Epigenetic Biomarker Profile Clinical White Paper

Metabolomic Biomarkers as Indicators of
Epigenetics and Personalized Nutrition

Clifford Morris, Ph.D.

Director of Research and Development Physicians Lab, Inc.

Metabolomic Biomarkers as Indicators of Epigenetics and Personalized Nutrition

Introduction

Science is well on its way to establishing fundamental understanding of how genetic variation, epigenetics and metabolism alter in response to diet, lifestyle and the environment.1,2 Much of what humans are made from is shared amongst everyone, however, the expression of your genes, proteins and metabolism varies considerably from person to person.3 Small and subtle genetic and cellular modifications cause these variances between humans and the reason every human is completely unique.

Studying and understanding these molecular differences is the goal of the ‘omics’ sciences, including genomics (genes), proteomics (proteins) and metabolomics (metabolism). Genomics is the sequencing of your genetic information.4 It is hardcoded and non-modifiable. Epigenetics means “above genetics” and it was originally conceived by Conrad Waddington himself about 50 years ago to describe the existence of mechanisms of inheritance in addition to (over and above) standard genetics.5 Epigenetics is the study of heritable phenotype changes that do not

involve alterations in the DNA sequence. Epigenetics implies features that are in addition to the traditional genetic basis for inheritance. Epigenetics most often involves changes that affect gene activity and expression, but the term can also be used to describe any heritable phenotypic change. In short, epigenetics looks at factors that turn genes on or off, including diet, lifestyle and environment, and many more. This has allowed for a strong understanding of how genetic variation and epigenetic events alter requirements for, and responses to, nutrients.1 At the same time, methods for profiling almost all of the products of metabolism in a single sample of blood or urine are studied, known as metabolomics.6 Metabolomics is a fast and effective way to understand what is happening to the body as a result of epigenetics, nutrition and the environment around you. It is a comprehensive way of profiling the downstream products of gene expression and how your body is performing at a cellular level.7 Genes set the stage for what happens in the cell, but the most important activity is at the

Figure 1. Systems metabolomics. Complex interplay between molecular omics, environment and the phenotype.

The different layers of the biological system  are  nowadays  well  covered  by  various  omics  technologies.  The  metabolome is of special interest, since it integrates all molecular and environmental effects.

metabolite level, such as nutrient signaling, energy production, cell repair, and immune health. These metabolites are a much more accurate way of assessing cell health compared to genes, because it closely reflects what is happening within the cell at that exact time.1,3,5 It directly reflects everything your body is currently experiencing including changes to diet and nutrition, inflammation, toxin exposure, and lifestyle changes.8

Along the pathway of gene expression and metabolism, there are places where the road is rocky. By identifying predispositions to bumps in the road and making recommendations on how to modify both epigenetic expression and metabolism, practitioners have a road map to keep each patient on smooth pavement. Large clinical studies are leading the way for personalized nutrition by exposing the variance that occur between people and developing testing methods sensitive enough to detect these changes so that dietary and lifestyle recommendations can be precisely

targeted.9,10 Immune response, Inflammation, methylation, energy production, hormonal health, GI health and detoxification continue to emerge as the cornerstones of health and wellness.6,11,12 These cornerstones are all founded in how patients express their genes and metabolize the products. Currently, nutritional recommendations are set for a wide population and to address the nutrition of the average human. However, we are aware that large variances occur from person to person and the one-size-fits-all approach is far from suitable for patients seeking epigenetic and metabolic optimization. This strategy thereby avoids having patients over or under consume specific nutrients in a quantifiable manner aiming for high metabolic performance and preventative health.

Recent advances in the omics field

In the past, analytical limitations made life relatively simple, and the nutritional biochemist dealt with perhaps a half-dozen metabolites, developed an integrated theory

A close up of a map Description automatically generated

Figure 2. A schematic of the -omics indicating how greater phenotypical information is gained from left to right.

for how they related to each other, and predicted the effects on cell function or disease. Nowadays with the advent of new technologies and smart software, scientists have the ability to simultaneously assess thousands of metabolites.13,14 This greatly improves the ability to resolve even the most subtle metabolic differences that exist between individuals and provide information on how to address imbalances, deficiencies and disease. High-throughput genotyping methods gave rise to large-scale Genome-Wide Association Studies (GWAS) over a decade ago, with the promise to elucidate the genetic basis of complex diseases. Many traits have since been correlated with single nucleotide polymorphisms     (SNPs),          including metabolomics measurements from human cohorts.9,15 This provides a detailed in-vivo picture of the influence of genetic variation on the real world-implications. Not only are these systems allowing for diagnostics of disease, they offer the ability to closely monitor metabolic performance to maintain optimal nutrition, proactively prevent disease and improve overall quality of life.

The relationship between genes, metabolism and nutrition

The concept of epigenetic nutrition looks at how diet, supplements, lifestyle and environment contribute to altering gene expression through methylation and modification. Epigenetic gene modification is very different from person to person, and dependent on the molecular machinery involved in gene expression resulting in the individuals’ phenotype. As such, the nutrient requirements are also dependent on these mechanisms. Through epigenetic biomarker analysis, one can take advantage of knowing which parts of the molecular machinery are not functioning correctly and rectify it by implementing metabolic correction through personalized nutrition, lifestyle and environmental changes. These epigenetic changes can result in modifications that affect

metabolism in humans.16 Relations between diet, epigenomic and metabolomic profiles and between those profiles and health have become important components of research that have revolutionized clinical practice in nutrition and preventative health.14 We know that metabolic alterations produced by excessive intake of some nutrients, drugs and chemicals directly impact epigenetic regulation and corelate how metabolic pathways are modified by environmental and genetic factors, providing novel insights for the treatment of metabolic imbalance and diseases. For example, elevations in the levels of 6 amino acids predicts a significantly high risk of diabetes up to 10 years in advance.1,17,18 Integration of multi-omic assessments are allowing scientists to link together all the different systems involved in human metabolism and quickly making connections to nutrient absorption, elimination and physiological function.

There are 3 major conceptual groupings for thinking about nutrient-gene-metabolism interactions: i. direct interactions: nutrients, sometimes after interacting with a receptor, behave as transcription factors that can bind to DNA and acutely induce gene expression; ii. epigenetic interactions: nutrients can alter the structure of DNA (or of histone proteins in chromatin) so that gene expression is chronically altered; and iii. common genetic variations, SNPs, can alter the expression or functionality of genes.1,5 All of these mechanisms can result in altered metabolism of and altered dietary requirements for nutrients. Diet and nutrition play a role in epigenetic modification by causing by sustained effects of gene expression, mainly by a process called methylation.19 When methylation occurs in gene promoter regions, expression is altered. Increased methylation usually results in gene expression reduction or silencing. Epigenetic modifications can result in changes in gene expression that can last throughout a person’s life and can even persist across generations.

Figure 2.  DNA  methylation  can  silence  gene  expression.  Methylation  of  cytosine  located  in  cytosine-  guanosine groupings in gene promoter regions (called 5’-CpG-3’ islands) attracts capping proteins that hinder access to the gene for the transcription factors that normally turn on gene expression and formation of messenger RNA (mRNA). When the transcription factor does not bind to the promoter area of the gene, transcription of mRNA does not occur, and the gene is silenced.

It has been shown that nutritional factors have a major influence on gene expression and metabolism. Changes to mRNA and expression profiling and the corresponding proteins regulate the transport of certain nutrients and metabolites. As a result, a clinically actionable approach can focus upon nutrition and other modifiable lifestyle factors to achieve optimal gene expression, and therefore improved optimal health. Due to metabolic individuality, sub-groups of a population may respond differently to dietary intervention. An example of this is the use of folic acid in a population lacking the methylene tetrahydrofolate reductase (MTHFR) gene, this population would need the activated form of folic acid in order to benefit from this supplement, since they cannot convert it naturally.20,21 Further, genetotrophic disease occurs from suboptimal consumption of nutrients necessary to meet the epigenetically determined requirements of the individual. Quantitatively identifying these deficiencies and supplementing them through personalized nutrition affords optimal metabolic performance and disease prevention. Examples of physiologically critical nutrients include micronutrients such as vitamins and minerals, which exist to enhance enzyme efficiency.

Vitamins  are   co-factors   for   enzymes,   and enzymes are proteins that may  be altered due to epigenetic changes thereby affecting their intended function. In order to have enzymes functioning at their maximal capacity sufficient active cofactors must be present, and deficiencies can be addressed through specific nutritional interventions. Maintaining the correct balance of nutrients will ensure that the complex epigenetic molecular machinery of the body is running at optimal efficiency via metabolic correction.

Amongst all the omics, metabolomics plays a special role. The metabolome is the set of all small molecules, such as amino acids, sugars and lipids, in a biological system. It is considered to be an endpoint of biological processes and carries an imprint of all genetic, epigenetic and environmental factors.2,3,14 It has therefore also been referred to as the ‘link between genotype and phenotype’.4 Epigenetics is linked to metabolomics in response to the cellular microenvironment. The metabolism of various biochemicals such as amino acids, organic acids and fatty acids are critical in this linkage between  epigenetics and metabolism.

As such, metabolomics is a direct representation  of  the  outcome   of epigenetic modifications in the human body – it quantifies the metabolic response of pathways dependent on epigenetics; diet and environmental factors. Genomics and proteomics provide you with information about what might happen, whereas metabolomics provides you with information about what is happening. Individual metabolites have already been used as epigenetic biomarkers for years. Elevated estrogen, for instance, is indicative of a genetic modification affecting the aromatase enzyme which is responsible for conversion of testosterone to estrogen.22,23 Another example, 8-OH-dG, is a metabolite produced from DNA damage and oxidative stress.24–26 Metabolomics enables the identification of such biomarkers based on epigenetic and environmental disruptions to the biochemical pathways that are up/down- regulated in unison. Assessing epigenetic dependent metabolites provides in-depth and actionable information allowing for personalized treatment and nutrition strategies to address imbalances and restore optimal cellular health. Since the metabolic profile of a patient is a consequence of many contributing biological systems, the dynamic balance of the body’s biochemical pathways can be mapped out with high precision and a fundamental understanding of their specific roles in being healthy.6,27 Further, metabolomics affords rich data by repeated sampling that clearly shows how all the different metabolites can change, and be measured.

Just as the published Human Genome Project is an average representation of genes in humans, there is the Human Metabolome Data Base (HMDB) which is an accurate representation of every metabolic pathway possible for humans.10,28 However, there is significant variation from the average in both genome and metabolome in any given individual. Metabolomics can identify mechanisms that underlie individual variations in dietary requirements as well as in the capacity to respond to food-based interventions in a spatial and time-dependent manner more than any other omic alone. Each

metabolite can be traced back to specific function thereby allowing for clinically actionable, outcome-based solutions to promote metabolic balance. Doctors, clinicians and nutritionists are rapidly harnessing the power of metabolomics with great success in treating many of the chronic disease responsible for 88% of deaths per year.26 Metabolomics has the potential to transform the health of entire populations through integrative and preventative health strategies by guiding diet, supplementation and lifestyle changes. Nutrition is one of the best ways to prevent a variety of diseases and health issues in the body.4,30 Optimizing nutrition is done by identifying subtle imbalances, deficiencies and metabolic issues. It can help a practitioner target specific nutritional requirements to alleviate current symptoms and lower risks of certain diseases in the future.6 A balanced, personalized diet will ensure a person is receiving the correct amount of nutrients such as vitamins and minerals, amino acids, fatty acids and much more. This all works to keep the body running at maximum efficiency with more energy, improved metabolism, stable weight, less inflammation and a healthy heart and brain.

Using the omics to tailor personalized nutrition

Epigenetic dependent metabolism involves a vast array of chemical reactions; of particular importance are those involved in the transfer of functional groups. When a methyl group, for example, is added to a particular molecule, it may change its activity. This simple chemical reaction allows cells to use metabolic intermediates to carry chemical groups between different reactions. These group transfer intermediates are called cofactors or coenzymes. Each transfer reaction is facilitated by a particular coenzyme. These coenzymes are continuously being made, consumed and then recycled. Nutrition has a sequential effect on the epigenome and metabolome which can produce either a healthy physiological state or fundamental metabolic disruption such as excessive inflammation, oxidation, neurological and metabolic stress.31 Dietary constituents have been shown to alter gene expression in a

Table 1. Examples of Epigenetic and Nutritional Dependent Biomarkers.17,32-43

number of ways: i. Acting as ligands for transcription factor receptors; ii. by being metabolized in primary or secondary metabolic pathways thereby altering concentrations of substrates or intermediates; and iii. by altering signal transduction pathways.27 Although epigenetics does not result in changes to the nucleotide sequence, it does comprise molecular modification of DNA and histones. Since information flows in both directions (i.e., from genetics to metabolites and vice versa),

gene expression can also be activated or deactivated by signals from the environment.

Dietary factors, such as the daily intake of folate, can be considered as an environmental stimulus with the potential to affect gene activation through, for instance, a change in the DNA methylation status. Accordingly, genes alone do not determine biological fate; it is the response to environmental and nutritional stimuli that actually determine the gene expression.

Every human is completely unique and requires specific nutritional considerations for optimal health. These needs are dependent on a variety of factors including genetics, diet, lifestyle and environmental influences. Through complex software and analysis, scientists have been able to elucidate and map out the patterns and dynamic balance of metabolic pathways, eg: groupings of related genes that regulate metabolic pathways and then groupings of related metabolic pathways. In order to rectify epigenetic and metabolic issues, one must address the symptoms of nutritional, metabolic and detoxification issues. Providing personalized nutrition for individuals allows for an optimal diet, improved metabolic performance and disease prevention.32 For this reason, most nutrition scientists immediately grasp the advantages gained from being able to measure many metabolites rather than a few. In the past, analytic limitations made life relatively simple, and the nutritional biochemist dealt with perhaps a half- dozen metabolites, developed an integrated theory for how they related to each other, and did an excellent job in predicting the effects on cell metabolism. Nowadays, advances in biotechnology instruments permit the analysis of thousands of metabolites simultaneously and massively increases diagnostic power and accuracy. Integrated platforms that combine the facets of genes, epigenetic modification and metabolism are essential to understanding their roles in the human body.33,34

The Epigenetic Biomarker Panel (EPB) from Physicians Lab is a simple and fast urinary  metabolomics  panel  quantifying over

60 epigenetic and nutrient dependent metabolites using the gold standard of bio- analytical equipment, Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS). The results provide detailed information on how the body is metabolically and nutritionally balanced thereby allowing for highly specific, personalized therapeutic strategies. Metabolism is a complex process, and through the Metabolic Performance Profile, one can identify how nutrients are used to fuel critical biochemical reactions within the body. The Metabolic Performance Profile quantifies key categories which affords the determination  of

personalized nutrient and supplement recommendations. The results are driven by algorithms to determine outcome-based recommendations which are supported by peer-reviewed clinical literature and population wide association studies.

Most traditional nutritionists and dieticians believe that the requirements for minerals are met simply with a conventional balanced diet, however this may ignore biochemical individuality. The optimum intake of micronutrients for each person will vary according to age, genetic makeup, diseases, and exposure to stress and toxins. The failure to apply the concept of biochemical individuality may negatively impact an individual’s efforts to reach metabolic optimization. Conversely, it has been proposed that a metabolic tune up could produce a marked increase in health in certain individuals. Every physiological component must be considered in order to perform at peak efficiency. Metabolic processes can be viewed as links in a chain. The strength of the entire chain can be compromised by only one weak link. However, a significant portion of the American population does not even reach the Recommended Daily Allowance (RDA) of some critical nutrients from their diet.45 A state of subclinical deficiency or dietary insufficiency seems prevalent and may have serious health consequences. Supplementation with specific nutrients has been estimated to be cost effective in preventing diseases. Food alone may not provide sufficient micronutrients for preventing deficiency or insufficiency. Many older adults do not consume sufficient amounts of numerous necessary nutrients from foods alone. Supplements compensate, but only an estimated half of this population uses them daily, so having an easy daily solution is key to addressing the issue. Scientific formulations capable of

multifunctional synergy with the ability to support healthy epigenetics and metabolomics should be an area of focus. This approach can produce a partnership with healthcare professionals that will allow individuals to optimize their health regardless of their underlying genetics.

Future Prospects

There are a number of medical and economic implications to the development of nutritional epigenetics and metabolism optimization strategies: i. to prevent diseases is economical, since prevention is less expensive than treatment; ii. to lessen the cost of healthcare is imperative, since healthcare or better yet disease- care cost is too high and continues to increase; iii. to educate the public in this new health paradigm, so they can start taking better care of themselves; iv. to make science accessible, in all terms, especially economically; so that medication, supplementation and metabolic optimization costs can be lower.

It is apparent that a niche will exist for health professionals who can effectively place the new information generated by metabolomic profiling into a context that enables integration, interpretation, and, subsequently, individualized dietary recommendations. Thus, the field of nutrition would benefit by establishing itself as the predominant discipline using this knowledge in clinical and public health practice.

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