A chloroformate based method for measuring rapidly and stably the δ¹⁵N values of glutamine: implications to position-specific isotope analysis of polynitrogenous amino acids for biogeochemical studies

抄録

The analysis of stable nitrogen isotope ratios of amino acids (δ¹⁵N_AA) has been employed as a powerful tool to estimate the trophic position of organisms and to illustrate the trophic relationship among them in diverse ecosystems. However, applicability of this analysis is somewhat doubtful in the estimation of the trophic position of marine mammals. Unusual small values of the trophic elevation in the δ¹⁵N_AA value have been apparently expected for marine mammals in previous studies, which may be affected by isotopic alteration during the ammonia recycling through glutamate - glutamine exchange associated with the urea-excretion. In the present study, we developed a chloroformate based method (to form isopropoxy isopropyl ester derivative) for separately measuring the δ¹⁵N values of two amino groups within a single authentic glutamine molecule, as a first step accurately to evaluate the isotopic alteration during the ammonia recycling in wild animals. The isopropyl derivative of glutamine used in the present study can be preserved for longer than 47 days at −20ºC (or for up to 5 days at room temperature), unlike methyl and ethyl derivatives used in previous studies are quickly degraded within several days even at −20ºC. Moreover, together with the hydrolysis of glutamine to form glutamate, the δ¹⁵N values of two amino groups within a single glutamine were measured. Thus, the chloroformate based method developed can be useful to compare the δ¹⁵N values between two amino groups within a single glutamine molecule, and will provide further evaluation of the isotopic alteration on the δ¹⁵N_AA values during the urea-excretion and related processes in marine mammals.