Pigs were raised on six isotopically controlled diets to examine the dietary macronutrients used in the synthesis of bulk bone biochemical components (apatite, collagen and lipids) and individual compounds (bone fatty acids, cholesterol and amino acids from collagen). δ 13 C values of apatite and bulk bone lipids reflected those of the whole diet, with 13 C apatite-whole diet = 10.2 ± 1.3‰ and 13 C bone lipids-whole diet = −2.4 ± 0.7‰. A wide variation observed in the 13 C collagen-whole diet values (0.5 to 6.1‰) was hypothesized to reflect the relative importance of (i) the direct incorporation of essential amino acids, and (ii) the balance between direct incorporation and de novo synthesis of non-essential amino acids. Linear regression (n = 6) was used to assess the relationship between the δ 13 C values of whole diet and bulk bone components and individual compounds. Whole diet δ 13 C values showed a strong correlation with those of bone cholesterol (R 2 = 0.81) and non-essential fatty acids (0.97 ≤ R 2 ≤ 0.99). Not surprisingly, bone linoleic acid δ 13 C values correlated well with dietary linoleic acid (R 2 = 0.95). Mass balance calculations using the δ 13 C values of single amino acids accurately predicted the δ 13 C value of whole collagen. The δ 13 C values of whole diet were well correlated with those of the non-essential amino acids, alanine (R 2 = 0.85) and glutamate (R 2 = 0.96) in collagen. The essential amino acids leucine ( 13 C collagen leu-diet leu = 0.5 ± 1.2‰) and phenylalanine ( 13 C collagen phe-diet phe = −0.6 ± 0.6‰) showed little isotopic fractionation between diet and bone collagen.

The diets of laboratory rats were isotopically and nutritionally manipulated using purified C 3 and/or C 4 macronutrients to investigate the routing of dietary carbon to bone collagen biosynthesis. Diets were formulated with purified proteins, carbohydrates and lipids of defined composition and natural abundance stable isotope ratios. Bulk protein and constituent amino acid d 13C values determined for whole diet and bone collagen provided the basis for assessing isotopic fractionation and estimating the degree of routing versus synthesis de novo of essential, non-essential and conditionally indispensable amino acids. Essential and conditionally indispensable amino acids were shown to be routed from diet to collagen with little isotopic fractionation whereas non-essential amino acids differed by up to 20‰. Mathematical modelling of the relationships between macronutrient and tissue d 13 C values provided qualitative and quantitative insights into the metabolic and energetic controls on bone collagen biosynthesis. Essential amino acids comprise 21·7 % of the carbon in collagen, defining the minimum amount of dietary carbon routing. Estimates of 42 and 28 % routing were shown for the non-essential amino acids, glycine and aspartate, respectively. In total, the routing of non-essential and conditionally indispensable amino acids was estimated to equal 29·6 % of the carbon in collagen. When the contribution of carbon from the essential amino acids is also considered, we arrive at an overall minimum estimate of 51·3 % routing of dietary amino acid carbon into bone collagen.

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.

Cladistic analyses of 17 wild and cultivated pea taxa were performed using morphological characters, and allozyme and RAPD (random amplified polymorphic DNA) markers. Both branch-and-bound and bootstrap searches produced cladograms that confirmed the close relationships among the wild species and cultivars of Pisum proposed by a variety of systematic studies. Intraspecific rankings were supported for northern P. humile, southern P. humile, P. elatius and P. sativum, which together comprise a single-species complex. P. fulvum, while clearly the most divergent of the pea taxa, could also be assigned to the same species complex without violating the hierarchial logic of the cladogram. Its inclusion or exclusion depends on whether the level of interfertility it displays with other pea taxa or its overall morphological and chromosomal distinction are emphasized. As suggested by previous studies, northern P. humile was the most likely sister taxon to cultivated P. sativum; although, rigorous phylogenetic evaluation revealed a close genealogical affinity among P. elatius, northern P. humile and P. sativum. Despite their limited number, the 16 morphological characters and allozyme markers used precisely organized the pea taxa into established taxonomic groupings, perhaps in part reflecting the role morphology has played historically in pea classification. The RAPD data also generally supported these same groupings and provided additional information regarding the relationships among the taxa. Given that RAPDs are relatively quick and easy to use, are refractory to many environmental influences, can be generated in large numbers, and can complement traditional characters that may be limited in availability, they provide a valuable new resource for phylogenetic studies.

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