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Gluconeogenesis in isolated rat hepatocytes evaluated by gas chromatography/mass spectrometry using deuterated water
Author(s) -
Arnoldi L.,
Valsecchi G.,
Magni F.,
Monti L. D.,
Piatti P. M.,
Costa S.,
Galli Kienle M.
Publication year - 1998
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199805)33:5<444::aid-jms651>3.0.co;2-b
Subject(s) - chemistry , gluconeogenesis , isotopomers , mass spectrometry , chromatography , gas chromatography , metabolic flux analysis , gas chromatography–mass spectrometry , flux (metallurgy) , biochemistry , metabolism , organic chemistry , molecule
Tritiated water and radioactive tracers have been used to monitor glucose production by primary cultures of hepatocytes. More recently, 3 H 2 O has been replaced for by 2 H 2 O in ‘ in vivo ’ studies addressed at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2 H 2 O to determine the ratio between the glucogenic flux and the overall flux through glucose 6‐phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rats were incubated with a medium containing 6, 12 and 25% of 2 H 2 O in the presence of either 2 or 20 m M pyruvate. Isotopomer analysis of six different mass clusters ( m / z 328, 314, 242, 212, 187 and 145) was carried out by gas chromatography/mass spectrometry (GC/MS) of glucose aldonitrile pentaacetate. For each cluster, ions at m / z +1, +2, +3 and +4 were monitored. From the combination of different clusters the enrichment at C‐6 and C‐2 of glucose was computed and the C‐6/C‐2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obtained, conditions selected to be optimum for the use of the method in studies on the modulation of gluconeogenesis were as follows: incubation of hepatocytes with 20 m M pyruvate in 12% 2 H 2 O followed by GC/electron ionization MS analysis of the clusters of ions at m / z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C‐2 and C‐6 positions of glucose. © 1998 John Wiley & Sons, Ltd.