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.

Model for 15N in the 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)

~3 per mil for every trophic level

Trophic Ecology

Amino Acids in Trophic Ecology

• Bimodal 15N distribution• Source amino acids (essential)• Trophic amino acids (nonessential)

Martinez del Rio et al. 2009

Trophic Ecology

But lots of variation.

Why?

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

↑ %N

↑ uric acid w/ ↑ 14N

in

out

↑ tissueδ15N

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

data

=

+

Results

4.74‰ to 4.79 ‰ enrichment

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

Summary

1. Animals retain 15N, excreting 14N preferentially (~6‰)1. Useful in trophic ecology

2. Differences between source and trophic amino acids

2. Discrimination affected by:1. Protein quality and quantity

2. Aridity affects food chain, not physiology

3. Starvation increases δ15N