Use of (2)H(2)O for estimating rates of gluconeogenesis: determination and correction of error due to transaldolase exchange

Abstract

The use of deuterated water as a method to measure gluconeogenesis has previously been well validated and is reflective of normal human physiology. However, there has been concern since the method was first introduced that transaldolase exchange may lead to the overestimation of gluconeogenesis. We examined the impact of transaldolase exchange on the estimation of gluconenogenesis using the deuterated water method under a variety of physiological conditions in humans by using the gluconeogenic tracer [U-(13)C]propionate, (2)H(2)O, and (2)H/(13)C nuclear magnetic resonance (NMR) spectroscopy. When [U-(13)C]propionate was used, (13)C labeling inequality occurred between the top and bottom halves of glucose in individuals fasted for 12-24 h who were weight stable (n = 18) or had lost weight via calorie restriction (n = 7), consistent with transaldolase exchange. Similar analysis of glucose standards revealed no significant difference in the total (13)C enrichment between the top and bottom halves of glucose, indicating that the differences detected were biological, not analytical, in origin. This labeling inequality was attenuated by extending the fasting period to 48 h (n = 12) as well as by dietary carbohydrate restriction (n = 7), both conditions associated with decreased glycogenolysis. These findings were consistent with a transaldolase effect; however, the resultant overestimation of gluconeogenesis in the overnight-fasted state was modest (7-12%), leading to an error of 14-24% that was easily correctable by using either a simultaneous (13)C gluconeogenic tracer or a correction nomogram generated from data in the present study.

Figures

Fig. 1.

Mechanisms by which unequal enrichment in the top and bottom halves of glucose can occur when a gluconeogenic tracer is given. Under such conditions, the tracer enters the gluconeogenic pathway as phosphoenolpyruvate (PEP). A: transaldolase (TA) is an integral enzyme of the pentose phosphate pathway that can exchange the bottom 4 carbons of sedoheptulose 7-phosphate (C4–C7) and, more importantly, the bottom 3 carbons of fructose 6-phosphate (F6P; C4–C6) with glyceraldehydes 3-phosphate (GAP) from the triose pool (23). In order for TA exchange to dilute enrichment in the top half of glucose, an unlabeled pool of glucose must be available. Under the conditions of study, only glycogen could provide such a pool. Given that glucose derived from glycogen is in equilibrium with F6P, TA exchange would serve to enrich glucose carbons 4, 5, and 6, yielding higher enrichments in the bottom half of glucose relative to the top half. This effect should be minimal or absent under conditions of glycogen depletion. B: triose phosphate isomerase (TPI) is responsible for the isomerization of GAP to dihydroxyacetone phosphate (DHAP) and vice versa. When a gluconeogenic tracer is used, it is assumed that TPI provides rapid equilibration of the label in GAP to DHAP and therefore equivalent enrichment in the top and bottom halves of glucose. Incomplete equilibration of the label in GAP would lead to greater enrichment in glucose carbons 4, 5, and 6 compared with carbons 1, 2, and 3. The unlabeled DHAP is derived, in part, from glycerol.

Fig. 2.

Glucose isotopomers derived from [U-13C]propionate and their analysis by 13C-NMR. After ingestion, propionate is avidly taken up by liver and enters the TCA cycle as [1,2,3-13]succinyl-CoA. This labeled intermediate can take several routes on its way to becoming a triose (GAP and/or DHAP), resulting in formation of a variety of triose isotopomers that are then combined to form the top and bottom halves of glucose. Assuming that there is no TA exchange and that the 13C label is equilibrated between the trioses by TPI, 13C enrichment in the top and bottom halves of glucose should be similar. Due to spin-spin coupling, the 13C resonance of glucose C2 and C5 after [U-13C]propionate ingestion is a multiplet composed of a singlet (S), two doublets (D), and a quartet (Q). Together, the multiplet provides information regarding the 13C enrichment of glucose C2 and C5 and allows isotopomers derived from the gluconeogenic tracer to be easily distinguished from primarily natural abundance 13C. Use of the [3,4-13C]glucose tracer did not interfere with this analysis.

Fig. 3.

Correction of the relative rate of gluconeogenesis determined by the deuterated water method for TA exchange. A: In the presence of deuterated water, the ratio of 2H enrichment at glucose carbon 5 to that of carbon 2 (H5:H2) provides an estimate of the rate of gluconeogenesis relative to glucose production. The measured H5:H2 ratios for all data points (n = 44) were corrected for TA exchange using 2 different metrics and the measured values plotted against the corrected values. B: the relationship between measured and corrected H5:H2 ratios was derived using linear regression and used to create a correction nomogram. The unity line (x = y) is provided for reference. The slopes of the 2 regression lines were similar (P = 0.331) but differed significantly from unity (P < 0.001). The adjusted means of the 2 regression lines also differed significantly (P = 0.035).

Fig. 4.

Addition of protons to the carbons of glucose by TPI. The trioses DHAP and GAP are numbered according to their final position in glucose. In isomerization of GAP to DHAP, a proton is added to what will become glucose carbon 3 (arrow). In isomerization of DHAP to GAP, a proton is added to what will become glucose carbon 5 (arrow). In the presence of 2H2O, 2H can be added to these carbons during isomerization via TPI, thereby enriching carbons 5 and 3 of glucose, respectively. Likewise, when hexose tracers with 2H at carbons 3 and 5 are broken down to the level of a triose, the 2H label should be lost equally due to the isomerization reaction. However, enzyme kinetic studies have shown that the free energy of adding 2H to DHAP is high, leading to preferential protonation of this carbon by TPI (12). The free energy of adding 2H and 1H to GAP is similar, and TPI demonstrates no preference for protonation or deuteration of this carbon. As a result, the ratio of 2H enrichment at glucose carbon 3 (H3) to that of carbon 5 (H5) with the deuterated water method is nearly always less than 100% (see Table 6). Conversely, this effect will lead to preferential retention of 2H on carbon 3 when a [3, 5-2H]hexose tracer is used and contribute to the overestimation of TA exchange.