Carbon Isotopes in IndividualCompounds

03 February 2010

GOOD CHROMATOGRAPH SEPARATION

What Compounds?

Amino Acids - 13C signatures

Fatty Acids - lipidbiomarkers

Diane’s Diagram: Follow the Carbon

Background & Fractionation Informationfor you comprehending pleasure

• Mechanistic understanding of the biochemicalfactors that underpin stable isotope signals

• Links biochemistry to stable isotope composition

The Idea:

Compound-specific isotopesare useful

1. Different biochemical components can possess differentstable isotope values

2. Structurally similar biochemical components of ecologicalmaterials can derive from a range of sources potentiallyexhibiting different signatures

3. Biogenic organic matter can change in chemical composition4. Reveals contributors mediating processes that would

otherwise be masked by in bulk5. Biomarkers together with compound specific isotopes

information on biological processes6. Biochemical components posses significantly different

turnover times7. 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

HydrolysisDerivatization

Free Lipids

ChromatographyDerivatization

Extraction

Vaporize!

Why so much preparation?Most compounds of interest must be

modified, usually of compoundscontaining polar functional groups, toenhance their volatility prior toGC/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 patternsof 13C signatures

Tool:New approach for tracing amino acid exchange in

symbiotic and trophic relationships

Lysine and Leucineexhibited significantdifferences

Phenylalanine leastvariable among taxa

Differences in amino acid 13Cvalues between the three mostinformative essential amino acids

Significant difference ofnon-normalized essentialamino acids, distinctisotope clusters

Lichen identified as fungi, whatrole is each organism playingbiochemically?

Does mostly well identifyingwhat the insects were eating

To Summarize:

•13C fingerprinting of amino acids could provide as apowerful in situ assay of amino acid sources interrestrial ecosystems in

-identifying the primary contributors of aminoacids in animals-understanding symbiotic associations betweenanimals and microorganisms

•Greatest accuracy is from the essential amino acidsmeasured based on their more complex biosyntheticpathways

Frolicking for food!

Carbon values getheavier fromsouth to north by6 per mil

Phe preservesbulk isotopevalue

Phe, Lys, Argstrong correlationto bulk alonglatitude

The Study: Diets formulated for Pigs to contain 20% protein and widerange in δ13C values

The Idea:Relationship b/w tissuebiochemical compounds anddiet δ13C values

Relationship b/w δ13Cvalues of bone collagen andits 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 fattyacids

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

δ13C values of non-essential amino acids were distributed across 10‰, reflectingdifferences 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 δ13C 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 theseamino 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 wholecollagen

• The δ13C values of non-essential amino acids,alanine and glutamate, from bone collagen correlatewell with whole diet

• The essential amino acids leucine and phenyalanineshowed little isotopic fractionation between diet andbone collagen

Fatty Acid Routing(Jim et al. 2003 Lipid)

Those damnrats!

Fatty Acid Routing(Jim et al. 2003 Lipid)

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

Oh Dear God!Not the ratsagain.

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

C3P/C4E

C4P/C3E

Amino Acid Routing(Jim et al. 2006 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 orleucine), or amino acids that behave as if they are essential(proline).

4. Routing between dietary protein and bone protein issubstantial for animals on protein-rich (20%) diets. It hasnot been tested for animals on lower protein diets.

5. Lipid routing is also dependent on the concentration of theparticular lipid in the diet.