Saturday, August 3, 2019

Nitrogen Isotope Biogeochemistry

         In September 1982, my first postdoctoral fellow, Dr. Steve Macko, arrived from UTMSI. He brought with him not only boundless enthusiasm and a plethora of ideas, but the technical wherewithal to measure nitrogen isotopes in organic matter.  Macko quickly set up sealed tube methods for analyzing organic matter at the Geophysical Lab. Steve came with a host of marine science projects, collaborators from around the globe, as well as a zeal to learn as much as he could. His main project was to culture microorganisms, then separate and purify amino acids for isotopic analysis, similar to the original work of Abelson and Hoering (1961).
         This work involved all three staff members in biogeochemistry: me for culturing and biological aspects; Ed Hare for separation of the amino acids; and Tom Hoering for IRMS and other isotope support.  To carry out these experiments, I needed to ramp up my culturing ability in order to grow the substantial amounts of material needed for amino acid isotope analyses. Ed Hare needed to produce grams of the special ion exchange resin required to separate milligram quantities of amino acids. Hare and Macko worked hard in order to have the amino acid analyzers going full time to assay compounds as they eluted from the separation column. We used two IRMS instruments for the isotope measurements: a newer Nuclide 6” IRMS for carbon and for nitrogen a much older IRMS constructed at the Geophysical Laboratory by Hoering and others.
         It took several weeks to grow up the bacterial and algal cultures. We analyzed the bulk isotope values, then hydrolyzed the sample to produce hydrochloride salts of amino acids.  Individual amino acids in the hydrolysate were separated using column chromatography which took 24 to 48 hours to complete.  The fact that amino acids do not separate at regular or specific time intervals was a problem. The trick was to use a separate liquid chromatography system to determine when each amino acid started to elute and when it finished eluting.  In this way we could to isolate and capture each amino acid in its entirety but separate it from other amino acids in the sample. Fractions were collected from the eluant every few minutes. After analysis via liquid chromatography, fractions were pooled to capture the entire amino acid eluting from the column.  This result resulted in about 13-15 samples, each containing the hydrochloride salts of a single amino acid. The samples were then dried by evaporation, and the amino acids were weighed into quartz tubes, sealed, and combusted. The final steps were purification of gases and mass spectrometric analysis.
         This was an elaborate and slow process where each sample took a total of about 4 to 6 weeks from start to finish, depending on whether everything was working in tip-top order (Macko et al., 1987). We were the only research group in the world doing these detailed, complicated analyses. The work we did in the early 1980s was twenty years ahead of its time. Today, experiments of this sort can be accomplished with much less material (10 mg), and by automated analysis, where five to ten samples can be measured for both nitrogen and carbon isotopes in a week. This laborious work established the framework we established in the 1980s was important for subsequent measurements of nitrogen isotopes in amino acids from animals, a critical method for studying the mechanisms that are required to study ecological trophic levels.
         The carbon isotope patterns of the amino acids in both the cyanobacteria and the Vibrio cultures were remarkably similar (Macko et al., 1987; Macko and Estep, 1985). More recently, scientists have used linear discriminant analyses to create isotopic “fingerprints” of primary producers (Larsen et al. 2009, 2012). These are helpful for determining the relative importance of different primary producers in fueling food webs, and for identifying dietary resources for animals (e.g., detritivores) for which traditional proxies are not useful. The isotope fractionation patterns we measured back in 1982 (and published formally five years later) were related to the biosynthetic pathways that created them. In subsequent reviews (e.g. Hayes, 2001), carbon isotope patterns were related to specific biosynthetic pathways: glycolysis, the TCA cycle, or branched-chain amino acid pathways. We also determined that the range in carbon isotope compositions of amino acids is almost twice as large in primary producers as it is in secondary consumers (i.e., bacteria). It was not until we extended these measurements to animal tissues that their power became evident (Hare and Estep, 1983) for distinguishing the input of essential amino acids originating from the base of the food chain.
         Another pivotal postdoctoral fellow from this time was Michael Engel. Engel and Macko became fast friends and colleagues, a relationship that has lasted over 30 years. Michael Engel and his major professor Bart Nagy at the University of Arizona measured an enantiomeric excess of some acids found in the Murchison meteorite (Engel and Nagy, 1982). Engel came to work with Ed Hare to learn as much as he could about amino acid analytical techniques. Macko and Engel published their first continuous flow paper in 1991 (Silfer et al., 1991), with multiple papers published in the coming decade on isotopic compositions of amino acids from meteorites, which confirmed Engel’s earlier assertion that the enantiomeric excess he measured and published with Nagy in 1982 was in fact correct. 
         While Engel and Macko were at the Geophysical Laboratory, we conducted experiments measuring nitrogen isotopic fractionation by the glutamate-aspartate transaminase (Macko et al., 1986). This was my second enzyme isotope fractionation experiment and for several reasons was much easier than working with Rubisco. First, we could purchase the enzyme directly from Sigma Chemical Company. The reactants (aspartate, glutamate, α-ketoglutarate and oxaloacetate) were all inexpensive and readily obtainable. We determined the isotope fractionation for the reaction from glutamate to aspartate, one of the most basic and important reactions in producing amino acids in all organisms. 
            In addition to my earlier work on compound specific amino acid studies, having the capability for measuring nitrogen, carbon, and hydrogen isotopes in my laboratory opened up the entire world of isotope biogeochemistry for me. Every experiment that we could think of was new, and the outcomes unknown. At that time, the literature on stable isotopes was so small that you knew each publication that came out, memorized its contents, and thought about it long and hard. The work on amino acids quickly progressed to investigating fossil collagen samples and lab grown animals, a field that was dormant for 20 years, before resurging as continuous flow methods became more robust.

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