Positional isotopic analysis of13C-labelled glucose by mass spectrometry: Applications to the study of gluconeogenesis in liver cells

Abstract

The aim of the present investigation was to ascertain whether mass spectrometric analysis of glucose allows determination in small samples (0.01 nmol) of the sites and the extent of labelling of glucose produced by isolated liver cells from various gluconeogenic labelled precursors. The electron impact spectrum of the methyloxime pentatrimethylsilyl derivative of natural glucose affords fragment ions retaining specific carbon atoms, i.e. 1-2 (m/z 160), 1-2-3 (m/z 262), 3-4-5-6 (m/z 319), 4-5-6 (m/z 217), 5-6 (m/z 205), 6 (m/z 103). The mass fragmentography analysis of the same derivative of commercially available labelled glucose molecules (1-13C, 6-13C, 2-2H, 3-2H, 6,6-2H2) permitted evaluation of the degree of specificity of these fragment ions, and development of a calculation method for isotope incorporation. Using this methodology we found that incubation of hepatocytes with (2-13C)glycerol, (1,3-13C)glycerol or NaH13CO3 plus pyruvate or lactate produced (2,5-13C)glucose, (1,3,4,6-13C) glucose or (3,4-13C)glucose, respectively. The extent of labelling was measurable on individual carbon of the glucose molecule except for carbon 1. The lowest enrichment detectable on carbon 1-3 or 3 was found to be 0.5%. In conclusion, gas chromatography mass spectrometry is a reliable method for positional isotopic analysis of 13C-labelled glucose, and appears useful in the study of the gluconeogenic pathway.

A re-investigation of the path of carbon in photosynthesis utilizing GC/MS methodology. Unequivocal verification of the participation of octulose phosphates in the pathway

“…In support of an earlier study of gluconeogenesis in liver cells (Desage et al. 1989) it has, however, been shown here that GC/MS/SIM offers a viable alternative methodology for the study of positional isotope analysis of intermediates formed from stable-isotope labelled precursors in complex biological systems.…”

Analysis of [U‐13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

“…Many clinical and metabolic studies have used [1‐ 13 C]glucose,3, 4 [U‐ 13 C 6 ]glucose5 or [6,6‐ 2 H 2 ]glucose6–9 to measure glucose turnover. The various methods to determine plasma glucose enrichments involve different cleanup and derivatization techniques, such as trimethylsilyl,6, 7, 10–12 pentaacetate,13, 14 butylboronic acid15–17 and aldonitrile pentaacetate18, 19 derivatization in combination with gas chromatography/mass spectrometry (GC/MS) measurement in electron ionisation (EI)4 or chemical ionisation (CI) mode 20, 21. Plasma glucose concentration and isotopic enrichment are usually measured by different methods, using separate aliquots of the same sample 22.…”

Growth hormone improves protein catabolism and growth in prepubertal children with HIV infection

“…Determination of leucine Ra from serum was measured by a gas chromatograph mass spectrometer. Plasma samples were derivatized with methoxamine an N,O‐bis‐(trimethylsilyl N) trigluoroacetamide, as described by Desage 19 . Gas chromatographic separation was accomplished using a Hewlett‐Packard HP‐5 capillary column (Hewlett‐Packard, Valley Forge, PA).…”