Nitrogen Isotopes in Animals:
Systematics
http://www.zuropak.com/photogallery/2008-favourites/slides/Yellow-rumped-Warbler-214.html
Timothy Lambert (adapted from 2007 presenter)
Earth 229, Winter 2010
Roadmap
Why are animals enriched in 15N?
1. Physiology
2. Model
What causes variability in this discrimination?
1. Dietary protein
2. Environmental controls
3. Growth vs. catabolism
N Cycle
(human)
Enzymes break
dietary protein into
amino acids.
protein turnover
•Some proteins
turnover faster than
others
amino acid pool
•throughout body
•significant
mixing
N Cycle
(human)
Amino acid
pool
Fate #1:
Body protein
Fate #2:
Metabolized
-N excreted as
ammonia/urea
-C skeleton
converted to
fat/glucose
Nitrogen excretion
Ammonia NH3
•Simplest form, but toxic
•Bony fish, amphibian larvae
Urea (NH2)2CO
•More complex but still toxic
•Mammals, some herps (frogs),
cartilagenous fish
Uric Acid C5H4N4O3
•Least toxic
•Birds, insects
Water efficiency
Moving N in the body:
Transamination
α-keto acids
Amino acids
Transfer of amine group
http://molbio.med.miami.edu/Medical/Werner/Pdf-Files/MBL%2039.pdf
Deamination
First transfer amine group to carrier
Ketoglutarate → Glutamate
Deamination fractionates N!
Ammonia product is
depleted in 15N.
in liver or kidney
Then deaminate Glutamate
to produce ammonia
The Urea Cycle
• Requires CO2, NH3, and
aspartate
•Glutamate = source of NH3 and
aspartate
•Glutamate fractionates N
(14N is preferentially reacted)
N in the Body
Kinetic Fractionation, Open System
-6 per mil
Diet
Body
(enriched in 15N)
Urea
(depleted in 15N)
Hair, milk,
feces…
Animals are enriched in 15N relative to diet because
urea is depleted in 15N relative to body.
Model for 15N in the Body
Llama Study (Sponheimer et al. 2003)
Diet = Excretion Products
No significant depletion of waste
products relative to diet
• Claimed it contradicted theory
of enrichment due to depleted
15N in urea
Explanations?
• Urea is not urine (contains
creatinine, etc.)
• High protein diet
• In equilibrium, inputs = summer
outputs (always!)
Body tissue is elevated relative
to diet, urea is depleted relative
to body.
Pretty
llama
pictures
Trophic
Ecology
~3 per mil for
every trophic
level
Amino Acids in Trophic Ecology
Martinez del Rio et al. 2009
• Bimodal 15N distribution
• Source amino acids
(essential)
• Trophic amino acids
(nonessential)
Trophic
Ecology
But lots of variation.
Why?
What causes
variability in N
isotope
fractionation?
1. Protein in the Diet
Low vs. high protein
Herbivore vs. carnivore
↑ quality decreases
fractionation
↑ quantity increases
fractionation
Koch 2007
Effects of elemental composition on the incorporation
of dietary nitrogen and carbon isotopic signatures in
an omnivorous songbird.
(Pearson et al., 2003)
• High vs. low protein
diets
• Food: Bananas and
insects in varying
proportions
•Sampling of mass,
blood, feathers
Yellow-rumped warbler
Diets: %Insect, Isotopes, &
Concentrations
Attempted to create diets along a linear continuum of increasing
a) isotopic signature (didn’t quite work for 15N)
b) elemental concentration
by increasing the % insect protein in diet
Diets: %Insect, Isotopes, &
Concentrations
Only 0.12‰ difference in δ15N values among diets.
Diet containing most insects did not have highest δ15N value
(diet with lowest proportion of insects did not have the lowest δ15N value)
Turnover Rates: Half-life Plasma & Blood
Half-life estimates plasma: δ13C 0.4-0.7 days
Half-life whole blood:
Whole blood is variable!
δ13C ~4-6 days (diet 1=33 days!)
δ15N: 0.5-1.7 days
δ15N 7.45-27.7 days
Discrimination: Plasma, Feather, and Blood
15N values plasma & whole blood
enriched 1.7 to 3.0‰
“Apparent” fractionation factor for
feathers
15N enriched (3.2-3.6‰)
Fractionation factors
increased linearly with
elemental concentration
in diet for N
↑ %N
in
↑ tissue
δ15N
out
↑ uric acid w/ ↑ 14N
High Protein = Large Fractionation
Due to larger loss of
15N-depleted urea
Results
1. Diet:
Linear relationship
between elemental
concentration and
fractionation factor.
2. Tissue:
Discrimination and
turnover rates vary.
%N in diet
Solution: Concentration
dependent, multi-compartment
mixing models
What causes variability in N isotope
fractionation?
1. High vs. low protein diets
2. Water availability?
Correlation between
bone collagen 15N and
aridity
Why does ↓ Water availability
↑ δ15N in Animal Tissue?
1. Diet/plant δ15N increases in arid habitats
– ↑ aridity = larger relative 14N-rich gas loss (soil denitrification)
2. Metabolic enrichment theories
– ↑ urine excreted is isotopically heavy (rich in δ15N) (Ambrose & DeNiro 1986,
Sealy 1987)
– ↓ protein diets in arid regions promote urea recycling for N
Kangaroo metabolism does not cause the relationship
between bone collagen δ15N and water availability
(Murphy & Bowman, 2006)
Motivating question: Can ↑ δ15N be explained by herbivore diet alone?
Methods
Big study!
• 779 road killed roos
– 15N, 13C of bone collagen
– Macropus spp, grazers, small ranges
• 173 grass collections
– 3-4 primary spp at each site, 15N
• Water Availability Index
+
=
data
Results
4.74‰ to 4.79 ‰ enrichment
What about C3 vs C4 grasses?
Q: Can dietary C3:C4 explain the δ15N vs. water availability trend?
• δ13C of bone collagen as proxy
• Negative and weak relationship
• Lower δ15N in C4 plants
(1.1‰)
• Both C3 and C4 plants show
decreased δ15N with increased
water availability.
A: No! Can’t explain isotope
trend by differences in C3:C4.
C3
C4
C3
C4
Summary
•
Strong negative relationship of herbivore δ15N bone
collagen and water availability.
•
Near identical negative pattern of δ15N in grass and
kangaroo bone collagen with water availability
•
Plant δ15N is main cause, with no change in metabolism
• Huge support for historic trophic ecology and past
climate change data that rely on direct
relationship between herbivores and plants which
not confounded by animal metabolism
What causes variability in N isotope
fractionation?
1. High vs. low protein diets
2. No aridity effects (but
understand environmental
effects on 15N of the food
chain’s base!)
3. Starvation!
Growth vs. catabolism
Nitrogen Balance: Starvation
Kinetic Fractionation, Closed System
• Generalization: Starvation
increases 15N of tissue.
-6 per mil
Diet
Body
Urea
(depleted in 15N)
(enriched in 15N)
Hair,
milk,
feces…
Body
• Inconsistent results
6‰ Urea
(Martinez del Rio et al.)
• Assumes well-mixed pool
• Reality: tissues vary in growth
Some continue protein synthesis (e.g. splanchnic
organs, liver), others shut off (e.g. muscle)
Body Mass Lost
• Solution: Multiple compartments
Nitrogen Balance: Starvation
Kinetic Fractionation, Closed System
• Generalization: Starvation
increases 15N of tissue.
• Inconsistent results
Body
(Martinez del Rio et al.)
• Assumes well-mixed pool
6‰ Urea
• Reality: tissues vary in growth
Amino acid pool becomes enriched;
Some tissues continue protein synthesis (e.g.
liver), others shut off (e.g. muscle)
• Solution: Multiple compartments
Body Mass Lost
Summary
1.
2.
Animals retain 15N, excreting 14N preferentially (~6‰)
1.
Useful in trophic ecology
2.
Differences between source and trophic amino acids
Discrimination affected by:
1.
Protein quality and quantity
2.
Aridity affects food chain, not physiology
3.
Starvation increases δ15N