Journal of Applied Physiology AJP: Regulatory, Integrative and Comparative Physiology
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J Appl Physiol 104: 1854-1855, 2008; doi:10.1152/japplphysiol.90521.2008
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LETTER TO THE EDITOR

Reply to Burgess, Chandramouli, Browning, Schumann, and Previs

TO THE EDITOR: Burgess et al. (1) provided 2H-enrichment at various glucose carbons (2H-NMR) and compared 2H-enrichments at C-5 with the average 2H-enrichments of C-1, C-3, C-4, C-5, and C-6 (2H-NMR) and suggest that 2H-enrichment differences exist among glucose carbons that may impact quantitation of gluconeogenesis. Unfortunately no reproducibility data [coefficient of variation (CV)] at different carbons are provided to determine if the differences reported are real or within the error of the measurements particularly at low enrichment, considering signal-to-noise ratio, operator error, and precision sealing of tubes. The glucose deuterium peak intensities in 2H-NMR spectra reported in 6-h and 24-h-fasted rats and ~15-h-fasted humans (2, 8) do not exhibit major differences by inspection among glucose carbons except on C-2 and C-3. The variance (CV) in fractional gluconeogenesis in 6-h-fasted rats (n = 6) was 8.2% [C-5 hexamethylenetetramine (HMT)] and 15.5% (2H-NMR) (2). Table 1 below suggests considerable uncertainty in 2H-measurement using 2H-NMR at C-3. Therefore, it is unclear whether the differences in enrichments measured by 2H-NMR at different carbons are due to unequal labeling or difference in spatial environment.


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  		Table 1. Coefficient of variation of 2H-NMR measurements calculated using the data (n = 4) supplied in Fig. 1 of Burgess et al. (1)

 
Relative 2H-enrichment rather than absolute enrichments introduces even more uncertainty (1, 2). In measuring absolute 2H-enrichment at low levels, Jones et al. (7) relied on an external reference/correction factor instead of an intramolecular reference as intended. Therefore, 2H-NMR/HMT methods used to suggest unequal 2H-labeling could alternatively be the result of variation in absolute reproducibility and/or complex chemical/enzymatic degradation reactions with potential contamination/interference particularly at low enrichments.

Various investigators have reported measurements of gluconeogenesis based on 2H-enrichment at C-3, C-5, or C-6 (5, 6, 9, 10). The 2H-NMR spectra of glucose synthesized from fructose-6-phosphate and dihydroxyacetone phosphate in 2H2O appear to have comparable deuterium labeling in glucose (C-1, C-3, C-4, C-5, and C-6) and in glucose (C-3, C-4, and C-5), respectively (14). Deuterium labeling at the same or different carbons by a single or multiple substrates (6, 8–10, 12) and the cycling of substrates facilitates an even distribution of labeling among glucose carbons during gluconeogenesis. The series of isomerization and equilibration reactions involved in the gluconeogenic pathway should result in a more equal distribution of deuterium labeling on glucose C-1, C-3, C-4, C-5, and C-6, thus providing a better estimate of gluconeogenesis based on average enrichment. We demonstrated that our method provided results comparable to the C-5 HMT method with a CV of <3% (3), despite the differences in substrate availability under conditions of short-/long-term fasting and total parenteral nutrition providing various substrates at high concentration and at high, intermediate, and low fractional gluconeogenesis. Considering the above issues, we do not believe that this is a "fortunate concurrence" (1).

Despite reports of preferential 2H-labeling at C-1 during glycogenolysis [4], 2H-NMR spectra of glucose isolated from short-term fasted rats (6 h) and humans (~15 h) (2, 8) do not show major differences in peak intensity at C-1 when compared with C-4, C-5, and C-6. Our average data from overnight fasting humans are in excellent agreement with our data obtained by the C-5 HMT method (3). Interestingly, 2H-enrichment at C-5 (2H-NMR) and average enrichment (2H-NMR) in 24-h-fasted humans gave similar results (1).

In contrast to nonphysiological in vitro conditions, we demonstrated that our method is not affected by nongluconeogenic exchange reactions/isotope effects (11–13) by analyzing samples from two children with glycogen storage disease type-Ia. We observed that fractional gluconeogenesis was essentially zero at this level of deuterium enrichment.

In summary, no gold standard exists for determining absolute rates of gluconeogenesis or methods to measure accurately the slight 2H-enrichment differences among glucose carbons. It remains to be determined whether slight differences, if real, impact meaningfully measurements of gluconeogenesis. Therefore, our highly reproducible and simple/sensitive method requiring less sample/complex analyses is an excellent method that is now available to most investigators.

FOOTNOTES


Address for reprint requests and other correspondence: M. W. Haymond, Dept. of Pediatrics, USDA/ARS Children's Nutrition Research Center, 1100 Bates St., Houston, TX 77030-2600 (e-mail: mhaymond{at}bcm.tmc.edu)

REFERENCES

  1. Burgess SC, Chandramouli V, Browning JD, Schumann WC, Previs SF. Complicating factors in the application of the "average method" for determining the contribution of gluconeogenesis. J Appl Physiol. doi: 10.1152/japplphysiol.90406.2008.[Free Full Text]
  2. Burgess SC, Nuss M, Chandramouli V, Hardin DS, Rice M, Landau BR, Malloy CR, Sherry AD. Analysis of gluconeogenic pathways in vivo by distribution of 2H in plasma glucose: comparison of nuclear magnetic resonance and mass spectrometry. Anal Biochem 318: 321–324, 2003.[CrossRef][Web of Science][Medline]
  3. Chacko SK, Sunehag AL, Sharma S, Sauer PJJ, Haymond MW. Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide. J Appl Physiol 104: 944–951, 2008.[Abstract/Free Full Text]
  4. Chandramouli V, Ekberg K, Schumann WC, Wahren J, Landau BR. Origins of the hydrogen bound to carbon 1 of glucose in fasting: significance in gluconeogenesis quantitation. Am J Physiol Endocrinol Metab 277: E717–E723, 1999.[Abstract/Free Full Text]
  5. Jin ES, Jones JG, Mathew M, Merritt M, Burgess SC, Malloy CR, Sherry AD. Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative. Anal Biochem 327: 149–155, 2004.[CrossRef][Web of Science][Medline]
  6. Jones JG, Carvalho RA, Sherry AD, Malloy CR. Quantitation of gluconeogenesis by 2H nuclear magnetic resonance analysis of plasma glucose following ingestion of 2H2O. Anal Biochem 277: 121–126, 2000.[CrossRef][Web of Science][Medline]
  7. Jones JG, Perdigoto R, Rodrigues TB, Geraldes CFGC. Quantitation of absolute 2H enrichment of plasma glucose by 2H-NMR analysis of its monoacetone derivative. Magn Reson Med 48: 535–539, 2002.[CrossRef][Web of Science][Medline]
  8. Jones JG, Solomon MA, Cole SM, Sherry AD, Malloy CR. An integrated 2H and 13C NMR study of gluconeogenesis and TCA cycle flux in humans. Am J Physiol Endocrinol Metab 281: E848–E856, 2001.[Abstract/Free Full Text]
  9. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC. Contributions of gluconeogenesis to glucose production in the fasted state. J Clin Invest 98: 378–385, 1996.[Web of Science][Medline]
  10. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC. Use of 2H2O for estimating rates of gluconeogenesis. J Clin Invest 95: 172–178, 1995.[Web of Science][Medline]
  11. Leadlay PF, Albery WJ, Knowles JR. Energetics of triosephosphate isomerase: deuterium isotope effects in the enzyme catalyzed reaction. Biochemistry 15: 5617–5620, 1976.[CrossRef][Web of Science][Medline]
  12. Rognstad R, Clark DG, Katz J. Glucose synthesis in tritiated water. Eur J Biochem 47: 383–388, 1974.[Web of Science][Medline]
  13. Rose IA. Mechanism of the aldose-ketose isomerase reactions. Adv Enzymol Relat Areas Mol Biol 43: 491–517, 1975.[Web of Science][Medline]
  14. Schleucher J, Vanderveer PJ, Sharkey TD. Export of carbon from chloroplasts at night. Plant Physiol 118: 1439–1445, 1998.[Abstract/Free Full Text]

Shaji K. Chacko1
Agneta L. Sunehag1
Susan Sharma1
Pieter J. J. Sauer2
Morey W. Haymond1
 1Department of Pediatrics, Baylor College of Medicine, Children's Nutrition Research Center, U.S. Department of Agriculture/Agricultural Research Service, Houston, Texas; and 2Department of Pediatrics, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands





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