Tuesday, October 1, 2019

Hydrogen isotopes in Amino Acids tell about an organism’s food and water

Student Patrick Griffin at the Geophysical Lab, circa 2008

In 2008, I received a grant from the W. M. Keck Foundation that provided funding for a new isotope system and the ability to measure the hydrogen isotopes of individual compounds. All of the papers published with hydrogen isotopes in single compounds were focused on lipids (i.e. fats) or hydrocarbons from plants, microbes, and sediments. I wanted to try my hand at measuring them in amino acids, the building blocks of proteins. I wanted to know more about how hydrogen isotopes in animal tissues related to geographic location and diet.
Biochemical Pathways of Amino acid synthesis--complicated!

         I began this work with postdoc Seth Newsome and a graduate student, Patrick Griffin. Patrick, now a student at Indiana University, was training in Steelie’s lab as a microbiologist. Famous for his outrageous, funny, off-color one-liners, Patrick has an interest in astrobiology, broadly, and microbiology, specifically. He kept unusual work hours, often working into the wee hours of the night at the lab bench, harvesting microbes, setting up experiments, and reading scientific papers. I didn’t necessarily approve of this behavior, but Patrick had glimmers of brilliance that we hoped to nurture.
         Although hydrogen is one of the major elements in living organisms, it is not fully understood how organisms incorporate hydrogen from their surroundings into the biochemical compounds that comprise them.  Living systems derive their hydrogen from two primary sources--food and water---and a full understanding of how hydrogen moves through an organism or an ecosystem must consider studies of both sources.  To determine how hydrogen from water is incorporated into living matter, Patrick cultured the common bacteria E. coli in nutrient broths composed of waters of differing isotopic compositions. To determine the influence of the dietary source on hydrogen, we designed experiments with a glucose-based culture broth, as well as a one based on the protein digest, tryptone. Together with Patrick and Seth Newsome, our experiments showed that roughly 25-35% of the hydrogen in E. coli cells originated from the water in which it is grown, but the remainder transfers directly from the diet to cellular biomass (Fogel et al., 2016). It made sense that more hydrogen originates from media water in E. coli grown on the glucose-based medium than from organisms grown on tryptone, because the bacteria had to synthesize more of its cellular biomass with just glucose as its “food”. 
 
         Our first set of measurements we made with hydrogen in amino acids was in the single protein, tryptone, that we used to feed the E. coli. We were surprised to find that the hydrogen isotopes in the different amino acids varied by a huge amount--nearly the entire natural range of hydrogen isotopic compositions of living organisms on Earth. Amino acids extracted from our E. coli cultures showed even more extreme variations with some amino acids having identical isotopic compositions to those in tryptone and others being enriched or depleted in the heavier isotope of hydrogen. 
         Our results from these simple experiments with E. coli grown on tryptone provided the basis for examining complex organisms, such as birds, mice, and fish.   Tryptone is essentially food for the microbes, and our data showed that the more complex branch-chained amino acids (e.g., isoleucine, valine, and leucine) could be incorporated, or routed, directly into cellular protein. Less than 10% of the hydrogen in these amino acids was sourced from water. Mathematical models suggested that ~40-50% of the hydrogen in one of the simplest amino acids, alanine, was sourced from media water. We had discovered that by analyzing hydrogen isotope values in a suite of amino acids, we could tell what an animal was eating as well as where its drinking water came from.
         We went on to measure the hydrogen isotopes of amino acids in bird feathers. Using some of the Black-throated Blue warbler samples measured previously, we found that there were similar patterns of isotope discrimination among the hydrogen isotopes of individual amino acids similar to those in the bacteria. Amino acid isotope values from feathers in warblers from North Carolina showed that these birds lived in a more southern climate than those amino acids in feathers from warblers collected in Ontario, Canada, showing the influence of geographical location. 
Nathan Wolf mixing up fish diets

         Seth Newsome, then at the University of Wyoming, and his grad student Nathan Wolf carried out a defined diet experiment in which tilapia fish (Oreochromis niloticus) were grown in tanks where the hydrogen isotopes of environmental water and the isotopes of each of the major diet macromolecules of carbohydrates, proteins, and fats was known. We had nine experimental tanks: 3 different isotopically-flavored diets, each tested with three different isotopically labeled waters. About 25% of the hydrogen in fish tissues originated from water. Of the remainder, 34-44% came from dietary protein, 25-30% from carbohydrates, and <1% from lipids (Newsome et al., 2017).
         The individual amino acid data was more complicated. Interpreting the hydrogen isotope composition of individual amino acids required us to measure the carbon isotope composition of the amino acids as well to tease apart our data. Since corn is a C4 plant, and the casein was derived from C3-based milk protein, we could use the carbon isotope values to roughly estimate which proportion of the fish’s amino acids came from C4 plants and which from C3 based casein. We anticipated that the simplest amino acids would show the strongest relationship to the isotopic composition of the water in the tank. Based on carbon incorporation proportions we expected the more complicated amino acids, including those essential for the fish, to be more influenced by the hydrogen isotopes of food rather than water.
The hydrogen isotopes of the tank water significantly influenced only alanine, glycine, and serine—three simple amino acids-- for all of the nine experimental tanks. The remainder of the amino acids had little to no isotope relationship between water and the amino acid. We then linked the carbon isotope data with the hydrogen isotope data to understand and possibly tease out the source of hydrogen to all of the amino acids. What is striking about our results is the fact that very few of the amino acids in the tilapia came from only one dietary carbon or hydrogen source. Interestingly, the origin of essential amino acids was as variable as the non-essential amino acids. Microbial synthesis in the gut must have been a critical component supporting protein synthesis in the fish.  
Tilapia experimental tanks

Publishing this data has been put on hold, because pretty much all of our results point to a greater complexity than we’d originally thought. Seth’s undergraduate student Mariel Curras did another experiment, this time changing the amount of protein in the experimental animal’s diet. He grew some mice, and my student Bobby Nakamoto analyzed the isotopes in the amino acids. We’ve submitted a nice paper explaining the very basics and hopefully, when that manuscript is accepted, it will open up the floodgates for all of the data we’re been collecting for the past 6 years! Scientific research often begins with a simple premise or hypothesis but more often than not, the results lead you in a different, unanticipated direction. In my career, my best work has followed this pathway.

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