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Carbon Isotopes in Individual Compounds. 03 February 2010. GOOD CHROMATOGRAPH SEPARATION. Amino Acids - 13 C signatures. Fatty Acids - lipid biomarkers. What Compounds?. Diane’s Diagram: Follow the Carbon .

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Presentation Transcript
what compounds
Amino Acids - 13C signatures

Fatty Acids - lipid biomarkers

What Compounds?
background fractionation information for you comprehending pleasure
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
Compound-specific isotopes are useful
  • Different biochemical components can possess different stable isotope values
  • Structurally similar biochemical components of ecological materials can derive from a range of sources potentially exhibiting different signatures
  • Biogenic organic matter can change in chemical composition
  • Reveals contributors mediating processes that would otherwise be masked by in bulk
  • Biomarkers together with compound specific isotopes information on biological processes
  • Biochemical components posses significantly different turnover times
  • Kinetic fractionation can only be determined at the level of the biochemical component and specific pathway
slide7
Before we can burn our samples up, preparation for compound-specific stable isotopes via GC/C/IRMS follows:

Sample

Extraction

Total Lipid Extract

Residue

Chromatography

Derivatization

Hydrolysis

Derivatization

Free Lipids

Monosaccharides

&

Amino Acids

Vaporize!

why so much preparation
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
Applications
  • Fingerprinting
  • Nutritional Linkages
  • Biomarkers
slide10

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

slide11

Lysine and Leucine exhibited significant differences

Phenylalanine least variable among taxa

slide12

Significant difference of non-normalized essential amino acids, distinct isotope clusters

Differences in amino acid 13C values between the three most informative essential amino acids

slide13

Lichen identified as fungi, what role is each organism playing biochemically?

Does mostly well identifying what the insects were eating

slide14

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
slide18

Phe preserves bulk isotope value

Phe, Lys, Arg strong correlation to bulk along latitude

slide19

(2003)

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
slide20

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

slide21

Result for pig on diet 3

Pork Fat

Non-essential fatty acids

correlated with whole diet values

(0.98<r2<0.99). Better than correlation

with dietary fatty acid

Essential

FA

Good correlation b/w cholesterol

and whole diet d13C values (r2 = 0.81)

slide22

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

slide23

13Cvalues 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.

slide24

Amino Acid-Diet Correlations

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.

to summarize
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
slide26

Fatty Acid Routing

(Jim et al. 2003 Lipid)

Those damn

rats!

slide27

Fatty Acid Routing

(Jim et al. 2003 Lipid)

slide28

Amino Acid Routing

(Jim et al. 2006 British J. of Nutrition)

Oh Dear God!

Not the rats

again.

slide29

C4P/C3E

C3P/C4E

Amino Acid Routing

(Jim et al. 2006 British J. of Nutrition)

slide30

Amino Acid Routing

(Jim et al. 2006 British J. of Nutrition)

the bottom line s
The Bottom Line(s):
  • If you want to measure the isotopic composition of bulk diet, use apatite, cholestrol, alanine or glutamate.
  • If you want to measure the isotopic composition of the lipid component of diet, measure essential fatty acids (e.g. Linoleic Acid).
  • 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).
  • 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.
  • Lipid routing is also dependent on the concentration of the particular lipid in the diet.