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

Abstract

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 H,O, C, andN 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 N andC from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO and N 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.

Position‐specific 13C/12C analysis of amino acid carboxyl groups – automated flow‐injection analysis based on reaction with ninhydrin

“…A number of previous studies have described the stable carbon isotopic analysis of individual amino acids, typically derived by hydrolysis of proteins, from various biological sources . This compound‐specific isotope analysis (CSIA) is achieved either by gas chromatography of derivatised amino acids coupled with isotope ratio mass spectrometry (IRMS) or by high‐pressure liquid chromatography (HPLC) of underivatised amino acids coupled to IRMS using chemical oxidation to convert the carbon present in organic compounds into CO 2 (LC/CO/IRMS) . The use of LC/CO/IRMS for CSIA is now a relatively mature technique, especially with regard to the separation of underivatised amino acids using mixed‐mode HPLC columns and aqueous mobile phases .…”

“…[4][5][6][7][8][9] This compound-specific isotope analysis (CSIA) is achieved either by gas chromatography of --------------------------------------------------------------------------------------------------------------------------------derivatised amino acids coupled with isotope ratio mass spectrometry (IRMS) or by high-pressure liquid chromatography (HPLC) of underivatised amino acids coupled to IRMS using chemical oxidation to convert the carbon present in organic compounds into CO 2 (LC/CO/IRMS). 10,11 The use of LC/CO/IRMS for CSIA is now a relatively mature technique, especially with regard to the separation of underivatised amino acids using mixed-mode HPLC columns and aqueous mobile phases. 12 Recent research has shown that the CSIA δ 13 C values of underivatised amino acids, derived from hydrolysis of hair samples, can indicate metabolic trends associated with factors such as age and obesity in humans.…”

The study of isotopic enrichment of water in human plasma and erythrocyte

“…It is now known that distributions of 13 C and 15 N stable isotopes in metabolites are, in part, caused by isotope effects. 8 Tea et al showed that the natural isotope abundance of 13 C and 15 N exhibit isotopic differences in healthy and breast cancer biopsy tissues. 9 Such isotopic differences were also observed with water.…”

“…We speculate the hypo-isotopic enrichment of water in ESRD may be a consequence of upregulated AQP1 expression in the erythrocyte, or the structure of AQP1 could be a deficiency. In addition, water isotope effects 46 might be another factor that may associate with kidney tissues in conditions of abnormal metabolisms or inflammations 8,47 that may also lead to hypo-isotopic water in ESRD.…”

Metabolic Flux Analysis—Linking Isotope Labeling and Metabolic Fluxes

“…This means the enzymes and transporters do not discriminate between unlabeled and labeled substrates. In another word, there is no kinetic isotope effect (KIE) [68][69][70][71][72]. As Liuni et al [69] has shown, 12 C/ 13 C KIE is typically < 1.1 so this assumption is largely valid in carbon labeling experiments.…”