A method for the quantitative detection of 13C labelled amino acids and aldoses in marine sediments and fauna
Introduction
Studies that aim to characterise the biochemical alteration of sedimentary organic matter (OM) during processing by fauna, decay and burial are essential to improving our understanding of the cycling of OM in seafloor sediments and of the processes which eventually produce mostly uncharacterisable, buried OM. Both digestion and decay have been investigated using the natural biochemical compositions of sedimentary organic matter (e.g. Bradshaw et al., 1989, Cowie and Hedges, 1994, Cowie and Hedges, 1996, Dauwe and Middelburg, 1998). Digestion studies usually involve feeding OM to chosen animals, collecting faecal pellets and comparing food and pellet compositions (Bradshaw et al., 1990a, Cowie and Hedges, 1996). The problem with such studies is that the biochemicals in the food material are not distinguishable from their equivalents in the fauna and the ambient sediment. Therefore it is not possible to measure directly, or to quantify, processes such as assimilation, and distinguishing between anabolism and catabolism is impossible.
The use of stable isotopic labels provides a way around some of these shortcomings. Uptake and metabolism of different compound classes by marine fauna and bacteria have been studied using stable isotopic labels. The types of biochemicals studied in this way include lipids (Bradshaw et al., 1991, Sun et al., 1999, Sun et al., 2002, Sun, 2000), amino acids (Berthold et al., 1991, Thomas and Blair, 2002), and bacterial biomarkers, including d-alanine and bacterial phospholipid fatty acids (PLFAs) (Boschker et al., 2001, Boschker and Middelburg, 2002, Veuger et al., 2005, Veuger et al., 2006). Gas chromatography–mass spectrometry (GC–MS) techniques (as opposed to gas chromatography–combustion–isotope ratio mass spectrometry (GC–c–IRMS) and liquid chromatography–isotope ratio mass spectrometry (LC–IRMS) techniques) for the tracing of labelled biochemicals face quantification difficulties, as standards with the correct level of isotopic labelling are either prohibitively expensive or unobtainable. For this reason, previous studies have mainly traced labelled biochemicals in a qualitative, or relatively quantitative (i.e. mole percentage) sense (Berthold et al., 1991, Thomas and Blair, 2002). This problem is however surmountable using a calibration technique devised for tracing isotopically labelled lipids (Sun, 2000).
Amino acids and carbohydrates constitute significant proportions of both living biomass and sedimentary OM in the marine environment. Both groups of biochemicals have been shown to undergo compound selective degradation (Lee and Cronin, 1984, Hamilton and Hedges, 1988, Cowie and Hedges, 1996, Dauwe and Middelburg, 1998). Therefore, the ability to study the fate of amino acids and carbohydrates during faunal digestion and sedimentary OM decay would add greatly to our knowledge of the processes and factors that combine to determine OM burial efficiency.
In this study, methods for quantitative measurement of 13C labelled amino acids and aldoses in sediments and fauna are described. These involve the combination of previously developed protocols for derivatisation and separation by GC, with quantification using MS and a Sun (2000) type isotope calibration. In order to demonstrate the methods, example results are presented from the analysis of samples taken from a set of polychaete feeding experiments.
Section snippets
Amino acids
The methods used for amino acid extraction and derivatisation are described elsewhere (Mabbott, 1990, Cowie and Hedges, 1992) and summary descriptions are given here.
Decarbonated samples of freeze dried sediment (∼100 mg) or faunal tissue (∼7 mg) were placed in ampoules with N2 flushed 6 N HCl, which were then flushed with Ar and flame sealed. Hydrolysis proceeded at 150 °C for 70 min. An appropriate amount (∼16 μl of 2.5 mM mixed internal standard solution per 10 μg of organic N) of a mixture of
Method theory
The detection of 13C labelled biochemicals was based on the fact that a compound in which n of the natural 12C atoms in the carbon skeleton are replaced with 13C atoms will be heavier than the naturally occurring form by n mass units. Thus, by simultaneously measuring the abundance of a mass fragment with only 12C atoms (m/zM), and the equivalent mass fragment in which some of the atoms are 13C (m/z M+n), it is possible to simultaneously quantify naturally occurring and labelled forms of the
Conclusions
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The quantitative tracing of 13C labelled amino acids and aldoses in sediment and fauna samples has been demonstrated.
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The relative standard deviations produced during replicate analyses show that the techniques are sufficiently precise to generate useful data.
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The techniques generate a different kind of data compared to GC–IRMS methods, as they have the potential to elucidate biochemical transformations.
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Example data from feeding experiments shows that taxon specific compound selective
Acknowledgements
The authors would like to thank Stephen Mowbray and Pieter van Rijswijk for their assistance in the laboratory and field, respectively. Feeding experiments were performed at the Netherlands Institute of Ecology. In addition Dr. Tom Preston and Dr. Ming-Yi Sun are thanked for their helpful correspondence. Three anonymous reviewers are thanked for the time and effort they spent producing careful and thorough reviews, which helped the authors to make significant improvements to the manuscript.
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2014, Geochimica et Cosmochimica ActaCitation Excerpt :Isotope tracing techniques provide a way to quantify assimilation and retention of C. Initially, 14C was used to determine feeding rates and assimilation efficiencies for bulk carbon (e.g. Amouroux et al., 1989, 1997; Charles et al., 1995). Further techniques have recently become available that allow stable isotopes, which are easier to work with, to be reliably detected both in bulk OM, and in individual biochemicals including fatty acids, amino acids, and aldoses (Sun, 2000; Boschker et al., 2008; Woulds et al., 2010). Considering the methodological limitations of previous work, the aim of this study was to apply compound-specific stable isotope tracing techniques to study the uptake and losses of aldoses and fatty acids during macrofaunal gut passage, and, for the first time, to link this to the organic geochemical changes observed during OM decay.
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2012, Geochimica et Cosmochimica ActaCitation Excerpt :Preservation of these large ions required the use of a relatively low ion source temperature (154 °C). For details see Woulds et al. (2010). Naturally present (unlabelled) amino acids in each sample were quantified using the internal standards and a single external standard containing known amounts of all compounds.