Underestimation of gluconeogenesis by the [U-¹³C₆]glucose method: effect of lack of isotope equilibrium

Abstract

13 C3]lactate and the delay in M3 glucose equilibrium estimated from the isotopic steady-state value determined by modeling M3 glucose to a single-exponential fit. We found that, even with the addition of [U-13 C3]lactate infusion, the M3 glucose enrichment of the last timed sample was ϳ20% less than the isotopic steady-state value. Thus the lack of isotopic equilibrium of the glucose compartment potentially accounts for 20% of the underestimation of gluconeogenesis. The underestimation of gluconeogenesis using [U-13 C6]glucose without the additional infusion of [U-13 C3]lactate in previous publications is expected to be even greater because of the lack of isotope equilibrium in both the lactate and glucose compartments. These findings are consistent with the results from our computer simulation. mathematical modeling; mass isotopomer distribution analysis, stable isotopes THE RECOGNITION THAT [U-13 C 6 ]GLUCOSE has the property of being both a "nonrecyclable" and a "recyclable" tracer (9) has led Tayek and Katz (15,16) to propose a method of estimating gluconeogenesis by the use of [U-13 C 6 ]glucose. During an infusion of [U-13 C 6 ]glucose (glucose labeled in all six positions, M6 glucose), glycolysis leads to the production of lactate labeled in all three carbon positions (m3 lactate). When 13 C carbon atoms are recycled in gluconeogenesis, glucose molecules with 1, 2, or 3 13 C substitutions (M1, M2, and M3 glucose) are produced. The appearance of mass isotopomers M1, M2, and M3 of glucose provides a measure of the rate of gluconeogenesis. Because the chance of two labeled triose phosphates combining to form glucose is negligible, M6 glucose behaves as a nonrecyclable tracer, and the steady-state enrichment of M6 glucose in plasma allows the determination of the hepatic glucose production rate. Thus the [U- 13 C 6 ]glucose method has the advantage of being able to estimate simultaneously hepatic glucose output and fractional gluconeogenesis from the analysis of mass isotopomers in glucose. However, Landau et al. (7) have shown that, when [U- 13C 6 ]glucose is administered as a primed constant infusion, this method grossly underestimates the rate of gluconeogenesis. This underestimation could be attributed to dilution by other unlabeled gluconeogenic substrates and/or the lack of label steady state in m3 lactate and M3 glucose by the end of the study period. In the absence of experimental data, the magnitude of underestimation of gluconeogenesis by the [U-13 C 6 ]glucose method attributable to each of these sources of error remains controversial (2,5,11,12).Because the 13 C label from M6 glucose is recycled through an intermediate compartment, namely lactate, before gluconeogenesis, the precursor-product relationship of labeled substrate from M6 glucose to newly synthesized glucose is complex. A basic principle of precursor-product relationship dictates that the isotope in each precursor pool must be in isotopic steady state before the subsequent pool can reach its plateau enrichment. Therefore, the time to...

Integration of [U‐13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis

“…These fluxes can now be measured in a standard clinical setting by ingestion of 2 H 2 O 1-4 followed by an isotope dilution measurement of glucose turnover once plasma glucose 2 H-enrichment from 2 H 2 O has reached steady state. 1,2,4 Glucose turnover is obtained by a primed infusion of a non-recyclable glucose tracer such as [1,[6][7][8][9][10][11][12][13] C 2 ]glucose, 2 or [6,6-2 H 2 ]glucose. 1,4 Enrichment of plasma glucose from both tracers is then resolved and quantified from a single set of blood samples obtained at the end of the glucose turnover measurement.…”

“…Note that 2 H NMR cannot resolve deuterium isotopomers of glucose, thereby precluding measurement of GP with [6,6-2 H 2 ]glucose in the presence of glucose enrichment from 2 H 2 O. * However, 2 H enrichment and 13 C isotopomers can be resolved by NMR, allowing GP to be quantified with an alternative 13 C-tracer such as [1,[6][7][8][9][10][11][12][13] C 2 ]glucose. 2 This approach would be considerably simplified if all 2 H enrichment and 13 C isotopomer information could be obtained from a single MAG derivative.…”

“…2 This approach would be considerably simplified if all 2 H enrichment and 13 C isotopomer information could be obtained from a single MAG derivative. This cannot be accomplished with [1,[6][7][8][9][10][11][12][13] C 2 ]glucose because spin-spin coupling between carbons 1 and 6 of glucose is abolished following its conversion to MAG. However, MAG features 13 C- 13 C couplings that are favorable for resolving and quantifying a number of other glucose 13 C-isotopomers including [U-13 C]glucose, a widely used 13 C tracer of GP.…”

Measurement of Fractional Whole-Body Gluconeogenesis in Humans From Blood Samples Using 2H Nuclear Magnetic Resonance Spectroscopy

“…Previous methods for measuring gluconeogenesis yielded a wide range of estimates in humans, which may relate to biochemical limitations of those methods (2)(3)(4)(5). Gluconeogenesis can be also calculated from the difference between the rates of EGP determined with [6-3 H]glucose and of liver glycogen breakdown determined by in vivo 13 C-nuclear magnetic resonance spectroscopy (MRS) (1,6).…”

Unveiling the Metabolic Changes on Muscle Cell Metabolism Underlying p-Phenylenediamine Toxicity

“…Measurement of glucose released from glycogen was done by using the isotopomer [U-13C6-1,2,3,4,5,6,6-D7]D-glucose (or [U-13C-D7]-glucose as shorter name) as recovery standard and internal standard quantification procedures. The hydrolyzed glucose was isolated using ion exchange chromatography (Mao et al, 2002) and was derivatized to its aldonitrile acetate form using hydroxyl-amine in pyridine and acetic anhydride. We monitored the ion cluster around the m/z 328 (C1–C6, chemical ionization; Wamelink et al, 2008).…”