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Nitrogen Isotopes in Animals: Systematics
Timothy Lambert (adapted from 2007 presenter)Earth 229, Winter 2010
http://www.zuropak.com/photogallery/2008-favourites/slides/Yellow-rumped-Warbler-214.html
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)
amino acid pool•throughout body•significant mixing
protein turnover•Some proteins turnover faster than others
Enzymes break dietary protein into amino acids.
N Cycle(human)
Fate #2: Metabolized-N excreted as ammonia/urea-C skeleton converted to fat/glucose
Fate #1: Body protein
Amino acid pool
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
http://molbio.med.miami.edu/Medical/Werner/Pdf-Files/MBL%2039.pdf
α-keto acids Amino acids
Transfer of amine group
First transfer amine group to carrierKetoglutarate → Glutamate
Deamination
Then deaminate Glutamateto produce ammonia
in liver or kidney
Deamination fractionates N!
Ammonia product is depleted in 15N.
The Urea Cycle
• Requires CO2, NH3, and aspartate
•Glutamate = source of NH3 and aspartate
•Glutamate fractionates N (14N is preferentially reacted)
N in the BodyKinetic Fractionation, Open System
Urea(depleted in 15N)
Hair, milk, feces…
Diet
-6 per mil
Body(enriched in 15N)
Animals are enriched in 15N relative to diet because urea is depleted in 15N relative to body.
Diet = Excretion ProductsNo 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
Llama Study (Sponheimer et al. 2003)
Amino Acids in Trophic Ecology
• Bimodal 15N distribution• Source amino acids (essential)• Trophic amino acids (nonessential)
Martinez del Rio et al. 2009
What causes variability in N
isotope fractionation?
Low vs. high proteinHerbivore vs. carnivore
↑ quality decreases fractionation
↑ quantity increases fractionation
1. Protein in the Diet
Koch 2007
Effects of elemental composition on the incorporation of dietary nitrogen and carbon
isotopic signatures in an omnivorous songbird.(Pearson et al., 2003)
Yellow-rumped warbler
• High vs. low protein diets
• Food: Bananas and insects in varying proportions
•Sampling of mass, blood, feathers
Diets: %Insect, Isotopes, & Concentrations
Attempted to create diets along a linear continuum of increasinga) isotopic signature (didn’t quite work for 15N)b) elemental concentration
by increasing the % insect protein in diet
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)
Diets: %Insect, Isotopes, & Concentrations
Turnover Rates: Half-life Plasma & Blood
Half-life estimates plasma: δ13C 0.4-0.7 days δ15N: 0.5-1.7 days
Half-life whole blood: δ13C ~4-6 days (diet 1=33 days!) δ15N 7.45-27.7 daysWhole blood is variable!
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
High Protein = Large FractionationDue to larger loss of
15N-depleted urea
%N in diet
Results1. Diet: Linear relationship
between elemental concentration and fractionation factor.
2. Tissue: Discrimination and turnover rates vary.
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
What about C3 vs C4 grasses?
• Both C3 and C4 plants show decreased δ15N with increased water availability.
• δ13C of bone collagen as proxy
• Negative and weak relationship• Lower δ15N in C4 plants (1.1‰)
C4C3
C4C3
A: No! Can’t explain isotope trend by differences in C3:C4.
Q: Can dietary C3:C4 explain the δ15N vs. water availability trend?
• 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
Summary
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
Body Mass Lost
Urea
Body
6‰
• Generalization: Starvation increases 15N of tissue.
• Inconsistent results (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)
• Solution: Multiple compartments
Kinetic Fractionation, Closed System
Urea(depleted in 15N)
Hair, milk, feces…
Diet-6 per mil
Body(enriched in 15N)
Nitrogen Balance: Starvation
Body Mass Lost
Urea
Body
6‰
• Generalization: Starvation increases 15N of tissue.
• Inconsistent results (Martinez del Rio et al.)
• Assumes well-mixed pool
• 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
Kinetic Fractionation, Closed System