|Sauer, Pieter j|
Submitted to: Journal of Applied Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/8/2007
Publication Date: 1/10/2008
Citation: Chacko, S.K., Sunehag, A.L., Sharma, S., Sauer, P.J.J., Haymond, M.W. 2008. Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide. Journal of Applied Physiology. 104:944-951. Interpretive Summary: Glucose is produced in the body by two major processes: one being the breakdown of stored glycogen, and the other by the de novo synthesis of glucose. Glucose stored in a complex starch form as glycogen is generally from carbohydrate that is consumed during meal absorption, whereas during periods of fasting or at other times glucose can be made from other compounds such as lactate, glycerol, and amino acids to form glucose providing essential nutrient for brain metabolism. In the past, it has been difficult to measure the process of gluconeogenesis to be able to determine what factors may or may not regulate glucose production from gluconeogenesis. In this manuscript we report a new method to measure the fraction of glucose derived from gluconeogenesis using a very simple stable isotope tracer, deuterated water, and looking at the average enrichments of deuterium in the glucose form by the process of gluconeogenesis. We have found that this method is very precise, easily reproducible, can be done on small amounts of plasma, and provides data of rates of gluconeogenesis that are not different from a very complex and difficult method called the C5HMT method. As a result, we believe that this new method makes measurements of gluconeogenesis available and affordable to a large number of investigators under conditions of low and high fractional gluconeogenesis in all subject population and in many animal models.
Technical Abstract: We report a new method to measure the fraction of glucose derived from gluconeogenesis using gas chromatography-mass spectrometry and positive chemical ionization. After ingestion of deuterium oxide by subjects, glucose derived from gluconeogenesis is labeled with deuterium. Our calculations of gluconeogenesis are based on measurements of the average enrichment of deuterium on carbon 1, 3, 4, 5, and 6 of glucose and the deuterium enrichment in body water. In a sample from an adult volunteer after ingestion of deuterium oxide, fractional gluconeogenesis using the "average deuterium enrichment method" was 48.3 +/- 0.5% (mean +/- SD) and that with the C-5 hexamethylenetetramine (HMT) method by Landau et al. (Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC; J Clin Invest 98: 378–385, 1996) was 46.9 +/- 5.4%. The coefficient of variation of 10 replicate analyses using the new method was 1.0% compared with 11.5% for the C-5 HMT method. In samples derived from an infant receiving total parenteral nutrition, fractional gluconeogenesis was 13.3 +/- 0.3% using the new method and 13.7 +/- 0.8% using the C-5 HMT method. Fractional gluconeogenesis measured in six adult volunteers after 66 h of continuous fasting was 83.7 +/- 2.3% using the new method and 84.2 +/- 5.0% using the C-5 HMT method. In conclusion, the average deuterium enrichment method is simple, highly reproducible, and cost effective. Furthermore, it requires only small blood sample volumes. With the use of an additional tracer, glucose rate of appearance can also be measured during the same analysis. Thus the new method makes measurements of gluconeogenesis available and affordable to large numbers of investigators under conditions of low and high fractional gluconeogenesis (approx.10 to approx.90) in all subject populations.