TO THE EDITOR: We are writing with regard to the study by Chacko et al. (3). Although we commend Dr. Haymond's team for addressing a difficult and important problem, the concept of using an “average” label incorporation requires additional consideration. We are concerned about whether the hydrogen/deuterium bound to carbon 1, 3, 4, 5, and 6 of glucose exchange equally during gluconeogenesis, as required for the validity of this method (3), especially since nongluconeogenic exchange reactions and/or kinetic isotope effects influence these sites differently (4, 9-11). Although Chacko et al. found agreement between the hexamethylenetetramine (HMT) method and their “average method” (implying equal exchange in the various positions), the limited range of physiological conditions is understated (3). For example, Katz and coworkers (10) demonstrated that the positional labeling of newly made glucose is affected in a substrate-specific manner.
We have found that the labeling of hydrogen/deuterium bound to carbon 1 of glucose is 1.3-1.8 times that bound to carbon 6 in humans (4), and we observed time-dependent changes in the labeling ratios, e.g., the hydrogen/deuterium labeling bound to carbon 1 > carbon 5 at 14 h of fasting but approached that of carbon 5 as the fasting reached 42 h (4). Those studies relied on the HMT method (4), which in our experience (6) has better precision than that reported by Chacko et al. (3).
Nuclear magnetic resonance (NMR) spectroscopy can quantify the enrichment at all positions of glucose with an accuracy similar to the HMT method (1). Using NMR, we observed that the 2H-labeling of position 1 > position 5 > positions 3, 6R, and 6S in human subjects after enrichment of body water to 0.5% 2H2O (2, 5, 7). We demonstrate, with ample precision, that the enrichment in these positions is significantly different in 24- and 48-h-fasted humans (Fig. 1). The reasons for differential enrichment in the various positions of glucose are well described, i.e., 1) the hydrogen on carbon 1 is exchanged during glycogenolysis and gluconeogenesis (4), 2) the hydrogen on carbon 3 is subject to a severe isotope effect during the triose isomerase reaction (9), and 3) the hydrogens on 6R and 6S do not include gluconeogenesis from glycerol (8). Therefore, one expects that 2H-labeling on position 1 > position 5; and that on positions 6 and 3 are each < position 5.
The fact that the “average” method of Chacko et al. matches the 5/2 ratio (3) seems to be a fortunate concurrence, reflecting the overestimation of gluconeogenesis by position 1 and the underestimation of gluconeogenesis by positions 3, 6R, and 6S. We suspect that the “average” method is susceptible to systematic errors to the extent that factors controlling the enrichment of positions 1, 3, 4, 6R, and 6S are independent of gluconeogenesis, e.g., as gluconeogenesis approaches 100%, the accuracy of the two methods diverge (Fig. 1).
A final remark concerns the reliance of the “average method” on body water 2H2O enrichment as a surrogate for C2 enrichment. Under nonisotopic steady-state and/or “glucose-insulin” clamp conditions, the 5/2 ratio remains a valid measure of gluconeogenesis since enrichment in both positions is altered to the same degree. However, without a correction factor(s) the “average method” will underestimate gluconeogenesis because 2H2O enrichment does not change as glucose enrichment is diluted.
In summary, we concur with Chacko et al. (3), in that the HMT or NMR method requires extra consideration/instrumentation, and we commend Dr. Haymond's team for developing an alternative. However, we caution against the general application of the “average method” without broad consideration for its limitations.