Transcript Document

Carbon Isotopes in Individual
Compounds
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03 February 2010
GOOD CHROMATOGRAPH
SEPARATION
What Compounds?
Fatty Acids - lipid
biomarkers
Amino Acids - 13C signatures
Diane’s Diagram: Follow the Carbon
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Background & Fractionation Information
for you comprehending pleasure
The Idea:
• Mechanistic understanding of the biochemical factors
that underpin stable isotope signals
• Links biochemistry to stable isotope composition
Compound-specific isotopes
are useful
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2.
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7.
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
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
Monosaccharides
&
Amino Acids
Free Lipids
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
-NH2
Mechanism
tBDMS
Reagent
MTBSTFA
Product
Applications
 Fingerprinting
 Nutritional Linkages
 Biomarkers
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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
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Lysine and Leucine
exhibited significant
differences
Phenylalanine least
variable among taxa
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
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Lichen identified as fungi, what
role is each organism playing
biochemically?
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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
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Phe preserves
bulk isotope
value
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Phe, Lys, Arg
strong correlation
to bulk along
latitude
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(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
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<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)
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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.
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:
• 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)
Those damn
rats!
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Fatty Acid Routing
(Jim et al. 2003 Lipid)
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Amino Acid Routing
(Jim et al. 2006 British J. of Nutrition)
Oh Dear God!
Not the rats
again.
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Amino Acid Routing
(Jim et al. 2006 British J. of Nutrition)
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C4P/C3E
C3P/C4E
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Amino Acid Routing
(Jim et al. 2006 British J. of Nutrition)
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The Bottom Line(s):
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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.