Application of 31P NMR spectroscopy and chemical derivatization for metabolite profiling of lipophilic compounds in human serum

Abstract

New methods for obtaining metabolic fingerprints of biological samples with improved resolution and sensitivity are highly sought for early disease detection, studies of human health and pathophysiology, and for better understanding systems biology. Considering the complexity of biological samples, interest in biochemical class selection through the use of chemoselective probes for improved resolution and quantitation is increasing. Considering the role of lipids in the pathogenesis of a number of diseases, in this study fingerprinting of lipid metabolites was achieved by (31)P labeling using the derivatizing agent 2-chloro-4,4,5,5-tetramethyldioxaphospholane. Lipids containing hydroxyl, aldehyde and carboxyl groups were selectively tagged with (31)P and then detected with good resolution using (31)P NMR by exploiting the 100% natural abundance and wide chemical shift range of (31)P. After standardizing the reaction conditions using representative compounds, the derivatization approach was used to profile lipids in human serum. The results show that the (31)P derivatization approach is simple, reproducible and highly quantitative, and has the potential to profile a number of important lipids in complex biological samples.

Figure 1

¹H NMR spectrum of a serum extract from a healthy individual (commercial sample). Assignments for the numbered peaks are listed in Table 1.

Figure 2

A portion of the ³¹P NMR spectrum of a mixture of six derivatized representative compounds.

Figure 3

A portion of the ³¹P NMR spectrum of derivatized serum from a healthy individual. Derivatized compounds include: free fatty acids 1; cholesterol 2; lyso lipids 3; possible fatty aldehydes 4; phosphotidyl mono glycerides (primary) 5; free n-alkanol 6; 1,2-diacylglycerol 7.

Figure 4

A portion of the ³¹P NMR spectrum of derivatized serum from 5 healthy individuals with added cyclohexanol (2µl; 145.2 ppm) as an internal standard in the stock solution. Impurities arising from cyclohexanol are marked with an asterisk.

Scheme 1