Nitrogen analysis stable isotope

We proposed to study diet and health by combining bone chemistry and histomorphometry. Diet would be determined by analysis of stable isotopes of carbon and nitrogen in bone protein and some preserved hair. In addition, trace elements would be quantitatively analyzed in preserved bone mineral. Abonyi (1993) participated in the study by reconstructing the diet from historical sources and analyzing various foods. Having analyzed human tissues for stable isotopes and trace elements, and foods for the same variables, we hoped to learn more about 19th century diet in southern Ontario, and at the same time, learn more about paleodiet reconstruction. 

Stable isotope analyses of the organic fraction of bone and of food samples was carried out on a Micromass Prism Mass Spectrometer in the Stable Isotope Laboratory, Department of Physics, University of Calgary, under the direction of H.R. Krouse. Collagen samples were combusted in a Carlo Erba gas analyser which provides information on the carbon and nitrogen content of the samples andintroduces Nior CO gases into the mass spectrometer for analysis of nitrogen or carbon stable isotopes, respectively. 

A smaller number of individuals, 132 samples, was analyzed for stable isotopes of nitrogen. There were two objectives to the nitrogen isotope analysis 1) to determine the amount of animal protein in the diet and to look for variation between the sexes and 2) to determine when infants were weaned from the breast. These objectives were addressed by selecting at least ten individuals, when available, from a number of age classes. Figure 1.1 illustrates the age distribution of the sample. Table 1.3 shows the results of these analyses. Individual 5 N values are provided in Herring et al. (1998). 

Katzenberg, M.A. 1991 Analysis of stable isotopes of carbon and nitrogen. In Pfeiffer, S. and Williamson, R.F., eds.. Snake Hill An Investigation of a Military Cemetery from the War of 1812. Toronto, Dundrun Press 247-255. 

MS is the method used in stable isotope analysis. By enriching samples with a particular isotope, that sample or isotope can be followed as it passes through the environment. This technique has been particularly useful in studying the fate of nitrogen as 15N in environmental studies. 

Gas chromatography/combustion/isotope ratio mass spectrometry (CSIA) was used to determine the stable isotope composition of amino acid enantiomers by nitrogen isotope analysis [660]. 

Brand W (2002) Mass spectrometer hardware for analyzing stable isotope ratios. In P de Groot (ed.) Handbook of stable isotope analytical techniques. Elsevier, Amsterdam Brandriss ME, O Neil JR, Edlund MB, Stoermer EF (1998) Oxygen isotope fractionation between diatomaceous silica and water. Geochim Cosmochim Acta 62 1119-1125 Bremner JM, Keeney DR (1966) Determination and isotope ratio analysis of different forms of nitrogen in soils. III. Soil Sci Soc Am Proc 30 577-582 Brenninkmeijer CAM (1993) Measurement of the abundance of CO in the atmosphere and the and 0/ 0 ratio of atmospheric CO with applications in New Zealand and Australia. J Geophys Res 98 10595-10614... 

Several techniques have been described in the past for the analysis of VLCFA, pristanic acid and phytanic acid [1, 10]. In our hands gas chromatography-mass spectrometry (GC-MS) analysis after derivatisation with N-methyl-N-(tert-butyldi-methylsilyl) trifluoroacetamide (MTBSTFA), is a robust and reliable method for the quantitative analysis of VLCFA, pristanic acid and phytanic acid, especially when combined with stable isotopes for C26 0, C24 0, C22 0, phytanic acid and pristanic acid [13]. In order to allow measurement of the total pool of VLCFA, pristanic acid and phytanic acid, samples need to be subjected to both acidic and alkaline hydrolysis, followed by extraction into hexane. After the hexane phase is washed once more, the sample is dried under nitrogen followed by addition of pyridine and MTBSTFA and heating of the samples at 80°C. The sample is subsequently dried again under nitrogen and taken up in hexane, followed by GC-MS analysis. 

Minor element contents and isotopic ratios are used for physiological purposes. Sr/Ca ratios in bone and teeth are said to reflect the diet of the animal (herbivores versus carnivores). Stable isotope analysis of teeth and bones provides direct information on the lifetime diets the nitrogen isotopes reflect the trophic level of the protein that has been consumed. Within an ecosystem, they can identify herbivores and carnivores, while the carbon isotopes tell mainly about the amount of protein in the diets from terrestrial versus marine ecosystems. 

Two analytical approaches have been adopted in attempts to obtain stable-isotopic information on insoluble organic matter in carbonaceous chondrites stepped-combustion analysis (e.g., Kerridge, 1983 Swart et al, 1983) and CSIA of pyrolysis products (e.g., Sephton et al, 1998). Stepped-combustion analysis has proved to be more successful in providing information on the major-elemental constituents of chondritic organic matter, i.e., carbon, hydrogen, nitrogen, and oxygen, whereas CSIA has started to yield detailed carbon isotopic and structural information. 

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

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