Nutri Report

CONTENTS: INTRODUCTION  REVIEW OF NUTRIGENOMICS  BACKGROUND AND PREVENTIVE HEALTH  CONCEPT IN NUTRIGENOMICS  WHY IS NUTRIGENOMICS IMPORTANT



COMMERCIAL IMPLICATIONS

 FOOD AND DRUGS AFFECT INDIVIDUALS DIFFERENTLY



PHARMACOGENOMICS IS SIMILAR TO NUTRIGENOMICS



PERSONALIZED MEDICINE INCLUDES PERSONALIZED NUTRITION

 GENE-DIET-DISEASE INTERACTION  INFLUENCE OF HUMAN GENETIC VARIATION ON NUTRITIONAL REQUIREMENTS  BUSSINESS MODEL OF NUTRIGENOMICS  FIRST FOOD DERIVED NUTRIGENOMICS PRODUCT



ADDITIONAL STRUCTURAL-FUNCTIONAL CLAIMS FOR SUPPORTING IMMUNE SYSTEM DEFENSE

 ASPECTS OF NUTRIGENOMICS



APPLICATION OF NUTRIGENOMICS FOR PERSONAL AND PUBLIC HEALTH



NUTRIGENOMICS AND CANCER



DIET NUTRITION AND CANCER PREVENTION



INSULIN RESISTANCE



FOLIC ACID DEFICIENCY



LOW VITAMIN-D



GLUTEN SENSITIVITY

 NUTRIGENOMICS-THE RESEARCH

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BIOMIC TECHNOLOGIES



HUMAN GENOME PROJECT

 PERSONALIZED NUTRITION TESTING  TESTING FOODS IN THE LABORATORY  USING LABORATORY MODELS TO TEST THE EFFECTS OF FOOD



USING REPORTER GENES IN RESEARCH WITH CELL LINES

 FIVE TENETS OF NUTRIGENOMICS  WHY IS NUTRIGENOMICS NOW GROWINGb PROMISE OF NUTRIGENOMICS  THE STATE OF THE FIELD AND NEEDS  NUTRIGENOMICS AND GENOME HEALTH  RATIONALE AND AIMS OF NUTRIGENOMICS

 CONCLUSION

NUTRIGENOMICS-The future of health Nutrigenomics is one of the most exciting new areas in the food and wellness. It is a very new word in the food and scientific fields. It is a now emerging as a business. The term

“genomics”

represents

genes

and

similar

words,

an

example

being

pharmacogenesis. Nutrigenomics is still an emerging science only named in 1999. But as knowledge in the field grows, it looks set to only offer new insight into how genetics shape our nutritional needs but also significantly improve understanding of the molecular mechanism of the food components. It is the study of how DNA and our genetic code affect our need for certain nutrients and help maintain optimal heath throughout our life. Nutritional system biology is the popular word for nutrigenomics.

First it was smart drugs. Now it's smart diets Nutrigenomics is a genetically based nutrition and dietary intervention approach that will result in dietary supplements, functional and medicinal food that maximize the health of each individual. It is the study of food and nutrition focused mainly on the interactions with diseases and human specific genes. Nutrigenomics is the science that could bring real advances for the functional food industry within the next 2 or 3 years. This word refers to the study of our food and how it influences our health through interactions with our personal genetic make-up. It is suggested in the hand-waving way of futurist commentators in this field, that one day many of our ailments might be treated not with drugs but with special diets. However, the complexity of the factors influencing health not merely diet and heredity, but also

economic and social conditions, culture and behavior -are likely to make it difficult to isolate the influences of food from all other.

BACKGROUND AND PREVENTIVE HEALTH Throughout the 20th century, nutritional science focused on finding vitamins and minerals, defining their use and preventing the deficiency diseases that they caused. As the nutrition related health problems of the developed world shifted to over nutrition, obesity and type two diabetes, the focus of modern medicine and of nutritional science changed accordingly. In order to address the increasing incidence of these diet-related-diseases, the role of diet and nutrition has and is been extensively studied. To prevent the development of disease, nutrition research is investigating how nutrition can optimize and maintain cellular, tissue, organ and whole body homeostasis. This requires understanding how nutrients act at the molecular level. This involves a multitude of nutrient-related interactions at the gene, protein and metabolic levels. As a result, nutrition research has shifted from epidemiology and physiology to molecular biology and genetics and nutrigenomics was born. The emergence and development of nutrigenomics has been possible due to the development in genetics research. Inter-individual differences in genetics, or genetic variability, which have an effect on metabolism and on phenotypes, were recognized early in nutrition research, and such phenotypes were described. With the progress in genetics Biochemical disorders with a high nutritional relevance were linked to a genetic origin. Genetic disorders, which cause pathological effects, were described. Such genetic disorders include the polymorphism in the gene for the hormone Leptin which results in gross obesity. Other gene polymorphisms were described with consequences for human nutrition. The folate metabolism is a good example, where a common polymorphism exists for the gene that encodes the methylene-tetrahydro-folate reductase (MTHFR).

It was realized however, that there are possibly thousands of other gene polymorphisms which may result in minor deviations in nutritional biochemistry, where only marginal or additive effects would result from these deviations. The tools to study the physiological impact were not available at the time and are only now becoming available enabling the development of Nutrigenomics. Such tools include those that measure the transcriptome - DNA microarray, Exon array, Tiling arrays, single nucleotide polymorphism arrays and genotyping. Tools that measure the proteome are less developed. These include methods based on gel electrophoresis, chromatography and mass spectrometry. Finally the tools that measure the metabolome are also less developed and include methods based on Nuclear magnetic resonance imaging as well as gas and liquid chromatography.

CONCEPT IN NUTRIGENOMICS Nutrigenomics or nutritional genomics is a multi-interdisciplinary science that combines information

from

genetics,

nutrition,

physiology,

pathology,

molecular

biology,

bioinformatics, biocomputation, sociology, ethics and other disciplines. We believe that understanding diet-nutrition interactions in different individuals will help explain and help alleviate health disparities hence; this knowledge applies to national and international societal issues in personal and public health. Department of nutrigenomics seeks to provide a molecular genetic understanding how common dietary chemicals (i.e., nutrition) affect health by altering the expression and/or structure of an individual’s genetic make up. The fundamental concepts of the field are that the progression from a healthy phenotype to a diseased phenotype must occur by changes in gene expression or by differences in activities of proteins, enzymes, and that dietary chemicals directly or indirectly regulate the expression of genomic information. Nutrigenomics is an emerging inter-disciplinary niche science that may have real go-go potential. The gist of the concept is that the foods we eat provide a feedback stimulus to our very DNA, triggering our genes to either salubrious or unhealthy responses. An example is the effect of infesting junk foods (bad fats, processed carbohydrates), which trigger genes to send out messages that slow the body’s metabolism and actually promote weight gain, inflammation, diabetes and other unpleasantness. Applying the concepts of nutrigenomics to individuals is challenging because of the diversity of genetically individual responses to the complexity of foods, and the study design of health or disease processes.

WHY IS NUTRIGENOMICS IMPORTANT? While some DNA is common to all people, there are variables within genes known as polymorphisms. When they occur they may be responsible for differences in the protein product of that gene, proteins that play key roles in important body processes. Current research has determined how genetic variation affects our body’s ability to utilize nutrients and remove toxins. One’s health is a result of how these processes interacts with environmental and lifestyle factors such as diet, exercise, stress, smoking and alcohol.

By learning about the specific nature of genes, we will find out how their variations (polymorphisms) affect the way in which our body manages its nutrition and detoxification processes. By testing for these variations one can learn if these body processes are below their optimum level. Every individual has a predetermined susceptibility to disease based on his/her genetic profile.

Commercial implications: As with our understanding of the very nature of DNA, nutrigenomics will find its way into our life by evolution not by revolution. We envision two key phases of products that could be separated by as much as 10 years: the “biomarker phase” and the “genetic phase”.

The biomarker phase is upon us and will be based on genetic tools that are presently available. It must be based on the impacts of bioactive on biomarkers (such as cholesterol or blood pressure) and end points (such as pain or mobility). With an estimated 20,000-25,000 gene in the body, it will be many years before we understand the function of each gene and the involvement of each gene in chronic human diseases. However, with our present state of knowledge it is still possible to use gene expression screening to identify bioactive that can impact genes involved in human diseases. For example, it has been demonstrated by researchers at Rutgers University that theaflavins from tea impact expression of multiple genes involved in arthritis. When mice are fed a theaflavin-enriched tea plant extract, inflammatory cytokines are greatly reduced. Also, these mice have greatly reduced symptoms of induced arthritis. Hence, genetic screening technology can be used to identify bioactive that have a direct impact on treatment or prevention of a disease through regulation of associated genes. To determine if the product works in people is rather simple. If mobility is improved and swelling is reduced after taking the product, its benefit, especially in those who suffer daily from arthritis, will be obvious. All natural plant-based extracts appear to be ideal for the development of products in the biomarker phase. Natural products are often composed of mixtures of related compounds that could have impact on several genes in associated biochemical pathways. For example, research has demonstrated that consumption of cocoa could prevent obesity in rat fed a high fat diet. These rats had altered gene expression of several genes involved in fatty acid synthesis and fatty acid transport. As target genes are identified, gene expression screening will be a valuable tool to identify whether foods and extracts have an effect on such genes and will enable the discovery of bioactive that alter biomarkers associated with human disease. The genetics phase will be based on genetic testing in which the DNA is analyzed. The result of a test will be a personalized road map for disease prevention. If one knows that what diseases one is likely to contract, one can supplement one’s diet with personalized preventive products. Such DNA tests exist today, but only for woefully small number of genes. These present day tests provide only a glimpse of the future. The MTHFR gene, with a T instead of a C at a specific position in the DNA, makes an enzyme with an

altered amino acid (valine instead of alanine) that has altered enzyme activity. It has been estimated that 10% of Europeans have two copies of the mutated MTHFR. When present in two copies, this mutation results in higher plasma levels of homocysteine. High plasma level homocysteine level is correlated with increased risk of cardiovascular disease. Present day test for the MTHFR gene recommend increased consumption of folic acid for those with the mutated MTHFR gene. Because it has been shown to reduce homocysteine levels. The genetics phase will entail nutrigenetics, a subset of nutrigenomics, which will enable dietary recommendations for individuals with specific, chronic disease genotypes. Nutrigenetics refers to how a person’s genetic make-up predisposes them to dietary components. Genetic variation (genotype) dictates the response of individuals to medicines (pharmacogenomics), as well as to food and supplements to interact. While nutrigenomics explores how food chemicals alter the expression of genetic material, genetic differences called polymorphisms (or SNP’s) affect an individual’s biological response.

Because

not

everyone

is

genetically

identical,

ultimately

through

nutrigenetics, recommendations of dietary supplements and whole foods can be made based on the specific genes of these individuals. For example it is possible that a single allele variation can alter whether polyunsaturated fatty acids or soluble oat can help reduce cholesterol.

FOOD AND DRUGS AFFECT INDIVIDUALS DIFFERENTLY Humans can metabolize a wide variety and range of amounts of food chemicals. The flexibility in metabolic response to changes in type and concentration of dietary chemicals demonstrates an important clue for understanding the effects of diet on health. It is the interactions of dietary chemicals with genetic machinery and information (diet X genotype interactions) that play a key role in maintaining health and preventing diet-influenced chronic diseases.

Pharmacogenomics is Similar to Nutrigenomics The advances and concepts of pharmacogenomics underscore the importance of genotype X environment interactions by showing how individual genetic variation in human populations can affect a drug's efficacy and severity of undesirable side effects. Genotyping is now being incorporated into clinical trails to predict drug safety, toxicity, and efficacy. By relating phenotype to genotype, drug companies are designing and developing better drugs with less adverse side affects. By identifying the nonresponding sub-populations, pharmacogenomics can also develop new drugs from compounds previously thought too toxic for human use.

Personalized Medicine includes Personalized Nutrition The concept of “personalized” medicine is now being extended to the field of nutrition. It is now accepted that nutrients (i.e., macronutrients, micronutrients) alter molecular processes such as DNA structure, gene expression, and metabolism, and these in turn may alter disease initiation, development, or progression. Individual genetic variation can influence how nutrients are assimilated, metabolized, stored, and excreted by the body.

GENE-DIET-DISEASE INTERACTION 97% of the genes known to be associated with human diseases result in monogenic diseases, i.e. a mutation in one gene is sufficient to cause the disease. Modifying the dietary intake can prevent some monogenic diseases. One example is phenylketonuria, a genetic disease characterized by a defective phenylalanine hydroxylase enzyme, which is normally responsible for the metabolism of phenylalanine to tyrosine. This results in the accumulation of phenylalanine and its breakdown products in the blood and the decrease in tyrosine, which increases the risk of neurological damage and mental retardation. Phenylalanine-restricted tyrosine-supplemented diets are a means to nutritionally treat this monogenic disease.

In contrast, diseases currently in the world, e.g. obesity, cancer, diabetes, and cardiovascular diseases, are polygenic diseases, i.e. they arise from the dysfunction in a cascade of genes, and not from a single mutated gene. Dietary intervention to prevent the onset of such diseases is a complex and ambitious goal. Recently, it was discovered that the health effects of food compounds are related mostly to specific interactions on molecular level, i.e. dietary constituents participate in the regulation of gene expression by modulating the activity of transcription factors, or through the secretion of hormones that in turn interfere with a transcription factor.

INFLUENCE OF HUMAN GENETIC VARIATION ON NUTRITIONAL REQUIREMENTS Gas chromatography-mass spectrometry, Liquid chromatography-mass spectrometry and.Genetic variation is known to affect food tolerances among human subpopulations and may also influence dietary requirements, giving rise to the new field of nutritional genomics and raising the possibility of individualizing nutritional intake for optimal health and disease prevention on the basis of an individual's genome. However, because gene-diet interactions are complex and poorly understood, the use of genomic knowledge to adjust population-based dietary recommendations is not without risk. Whereas current recommendations target most of the population to prevent nutritional deficiencies, inclusion of genomic criteria may indicate subpopulations that may incur differential benefit or risk from generalized recommendations and fortification policies. Current efforts to identify gene alleles that affect nutrient utilization have been enhanced by the identification of genetic variations that have expanded as a consequence of selection under extreme conditions. Identification of genetic variation that arose as a consequence of diet as a selective pressure helps to identify gene alleles that affect nutrient utilization. Understanding the molecular mechanisms underlying gene-nutrient interactions and their modification by genetic variation is expected to result in dietary recommendations and nutritional interventions that optimize individual health.

BUSSINESS MODEL OF NUTRIGENOMICS Nutrigenomics is surely expected to be the next wave for food industry, even though only a few practical ideas have emerged.  One business model is the development of customized nutraceuticals based on specific genetic profiles. Nutraceutical is a portmanteau of "nutrition" and "pharmaceutical" and refers to foods claimed to have a medicinal effect on human health. Such foods are also called functional foods. It can also refer to individual chemicals present in common foods. Many such nutraceuticals are phytonutrients.The name was coined by Dr. Stephen Defelice in 1989. Examples of claims made for nutraceuticals are red wine (resveratrol) as an antioxidant

and

an

anticholesterolemic,

broccoli

(sulforaphane)

as

a

cancer

preventative, and soy and clover (isoflavonoids) to improve arterial health in women. Such claims are being researched and many citations are available via PubMed to ascertain their veracity. Several nutraceuticals are known. Some examples are flavinoids, antioxidants such as gamma-linolenic acid, beta carotene, anthocyanins, etc. With the Dietary Supplement Health and Education Act (DSHEA), USA, several other compounds were added to the list of supplements originally mentioned in FDA notification. Thus many botanical and herbal extracts such as Ginseng, garlic oil,etc found place as nutraceuticals. Nutraceuticals are often used in nutrient premixes or nutrient systems in the food and pharmaceutical industries.  Another model may be foods for specified health use, which are already on the Japanese market. In Japan, the Japanese Ministry of Health, Labor and Welfare has approved about 350 items as food for specified health use. Each item has a specific health claim, such as food for hypertension, high cholesterol, diabetes, etc. based on clinical studies. All products have been developed based on

scientific analysis and data, even though not yet on the genetic level yet. The foods are expected to expand the field of nutrigenomics.

FIRST FOOD DERIVED NUTRIGENOMIC PRODUCT First food derived nutrigenomic product could be on the market as early as next year, if research continues according to plan and suitable partner can be found. After ten years of research and development, the first product is likely to be theaflavins from black tea, which have been shown to turn off a number of genes involved in inflammation. The leveraging technology and the scientists are trying to find new bioactive compounds that could have a significant effect in response to human disease and to take the resulting ingredients to market. Theaflavin have already been shown to turn off genes involved in inflammation in mice and horse models. The next step is to carry out pre-clinical trials in animals and then human clinical trials. There may also be applications for the theaflavins technology in the area of sports, as minor aches and pains are caused by inflammation, which could be reduced by the down-regulation of the genes responsible. Inflammation is a factor in a number of other health conditions including psoriasis, certain cancers, and cardiovascular disease and to some extent obesity; the theaflavins would be ideally delivered in supplement form. Anther viable approach could be to put the theaflavins back into tea to deliver a higher concentration to consumers. Theaflavins are already available as food and supplement ingredients through a number of suppliers including Applied Food Sciences and Naschai Biotech, but Wellgen’s will be the first developed according to nutrigenomics principles. In the regulatory environment, it is advantageous that we start with food as a source material, as unlike many chemicals it has a history of safe use.

Another keen area of investigation is chemicals found in the peel of certain species of orange, which have been found to turn on a gene involved in cancer protection. However it remains to prove how they can prevent cancer in humans, as this will involve clinical trials over a long period of time. WellGen, Inc. has announced that it has completed a pivotal study demonstrating that its nutrigenomics technology platform can successfully predict biological activity of natural products in humans. WellGen is a biotechnology company that uses nutrigenomics to discover and develop proprietary functional food ingredients. Using a proprietary ingredient standardized for certain theaflavins in black tea, WellGen’s study has proven that the ingredient’s inflammation-fighting properties can be identified and quantified by the company’s technology such that the findings can be effectively and efficiently translated to consumer applications. WellGen’s proprietary ingredient, WG0401, provided protection to healthy volunteers who were given a potent inflammatory challenge. Healthy volunteers treated with the WellGen extract had inflammatory biomarker levels ranging between two-to-six fold less than the placebo group when challenged with an inflammation-inducing bacterial lipopolysaccharide.

Study Leads to Additional Structure-Function Claims for Supporting Immune System Defense In addition to the potent anti-inflammatory benefits already known for the ingredient, the human study of WG0401 demonstrated immune system defense support. Based on the analysis of the biomarkers used in the studies, WellGen, Inc. discovered that its WG0401 enhances the response of the immune system to a bacterial challenge. Thus these results indicate that WG0401 can be beneficial in immunoregulation. WG0401, a food ingredient that is derived from black tea and is characterized by enriched theaflavin content, will be available commercially this year. WellGen’s inflammation program, its first research initiative, was set up to develop proprietary ingredients with meaningful benefits for functional food applications. WellGen is also in late stage development of a model to prove the same lab-to-man paradigm with a program focused on developing proprietary ingredients for foods that can affect the genes involved in adipocyte (fat cell) development. The objective is to discover functional food ingredients that will combat obesity. WellGen has employed a process that includes gene expression analysis, cell based assays, animal studies and ex vivo human studies to identify potential candidates that will interfere with fat cell development. The lead ingredient in this program has successfully completed that process and will enter a human biomarker study in the third quarter of 2007. WellGen is committed to developing proprietary ingredients called “therapeutic nutrition ingredients” using its patented nutrigenomic screening process and its proprietary research models for validating health benefits in humans. As a proof of efficacy in humans, WellGen uses quantitation of biomarkers of inflammation to demonstrate the biological activity of its proprietary ingredients that have shown positive results in laboratory and animal tests. WellGen uses relevant and quantifiable biomarkers to streamline an ingredient’s evaluation in humans by reducing the duration of human trials and obtaining objective data. Examples of validated biomarkers include glucose levels as related to diabetes or cholesterol levels related to heart health/cardiovascular risk.

ASPECTS OF NUTRIGENOMICS

Application of Nutrigenomics for Personal and Public Health The application of nutritional genomics to personal and public health poses ethical issues similar to those of pharmacogenomics, particularly with respect to genetic privacy. In addition, some believe that the predictive power of a genetic test for diet advice is too low to be of concern. However, as we decipher the complex biology of gene – nutrient and gene – gene interactions, the probability of identifying disease susceptible increases: genetic testing may identify individuals with predisposition to diet induced disease. The biggest ethical and practical issue facing the application of the results of nutrigenomics research is timing: when is there a sufficient amount of results and knowledge available to interpret genetic tests. These issues require dialogue among all stakeholders. Nutrigenomics and cancer Nutrigenomics as the study of how different foods may interact with specific genes to increase the risk of common chronic diseases such as type 2 diabetes, obesity, heart disease, stroke and certain cancers. Nutrigenomics seeks to provide a molecular understanding of how common chemicals in the diet affect health by altering the expression of genes and the structure of an individual's genome. The premise underlying nutrigenomics is that the influence of diet on health depends on an individual's genetic makeup.

Diet nutrition and cancer prevention According to the European Prospective Investigation into Cancer and Nutrition (EPIC), a few of the emerging results found in the link between diet and cancer are:  Consumption of meat sharply increased risk of stomach cancer and esophageal cancer. For every 100 grams of meat consumed by subjects, risk for stomach cancer more than tripled. The association between meat intake and stomach cancer was considerably stronger among subjects with populations of H. pylori bacteria in their stomachs.  Two indicators of abdominal obesity, waist circumference and waist-to-hip ratio, were strongly associated with colon cancer risk in both sexes. Men with the largest waist circumference had 39 percent higher risk of colon cancer than men with the smallest, for example, while women in the study with the largest waist circumference has a 48 percent higher risk than women with the smallest waists.  Blood samples of women with breast cancer were compared to blood samples of women without breast cancer. Women over 60 whose blood was given under non-fasting conditions, high levels of serum C-peptide, that could reflect insulin resistance -- long suspected of contributing to cancer risk -- was associated with a doubling of breast cancer risk.  The risk for oral and pharyngeal cancers drop by 9 percent for every 80 grams of fruits and vegetables consumed per day. On conclusion it clearly shows that globally, diets that are high in fruits, vegetables, fiber and fish are associated with greater cancer prevention -- with obesity and sedentary lifestyles much larger factors in increasing cancer risk.

Insulin resistance: ‘the obesity disease.’ A diet of refined sugars and carbohydrates—such as bread, white rice and flour products—leads to a rapid rise in blood sugar and a spike in insulin (a hormone that controls the metabolism of carbohydrates). Over time, one can become resistant to insulin’s good effects and thus need more to do the same job. Insulin resistance is a major cause of weight gain, heart disease, cancer and dementia, and it often leads

to diabetes. It also causes hidden inflammation throughout the body, which, like a smoldering fire, damages our cells and organs and accelerates most of the diseases of aging. One may have insulin resistance if one has a family history of abdominal obesity, diabetes, gestational diabetes, early heart disease, high triglycerides or low HDL cholesterol. Prevention: One can prevent or reverse insulin resistance by eating unprocessed food—fruits and vegetables, beans, nuts, seeds and whole grains. Include in one’s diet wild fish such as small salmon, sardines and herring. Avoid foods with added salt. Stay away from highly processed foods,

particularly those containing

high-fructose

corn

syrup

and

hydrogenated fats. This diet will “turn off” the genes that promote insulin resistance, obesity and inflammation and turn on the genes that restore weight and metabolism to normal for most people.

Folic acid deficiency: The gene that increases the need for folate (or folic acid) affects up to half of Americans. Inadequate levels of folate can lead to dementia, many cancers, heart disease, osteoporosis, birth defects, autism and depression. One may have folate deficiency if one has a family history of heart disease, dementia, breast, colon or cervical cancer, spina bifida, Down syndrome or depression. Prevention: - Eat a diet rich in folic acid. Good sources include dark-green leafy vegetables—such as spinach, collards, kale and arugula—whole grains, asparagus and beans. Coffee, alcohol and smoking deplete folate and raise homocysteine levels. About 800 mcg (micrograms) a day of folic acid is sufficient for most people. Vitamins B6 and B12 also are recommended to keep homocysteine at an ideal balance.

Low vitamin D: a result of lives spent indoors. Vitamin D is not only important for bone health, but recent research has also linked vitamin D deficiency to conditions as diverse as colon, prostate and breast cancers, multiple sclerosis, type 1 diabetes, heart disease, autoimmune diseases, Graves’ disease, seasonal affective disorder (SAD) and osteoporosis. While our ancestors were foraging and hunting, their skin produced the equivalent of nearly 10,000 IU (international units) of vitamin D a day. Now, when many of us spend most our days indoor, our bodies produce dramatically less: Even the average multivitamin contains only 400 IU— and many people don’t even get that much. One recent study found that 40% of Americans were deficient in vitamin D. Plus, as we age, deficiency increases: 70-year-old skin produces only 25% of the vitamin D of 20-yearold skin. Increased vigilance against overexposure to the sun’s UV rays —which stimulate the skin to produce vitamin D—also has made it more difficult to get enough of this important nutrient. Sun block prevents its production by the skin. One may have vitamin D deficiency if one is dark-skinned. One’s melanin may prevent absorption of ultraviolet radiation, which helps the body manufacture this vitamin. There is a blood test for vitamin D deficiency. Prevention: It is recommended that one should take up to 2000 IU of vitamin D a day. Dietary sources include oily fish such as wild salmon, mackerel and sardines, but supplements are essential.

Gluten sensitivity: the great masquerader. Most of us eat large quantities of gluten, which is the protein found in such grains as wheat, barley, rye, spelt and oats. But 30% of Americans may develop some form of

sensitivity to gluten. That’s because they carry the genetic marker for celiac disease, which is an autoimmune disorder related to the consumption of gluten. (About 1% of our population has active celiac disease.) This condition is dramatically under diagnosed because it masquerades as many other diseases, including nearly all inflammatory and autoimmune diseases, arthritis, irritable bowel syndrome and other digestive disorders, anemia, osteoporosis, cancers, neurologic disease, depression, migraines, infertility, liver disease and more. One may have gluten sensitivity if one has a family history of celiac disease, irritable bowel syndrome, autoimmune diseases or thyroid diseases. Prevention: -. If one is tested positive for gluten sensitivity or celiac disease, a glutenfree diet usually will completely relieve the symptoms. Many gluten-free products can be found in health food and specialty stores.

NUTRIGENOMICS- The research The interaction between the human body and nutrition is an extremely complex process involving multi-organ physiology with molecular mechanisms on all levels of regulation (genes, gene expression, proteins, and metabolites). Only with the recent technology push have nutritional scientists been able to address this complexity. Both the challenges and promises that are offered by the merge of 'biomics' technologies and mechanistic nutrition research are huge, but will eventually evolve in a new nutrition research concept: nutritional systems biology. This review describes the principles and technologies involved in this merge. Using nutrition research examples, including gene expression modulation by carbohydrates and fatty acids, this review discusses applications as well as limitations of genomics, transcriptomics, and proteomics, metabolomics, and systems biology. Furthermore, reference is made to gene polymorphisms that underlie individual differences in nutrient utilization, resulting in, e.g., different susceptibility to develop obesity.

Biomics technologies

The recent advances in nutrigenomics studies are owed to the completion of human genome project and the new biomics technologies that provide means for the simultaneous determination of the expression of many thousands of genes at the mRNA (transcriptomics), metabolites (metabolomics) and protein (proteomics) levels. Genomic and Transcriptomic studies are mostly conducted by DNA microarray technologies. Proteomics and metabolomics have no standardized procedures yet, but usually, proteome analysis is done by two-dimensional gel electrophoresis and Liquid chromatography-mass spectrometry, while metabolome analysis is conducted through Gas chromatography-mass spectrometry, Liquid chromatography-mass spectrometry and Liquid chromatography -Nuclear magnetic resonance. Usually, these technologies are applied in a “differential display” mode, i.e. by comparing two situations (e.g. diseased versus healthy) in order to reduce the complexity in data by examining only differences. The Human Genome Project (HGP) is a project undertaken with a goal to understand the genetic makeup of the human species by identifying all the genes in the human genome and mapping how individual genes are sequenced. The project began in 1990, and by some definitions, it was completed in 2005.

The cells on the left is normal is normal but the one on the right shows signs of genetic damage. The damaged DNA appears as six micronuclei in the cell.

Post-genomic technologies take a more holistic perspective and use the information provided by the sequencing of the human genome, allowing us to measure how what we eat interacts with our bodies, or more specifically our genes, proteins and metabolism. In the past, we have been limited in our scientific exploration to a few dietary compounds, a handful of relevant biochemical pathways and perhaps latterly a small number of genes that might be pertinent to the disease in question. The result has been some specific examples of benefit provided by individual/ groups of food compounds.

We are exposed to a complex mixture of foods throughout life and intricate biochemical processes extract energy and use nutrients and other compounds to enable us to grow and function properly. In the past, many food compounds were dismissed as being unimportant, having no obvious nutritional role. However, dietary intake is not just about avoiding deficiency diseases but also optimal health or the avoidance of age-related disease, and what is obvious is that the benefits of some dietary choices are not the same for everyone, which is why nutrigenomics is such an exciting prospect for nutrition researchers.

TRANSCRIPTOMICS: The transcriptome is the complete set of RNA products that can be produced from the genome, and transcriptomics is the study of the transcriptome. Microarrays and chips represent powerful tools for studies of diet-gene interactions.

PROTEOMICS: -

Just as the entire DNA sequence is called the human genome so the full complement of proteins in a cell or tissue is called the proteome. Subsets of proteins from the proteome will be produced by different cells and these will change in response to internal and external stimuli. Proteomics is the study of the proteome, and attempts to determine the role of the proteins found in cells, tissues or an organism at any one time. Proteomics is technically very challenging and the presence/ absence of protein is not necessarily indicative of metabolic change. METABOLOMICS: -

METABOLOMICS examines the whole metabolism, which ultimately reflects the behaviour of different patterns of genes. It is sometimes spelt metabolomics, but these are essentially the same thing. Metabolomics is making great advances in this complex approach to nutrition research. This is partly because nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are established techniques, but also because the application of pattern-recognition statistics, such as principle component analysis, is conventional in this field.

Metabol/nomics also has the advantages of offering more immediate information about our metabolism, which is not presented by changes in gene transcription or protein expression since both can occur without apparent metabolic consequences.

PERSONALIZED NUTRITION TESTING The science of nutrition and genomics is driving the emergence of nutrigenomic food products, some of which are unique and patentable, that crosses traditional pharma, biotech, and food industry boundaries. Within the new field of diagnostic tests that relate to diet and health, two applications have emerged: Nutrigenetic tests are being used for personalized nutrition testing, and tests are also being developed to predict risk or susceptibility for diet-related diseases.

The sequencing of the human genome and the ability to determine the genetic profile of individuals and their predisposition to diseases has coincided with the development of functional foods. These are products, which confer physiological benefits as well as nutritional or other health, and lifestyle benefits on consumers. One exciting development of this is the relationship between human genetics and functional foods.

There is increasing

research showing that foods and food components can trigger changes in gene expression in animals and humans, and that some of these may be beneficial in disease

and disorder prevention. From a different angle, the genetic make-up of people may provide clues as to what food may be beneficial or disadvantageous. This research area is known as nutritional genomics or nutrigenomics.

Food has different effects on different people Our bodies need to be able to absorb the right amount of different food components, store them in the right places, and use them at the right time. Our genes control all of this. A person's genes therefore affect what happens to the food that is eaten. As a result, two different people can eat the same food, but it has different effects on them. For example, two people can eat the same amount of fatty food, and one will put on weight a lot faster than the other.

Different people respond differently to different foods. This is partly because we all have different genes, and our genes can affect the way our bodies deal with food. Because of this, particular food components will be more helpful to some people than to others. Nutrigenomics is the study of how our food and our genes interact. The main aim is to use information about genes to work out the effects that foods can have on an individual’s health, performance, and risk of disease.

The research

The nutrigenomics research team will be trying to work out the best (and worst) foods for individual patients, based on their genetic profiles (The unique sequence of bases in someone's DNA code is their genetic profile). The researchers will also be trying to work out eating patterns that might help to reduce the risk that family members who have affected genes will also get the disease.

Collecting patient information The first task is to find out as much as possible about people who have the disease. This requires a group of volunteers who agree to participate in the study. The researchers will collect details about each person's physical characteristics, disease history, diet and lifestyle. They will also collect information about whether or not family members have had the disease – this would suggest a genetic link.

Studying genetic diseases: Finding out about the genes Certain diseases are commonly found in members of the same family. This indicates that a particular gene variation, or gene combination, might be involved. A person's genetic makeup is called their genotype. The genotype affects their physical characteristics, or phenotype. Researchers are now able to identify particular gene variations that might increase a person's susceptibility to getting a particular disease. The aim of this is to provide information to help with disease management. First the researchers need to study the DNA from a huge number of people who have a disease, and those who don’t. The DNA patterns between these two groups can then be compared. The researchers will be looking for particular DNA patterns that only appear in people who have the disease. Often the change only involves a single base in the DNA code. The DNA is collected from people who have agreed to participate in the study. Usually a blood sample is collected. The DNA is separated from the white blood cells and stored.

Often it will be separated into at least two containers, which are stored in different places (one can act as a back-up if necessary). The samples are also coded, so that anyone working with the DNA sample has no idea which person the DNA comes from. Once the DNA has been purified, millions of copies are produced using the polymerase chain reaction (PCR). This is important because a lot of DNA is needed for results of DNA tests to be seen – more than what is collected from the blood. Researchers can then use two different techniques to study the DNA and look for gene variants: gel electrophoresis and DNA sequencing.

Using gel electrophoresis to find gene variants associated with a disease To use gel electrophoresis, the DNA is first cut into pieces, called DNA fragments. Specific enzymes are used to do this. A selected enzyme will always cut the DNA from a particular person in the same places. It will cut the DNA from two different people in different places. This means that the sizes of the DNA fragments produced are unique for each person. The DNA fragments are then separated according to size using gel electrophoresis.

Using DNA sequencing to find gene variants associated with a disease DNA is composed of bases, called A, T, C and G. These bases are joined together in specific sequences. Each person has a unique sequence of bases. DNA sequencing is used to determine the base sequence of parts of the DNA samples. The sequences from people who have the disease are compared with those who don’t have the disease. Researchers look for specific sequences that are only found in people who have the disease.

DNA sequencing can be used to find genes associated with a disease In this simulated case, the researchers are looking for DNA sequences that are only found in patients who have a particular disease.

DNA sequencing results: Finding gene variations associated with a disease The picture shows parts of the DNA sequence from two different people – someone who has the disease (second sequence) and someone who doesn’t (top sequence). Can you see that the person who has the disease has a whole sequence of DNA missing? Because the DNA sequences of each person are unique, to make sure this particular difference is related to the disease, researchers would have to see if the missing sequence is also missing in other people who have the disease, and that it is not missing in people who don’t have the disease.

Susceptibility to a genetic disease Most diseases that have a genetic component are caused by variations in more than just one gene region. Environmental effects such as exercise, diet, exposure to radiation, etc. also may have an important influence. Having a certain sequence in a region of DNA therefore does not necessarily mean you will get the disease, but it may mean you are more susceptible to getting the disease. The next step will be to try to find patterns between people's genetic combinations and their dietary preferences. For example, patients with a particular genetic combination might all report that a particular food makes their disease symptoms worse. This will help the researchers to start thinking about specific foods for specific people.

TESTING FOODS IN THE LABORATORY

Foods are very complex substances and contain thousands of different molecules. Each food might contain molecules that are helpful, and molecules that are less helpful. Because of this, the foods must first be separated into components.

Separating out foods into component molecules

Food is made up of many different chemical components or parts, including vitamins, minerals, sugars, fibres, water, lipids, proteins, starches. In addition to these main nutrient components, many foods contain smaller amounts of biologically active chemicals. In plants, these are referred to as phytochemicals. Scientists can separate out all of these different components of foods.

Why separate food into its component parts? There are many reasons why scientists might want to separate out the component parts of food. For example, Nutrigenomics New Zealand (NuNZ) is investigating why different people respond differently to different foods. It seems that sometimes these variations arise because of differences in our genes. This is because our genes can affect the way that our bodies deal with the chemical components that make up a particular food. As a result, some food components might be particularly good for some people, and cause problems for others. The aim of the nutrigenomics project is to work out which specific food compounds are most helpful (or most harmful) to people with particular food-related diseases, like Crohn’s disease.

How can different food components be separated? A range of different methods can be used to separate specific molecules out of foods using a range of different methods. These methods include: Differential solubility, ion exchange chromatography, size exclusion chromatography, and selective adsorption chromatography. Differential solubility: Some molecules dissolve easily in water; other molecules will dissolve in hexane but not in water. This is because some molecules are more polar than others. Polar molecules dissolve in polar solvents, like water. Non-polar molecules dissolve in nonpolar solvents, like hexane. Molecules like sugar are polar, whereas fat molecules are non-polar.

Ion exchange

chromatography:

Molecules have different charges (positive and negative). This property can be used as the basis of separation. The food sample (as a solution) is passed through a column containing beads that are charged. If the beads have a positive charge, negatively charged molecules in the food will attach but positively charged molecules will run easily through the column and can be collected. Size

exclusion

chromatography:

This has a sieving effect. Because molecules have different sizes, they move through a size exclusion chromatography column at different rates. Larger molecules are collected first, smaller molecules are collected last. Selective

adsorption chromatography:

Because different molecules have different chemical properties, they are more or less able to adsorb (or stick) to materials such as silica gel. This means that they can be separated based on their polarity (degree of surface charge). For example, hydrocarbons (molecules with lots of carbon and hydrogen atoms) have no affinity for silica, whereas alcohols have strong affinity.

 Extraction and analysis of fruit components This experiment involving the extraction and analysis of fruit components was designed by Dr. Dawei Deng at HortResearch, Hamilton.

Samples: Kiwifruit, oranges and lemons

Reagents and Solutions: Ethyl acetate Hexane Ethanol Molybdophosphoric acid

Developing solution: 4:1 hexane/ethyl acetate (i.e., add together 80 ml of hexane and 20 ml of ethyl acetate, and mix). You will need 10 ml per student or group of students. Visualizing solution: 5% Molybdophosphoric acid solution in a spray bottle (i.e., add 5 g of molybdophosphoric acid to 100 ml of ethanol, stir until all the solid has dissolved and then allow to stand for a while before transferring the top, clear solution to a bottle with a spray).

Apparatus per student / group of students: Beaker(250ml) Capillary tubes(commercially available) Pre-coated silica gel plates (commercially available) – use strips approximately 4 cm x 8 cm Test tubes Measuring cylinder Knife

Apparatus per class: Aluminium foils Spray bottle Solvent containers and Oven

bottles

Procedure: Extraction: •

Take about 5 g (half a thumb size) of each fruit sample and chop to small pieces (the smaller the better).

•

Put the chopped samples into separate test tubes and label (for example “K” for kiwifruit).

•

Add ethyl acetate to the tubes (just covering all of fruit pieces), and shake for 1015 minutes.

Analysis using Thin Layer Chromatography (TLC): Preparing the TLC plate:

•

Get a pre-coated silica gel TLC plate that is approximately 4 cm wide and 8 cm long. Be careful to hold it on the sides, and not to get your fingerprints on the silica coat.

•

Use a pencil (not pen) and ruler to draw a straight line parallel to the shorter side of the plate, about 1.5 cm from one end of the plate. Then mark three equal interval points on the line. Mark “K” for kiwifruit, “O” for orange and “L” for lemon under the points.

Transferring the samples: •

Place a capillary tube into a test tube containing the solution of kiwifruit, and allow the solution to rise in the capillary. This will happen spontaneously.

•

Once the capillary is ‘loaded,’ hold it vertically just above the “K” point on pencil line on the plate.

•

Lower the capillary until its end just touches the plate. You will observe that some of the solution flows from the capillary onto the plate. Leave the capillary in contact with the plate only briefly so that the spot is no larger than 1 mm in diameter. Then lift the capillary from the TLC plate and allow the solvent to completely evaporate from the spot.

•

Make a second deposit on the same spot with the capillary and allow the solvent to completely evaporate.

•

Repeat 3-4 times.

•

Do the same for the solutions of orange and lemon on the “O” and “L” points on the pencil line.

•

Place the plate flat on a clean dry surface for 5 minutes and allow the solvent to completely evaporate.

Developing the TLC plate: •

Pour developing solution (4:1 of hexane/ethyl acetate) into a beaker or small wide-mouth glass bottle to a depth of about 5 mm.

•

Pick up, by hand or using tweezers, the TLC plate at the top (the end opposite where the pencil line is drawn). Place it carefully in the beaker as vertical as

possible. Make sure that the two vertical edges do not touch the sides of the beaker. •

Tightly cover the beaker with aluminium foil.

•

When the developing solution has moved to within 5-8 mm of the top of the plate, remove the plate from the beaker and quickly draw a pencil line marking the solvent front.

•

Place the plate flat on a clean dry surface and allow the solvent to completely evaporate.

Visualizing the

samples:

Check the plate. You may find some colour spots, which are visible components in the fruits. To see those invisible components, you need do the following: •

Spray 4% visualizing solution on the dry plate and place it in a 120oC oven for a few minutes.

•

Remove the plate, using tweezers, once dark spots appear on the plate.

Analyzing the results: •

Compare the dark spots for each fruit sample. Each spot represents a different molecule found in the fruit. Can you see that different fruits contain different molecules? Are there any that are the same?

•

Measure the distance of solvent movement (i.e. the distance from the bottom pencil line to the pencil line marking the solvent front). Also measure the distances that each of the component molecules has moved (i.e. the distance from the bottom pencil line to the centre of each spot).

•

Calculate the Rf value of each substance. This is the ratio of substance advance distance to solvent advance distance: Rf =

where

Ls

=

length

of

Ls/Ln the

distance

Ln = length of the distance of advance of the substance

of

solvent

advance

Different components will give different Rf values. Those components with the same Rf value will have the same, or very similar, chemical structures. The effects of the separated food components are tested in a laboratory. One of the ways that researchers can do this is using laboratory models like cell lines.

USING LABORATORY MODELS TO TEST THE EFFECTS OF FOOD One way of testing food compounds is by using laboratory models. As part of the nutrigenomics project researchers are trying to identify food compounds that are likely to be especially helpful (or harmful) for people with particular genetic combinations. One of the ways that they will do this is by using laboratory models.

Why are laboratory models needed? It would be inappropriate to directly test the effects of different food compounds using people. There are several reasons for this. Firstly, it would be difficult to monitor the effects of only the food compounds, and not any other environmental factors (the person would have to live in a controlled environment for the duration of the experiment). Secondly, all research needs to be repeatable. Because the way we respond to food is controlled partly by our genes, researchers would need to find several people who had exactly the same genotype. This would be impossible, as only identical twins have the same genes. Thirdly, there are thousands of food compounds. You would either need a huge number of willing participants (all with the same genotype) to be able to test all the foods, or you would need to involve each person in a very long-term study (to test all the food compounds in isolation). Then there are the ethical issues. No one would agree to participate in the experiment unless they were assured that the tests were unlikely to cause significant harm. The researchers would not be able to guarantee this unless they already knew a lot about the responses that the food compounds are most likely to cause.

What are cell lines? One way that the interactions between food and genes can be studied in a laboratory is by using a cell line. A cell line is a group of cells that grow and replicate continuously in vitro in a laboratory.

The cells in a cell line are grown in a lab along the bottom of a container. The cells can keep growing forever as long as they are provided with sufficient nutrients, but they will always only be individual cells. They will never be able to grow back into a whole organism. Cell based models are systems whereby human cells from a particular part of the body are grown in tissue culture, which means in a liquid medium which provides them all the nutrients they need to survive, and they are grown in an incubator which is kept at 37 degrees, in which the cells continue dividing. They will grow more or less indefinitely until they run out of nutrients. So if you keep feeding them, they are essentially immortal. They will grow forever.

Cell lines are made from cells that are immortal, or don't die. An example of an immortal cell is a cancer cell. It keeps growing and dividing uncontrollably. It does not have the triggers that cause it to die. Most cells from your body won’t grow indefinitely in tissue culture. If you put a single one of your cells, some of them will divide a few times and keep growing and then realize that they are not really supposed to be doing that. All cells in your body have a set lifetime, and they will just over time slowly die out. So all of our cell lines have had to be immortalized in one way or another.

Why are cell lines useful? Cell lines are useful for research because every cell in a cell line has identical genes (all the cells originate from one cell). This means that they should all respond in exactly the same way to a particular treatment, allowing for repeatability. In addition, different groups of cells from a cell line can be used for testing different molecules. In the case of nutrigenomics, the effects of food compounds are tested. By using a cell line, a huge number of different food compounds can be tested in a relatively short time.

How are cell lines used in nutrigenomics research? In the nutrigenomics project, cell lines will be used to test the effects of thousands of different food compounds. The cell lines need to behave as closely as possible to cells from a person who has a particular genetic predisposition. The New Zealand researchers are first studying the effects of different foods on people who have inflammatory bowel disease (IBD). The cell lines that are used to test the foods need to behave as much as possible like cells from someone who has IBD.

Genetic engineering techniques are used to insert copies of gene variants associated with IBD into the cells. Remember, though, that these cells will never be able to be anything other than individual cells. They cannot grow into an organism.

The need for reporter genes Cells are tiny and can usually only be seen with a microscope. It is even more difficult to tell what is going on inside cells. The researchers need to have a way of measuring the effects that the food compounds have on cells. A reporter gene is inserted into the cells. The reporter gene is selected because of its ability to produce a reaction that researchers can measure. Often the luciferase gene is used. This is a gene that is found in fireflies. It causes luminescence, or glowing, when it is activated. Researchers can tell the effect that a food compound has on a cell by whether or not the cells glow. The amount of glowing is measured by a machine called a luminometer.

Using reporter genes in research with cell lines Scientists need a way of measure the ways that cells respond to particular treatments. In the Nutrigenomics Project, cell lines have been developed that mimic a disease situation. Different food molecules are then added to the cells to see what effect they have. One way of measuring the effects of the food molecules on the cells is to add a 'reporter gene' to the cells. The reporter gene will be turned on or off depending on the cell's response to the food molecule. In this case, the reporter gene is a gene from fireflies. It is called the luciferase gene. When the gene is turned on, the cell will glow. The glowing tells researchers what sort of effect the food molecule has had on the cell. In these cell lines, increases in inflammation cause the luciferase gene to be turned on, and the cell to glow.

The luciferase gene codes for a protein that is produced by fireflies, that is able to convert energy back into light - so in a way, the opposite of what plants do. And so it’s the protein that causes fireflies to glow. We can put that protein - the gene that expresses that protein into mammalian cells in such a way so that when the food influences the gene that is associated with Crohns disease, the firefly gene is turned on or off. We can monitor the luminescence of our cells and therefore tell what effect foods have had on the gene

that

we’ve introduced.

Limitations of cell lines A cell line will not behave exactly in the same way as cells that are part of a whole body. However, the results from the cell line tests allow researchers to make better predictions about the effects food compounds might have on people with particular genes.

Other laboratory models Laboratory animals can also provide a useful way of testing the effects of foods. Rats and mice are particularly useful because they reproduce very quickly. Planned breeding programmes can ensure that the mice have particular genetic characteristics (something that can’t be done with human participants). Inbred strains allow for replication of experiments. Environmental factors can also be easily controlled (compared with controlling the environment of a human participant).

The Agouti Mouse Paradigm The agouti mouse can express a number of different phenotypes. It can be yellow and obese or brown and slim. It can have a mottled yellow or a brown coat. These differences, however, are not genetic in origin. These mice are genetically identical. The differences arise from variations in the expression of the agouti gene; and coat color expression can be controlled by varying the mother’s diet before, during, and after pregnancy. The agouti allele is normally expressed only in a mouse’s skin, creating a yellow fur wherever it is expressed, but in agouti mice the gene is expressed throughout the body. In the mouse’s brain, for example, The agouti protein blocks a feeding control center, leading the animal to overeat and become fat. The reason for the ubiquitous expression is that the gene has an IAP (Intracisternal A-particle) proximal enhancer (IPE) element inserted next to the gene. The IPE element acts as a promoter and switches on the gene everywhere, not just in the skin. Agouti gene expression can be silenced, however, by methylation of the IPE element at a cytosine-phosphate-guanine (CpG) site. This methylation shuts down the promotion effects of the IPE element. Methylation can inhibit expression of the agouti gene altogether, resulting in a mouse with brown fur and normal weight. A key point is that the methylation state can be passed from one replicative generation to the next or from parent to offspring, as in the case of the agouti mouse, causing the differences in phenotypic expression. In a 2003 experiment performed by Waterland and Jirtle at Duke University. Female mice were fed one of two different diets before, during, and after their pregnancies. Those mice on the normal (control) diet produced pups that had a yellow coat, developed obesity, and were susceptible to a number of cancers. Those given a diet high in vitamin B12, folic acid, and other supplements that promote methylation produced pups with a brown coat that maintained a normal weight and had no increased cancer risk.

This experiment demonstrated that the diet modulated methylation of the IPE element in the mouse genome, which repressed expression of the agouti gene for the lifetime of the offspring. “I think this is a very interesting experiment,” Jaenisch said. “It tells us that the environmental effect at a certain short stage early in life affects the gene expression pattern throughout life and has an enormous effect on phenotype.” It is, in short, a prototype for the sort of epigenetic actions important to nutrigenomics: diet, acting through the mechanism of methylation, affects gene expression in a stable, lasting way with clear consequences for the health of the individual

Mice, Yeast, Fruit flies, Worms Determining how nutrition affects health and the development of chronic diseases has proved challenging because humans are not good experimental subjects for most molecular genetic studies: each human has a unique genotype and it is difficult to control for environmental influences. In contrast, both of these variables can be controlled in studies with laboratory animals. Rats and mice are particularly useful for such studies because of the relatively short generation times and planned breeding can be done to test genetic hypotheses. The human and mouse genome projects demonstrate the relatedness of the two species and comparative genomic methods are being used to understand observed differences in physiology. Nutritionists have long used outbred or individual inbred strains of rats and mice for analyzing the effects of diet on health, (equivalent to studying, respectively, populations or individuals). Many nutritionists and geneticists are developing best practices for controlling genetic backgrounds, nutrient intakes by using defined diets, and nutritional status by timing eating during the experimental procedures. Mice of defined genotype are important for identifying gene – nutrient interactions, gene – gene interactions (epistasis), and regulation by epigenetics – the heritable changes in gene function that occurs without a change in the sequence of nuclear DNA.

In addition to studying basic molecular, nutritional, and genetic processes, inbred mouse strains often have differences in disease susceptibilities that are similar to those observed between individual humans. Altering the highly controlled environmental conditions, including diets, changes the regulation of genes that produce disease in susceptible mice but not in mice that are more resistant to the disease. Comparative genomic methods, therefore, are effective for understanding differences between species (e.g., mouse to human) but also offer the possibility of identifying genes responsible for susceptibility to chronic diseases, how they interact with each other, and with environmental factors. In addition to laboratory animals, nutrigenomics research is conducted in yeast (Saccharomyces cerevisiae), fruit flies (Drosophila sp.), and worms (Caenorhabditis elegans).

FIVE TENETS OF NUTRIGENOMICS

Nutritional genomic, or nutrigenomics, is the study of how foods affect our genes and how individual genetic differences can affect the way we respond to nutrients (and other naturally occurring compounds) in the foods we eat. Nutrigenomics has received much attention recently because of its potential for preventing, mitigating, or treating chronic disease, and certain cancers, through small but highly informative dietary changes. The conceptual basis for this new branch of genomic research can best be summarized by the following five tenets of nutrigenomics: •

Under certain circumstances and in some individuals, diet can be a serious risk factor for a number of diseases.

•

Common dietary chemicals can act on the human genome, either directly or indirectly, to alter gene expression or structure.

•

The degree to which diet influences the balance between healthy and disease states may depend on an individual’s genetic makeup.

•

Some diet-regulated genes (and their normal, common variants) are likely to play a role in the onset, incidence, progression, and/or severity of chronic diseases. Dietary intervention based on knowledge of nutritional requirement,

nutritional status, and genotype (i.e., "personalized nutrition") can be used to prevent, mitigate or cure chronic disease.

WHY IS NUTRIGENOMICS NOW GROWING? The prime reason for the expansion of nutrigenomics is the completion of Human Genome Project, with the focuses of genomic studies shifting to functional genomic analyses. The functional elucidation of specific genomes will yield not only the feasibilities of new drugs but also the ways of preventing specific diseases. Nutrition and food will then be recognized as important tools for disease prevention. Secondly, preventive medicine including nutrition and food should be discussed more scientifically throughout the world, especially in developed countries like the USA and Japan, where the aging of people and the pursuit of higher QOL (quality of life) become important issues for academia and industry. Nutrigenomics / nutrigenetics sessions have been set up at many international food-related symposia and congresses.

Promise of nutrigenomics: The promise of nutritional genomic is personalized medicine and health based upon an understanding of our nutritional needs, nutritional and health status, and our genotype. Nutrigenomics will also have impacts on society – from medicine to agricultural and dietary practices to social and public policies – and its applications are likely to exceed that of even the human genome project. Chronic diseases (and some types of cancer) may be preventable, or at least delayed, by balanced, sensible diets. Knowledge gained from comparing diet/gene interactions in different populations may provide information needed to address the larger problem of global malnutrition and disease

THE STATE OF THE FIELD AND NEEDS

Ideally dietary advice for optimal genome health maintenance should be provided on an individual basis given that the ability to absorb and metabolize nutrients and DNA repair capacity can vary considerably between individuals. At this stage our knowledge is not refined enough to provide advice based on genotype for example of DNA repair or folate metabolism genes however large data bases are being accumulated world-wide on the relationships of these genotypes with established biomarkers of genome damage such as the cytokinesis-block micronucleus assay and assays that specifically measure oxidized bases in DNA. There is also a need for in vitro modeling and placebocontrolled interventions to identify, which genotypes respond best to which dietary patterns and micronutrient supplements in terms of genome health maintenance. Therefore, there is also considerable scope for the emerging field of Genome Health Nutrigenetics but progress will only occur rapidly once a set of key genome health biomarkers are used uniformly in laboratories world-wide; the micronucleus, comet and DNA methylation and base damage assays are probably the best advanced for this purpose.

NUTRIGENOMICS AND GENOME HEALTH

Numerous cofactors are required for DNA synthesis and repair (e.g. Mg as co-factor for DNA polymerases, Zn as an integral part of the glycosylase OGG1 required for repair of oxidized guanine, vitamin B12 as cofactor for synthesis of tetrahydrofolate and methionine which are required ultimately for synthesis of dTTP and maintenance methylation of CpG respectively). Furthermore, dietary anti-inflammatory substances may help to minimize oxidative DNA damage induced endogenously by an overactive immune system (5). Importantly it has been shown that moderate differences in micronutrient concentration within the physiological range can cause as much genome damage as significant doses of ionizing radiation and that sensitivity to environmental genotoxins is enhanced when cells are deficient in key nutrients such as folate (3). The accumulating evidence indicates quite clearly that sub-optimal dietary choices may have significant impacts on genome damage rate in populations and in individuals. The field of study relating to how dietary patterns and/or specific dietary factors impact on genome maintenance is known as Genome Health Nutrigenomics (3). An ultimate goal of this field of study is to define the optimal daily dietary intake levels and upper safety limits for each micro- and macro-nutrient using validated genome damage biomarkers.

RATIONALE AND AIMS OF NUTRIGENOMICS Nutritional genomics has tremendous potential to change the future of dietary guidelines and personal recommendations. Nutritional genomics covers nutrigenomics, which explores the effects of nutrients on the genome, proteome and metabolome, and nutrigenetics, the major goal of which is to elucidate the effect of genetic variation on the interaction between diet and disease. Nutrigenetics has been used for decades in certain rare monogenic diseases such as phenylketonuria, and it has the potential to provide a basis for personalized dietary recommendations based on the individual's genetic makeup in order to prevent common multifactorial disorders decades before their clinical manifestation. RECENT

FINDINGS:

Preliminary

results

regarding

gene-diet

interactions

in

cardiovascular diseases are for the most part inconclusive because of the limitations of current experimental designs. Success in this area will require the integration of various disciplines, and will require investigators to work on large population studies that are designed to investigate gene-environment interactions. SUMMARY: Based on the current knowledge, we anticipate that in the future we will be able to harness the information contained in our genomes to achieve successful aging using behavioral changes, with nutrition being the cornerstone of this endeavor. In nutrigenomics, nutrients are seen as signals that tell a specific cell in the body about the diet. The nutrients are detected by a sensor system in the cell. Such a sensory system works like sensory ecology where by the cell obtains information through the signal, the nutrient, about its environment which is the diet. The sensory system that interprets information from nutrients about the dietary environment include transcription factors together with many additional proteins. Once the nutrient interacts with such a sensory system, it changes gene, protein expressionand metabolite production in accordance with the level of nutrient it senses. As a result, different diets should elicit different patterns of gene and protein expression and metabolite production. Nutrigenomics seeks to describe the patterns of these effects which have been referred to as dietary signatures. Such dietary signatures are examined in specific cells, tissues

and organisms and in this way the manner by which nutrition influences homeostasis is investigated. Genes which are affected by differing levels of nutrients need first to be identified and then their regulation is studied. Differences in this regulation as a result of differences in genes between individuals are also studied. It is hoped that by building up knowledge in this area, nutrigenomics will promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control which will then be used to prevent the development of chronic diet related diseases such as obesity and type two diabetes. Part of the approach of nutrigenomics involves finding markers of the early phase of diet related diseases; this is the phase at which intervention with nutrition can return the patient to health. As nutrigenomics seeks to understand the effect of different genetic predispositions in the development of such diseases, once a marker has been found and measured in an individual, the extent to which they are susceptible to the development of that disease will be quantified and personalized dietary recommendation can be given for that person. The aims of nutrigenomics also includes being able to demonstrate the effect of bioactive food compounds on health and the effect of health foods on health which should lead to the development of functional foods that will keep people healthy according to their individual needs. As of yet, nutrigenomics is in its infancy. The tools to study protein expression and metabolite production have not yet developed to the point as to enable efficient and reliant measurements. Also once such research has been achieved, it will need to be integrated together in order to produce results and dietary recommendations. All of these technologies are still in the process of development.

CONCLUSION: Nutrigenomics, the study of how nutrients and genes interact and how genetic variations can cause people to respond differently to food nutrients, is still in its infancy but scientists predict that their work could bring about radical changes in how food is grown, processed and consumed, and lead to personalized diets tailored to the genetic makeup. The new science of nutrigenomics and its ethical and societal challenges Gene-diet interactions--which underlie relatively benign lactose intolerance to lifethreatening conditions such as cardiovascular disease--have long been known. But until now, scientists lacked the tools to fully understand the underlying mechanisms that cause these conditions. In recent years, however, strides in human genomics and the nutritional sciences have allowed for the advancement of a new science--dubbed nutrigenomics. Although this science may lead to personalized nutrition and dietary recommendations that can mitigate, prevent, or cure sickness, current oversight mechanisms and regulations for emerging direct-to-public nutrigenomic tests are still in the infancy. Science, Society, and the Supermarket: The Opportunities and Challenges of Nutrigenomics discuss the many ethical, legal, and social challenges presented by nutrigenomics. Concerning itself with the basic uses of nutrigenomic research as well as its clinical and commercial aspects, this text sheds light on such issues as: * Opportunities and challenges for nutrigenomics * The science of nutrigenomics * The ethics of nutrigenomic tests and information both in a clinical setting and by private third parties * Alternatives for nutrigenomics service delivery * Nutrigenomics and the regulation of health claims for foods and drugs * Equity and access to nutrigenomics in industrialized and developing countries * Intellectual property issues

REVIEW OF NUTRIGENOMICS

Nutrigenomics and Beyond: Informing the Future CRITICAL EVENTS: GENOMIC PROGRAMMING AND REPROGRAMMING Genomics studies can identify the specific genes involved in the body’s response to nutrients and pinpoint the genetic variations responsible for differences in a given response among individuals, but such studies have little to say about how nutrient molecules interact with genes to modify their expression and why nutrients can affect genetic expression long after the interaction has occurred. To address these questions, a different approach is needed, one that delves into the molecular mechanisms underlying the genetic response to nutrients. Rudolf Jaenisch, who is a founding member of the Whitehead Institute, offered three definitions of epigenetics that provide different but complementary ways of viewing the same event. First, epigenetics is “the transmission of information through meiosis or mitosis that is not based on the DNA sequence.” That is, during cell division (which accompanies meiosis or mitosis) information encoded in the DNA sequence is passed from one stage of cell division to the next; epigenetics concerns itself with the remainder of that information. Second, epigenetics is “a mechanism for the stable maintenance of gene expression states that involve physically marking the DNA or its associated proteins.” Because gene expression depends not just on the state of the DNA but also on the state of the entire chromatin complex, including both the DNA and its protein scaffolding, epigenetics takes all of this into consideration. Third, epigenetics is “mitotically or meiotically heritable changes in gene expression that are not coded in DNA itself.” Gene expression states can be passed on from one cell generation to the next, which happens, for example, during embryonic development, when cells differentiate and then reproduce to form lines of specialized cells (e.g., nerve cells and muscle cells), each of which is defined by specific genes. These genes can be either

activated or silent. Gene expression states are heritable, making it possible to express traits that are not dependent on the expression of a given gene per se. In short, epigenetics describes the way in which cells store and pass on information that is not coded in the DNA sequence itself but rather in various modifications made to the DNA and, more generally, to the chromatin complex containing it. Epigenetics is thus an important tool for nutrigenomics because it offers a way of understanding how nutrients interact at the molecular level with the genome to create long-lasting effects. “Epigenetic regulation is a mechanism that allows the genome to integrate intrinsic signals and environmental signals. It is a way the genome interacts with the environment. So, what you ate for lunch has found its way to change in some very subtle way the epigenetic state of your DNA.” That, in turn, is related to how diet can affect health and, in particular, the risk of certain diseases. When gene-environment interactions alter the epigenetic state of the genome, they may affect the incidence of diseases with long latencies or late-stage onset, such as cancer and neurodegenerative diseases. Relatively little research that has applied the tools of epigenetics to nutrigenomics has been conducted to date; but epigenetics is a rapidly developing field, one that has been invigorated by the sequencing of the human genome. The growing arsenal of tools and techniques available from the study of epigenetics thus offers new and revolutionary ways of studying how nutrients interact with the genome. .

Bovine milk in human nutrition

Milk and milk products are nutritious food items containing numerous essential nutrients, but in the western societies the consumption of milk has decreased partly due to claimed negative health effects. The content of oleic acid, conjugated linoleic acid, omega-3 fatty acids, short- and medium chain fatty acids, vitamins, minerals and bioactive compounds may promote positive health effects. Full-fat milk has been shown to increase the mean gastric emptying time compared to half-skimmed milk, thereby increasing the gastrointestinal transit time. Also the low pH in fermented milk may delay the gastric emptying. Hence, it may be suggested that ingesting full-fat milk or fermented milk might be favourable for glycaemic (and appetite) regulation. For some person’s milk proteins, fat and milk sugar may be of health concern. The interaction between carbohydrates (both natural milk sugar and added sugar) and protein in milk exposed to heat may give products, whose effects on health should be further studied, and the increasing use of sweetened milk products should be questioned. The concentration in milk of several nutrients can be manipulated through feeding regimes. There is no evidence that moderate intake of milk fat gives increased risk of diseases.

Nutrigenomics Consortium The Nutrigenomics Consortium aims to increase our understanding of the events unfolding during metabolic stress, with the ultimate objective to discover and validate molecular biomarkers for the early detection of metabolic stress and to identify and develop novel food components for dietary management and prevention of metabolic stress. The Nutrigenomics Consortium will focus on diet-related metabolic stress, the early and reversible stage of metabolic syndrome. The phenotypes spanned by metabolic syndrome include obesity, insulin resistance, hyperlipidemia and hypertension. Metabolic syndrome greatly increases the chance of developing type 2 diabetes and coronary heart disease. With the increasing prevalence of metabolic syndrome, the health care expenditures associated with the treatment of this condition continues to rise. The Nutrigenomics Consortium envisions an important role of nutrition in the prevention of metabolic stress. The effects of nutrition on health and disease cannot be understood without a profound understanding of how nutrients act at a molecular and genetic level. The Nutrigenomics Consortium will combine molecular nutrition research with state-of-the-art genomic tools to obtain novel insights into the events unfolding during the development of metabolic stress. Early detection by using validated molecular biomarkers and adequate treatment of metabolic stress are considered of utmost importance to prevent metabolic syndrome and its severe clinical consequences.

Salt substitution: a low-cost strategy for blood pressure control among rural Chinese. A randomized, controlled trial. Dietary sodium and potassium consumption is associated with blood pressure levels. The objective of this study was to define a practical and low-cost method for the control of blood pressure by modification of these dietary cations in rural Chinese. Methods: This study was a double-blind, randomized, controlled trial designed to establish the long-term effects of a reduced-sodium, high-potassium salt substitute (65% sodium chloride, 25% potassium chloride, 10% magnesium sulphate) compared to normal salt (100% sodium chloride) on blood pressure among high-risk individuals. Following a 4-week run-in period on salt substitute, participants were randomly assigned to replace their household salt with either the study salt substitute or normal salt for a 12-month period. Results: The mean age of the 608 randomized participants was 60 years and 56% of them were female. Sixty-four percent had a history of vascular disease and 61% were taking one or more blood pressure-lowering drugs at entry. Mean baseline blood pressure was 159/93 mmHg (SD 26/14). The mean overall difference in systolic blood pressure between randomized groups was 3.7 mmHg (95% confidence interval 1.6-5.9, P < 0.001). There was strong evidence that the magnitude of this reduction increased over time (P = 0.001) with the maximum net reduction of 5.4 mmHg (2.3-8.5) achieved at 12 months. There were no detectable effects on diastolic blood pressure. Conclusion: Salt substitution produced a substantial and sustained systolic blood pressure reduction in this population, and should be actively promoted as a low-cost alternate or adjunct to drug therapy for people consuming significant quantities of salt.

Effect of commercial breakfast fibre cereals compared with corn flakes on postprandial blood glucose, gastric emptying and satiety in healthy subjects: a randomized blinded crossover trial Background: Dietary fibre food intake is related to a reduced risk of developing diabetes mellitus. However, the mechanism of this effect is still not clear. The aim of this study was to evaluate the effect of commercial fibre cereals on the rate of gastric emptying, postprandial glucose response and satiety in healthy subjects. Methods: Gastric emptying rate (GER) was measured by standardized real time ultrasonography. Twelve healthy subjects were assessed using a randomized crossover blinded trial. The subjects were examined after an 8 hour fast and after assessment of normal fasting blood glucose level. Satiety scores were estimated and blood glucose measurements were taken before and at 0, 20, 30, 40, 60, 80, 100 and 120 min after the end of the meal. GER was calculated as the percentage change in the antral crosssectional area 15 and 90 min after ingestion of sour milk with corn flakes (GER1), cereal bran flakes (GER2) or wholemeal oat flakes (GER3). Results: The median value was, respectively, 42% for GER1, 33 % for GER2 and 51% for GER3. The GER after ingestion of bran flakes was significantly slower compared to wholemeal oat flakes (p=0.023). The postprandial delta blood glucose level was significantly lower at 40 min (p=0.045) and 120 min (p=0.023) after the cereal bran flakes meal. There was no significant difference between the areas under the curve (AUCs) of the cereals as far as blood glucose and satiety were concerned. Conclusions: The result of this study demonstrates that the intake of either cereal bran flakes or wholemeal oat flakes has no effect on the total postprandial blood glucose response or satiety when compared to corn flakes, but intake of cereal bran flakes slows the GER. Since these products do not differ in terms of glucose response and satiety on healthy subjects, they should be considered equivalent in this respect.

Nutrigenomics and metabolomics 29.07.2002

The next step in understanding what the human genome is telling us, especially

Despite some cosmetic differences, we all have the same genetic makeup that evolved from primitive man. Unfortunately, the genes that were in place before the advent of the earliest civilizations were not designed to carry individuals through today’s typical age span, now approximately eight decades of wear and tear. Additionally, the multiple genetic mutations that could survive in ancient times more than likely surrender to the chronic disorders that can be attributed to metabolic stress today. Thus the dramatic increase of those age-related diseases in current times. Scientists have known that dietary patterns are strongly linked to the development of seven of the ten top causes of morbidity and mortality in the United States, primarily cardiovascular diseases, cancer, and diabetes. Consequently, a scientific and technological revolution has been going on in the areas of nutrition and biochemistry. This revolution has lead to significant new understandings of the role of food and nutrition in human health, and with the Human Genome Project, a new ability to understand the role of genetics in metabolism and health. The advancement of biotechnology into the development of genomics, proteomics (expression of proteins), and metabolomics provides new tools for establishing the role of food and nutrients in human health. This combination of various disciplines is called "Nutrigenomics," and provides a potential for early identification of those at high risk to metabolic stress and to understand the molecular basis of physiological defects. This has the potential for providing the tools for a more personalized and effective dietary intervention that in some cases may need to be combined with pharmacological therapy.

Nutrigenomics--2006 update. The Human Genome and Hap Map projects have provided the tools and information that will aid in understanding how nutrients alter the expression of an individual's genetic information and why individuals differ in metabolism of foods at the molecular level. The study of how genes and gene products interact with dietary chemicals to alter phenotype and, conversely, how genes and their products metabolize nutrients is called nutritional genomics or "nutrigenomics." This new field has received considerable attention in the last 6 years, most of which has been on the promise rather than on scientific results from nutrigenomic experiments. Funding for nutrigenomics research focused primarily on individual laboratory projects in the 1990s and early 2000s. The novelty of combining nutrition and genetics limited that funding to a relatively small number of laboratories. Only in the past 3 years have centers been funded to foster collaborations and conduct large-scale projects that are studying nutrient-gene interactions. The increase in interest and funding is beginning to generate the critical mass to realize the promise of nutritional genomics.

Nutrigenomics in Eye health There is growing evidence for a major implication of nutrition and genetics in the etiology of age-related eye diseases (age related macular degeneration (AMD), cataract and glaucoma) which are major causes of blindness worldwide. However the interest in nutritional risk factors for these diseases and the identification of the associated genes are still recent. As such, the interactions between nutritional and genetic factors have not yet been studied.

NEEDS AND OPPORTUNITIES IN THE FOOD AND AGRICULTURAL SCIENCES

No matter how great the potential of nutrigenomics to deepen the understanding of nutrition and to point the way to healthier eating is, that potential will not be realized without corresponding advances in other areas. One of those areas is agriculture and the food industry. Joseph Spence addressed the role that agriculture and the food industry can play in helping nutrigenomics meet its potential. Spence pointed out that that role will develop in a number of different areas; one of the most important will be modification of the nation’s food supply to reflect new understandings about nutritional requirements, something that is already being done on a limited scale. For example, the Agricultural Research Service (ARS) developed the heart-healthy NuSun sunflower as a variety high in oleic acid, a monounsaturated fatty acid. That variety now accounts for about 77 percent of the sunflowers produced for oil seeds in the United States.

FIGURE - The role of value-added products, e.g., from nutrigenomics, in improved outcomes,

particularly improved health, to consumers. The NuSun sunflower was developed by traditional breeding methods, but researchers are also using genetic engineering methods to create varieties with desired characteristics. Scientists at ARS have also produced transgenic tomatoes that contain four to eight times as much lycopene, a carotenoid known for its strong antioxidant properties, as nontransgenic tomatoes.

As nutrigenomics research reveals more details about the roles of various nutrients, the agriculture industry can modify food to take these findings into account for future research and development. Of particular importance will be demonstration of the clear nutritional benefits of these various nutrients for individuals, as illustrated in Figure above. “We cannot fall into the trap of saying this probably will have a beneficial effect,” Spence said. “We have to clearly identify the health benefits and get people to understand that these are long-term benefits.”

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