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Laboratory of Human Nutrition, School of Science, Massachusetts Institute of Technology, Cambridge, MA 02139
2 To whom correspondence should be addressed. E-mail: vryoung{at}mit.edu.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • metabolomics • system • interdisciplinary • challenges
I was honored to have been asked to help close what has been to me a truly exciting and informative TEMA 11. It was suggested that I try to link, including identifying some of the strengths and possible weaknesses of the contemporary research focus, the expanding knowledge on the basic biology of trace elements with concerns for the trace elements and nutrition of individuals and populations in various settings. This is a decided challenge and it may even be asked why it was offered to me because, other than for a relatively brief interlude during the 1980s (1– 3), I essentially abandoned a research focus on mineral metabolism and nutrition as soon as I had completed my Ph.D thesis (4– 6). One might speculate, however, that one reason for the invitation is that even for one who has been interested primarily in amino acid and protein metabolism, a link with trace elements can be identified even as far back as the beginning of life itself; thus, about 4 billion years ago, according to one general theory based on a hydrothermal origin of life on Earth, autotrophic metabolism of low molecular weight constituents, including amino acids and peptides, emerged in a pressurized iron-sulfur world or an environment of iron-sulfide and hot magmatic exhalations (7) for which there is growing supportive evidence (8). Indeed, this calls to mind the interesting studies of Ross and Eisenstein (9) on mitochondrial aconitase, which has an iron-sulfur [4Fe-4S] cluster and is considered to be an ancient enzyme (10). It catalyzes the conversion of citrate to isocitrate, and they (9) show it is a target for regulation by iron regulatory proteins; a nice example of a connection between a trace element and the macronutrient metabolism with which I have been mainly interested. Another reason might be that we all easily connect via the small world of scientific networks (11) that tie us together in the different domains of nutritional science. Finally, and perhaps realistically, it is now ever more important for all of us interested in advancing nutritional science, and its application, to interact broadly and actively as biological knowledge and the technological and informational bases upon which we depend for our livelihood and understanding continue to expand and develop at an extraordinary pace.
The vision and some of the challenges
Thus, I will attempt to create a picture of some of the challenges ahead, or a sort of vision. This is not meant to be a prediction but rather how the world of trace elements in man and animals (TEMA) could be and, in part, I will approach my task with due recognition of the many exciting advances that have been presented at TEMA 11 and as recorded in these proceedings. In particular, there has been a strong emphasis on the details of the molecular mechanisms responsible for trace element uptake, movement, homeostasis and function. However, considerably less attention than would have been desirable has been given to a more holistic representation of trace element biology or to a system-wide understanding of trace elements in human and animal function and well-being (12). I will later make a point about the need for a reconstructionist undertaking to compliment and exploit the reductionist paradigm that has been so successful, with the sequencing of the smallest genomes containing 
396 protein coding genes (13) to larger and larger genomes, including those of humans (14,
15) and of two subspecies of rice (16,
17), being prime examples. Indeed, as an editorial (18) asked; "What does the genome sequence mean for me, my research and my constitution?" and many attending this meeting have given this question obvious thought by focusing their research on the sequences of relevant genes and their functions. Parenthetically, it is also interesting that geneticists are now arguing that the noncoding output of the genome (e.g., noncoding RNAs) functions in gene regulating networks (19,
20) and it will be exciting to learn how trace elements might function via and/or interact with the "RNome" (21). Nevertheless, we need to appreciate and understand that an integration of "_omes and _omics" is ultimately the key to the resolution of complex trace element/nutritional problems. In
Table 1. some challenging areas of application for the newer knowledge of trace element biology and for further areas of research are listed. This is not meant to be an exhaustive detailed list but a somewhat general one that I have developed in relation to my interest as a nutritional biochemist and that I can use to make my points.
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Additionally, in terms of deficiencies we need to ask, for example, which of the multiple functions of zinc (e.g., 25) and/or mechanisms that account for the essentiality of zinc in the animal organism are more or less vulnerable to a change in the availability of this element. i) Is there an initial and well-defined acceleration of one or more apoptotic pathways (a group of genetically encoded cell death programs) (26, 27) in cells, such as precursor lymphocytes that can be obtained relatively easily for evaluation? ii) Is the formation and /or activity of one or many of the zinc-finger proteins (28) or of the 700+ genes (29) that encode these transcription factors affected early on in the development of the depletion of zinc pools; or iii) is DNA repair and replication (30) more immediately and profoundly altered and could this effect be detected and localized at the single cell level by new and modified comet assays (31)?
As already mentioned, the challenges here are even more interesting, as well as complex, when the interaction among trace elements and other nutrients is duly considered. To further emphasize: a) the activity of the DNA binding domain of the retinoic acid receptor (containing two zinc fingers) is diminished in a pro-oxidant environment with release of zinc from the zinc-finger motif (32). Specifically, does the zinc deficient phenotype differ under varying degrees of antioxidant nutrient status? b) There is a zinc "sensing" receptor involved in intracellular calcium mobilization and signaling (33, 34), and c) mobilization of calcium induces nitric oxide, which releases zinc from metallothionein (35). Hence, an early biochemical defect in zinc deficiency might be a defect in Ca channels, as the work of O'Dell and colleagues seems to suggest (36). The important point, once again, is that there is a great deal of cross talk among and interactions between trace elements, other minerals and nutrients. These have major ramifications for the way by which we might widen and improve approaches for assessment of trace element nutritional status and needs, especially in humans.
Requirements for trace elements. The foregoing leads us now briefly to the second entry on the list in Table 1, namely the determination of the requirements for trace elements by humans at different stages of life and under various physiological, dietary and environmental conditions. I think it would be agreed that, to date, the approaches used to determine requirements for trace elements (37), such as those indicated in Table 2, are far from ideal and in some cases quite unsatisfactory. Apart from a lack of information and difficulty in conducting human studies, a reason for this problem is that there has not been a concerted major research commitment to establish requirements for nutrients in initially healthy humans. Insufficient resources and a minimal commitment to improve upon prior estimates of requirements contribute to the current lack of sound quantitative nutrient requirement information, despite its critical importance for the planning and evaluation (38) of diets. As David Baker (39) said some time ago "Nutrition scientists often avoid projects entailing determination of nutrient requirements. These studies are generally considered routine, easy and lacking in creativity. Hence, peer review generally results in "low marks" for such endeavors, resulting in difficulty in obtaining outside funding." Although there is considerable truth to this view, I believe basic investigators and those with a more applied focus in the trace element arena should coalesce and integrate some of their research interests and efforts in and around the issue of defining trace element requirements and improving assessment of nutritional status in quantitative and more precise predictive terms. This would lead to significant and meaningful advances in trace element nutrition. Furthermore, it might also be time for those of us interested in this area to step back for a while and abandon immediate attempts to refine, for example, current values for the mean requirements in healthy adults for copper and molybdenum, which are 700 and 34 mg/d, respectively (37). Rather, it might now be more productive to begin collaborative studies designed to generate more complete mechanistically based information on the response (pattern of gene expression, activity of metabolic pathways, physiological system functions, such as those related to immune and stress resistance, trace element kinetics, for example) to different intake levels of a specific trace element within a given genotype/environmental context. The lack of good dose response biological data characterizes so much of the knowledge base of human nutrient requirements. This might be extremely difficult to achieve in any comprehensive way, but I don't see a major alternative or otherwise simple way forward. Such an effort would, furthermore, bring into sharper focus and contact a great deal of basic biology with the more applied research findings and this would measurably enhance knowledge on nutritional aspects of trace elements in man and animals. With this new knowledge a better understanding of the mechanistic basis for and extent of variation in requirements among otherwise apparently similar individuals would emerge. The variation in iron absorption, which accounts for a major portion of the difference in iron requirements among men and nonmenstruating women, might have a genetic origin and perhaps due, at least in part, to a specific gene that encodes duodenal cytochrome B (40), as I have speculated before (41). The real point is that a combination of genetic/cellular studies together with whole body investigations of iron uptake and dynamics would be both valuable and exciting and they would also test the validity of such an integrated research framework.
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3% of mammalian genes encode for zinc fingers that mediate interactions with DNA and protein-protein interactions. This raises the possibility of i) conducting differential enzyme displays, where as many as 40–50 enzymes can be measured for titer by immunoassay and activity measured with fluorescent substrates on easily accessible matrices such as blood, saliva, urine, synovial fluids, and/or ii) developing enzyme sensor signatures that, for example, assess the inflammatory response where lymphocyte ectopeptidases and/or convertases and apoptotic enzymes, and/or matrix, and/or metalloproteases could be determined. Together with the aid of informatic tools, this technology- and data-rich approach could be a new basis for exploring the consequences of altered trace element status and function and for identifying underlying mechanisms. Although all this needs development and testing, there are potentially many new challenges to metabolic phenotyping and real opportunities for developing and establishing new approaches and/or paradigms to characterize the trace element dependent metabolome. Thus, it is either now possible or with some further development it should be possible to: a) probe components of the metabolome with specific trace element dependent functions related to immune defense, apoptosis and energy transformation; b) compliment molecular, organelle and other reductionist approaches with essentially reconstructive paradigms; and c) provide information about trace-element needs, function and toxicity. However, a strategic research effort is now required to promote and enhance this framework. It is most important and it may be unnecessary to emphasize the importance of this research, but as Claude Bernard said, "When we wish to ascribe to a physiological quality its value and true significance, we must always refer it to its whole and draw our final conclusions only in relation to the effects in the whole."
Although the science of nutritional metabolomics is in its infancy, major advances for growth in this area can be anticipated. In short, the development and evolution of metabolic profiling technologies, including mass spectrometry (52) and nuclear magnetic resonance spectroscopy (53), coupled with advances in high throughput analytical and computing technologies, will revolutionize our ability to discover how trace-element nutrients determine the level and activity of specific gene products and affect genetic pathways. Particularly important will be the development of generic techniques, as has been accomplished in molecular and cellular biology, to monitor many hundreds of metabolites, possibly accomplished with the aid of novel biosensors, such as those exploiting fluorescence resonance energy transfer (FRET) (54, 55). Furthermore, metabolomics will inform us how trace-element nutrient-gene activity is integrated into the jigsaw of global gene activity. In reasonable contrast to the genome, and like the proteome, the metabolome is not stable over time. Thus, the comprehensive study of metabolomics requires that the quantitative dynamic features of the metabolome be defined and examined under various conditions and at specific times, using an array of techniques ranging from micro-total analytic systems (56) and tracer technology (46) to metabolic and functional imaging (57– 60) that can provide an anatomic and behavioral dimension to the metabolomic assessment of trace-element nutritional responses and requirements. To be fully successful in this context will require the elaboration and strong support of a major interdisciplinary enterprise, which is really what nutrition science is all about in my opinion.
Among the various challenges listed earlier in Table 1 were ways to improve intakes and balances among trace elements and other nutrients so as to enhance the role of diet in health promotion and maintenance. Space prevents a detailed discussion of this topic but I find the potential contribution of agricultural biotechnology (61) to be particularly important for improving trace element nutrition in human populations. To fully and effectively exploit this technology we need: i) a goal (criterion of trace element adequacy), ii) information about intakes of individuals, iii) information about individuals' requirements, iv) information about factors that affect requirements, v) information about individuals' nutritional status, vi) dietary characteristics (foods, sources, meals), as well as an understanding of the social, cultural, economic and political determinants of well-being. Much of this information depends on the success of the type of research effort alluded to above, with an especially strong focus on the systems biology of trace elements in man and animals.
A multidisciplinary enterprise for TEMA. It is clear to me, as we consider linking genotype to phenotype through the various biological levels of complexity at which trace elements exert their influence, that there are multiple and different kinds of challenges for us to face and resolve. These are related to research, its nature and the institutional framework in which it is conducted; the multidisciplinary approach must be fostered and this will require infrastructural changes in the way the science is investigated, including the nature of scientific collaboration and the need to acquire new skills (biomedical scientists in mathematics, statistics, kinetics) and so on.
With reference to the value of cross-disciplinary collaboration, I find the biology and function of boron to be an interesting case; recently the Institute of Medicine, Food and Nutrition Board (IOM/FNB) (37) concluded that it is still not possible to establish a clear biological function for boron in humans. Nevertheless, I learned recently that red wine is rich in rhamnogalacturonan II (RGII) (62). This is the most complex polysaccharide on Earth and is present in plant cell walls. It is composed of 11 kinds of sugar monomers and forms dimers through boron, and it is essential for plant growth. Furthurmore, boron rescues the effects of a defect in RGII cross-linked dimer content of an Arabidopsis mutant, mur-1 (63). It could be asked, therefore, what is the relationship between the structure of cell surface carbohydrates in eukaryotes and boron availability/levels. Also, if boron is involved in calcium function (64, 65), should we examine calcium-boron relations in soft tissues with particular reference to structure-function relationships.
A multidisciplinary approach will also require a collaborative spirit as a part of the process needed to solve practical trace-element nutritional problems. Such a spirit would help create new and exciting interdisciplinary programs, in this case with trace elements and nutritional well-being the primary focus. Also, there must now be careful consideration and reassessment of the nature of the training given to the coming generations of graduates and post graduates in areas relevant to TEMA's purpose. This is another challenge and it should not be underestimated.
Coda
In essence, it seems to me that for the field to move forward we need to be less insular as investigators and even more creative as mentors. There is a necessity for all participating parties, teachers, scientists, students and supporters, to understand and embrace the overall objectives of TEMA and to mobilize the necessary intellect and physical and financial resources. To borrow an idea from Bradshaw (66), it also seems to me that trace-element science and nutritional science, more broadly, is an excellent example of "The Blind Men and the Elephant." This poem, by the American poet John Godfrey Saxe (1816–1887), based on a fable told in India many years ago, reads in part:
Just as Bradshaw (66) commented about the future of the journal "Molecular and Cellular Proteomics" of which he was editor, it similarly appears that as long as those six blind men talk and interact wisely the elephant will be well cared for. In achieving this, trace-element and nutritional science will evolve effectively for the improved good of those individuals and populations most vulnerable to trace-element inadequacies, excesses or imbalances. It will be interesting to learn whether any of the ideas presented herein have merit or perhaps even find a place in the organization and/or content of TEMA 12, to be held approximately three years hence in Coleraine, N. Ireland.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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