Abstract
The carbon isotopic compositions of amino acids are increasingly measured to characterize diets and metabolic response to diets. We report a new high-resolution system to measure the stable carbon isotopic composition of carboxyl atoms within amino acids. The automated system used HPLC to separate amino acids followed by addition of ninhydrin for decarboxylation and transfer of the evolved CO2 to a stable isotope ratio mass spectrometer for δ13CCARBOXYL measurement. The ninhydrin reaction was conducted at acidic pH (1.5) and elevated temperature (160 oC) giving yields close to 100% for most common amino acids. Eight mammalian keratin samples from herbivores (kudu and caribou), omnivores (humans) and carnivores (bowhead and humpback zooplanktivorous whales) were analysed with this new system. The data provide an initial calibration of reference materials to be used in studies of this type and is the first report of carboxyl carbon isotope distributions in mammals. Results showed widespread 13C enrichments in both essential and non-essential amino acid carboxyl groups, likely linked to decarboxylation of amino acids during normal metabolism. Analyses of non-essential amino acid isotope profiles showed (1) consistent and general taxon-level metabolic differences between the herbivore, human and whale samples, (2) marked differences among individual humans, ruminants and whales (3) evidence for gluconeogenesis in the wildlife samples, and (4) extensive 13C enrichment likely associated with fasting in the humpback whale sample. Future mammalian research related to the metabolism of growth, reproduction, aging and disease may benefit from using this technique. Values obtained for internationally available samples USGS42 and USGS43 (Tibetan and Indian human hair) provide a first characterization of reference materials for δ13CCARBOXYL profiles.
Highlights
Carbon stable isotopes in bulk tissues such as muscle have been widely used in studies of human and animal diets and migration, tracing carbon flow from source inputs and regions
We have recently developed the first automated position-specific isotope analysis (PSIA) system, using a well characterized ninhydrin chemistry to decarboxylate amino acids for δ13CCARBOXYL isotopic analysis [9]
When combined with an HPLC eluent, this reagent caused three amino acids (Glu, Lys and Cys) partially to decompose and “overyield” CO2 with isotope values that did not solely reflect the isotopic composition of the carboxyl group. To overcome this problem we developed a ninhydrin reagent that functioned at acidic pH and did not cause glutamic acid (Glu) to decompose
Summary
Carbon stable isotopes in bulk tissues such as muscle have been widely used in studies of human and animal diets and migration, tracing carbon flow from source inputs and regions. Studies showed that in some cases, carbon isotopes are sensitive to metabolic variation so that, for example, small (1 to 2 ‰) 13C enrichments were shown in the 1970s to characterize cancerous versus normal tissues [1,2]. This metabolic “fingerprint” appears more pronounced when the isotope techniques are employed at higher resolutions, e.g. in studies of specific compound classes such as lipids [3], specific molecules such as amino acids [4] or specific positions within molecules such as carboxyl positions of peptides and amino acids [5,6]. Having obtained the first PSIA data for a suite of RMs it became possible to test an isotope model using comparative analyses of the amino acids derived from eight mammalian proteins
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