IsoLife: the meaningful role of stable isotopes in health-related food researchPublished: 04-02-2014
IsoLife focuses on actual cases of health research in which stable isotopes like 13C help improve the tracing and analysis of foods and their metabolites in the body, with enhanced efficiency, safety and comfort to the consumer and/or patient. Improvements in human health research are critical in view of the epidemic of health problems like overweight, obesity, metabolic syndrome, type 2 diabetes and intestinal cancers.
New technologies for tracing and analyzing healthy food components in the body are becoming increasingly available, and are finding use in routine diagnostics. We will illustrate four new applications of IsoLife‘s unique, stable isotope products in ongoing health-related projects involving several consortia. These project cases will show how stable isotopes help to improve the monitoring of health effects, with better traceability and diagnostic analysis in health research and diagnostics.
Apart from enhancing analysis of human health, the new stable isotope technology fits nicely with strategies aimed at improving health by primary and secondary prevention. It will increase sustainability through its contribution to public health, thereby increasing economic productivity and lowering healthcare costs.
Today’s modern sedentary lifestyle—often accompanied by an unlimited access to food—is known to be linked to an increased incidence of diseases like obesity, metabolic syndrome, type 2 diabetes, inflammatory diseases and cancers. Researchers have proposed various solutions to these problems, including changing lifestyles by introducing new, healthy foods.
De Visser explains: “For this purpose, IsoLife is collaborating with multidisciplinary research teams studying the fate of food in the human body. The goal is to increase our knowledge and support health claims. To gain insight into the fate of a particular food or food component in a living body—human or animal—we need to make the food component and its metabolites stand out against the background of endogenous metabolites, i.e., compounds already present in the body at the start of the experiment, originating from other sources, exogenous or endogenous.”
A safe way of marking a food without affecting its digestion or metabolism is by enriching it with stable isotopes, like 13C and 15N, found in nature in small percentages (1.1% 13C, 0.4% 15N). These isotopes may be increased in foods by up to 98%, are absolutely safe for humans and animals, and can be directly measured in the body (e.g., Lim et al., 2011). This is exactly what IsoLife‘s new technology is all about: enriching food and feed with stable isotopes, to be used as tools in health research. The following cases will demonstrate this.
Starches preventing colon cancer
Colon cancer is the second-most common type of cancer in both men and women. One project deals with stable isotope-labeled starches from wheat and maize, which are used as tools in colon cancer prevention research. Much research into the risk factors for colon cancer has provided evidence for the protective role of starch fermentation. During this fermentation, short-chain fatty acids (SCFAs) are formed. According to Gorissen, “Increased production of these SCFAs from resistant starches—in particular butyrate—has proven to protect against colon cancer.”
The mechanism of this protective action is not known, nor is it clear which SCFAs are formed by fermentation of different types of resistant starches originating from potato, maize or wheat, etc., and to what extent they contribute to the total protective action. In a feasibility study funded by the Dutch Government, the University Medical Center Groningen (UMCG) concluded that application of 13C-labeled starches is of great value for research towards prevention of colon cancer.”
Worldwide, IsoLife is the only commercial supplier of 13C starches from food crops, thanks to a unique “closed-system” facility designed for the uniform labeling of higher plants. Using 13C-labeled starches, researchers will be able to characterize the fermentation of starches, to determine the additional production of SCFAs from specific starches, to show which SCFAs are formed and to find the mechanism of uptake into the blood circulation. The objective of the research is to develop a diagnostic 13C starch test for measuring the risk of development of colon cancer in humans. This requires detailed information on the fermentation properties of starches from different crops taking place within the colon.
During the past few years, researchers have cleared several bottlenecks in the production of 13C-labeled corn and wheat starches. This opens up new avenues for developing a diagnostic 13C starch test for colon health, for instance, by using the recently developed oral ileocolonic delivery “ColoPulse” device (Schellekens et al., 2010) and the Stable Isotope Probing (SIP) technology for studying microbiological aspects of gut health (prize-winning paper by Kovatcheva-Datchary et al. 2009).
Such research fits within innovation programs on “Food and Health,” “Disease prevention,” “Biomarkers,” and “Bio-ingredients and functional foods” for enhancing SCFA production in the colon. New starches may also be developed to help prevent type 2 diabetes by increasing the proportion of resistant starch in our daily nutrition. The economic perspective includes an increased market share of Dutch potato breeders for cultivars with increased resistant starch levels, a profitable diagnostic test for UMCG, new 13C products and markets for IsoLife, and an increased health status and productivity for people. Within the EU association “Healthgrain Forum,” further initiatives may be developed.
This project will lead to better diagnostics (supporting prevention of colon cancer) and better disease prevention through healthier foods with improved nutritional value.
Broccoli Cress, a vegetable with anti-cancer properties
Innovative food companies like Koppert Cress are pioneering the development of health claims for their new products. With IsoLife, they partner on a project on metabolome research of bioactive compounds in healthy humans for supporting health claims of new micro-vegetables such as Broccoli Cress (see photo). Glucosinolates are secondary plant metabolites in Brassica vegetables like kale and broccoli exhibiting various important nutritional properties. Glucosinolates have been gradually discovered to be phytochemicals exhibiting anti-cancer properties; Zhang, Talalay, Cho and Possner (1992) identified sulforaphane (C6H11NOS2), originating from glucoraphanin in broccoli, as a strong inducer of quinine reductase, a phase II detoxification enzyme. In a recent review, researchers suggested that about three to five servings of broccoli per week seem to constitute a cancer preventive (Herra and Büchler, 2010).
According to new EC legislation, it is no longer permitted to claim health benefits of a product simply because it contains “bioactive” compounds, without proving its bioavailability and mechanism of action. In collaboration with the world-renowned UMCG, researchers are conducting a project to deliver the required scientific proof. The evidence should not only verify the presence of the components in the product and the proven absorption of these components in the gut, but must also demonstrate the arrival of the components and their metabolites at the target location in the body where their health benefits are effectuated.
Gorissen: “In the current food innovation project, we investigate various aspects of ADME (absorption, distribution, metabolism and excretion) that are required for underpinning health claims from the perspective of the consumer using the stable isotope 13C.” In addition to performing work regarding sensitive and relevant biomarkers, researchers are studying the effect of the food matrix on the bioavailability of components and their uptake in the bloodstream, as well as interactions between various components and inter-individual variation in ADME of bioactive compounds.
“The uniform 13C-labeling of sulforaphane makes it an effective tracer for bioavailability studies. Preliminary data on 13C-sulforaphane analysis in plasma indicate promising perspectives for oral administration of 13C-enriched broccoli in the ADME studies,” Gorissen explains.
“Concurrent with these ADME studies, we studied the source of glucoraphanin, the precursor of sulforaphane in broccoli seedlings or cress. Both 12C (99 atom% 12C) and 13C (98 atom% 13C) -enriched broccoli seeds were produced, and subsequently germinated and grown either in a 13CO2 or a 12CO2 environment. Afterwards, the 13C enrichment of sulforaphane in seeds and in seedlings was determined by HPLC–MS. We found that sulforaphane originates exclusively from seed reserves and that de novo biosynthesis in broccoli seedlings was less than 1%. This information will help breeders and growers to produce healthier cresses.” (Gorissen et al., 2011)
The use of 13C-labeled vegetables and potatoes for demonstrating ADME of bioactive plant components in a quantitative way is a highly innovative approach. In the end, this should result in scientifically supported statements on the actual contribution to our health.
Macular degeneration—breeding new food crops that help prevent age-related macular degeneration (AMD) by increasing the intake and bioavailability of lutein and zeaxanthin
AMD is a major cause of blindness and visual impairment in older adults. It results in a loss of vision in the center of the visual field (the macula) because of damage to the retina.
The National Eye Institute and others have suggested vitamin supplements with high doses of antioxidants—like the carotenes lutein and zeaxanthin—to slow the progression of AMD and, in some patients, improve visual acuity (Tan et al., 2008). Lutein, zeaxanthin and β-carotene belong to a large group of health-promoting compounds, since they function as antioxidants and as precursors of vitamin A. Synthesized in plants and some microorganisms, zeaxanthin is the pigment that gives bell peppers, corn, saffron, and many other plants and microbes their characteristic color.
Breeding new food crops can help prevent AMD by increasing the intake and bioavailability of the carotenoids lutein and zeaxanthin. Questions that still need to be answered are:
- Which foods contribute to the uptake of lutein and zeaxanthin by the gastrointestinal tract?
- How long does it take for vegetable carotenoids to reach the eye and how long do they stay there?
- What is the dose required for sustainable eye health?
Simply sampling blood plasma after consumption of the new high-carotenoid food does not yield reliable information on bioavailability since, once inside the body, the absorbed carotenes are mixed with carotenes already present and originating from other food sources. Again, ADME studies using 13C-enriched foods can help deal with these challenges. The 13C-enriched vegetables can be administered to volunteers as part of normal daily diets that may contain any other source of carotenes, since the 13C-labeled carotenes can be easily distinguished from non-labeled forms in exo- or endogenous pools. After consumption of the experimental meals, plasma samples of the volunteers are taken during a long period (up to a month) and the carotenes are quantified using specific liquid chromatography–mass spectrometry techniques. The kinetics of the 13C carotenes and their metabolites can thus be monitored in the blood circulation. Uptake efficiency of lutein and β-carotene was studied by these techniques using 13C-enriched kale (Brassica oleracea var acephala) as vegetable (Kurilich et al., 2003; Novotny et al., 2005). Isotopic labeling of kale provided the opportunity for studying the kinetics of the consumed lutein and β-carotene without confounding background pools of these compounds in the body. Novotny et al. (2005) concluded that the bioavailability of lutein is much higher than that of β-carotene, although their absorption appeared to be similar. IsoLife is now performing a similar study with private and public partners.
Inborn Errors of Metabolism
Diagnostics of rare metabolic diseases used to be out-of-reach for modern analytical techniques, because of a lack of sensitive and specific assays that require stable isotopes as internal standard. De Visser: “At Radboud University Nijmegen Medical Centre (RUNMC), researchers are conducting a project funded by The Netherlands Organisation for Scientific Research (NWO) entitled ‘High-resolution glycoprofiling in human medicine.’ RUNMC studies an important group of orphan diseases with abnormal protein glycosylation. In this project, research and diagnosis has now been accelerated by new developments such as high-throughput analytical tools (innovative nanoLC-mass-spectrometry) that are much more efficient by enabling the detection of a complete profile of glycans in one single analytical run, leading to more efficient screening of young patients, especially newborn babies.”
This technology forms an essential extension of the current infrastructure and enables highly sensitive and detailed fingerprinting of many different glycan classes that are present on proteins and lipids in body fluids or cells. Changes in the glycosylation of proteins and lipids are associated with a growing variety of disorders, including inherited glycosylation disorders. This type of structural information complements functional studies performed within the Nijmegen Glycobiology Platform.
For this new technology to be reliable, stable isotope internal standards (SIIS) are required, which IsoLife is currently developing from food plants like chicory and potato. Similar stable isotope technology, using IsoLife‘s internal standards, has been published and developed in the field of food science, e.g., at the University of Kaiserslautern (Germany), which, in this case, resulted in the improved analysis of polyphenols in fruit and human body fluids (Erk et al. 2009; Hagl et al. 2011).
Additional applications include the use of isotopically enriched food oligosaccharides to study the systemic effects by analyses in serum and urine. In addition, the uptake of (isotopically labeled) food sugars and their conversion into protein linked glycans can finally be investigated in high resolution.
New stable isotope internal standards: applications in food and pharma research
NextGen Metabolomics Inc. (Michigan) (http://nextgenmetabolomics.com/ ) has developed an automated metabolic fingerprinting technology, IROA (Isotopic Ratio Outlier Analysis) for metabolic phenotyping of tissues or bio-fluids. The company is now applying IsoLife‘s 13C-enriched internal standards for reliable quantification.
De Visser: “IROA is a type of metabolomics assessment in which both the control sample and experimental samples are isotopically labeled so both may be easily found with absolute assurance of their identity, and any artifacts may be removed. This leads to very clean, high-resolution data sets that clearly define the biological response of a biological system. In the case of a phenotyping application, where it is not possible to isotopically label the biological sample, the sample is collected at natural abundance and is mixed with a fully predefined ‘Internal Standard’ that has been isotopically labeled using 13C media or IsoLife‘s technology. The 13C-internal standards function as absolute yardsticks in the MS analyses. Degradation of metabolites during the analyses and variations in sample processing are accounted for by spiking the samples with U-13C-labeled standards before processing.”
De Visser: “The great advantages of 13C-enriched internal standards are accurate quantification of your analytes, independent of time, place, sample composition, processing method, operator or apparatus used. They do not only enable accurate quantification, but also facilitate identification and quality control of metabolites in natural, non-labeled samples analyzed by LC-MS or GC-MS.” (Figure 3)
The examples described in this article clearly show the potential of stable isotopes with respect to research in the “Nutrition and Health” domain. Stable isotopes will not only improve monitoring of health effects by enhancing traceability and diagnostic analysis, but will also contribute towards the substantiation of future health claims. If you are interested in collaboration with IsoLife, please contact Ries de Visser or Ton Gorissen.
http://www.umcn.nl/Research/Departments/metabolicandendocrinediseases/ Pages/geneticsandmetabolism.aspx http://www.glycobiologynijmegen.nl/ http://www.healthgrain.org http://www.isolife.nl/Award_Kovatcheva.pdf and http://www.tno.nl/content.cfm?context=overtno&content =nieuwsbericht&laag1=37&laag2=2&item_id=2009-05-22%2011:27:01.0&Taal=2 http://nextgenmetabolomics.com/
Erk T, H Bergmann, E Richling. 2009. A novel method for the quantification of quinic acid in food using stable isotope dilution analysis. Journal of AOAC International 92: 730-733.
Gorissen A, NU Kraut, R de Visser, M de Vries, H Roelofsen, RJ Vonk. 2011. No de novo sulforaphane biosynthesis in broccoli seedlings. Food Chemistry 127: 192-196.
Hagl S, H Deusser, B Soyalan, C Janzowski, F Will, H Dietrich, F Albert, S Rohner, E Richling. 2011. Colonic availability of polyphenols and D-(-)-quinic acid after apple smoothie consumption. Molecular Nutrition & Food Research 55: 368-377.
Herra I, MW Büchler 2010. Dietary constituents of broccoli and other cruciferous vegetables: Implications for prevention and therapy of cancer. Cancer Treatment Reviews, 36: 377–383.
Kovatcheva-Datchary P, M Egbert, A Maathuis, M Rajilic-Stojanovic, AA de Graaf, H Smidt, WM de Vos, K Venema. 2009. Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing. Environmental Microbiology 11: 914-926. (Award-winning publication; http://www.isolife.nl/Award_Kovatcheva.pdf).
Kurilich AC, SJ Britz, BA Clevidence, JA Novotny. 2003. Isotopic labeling and LC-APCI-MS quantification for investigating absorption of carotenoids and phylloquinone from kale (Brassica oleracea). Journal of Agricultural and Food Chemistry 51: 4877–4883.
Lim EL, KG Hollingsworth, FE Smith, PE Thelwall, R Taylor. 2011. Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes. American Journal of Physiology Endocrinology and Metabolism 301: E1155-E1162.
Novotny JA, AC Kurilich, SJ Britz, BA Clevidence. 2005. Plasma appearance of labeled β-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale. Journal of Lipid Research 46: 1896 – 1903.
Schellekens RCA, F Stellaard, GG Olsder, HJ Woerdenbag, HW Frijlink, JGW Kosterink. 2010. Oral ileocolonic drug delivery by the colopulse-system: a bioavailability study in healthy volunteers. Journal of controlled release 146: 334-340.
Tan JS, JJ Wang, V Flood, E Rochtchina, W Smith, P Mitchell. 2008. Dietary antioxidants and the long-term incidence of age-related macular degeneration: the Blue Mountain Eye Study. Ophthalmology 115: 334–341.
Zhang Y, P Talalay, CG Cho, GH Possner. 1992. A major inducer of anticarcinogenetic protective enzymes from broccoli: Isolation and elucidation of structure. Proceedings of the National Academy of Sciences USA 89: 2399–2403.