carbon isotopes in individual compounds 03 february 2010

Carbon Isotopes in Individual Compounds

03 February 2010

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GOOD CHROMATOGRAPH SEPARATION

What Compounds?

Amino Acids - 13C 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 Extract Residue

Monosaccharides&

Amino Acids

Hydrolysis

Derivatization

Free Lipids

ChromatographyDerivatization

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 Group Mechanism Reagent Product

-NH2 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 13C 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 13C values between the three most informative essential amino acidsQuickTime™ and a

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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:

•13C 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 13C values

The Idea: Relationship b/w tissue biochemical compounds and diet 13C values

Relationship b/w 13C 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 acidscorrelated with whole diet values(0.98<r2<0.99). Better than correlationwith dietary fatty acid

Good correlation b/w cholesteroland whole diet d13C values (r2 = 0.81)

EssentialFA

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

13C 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 13C 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 δ13C 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 δ13C

values correlated well with the whole diet• Bone linoleic acid δ13C values correlated well with

dietary linoleic acid• Mass balance calculations using δ13C values of

single amino acids accurately predicted the δ13Coh whole collagen

• The δ13C 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. 2003 Lipid)

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Fatty Acid Routing(Jim et al. 2003 Lipid)





Amino Acid Routing(Jim et al. 2006 British J. of Nutrition)

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C3P/C4E





C4P/C3E

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.

carbon isotopes in individual compounds 03 february 2010

Documents

carbon slide

latitude slide

mil slide

specific pathway slide

tbdms mtbstfa slide

amino acid metabolism

tracing amino acid exchange

c values