Measurement of hepatic glucose output from plasma glucose 13C-isotopomers has been successfully accomplished in 24-hour fasted anesthetized rats that underwent adenovirus loading with b-galactosidase, glucokinase, and glucose-6-phosphatase (G6Pase) genes. The animal was given a 0.5 mg/kg min infusion of [1,6-13C]glucose and 2.5 mg/kg/min of [U-13C] propionate over 2 hours. 13C NMR spectra were obtained with a 1.5 ml blood sample from a putative G6Pase overexpressing animal. The spectrum collection time was approximately 1 hour, demonstrating adequate sensitivity. In the 13C spectrum, the contribution of [1,6-13C]glucose can be differentiated from all other 13C glucose molecules, including the background natural abundance signal since it generates an unique doublet as a result of long-range 13C-13C-coupling between carbons 1 and 6. From the 13C and 1H NMR spectra, the fraction of [1,6-13C]glucose in the plasma, hence its dilution, can be measured. Hepatic glucose output is given by the product of the dilution and infusion rate. We are confident that the amount of [1,6-13C]glucose can be cut from 0.50 to 0.15 mg/kg/min without compromising its measurement by 13C NMR. Analysis of the C2b glucose multiplet components provides metabolic flux information at the level of PEP and the citric acid cycle. Assuming that glucose is derived quantitatively from PEP after 24 hours fasting, absolute flux through anaplerosis, pyruvate kinase and citrate synthase can be estimated by indexing the gluconeogenic PEP flux to hepatic glucose output. (Service 1) REPORT PERIOD: (09/01/97-08/31/98)
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