Reconstructing Diet Histories and Migration Pathways of Bluefin Tuna from Compound-Specific Stable Isotope Analyses of
Scales and Bone 
PI: Simon R. Thorrold and Leah A. Houghton
PROJECT DESCRIPTION:
We proposed to develop and apply isotope ratio monitoring gas chromatography mass spectrometry to the analysis of
compound-specific d13C and d15N values in amino acids, and conventional isotope ratio mass spectrometry to measure
dH and d18O , from scales and bone of Atlantic bluefin tuna (ABT) . Our focus was on scales, in particular, because
they can be collected without sacrificing the fish. This is critical if we are to correlate migratory pathways
determined using conventional and archival tagging approaches with independent information on natal origins of
individual fish determined from geochemical signatures in tissues. We plan to develop a technique to provide information
on dietary history and natal origins of an individual fish, based on dH, d13C , d15N and d18O, which could then be outfitted
with an archival tag and released.
PROGRESS thru 10/31/2006:
We have developed a method for measuring d 13 C in amino acids from fish scales and vertebrae. We initially extracted and
hydrolyzed individual amino acids from proteins that were then analyzed for d 13 C using irm-GC/MS. However, amino acids
must be derivitized to volatile compounds prior to analysis, a process that adds carbon atoms to the original molecule.
In the case of d 13 C measurements, derivatization isotopically biases the derivative carbon over the amino acids to differing
extents depending on the carbon skeleton of the amino acids. And critically for our application, irm-GC/MS has proved unsuitable
for quantifying isotope ratios where analyte quantities are limited.
Given these problems, we have focused our attention on the moving-wire interface that is used to introduce CO2 gas, a product of the combustion of organic compounds, to an isotope ratio mass spectrometer. Briefly, hydrolyzed amino acids are separated and purified using high performance liquid chromatography (HPLC). One microliter droplets of water containing individual amino acids are deposited on a moving nickel wire every 30 to 40 seconds and are transported through a heating element to evaporate the water. From there the sample is carried into a combustion furnace where the nonvolatile or semi volatile compounds are quantitatively combusted to CO 2 , H 2 O, and N 2 + NO x gas. A small portion of these gases is diverted in a stream of helium through a membrane dryer to remove H 2 O, and is then carried into the mass spectrometer.
Preliminary Data
We have focused initially on determining stable carbon isotopes in specific amino acids using moving wire irm-MS. Our first step was
to demonstrate that we would recover d 13 C values from amino acids, separated and purified using HPLC, with the moving wire interface.
Figure 1. Compound specific d13C values for glycine, serine and glutamine standards analyzed using
conventional irm-MS (squares) and moving wire irm-MS (circles) before (closed symbols) and after (open symbols)
passing the compounds through a separation and purification process required for scale and vertebra samples.
Preliminary results are very promising (Figure 1). We have successfully separated and purified the three most common amino acids found in ABT scales and vertebrae (glycine, serine and glutamine) using HPLC. More importantly, we have shown that this process doesn’t induce significant carbon isotope fractionation. Finally, we have demonstrated that, by using the moving wire interface, we can accurately quantify d13C values from nanogram levels of carbon in the individual amino acids. Carbon isotope values for each of the three amino acid match well with values obtained by an outside laboratory at UC Davis using conventional irm-MS.
PLANS FOR THE NEXT SIX MONTHS TO YEAR:
IIn the next 6 months we plan initially to modify the moving wire interface so that d13C and d15N values can be obtained from glycine, serine and glutamine. We will then focus our attention on scales from 1+ ABTs collected in the eastern Atlantic (Bay of Biscay) and the Northwest Atlantic (Gulf of Maine). We will analyze scale samples for bulk d18 using conventional irm-MS, and for d13C and d15N in glycine, serine and glutamine that will be separated and purified using HPLC and analyzed using moving wire irm-MS. Isotope-isotope plots will then be used to determine if it is possible to identify natal origins of ABTs based on isotope signatures in scales.
