Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Animal, and Human Tissues

The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of ²H, ¹⁸O, ¹³C, and ¹⁵N isotopes in metabolites. Therefore, compound-specific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for ¹⁵N and ¹³C from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO₂ and N₂ for IRMS analysis, derivatization and isotope effect measurements. It appears that despite the flexibility of instruments used for CSIA, there is no universal method simply because the chemical nature of metabolites of interest varies considerably. Also, CSIA methods are often limited by isotope effects in sample preparation or the addition of atoms from the derivatizing reagents, and this implies that corrections must be made to calculate a proper δ-value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application.