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.

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.

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A novel derivatization procedure, N-acetyl methyl (NACME) esterification, was developed to improve the accuracy and precision of amino acid delta13C value determination using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS). Standard mixtures of 15 protein amino acids were converted to NACME and N-acetyl-isopropyl (NAIP) esters; the latter established derivative was employed for comparison purposes. Both procedures yielded baseline-resolved peaks for all 15 amino acids when GC columns coated with polar stationary phases were employed. For NACME esters, the methylation conditions governed reaction yields, with highest yields observed when a 1 h, 70 degrees C methylation procedure (anhydrous MeOH/acetyl chloride, 25:4, v/v) was performed. The mean derivatization yields expressed relative to an underivatized coinjected standard (n-nonadecane) for both NACME and NAIP esters were identical. Likewise, the mean kinetic isotope effects (KIEs) were not significantly different (KIE(NACME) = 1.036; KIE(NAIP) = 1.038) and were shown in both cases to be reproducible. The mean reproducibility obtained from 15 replicates (3 x batches of 5) of both derivatives was strong (mean STDV(NACME) = 0.3 per thousand and STDV(NAIP) = 0.4 per thousand). The isotopic robustness of both derivatization procedures was observed over a concentration range of 52,500 microg of amino acid. NACME esters displayed low errors (+/-0.6 per thousand for phenylalanine to +/-1.1 per thousand for serine) due to the higher sample-to-derivative carbon ratio of this derivative. Finally, the integrity of the new NACME procedure was confirmed through analysis of diet and bone collagen amino acids of rats reared on C3 or C4 diets, which indicated the high degree of both accuracy and precision of the delta13C values obtained for individual amino acids.

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