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Positional isotopic analysis of 13 C‐labelled glucose by mass spectrometry: Applications to the study of gluconeogenesis in liver cells
Author(s) -
Desage Michel,
Guilluy Roger,
Brazier JeanL.,
Riou JeanP.,
Beylot Michel,
Normand Sylvie,
Vidal Hubert
Publication year - 1989
Publication title -
biomedical and environmental mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0887-6134
DOI - 10.1002/bms.1200181110
Subject(s) - chemistry , gluconeogenesis , glycerol , mass spectrometry , labelling , isotopes of carbon , carbon fibers , molecule , chromatography , ion , metabolism , biochemistry , organic chemistry , materials science , total organic carbon , composite number , composite material
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‐ 13 C, 6‐ 13 C, 2‐ 2 H, 3‐ 2 H, 6,6‐ 2 H 2 ) 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‐ 13 C)glycerol, (1,3‐ 13 C)glycerol or NaH 13 CO 3 plus pyruvate or lactate produced (2,5‐ 13 C)glucose, (1,3,4,6‐ 13 C) glucose or (3,4‐ 13 C)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 anlysis of 13 C‐labelled glucose, and appears useful in the study of the gluconeogenic pathway.