Chemical derivatization of classes of metabolites in biological systems for detection by NMR with enhanced sensitivity and resolution

Abstract

NMR spectroscopy is one of the two most important analytical tools used in metabolic profiling of biological systems due to its reproducibility and ability to provide spectroscopic information on a broad range of metabolites. 1H NMR analysis of complex biological samples combined with multivariate data analysis provides a well-established approach for a wide range of applications in metabolomics. In this dissertation, nuclei such as 13C and 31P are explored for improved NMR-based metabolic profiling. 13C NMR is not highly utilized in metabolomics due to issues of poor sensitivity and low natural abundance. The wide chemical shift range and reduced J-coupling are attractive features despite the low sensitivity. In a novel approach, this study demonstrates that chemical derivatization using 13C labeled reagents used to enhance the sensitivity and resolution of selected classes of metabolites in complex biological fluids. Amine containing metabolites were selectively detected from a complex mixture by derivatizing the metabolites with 13C labeled acetic anhydride. 1-D 13C and 2-D HSQC spectra of biological samples after reaction with 13C enriched acetic anhydride can be obtained in less than 10 min, and these spectra show dramatically improved sensitivity (∼100 fold) and resolution. This method was used in analyzing trace amounts of amino acids in serum and urine from patients diagnosed with inborn errors of metabolism and in tissue extracts. The 31P nucleus has a higher natural abundance and wider chemical shift range than the 13C NMR. Chemical derivatization of labile hydrogens in hydroxyl and carboxylic acid functionalities with 2-chloro-4,4,5,5-tetramethyldioxaphospholane ensured a good spectral separation upon derivatization as the products appeared in a different chemical environment and thus provided higher sensitivity and resolution. The derivatization procedure was fast, required minimal sample preparation, and was reproducible and cost effective. 31P labeling was then successfully utilized for analyzing lipophilic compounds with labile hydrogens in human serum. In sum, application of enhanced detection of metabolites in pathological tissue and biofluids by appropriate chemical derivatization methodologies provides a convenient and sensitive method for the qualitative and quantitative analysis of various classes of metabolites that may have good diagnostic utility and for the study of pathophysiology in various diseases