Carbon Isotopes in Individual Compounds 03 February 2010

GOOD CHROMATOGRAPH SEPARATION

What Compounds? Amino Acids - 13 C signatures Fatty Acids - lipid biomarkers

Diane’s Diagram: Follow the Carbon

Background & Fractionation Information for you comprehending pleasure Mechanistic understanding of the biochemical factors that underpin stable isotope signals Links biochemistry to stable isotope composition The Idea:

Compound-specific isotopes are useful 1.Different biochemical components can possess different stable isotope values 2.Structurally similar biochemical components of ecological materials can derive from a range of sources potentially exhibiting different signatures 3.Biogenic organic matter can change in chemical composition 4.Reveals contributors mediating processes that would otherwise be masked by in bulk 5.Biomarkers together with compound specific isotopes information on biological processes 6.Biochemical components posses significantly different turnover times 7.Kinetic fractionation can only be determined at the level of the biochemical component and specific pathway

Before we can burn our samples up, preparation for compound-specific stable isotopes via GC/C/IRMS follows: Sample Total Lipid ExtractResidue Monosaccharides & Amino Acids Hydrolysis Derivatization Free Lipids Chromatography Derivatization Extraction Vaporize!

Why so much preparation? Most compounds of interest must be modified, usually of compounds containing polar functional groups, to enhance their volatility prior to GC/C/IRMS injection. Ex: Amino Acid Functional GroupMechanismReagentProduct -NH 2 tBDMS MTBSTFA

Applications  Fingerprinting  Nutritional Linkages  Biomarkers

The Study: Investigation of differences in amino acid metabolism among plants, fungi and bacteria that generate unique patterns of 13 C signatures Tool: New approach for tracing amino acid exchange in symbiotic and trophic relationships

Lysine and Leucine exhibited significant differences Phenylalanine least variable among taxa

Differences in amino acid 13 C values between the three most informative essential amino acids Significant difference of non-normalized essential amino acids, distinct isotope clusters

Lichen identified as fungi, what role is each organism playing biochemically? Does mostly well identifying what the insects were eating

To Summarize: 13 C fingerprinting of amino acids could provide as a powerful in situ assay of amino acid sources in terrestrial ecosystems in -identifying the primary contributors of amino acids in animals -understanding symbiotic associations between animals and microorganisms Greatest accuracy is from the essential amino acids measured based on their more complex biosynthetic pathways

Frolicking for food!

Carbon values get heavier from south to north by 6 per mil

Phe preserves bulk isotope value Phe, Lys, Arg strong correlation to bulk along latitude

The Study: Diets formulated for Pigs to contain 20% protein and wide range in  13 C values The Idea:  Relationship b/w tissue biochemical compounds and diet  13 C values  Relationship b/w  13 C values of bone collagen and its constituents ( 2003 )

What do we want to know? (1) Direct incorporation of essential amino and fatty acids (2) Balance between direct incorporation and de novo synthesis of non-essential amino and fatty acids

Pork Fat Result for pig on diet 3 Non-essential fatty acids correlated with whole diet values (0.98<r 2 <0.99). Better than correlation with dietary fatty acid Good correlation b/w cholesterol and whole diet d13C values (r 2 = 0.81) Essential FA

Essential Fatty Acid: Linoleic Acid Cannot be synthesized de novo ; must be incorporated directly from diet. Strong correlation b/w the diet and bone linoleic acid: direct incorporation

 13 C values of non-essential amino acids were distributed across 10‰, reflecting differences in their assimilation, transport, and biosynthesis. Glycine (serine?) was 8.4‰ more enriched than whole diet values? Also, strong correlation between the stoichiometric and measured bulk collagen values. Estimated  13 C values were 1.4‰ more positive than observed values. Study did not include arginine (7.9%) and lysine (4.5%) of carbon to collagen, which are typically depleted in d13C relative to bulk collagen and other amino acids.

Strong correlation of alanine and glutamate with the δ 13C value of whole diet. Decent correlations between essential amino acids (leucine & phenylalanine) and these amino acids in diet. Amino Acid-Diet Correlations

To summarize: Bone cholesterol and non-essential fatty acid δ 13 C values correlated well with the whole diet Bone linoleic acid δ 13 C values correlated well with dietary linoleic acid Mass balance calculations using δ 13 C values of single amino acids accurately predicted the δ 13 Coh whole collagen The δ 13 C values of non-essential amino acids, alanine and glutamate, from bone collagen correlate well with whole diet The essential amino acids leucine and phenyalanine showed little isotopic fractionation between diet and bone collagen

Fatty Acid Routing (Jim et al Lipid) Those damn rats!

Fatty Acid Routing (Jim et al Lipid)

Amino Acid Routing (Jim et al British J. of Nutrition) Oh Dear God! Not the rats again.

Amino Acid Routing (Jim et al British J. of Nutrition) C3P/C4E C4P/C3E

Amino Acid Routing (Jim et al British J. of Nutrition)

The Bottom Line(s): 1.If you want to measure the isotopic composition of bulk diet, use apatite, cholestrol, alanine or glutamate. 2.If you want to measure the isotopic composition of the lipid component of diet, measure essential fatty acids (e.g. Linoleic Acid). 3.If you want to measure the isotopic composition of protein, measure essential amino acids (e.g., phenylalanine or leucine), or amino acids that behave as if they are essential (proline). 4.Routing between dietary protein and bone protein is substantial for animals on protein-rich (20%) diets. It has not been tested for animals on lower protein diets. 5.Lipid routing is also dependent on the concentration of the particular lipid in the diet.