My watch list  

A granular look at sugar

Use of novel analysis methods

Our experience with sugar starts in early childhood – since breast milk contains lactose. Human sensory perception alone is, however, not enough to properly assess the useful but also potentially harmful effects of an age-old sweetener (saccharose). Nutrition researchers and physicians have, for this reason, been focusing attention for some time on sugar compounds, particularly glucose.

Significant progress in metabolic research on sugar could, however, only be achieved through the availability of novel analytical methods. With its GC-MS (gas chromatography-mass spectrometry) systems Shimadzu opens new ways to measure glucose metabolism in humans and animals with high precision, based on analytical expertise developed over decades.

Much is already known about glucose, one of the components of household sugar. Daily glucose requirement of adults is approximately 180 g, of which the brain is the largest consumer’, requiring 80% of this amount for its energy supply. For this reason, glucose must be newly synthesized during short-term periods of starvation. This process is called gluconeogenesis. It takes place in the liver and in the renal cortex as well as in the intestine. Substrates are pyruvate, lactate, glycerol, alanine and, in ruminants, propionate. Excess glucose is stored in the body as glycogen and can be converted back to glucose when needed, for instance during physical activity.

Figure 1: Schematic representation of glucose metabolism

Glucose concentration of blood (normally 90 - 110 mg/mL in human adults) mirrors the ‘static’ component of glucose metabolism. However, the causes of increased or decreased glucose level remain unclear. The synthesis rate of glucose and its consumption for the production of energy or conversion to other metabolites is unknown. These processes can be elucidated using special labeling techniques that incorporate chemical probes, so-called ‘tracers’.

To monitor the dynamics of glucose metabolism (i.e. its synthesis and breakdown), it is useful to incorporate a labeled glucose tracer into the body’s metabolism. But how can such a glucose tracer be produced? For the elements hydrogen, carbon and oxygen contained in glucose (molecular formula: C6H12O6), nature offers different heavy elements, so-called isotopes, which are suitable for use as labels. For hydrogen there is the stable, non-radioactive protium isotope with mass 1 (symbol 1H) and deuterium with mass 2 (symbol 2H, or D in short). For carbon, there are two stable isotopes: 12C and 13C.

The natural abundance of these heavy stable isotopes in glucose is very low. Out of 1000 glucose molecules, only one contains deuterium bound to one of the six carbon atoms. By drinking a small amount of heavy water (D2O), the deuterium content in glucose molecules increases relative to the natural level, thus producing ‘labeled’ (‘enriched’) glucose that differs from ‘normal’ glucose. The so-called D2O (‘heavy water’) method outlined below shows how gluconeogenesis (in g glucose/day) can be determined after deuterium enrichment of glucose.

The D2O (‘heavy water’) method

The D2O method is based on the fact that, after oral intake of D2O (0.6 g per kg body weight) with a deuterium content of > 50%, a rapid distribution in the body water pool (equivalent to approximately 60% of the body weight) initially takes place. As a result of enzymatic reactions, deuterium atoms present in water molecules are subsequently incorporated in glucose molecules. The difference between the deuterium content at carbon atom C2 and the other C-atoms provides information on specific metabolic pathways. The deuterium content in the different molecular positions can be measured via the aldonitrile pentaacetate derivative of blood glucose using GC-MS.

The D-enrichment at C2, E(C2), is proportional to the total glucose produced in the body (GP), i.e. the sum of newly synthesized glucose and ‘old’ glucose generated from glycogen breakdown. The D-enrichment at carbon C5, E(C5), is an indicator of newly synthesized glucose. Due to enzymatic exchange reactions, D-enrichment at the other carbon atoms (with exception of C2) is similarly high. Consequently, the relative gluconeogenesis GNGrel (in % of the glucose production GP) and the absolute GNGabs can be calculated as follows:

Glucose production (GP) is determined in a separate study, for instance using deuterated glucose which possesses two D-atoms at carbon C6. The factor 3 in equation (1) results from a total of three H-atoms or three D-atoms being bound to glucose carbon atoms C5 and C6 (see figure 2).

Figure 2: Fragmentation of glucose aldonitrile pentaacetate

The D-enrichment E(C5)+E(C6) and E(C2) can be determined with relatively little analytical effort from the mass fragments m/z 145 (C5-C6), 187 (C3-C6) and 328 (C1-C6) of the derivatized glucose using GC-MS (Junghans et al., 2010). The peak intensities of the mass fragments with their so-called isotopomers M+1, M+2 and M+3 (incorporation of 1, 2 or 3 deuterium atoms per glucose molecule) required for the calculation, can be obtained from the EI and PCI spectrum of aldonitrile pentaacetate derivative of glucose (Figure 3). Interference of the m/z 145 fragment with another fragment is avoided by using a different glucose derivative.

Figure 3a: EI-GC-MS spectra of the aldonitrile pentaacetate derivative (AAc) of glucose

Figure 3b: PCI-GC-MS spectra of the aldonitrile pentaacetate derivative (AAc) of glucose

D2O method harmless to health and environment

Does the deuterium isotope in the D2O method constitute a health hazard to humans and animals? The answer is no! For two reasons:

  1. Chemical properties of glucose and its metabolism as well as glucose-related metabolism are not changed by deuterium labeling, as only approximately 1% of the glucose molecules are labeled.
  2. Isotopes of heavy and light hydrogen (D and 1H) are stable and, therefore, non-radioactive. They are completely harmless, even for small children and pregnant women.

The stable isotope labeling technique discussed above, in combination with sensitive and accurate GC-MS analysis methods, provides significant advantages over the radioactive labeling techniques used in the past. In-vivo gluconeogenesis can be determined without putting the study subject (human or animal) or the environment at risk. The sample material, with respect to labeling, is indefinitely stable, since no radioactive decay takes place.

Junghans P, Görs S, Lang IS, Steinhoff J, Hammon HM, Metges CC. (2010). A simplified mass isotopomer approach to estimate gluconeogenesis rate in vivo using deuterium oxide. Rapid Commun Mass Spectrom. 24:1287-95.

Facts, background information, dossiers
More about Shimadzu
  • White papers

    A granular look at sugar

    Our experience with sugar starts in early childhood – since breast milk contains lactose. Human sensory perception alone is, however, not enough more

    How physics discovered TOC

    The determination of algal biomass using TOC is a new method that has proven to be highly suitable in a laboratory environment for the measurement of biomass more

    New since 5000 years

    Although the ancient Egyptians already used inks 5000 years ago, inks and their properties have been continuously improved and refined over thousands of years. more

  • Products

    Irspirit Series: Little Footprint and High Possibilities

    IRSpirit Series associate one of the smallest footprint with high sensitivity and stability. IRPilot software make IRSpirit's use easier than ever more

    Shimadzu's New UV-1900 UV-VIS Spectrophotometer

    Shimadzu's new UV-1900 UV-VIS spectrophotometer is equipped with an ultrafast scan function that enables data acquisition in 29,000nm/min (it takes about three seconds to measure in visible region.), the fastest level in the industry more

    Groundbreaking (U)HPLC Technology – Shimadzu’s New Nexera Series

    Unique auto-diagnostics and auto-recovery capabilities, as well as remote mobile phase monitoring and integrated consumables management maximize reliability and uptime. more

  • Videos

    Shimadzu's New UV-1900 UV-VIS Spectrophotometer

    Shimadzu's new UV-1900 UV-VIS spectrophotometer is equipped with an ultrafast scan function that enables data acquisition in 29,000nm/min (it takes about three seconds to measure in visible region.), the fastest level in the industry. The UV-1900 features a large, easy-to-use color touch pa ... more

    New Standards in Gas Chromatography

    Nexis GC-2030 - The Next Industry Standard The GC-2030 NEXIS stands for ‘Next Industry Standard’ . Meeting this demand requires a clear view on present day GC requirements. Gas chromatography is used in all areas of research and development that ultimately enrich but also influence our dai ... more

    Groundbreaking (U)HPLC Technology – Shimadzu’s New Nexera Series

    The new Nexera series of Ultra High-Performance Liquid Chromatographs incorporates Analytical Intelligence, allowing systems to detect and resolve issues automatically. It makes lab management simple by integrating IoT and device networking, enabling users to easily review instrument status ... more

  • News

    Shimadzu acquires specialist for isotope labeled standards

    Shimadzu has joined forces with the France-based AlsaChim company, an independent contract research and development organization. AlsaChim specializes in stable isotope-labelled compounds, metabolites and pharmaceutical related substances, and analytical purposes. With immediate effect, Shi ... more

    Shimadzu announces completion of new quality center

    Shimadzu announces the completion of the new Quality Center building, which has been under construction at its head office (Sanjo Works) premises since February 2013. The Quality Center has been constructed to improve the quality of products developed and manufactured by Shimadzu. It consol ... more

    Grand opening of new laboratory world

    Shimadzu has created a new Laboratory World for its customers from all over Europe. The Laboratory World is located at Shimadzu’s European headquarters in Duisburg, Germany. On an area greater than 1,500 m2, testing facilities are available for Shimadzu’s entire product range – from chromat ... more

  • Companies

    Shimadzu Deutschland GmbH

    Shimadzu is a world-leading manufacturer of analytical instrumentation. Shimadzu develops and manufactures innovative products for laboratories in industry, science and governmental institutions. With our customer-oriented strategy and our high-quality service, we strive to be Europe's mark ... more

    Shimadzu Italia

    Shimadzu is a world leader in instrumental analysis. Shimadzu develops and produces innovative products for laboratories in industry, science and institutions. We want to be a leader in all European markets with our customer-oriented strategy and our highly qualified services. We offer nume ... more

    Shimadzu Scientific Instruments, Inc.

    Shimadzu provides a broad range of analytical instruments indispensable for research, development, and quality control in a variety of fields. Our high-level, sophisticated instruments include chromatographs, spectrometers, and elemental and surface analysis systems. We also provide a broad ... more

Your browser is not current. Microsoft Internet Explorer 6.0 does not support some functions on Chemie.DE