., 2006; Honch et al., 2006; Jay and Richards, 2006; Katzenberg, 2000; Richards et al., 1998). Several techniques have been developed to prepare bone samples for isotope analysis. Most of these consider and adjust for factors such as humic acids and lipids that might influence the reproducibility of the measurements (Bronk Ramsey et al., 2004; Brown et al., 1988; Collins and Galley, 1998; Garvie-Lok et al., 2004; Lide´n et al., 1995; Nielsen-Marsh and Hedges, 2000; Semal and Orban, 1995). Generally, following Longin (1971), the extraction methods involve dissolving the mineral matrix in a HCl solution, subsequent solubilisation of collagen at elevated temperature in a weak HCl solution, followed by lyophilisation of the remaining collagen. However, there are various modifications including the addition of a treatment step with NaOH to remove humic acids before solubilisation of the collagen (DeNiro and Epstein, 1981), or the use of ultra-filtration to purify the solubilised collagen (Brown et al., 1988). These recommendations are commonly applied as cleaning steps in order to measure the original collagen used in dietary studies. There are diverse chemical approaches in sample preparation used by different stable isotope laboratories. However, whatever the steps, the laboratories use each others results as references and comparison in dietary studies (e.g. Bayliss et al., 2004; Jørkov, 2002; Keegan, 1989). Previous studies of these extraction methods have examined the effects of ultra filtration in radiocarbon-dating and the contribution of lipids on the stable carbon isotope values (e.g. Bronk Ramsey et al., 2004; Lide´n et al., 1995). Results have shown that ultra-filtration may still leave larger contaminating particles and lipids may alter the carbon signals. We therefore thought it necessary to investigate whether the extraction methods and cleaning steps may influence the isotopic result and hence potentially the interpretation of dietary variation. The specific purpose of this study was to compare three sample preparation methods on well-preserved skeletal material by evaluating the resultant differences in d13C and d15N, collagen quality and collagen yield. The first method (A) includes the treatment with NaOH, the second method (B) includes both ultra-filtration and filtration with Ezee filter separators (5e8 mm) (Elkay Laboratory Product) before lyophilisation as described in Richards and Hedges (1999) and Mu¨ldner and Richards (2005). The third method (C) is a modified version of method B, by excluding the ultra-filtration step. This modified version of method B is also used in laboratories conducting stable isotope analysis in dietary studies (Honch et al., 2006). ۲٫ Materials and methods The bone samples selected for this study were chosen from a large well documented skeletal collection from the medieval cemetery Ahlgade 15e17 in Holbæk, Denmark (Asmussen, 1997). The cemetery was used between ca. 1100e1573 AD, and contained more than 700 skeletons buried in clay soil. The material was chosen because of its excellent state of preservation (the bones were macroscopically intact with a hard structure and feel (i.e. non-flaky)). Eight bone samples from five individuals were each treated with the three extraction methods. Details on the samples, including age and sex of the individuals are given in Table 1.