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Summary of Genomics and Medicine: Role in Cardiovascular Diseases
Author: Larry H. Bernstein, MD, FCAP
The articles within Chapters and Subchapters you have just read have been organized into four interconnected parts.
Genomics and Medicine
Epigenetics – Modifyable Factors Causing CVD
Determinants of CVD – Genetics, Heredity and Genomics Discoveries
Individualized Medicine Guided by Genetics and Genomics Discoveries
The first part established the
rapidly evolving science of genomics
aided by analytical and computational tools for the identification of nucleotide substitutions, or combinations of them
that have a significant association with the development of
cardiovascular diseases,
hypercoagulable state,
atherosclerosis,
microvascular disease,
endothelial disruption, and
type-2DM, to name a few.
These may well be associated with increased risk for stroke and/or peripheral vascular disease in some cases,
essentially because the involvement of the circulation is systemic in nature.
Part 1
establishes an important connection between RNA and disease expression. This development has led to
the necessity of a patient-centric approach to patient-care.
When I entered medical school, it was eight years after Watson and Crick proposed the double helix. It was also
the height of a series of discoveries elucidating key metabolic pathways.
In the period since then there have been treatments for some of the important well established metabolic diseases of
carbohydrate,
protein, and
lipid metabolism,
such as – glycogen storage disease, and some are immense challenges, such as
Tay Sachs, or
Transthyretin-Associated amyloidosis.
But we have crossed a line delineating classical Mendelian genetics to
multifactorial non-linear traits of great complexity and
involving combinatorial program analyses to resolve.
The Human Genome Project was completed in 2001, and it has opened the floodgates of genomic discovery. This resulted in the identification of
genomic alterations in
cardiovascular disease,
cancer,
microbial,
plant,
prion, and
metabolic diseases.
This has also led to
the identification of genomic targets
that are either involved in transcription or
are involved with cellular control mechanisms for targeted pharmaceutical development.
In addition, there is great pressure on the science of laboratory analytics to
codevelop with new drugs,
biomarkers that are indicators of toxicity or
of drug effectiveness.
I have not mentioned the dark matter of the genome. It has been substantially reduced, and has been termed dark because
this portion of the genome is not identified in transcription of proteins.
However, it has become a lightning rod to ongoing genomic investigation because of
an essential role in the regulation of nuclear and cytoplasmic activities.
Changes in the three dimensional structure of these genes due to
changes in Van der Waal forces and internucleotide distances lead to
conformational changes that could have an effect on cell activity.
Part 2
is an exploration of epigenetics in cardiovascular diseases. Epigenetics is
the post-genomic modification of genetic expression
by the substitution of nucleotides or by the attachment of carbohydrate residues, or
by alterations in the hydrophobic forces between sequences that weaken or strengthen their expression.
This could operate in a manner similar to the conformational changes just described. These changes
may be modifiable, and they
may be highly influenced by environmental factors, such as
smoking and environmental toxins,
diet,
physical activity, and
neutraceuticals.
While neutraceuticals is a black box industry that evolved from
the extraction of ancient herbal remedies of agricultural derivation (which could be extended to digitalis and Foxglove; or to coumadin; and to penecillin, and to other drugs that are not neutraceuticals).
The best examples are the importance of
n-3 fatty acids, and
fiber
dietary sulfur (in the source of methionine), folic acid, vitamin B12
arginine combined with citrulline to drive eNOS
and of iodine, which can’t be understated.
In addition, meat consumption involves the intake of fat that contains
the proinflammatory n-6 fatty acid.
The importance of the ratio of n-3/n-6 fatty acidsin diet is not seriously discussed when
we look at the association of fat intake and disease etiology.
Part 2 then leads into signaling pathways and proteomics.The signaling pathways are
critical to understanding the inflammatory process, just as
dietary factors tie in with a balance that is maintained by dietary intake,
possibly gut bacteria utilization of delivered substrate, and
proinflammatory factors in disaease.
These are being explored by microfluidic proteomic and metabolomic technologies that were inconceivable a half century ago.
This portion extended into the diagnosis of cardiovascular disease, and
elucidated the relationship between platelet-endothelial interaction in the formation of vascular plaque.
It explored protein, proteomic, and genomic markers
for identifying and classifying types of disease pathobiology, and
for following treatment measures.
Part 3
connected with genetics and genomic discoveries in cardiovascular diseases.
Part 4
is the tie between life style habits and disease etiology, going forward with
the pursuit of cardiovascular disease prevention.
The presentation of walking and running, and of bariatric surgery (type 2DM) are fine examples.
It further discussed gene therapy and congenital heart disease. But the most interesting presentations are
in the pharmacogenomics for cardiovascular diseases, with
volyage-gated calcium-channels, and
ApoE in the statin response.
This volume is a splendid example representative of the entire collection on cardiovascular diseases.
Cancer is one of the most devastating and widespread diseases today. The development of cancer is a multi-step process involving genetic or epigenetic changes often occurring over a longer period of time. Moreover, cancer occurs in more or less all organs and tissues and is characterized by extensive heterogeneity both concerning the type and aggressiveness of the disease. Although some substantial progress in some areas has been made, there are still huge unmet needs in treatment methods and the efficacy of currently available drugs. The pharmaceutical industry has struggled with the ever increasing costs in drug development and unfortunately novel drugs have not seldom demonstrated only marginal improvement in efficacy often at the cost of quality of life of the patients. For these reasons, new approaches are focusing on disease prevention instead of only treating the symptoms. Recently, much attention has been paid to prevention of the disease in parallel to continuous drug discovery.
Intervention in food intake has been demonstrated to play an enormous role in both prevention as well as treatment of diseases. Numerous studies indicate a clear link between cancer and diet. The substantial development of sequencing technologies has resulted in access to enormous amounts of genomics information, which resulted in the establishment of nutrigenomics as an emerging approach to link genomics research to studies on nutrition. Increased understanding has demonstrated how nutrition can influence human health both at genetic and epigenetic levels. It investigates the effects of nutrition and bioactive food compounds on gene expression. This approach has allowed the investigation of the effect on nutrition on individuals with specific genetic features. Moreover, it has provided the basis for nutritional intervention in prevention and treatment of disease and the inauguration of personalized nutrition. However, differences in types of cancer, the level of aggressiveness, and their occurrence at different stages of life have seriously complicated the understanding of the effect of nutrition on cancer prevention and treatment. Other individual variations such as the amounts of food consumed, digestion, metabolism and other factors like geographical, ethnic and sociological diversity has hampered the identification of which food components are most important for human health. Dramatic dietary modifications have proven essential in reducing risk and even prevention of cancer. Moreover, intense revision of diet in cancer patients has revealed significant changes in gene expression and also has provided therapeutic efficacy even after short-term application.
Obviously, a multitude of diets have been evaluated, but probably the common factor for achieving both prophylactic and therapeutic responses is to consume predominantly diets rich in fruits, vegetables, fish and fibers and reduced quantities of especially red meat. There are numerous examples of how dietary intake can promote health on both a preventive as well as therapeutic level. Radical change in diet has resulted in dramatic changes in gene expression in prostate cancer patients revealing that many of those genes involved in cancer development were down-regulated. The importance of nutrigenomics as a multi-task approach involving genomics, proteomics, metabolomics, et cetera has further provided novel possibilities to address the effect of nutrition on human health. Despite encouraging findings on how dietary modifications can prevent disease and restore health, there are a number of factors which complicate the outcome. There are variations in response to dietary changes depending on age and gender. However, the vast amount of accumulated nutrigenomics data should not overshadow the needs to take into account other important factors such as lifestyle, social, geographical and economic factors affecting diet and health.
Metabolomics: its Applications in Food and Nutrition Research
Reporter and Curator: Sudipta Saha, Ph.D.
Metabolomics is a relatively new field of “omics” research concerned with the high-throughput identification and quantification of small molecule (<1500 Da) metabolites in the metabolome. The metabolome is formally defined as the collection of all small molecule metabolites or chemicals that can be found in a cell, organ or organism. These small molecules can include a range of endogenous and exogenous chemical entities such as peptides, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, polyphenols, alkaloids, minerals and just about any other chemical that can be used, ingested or synthesized by a given cell or organism.
Metabolomics is ideally positioned to be used in many areas of food science and nutrition research including food component analysis, food quality/authenticity assessment, food consumption monitoring and physiological monitoring in food intervention studies. However, the potential impact of metabolomics is still limited by two factors: (1) technology and (2) databases. In terms of instrumentation, it is clear that significant improvements need to be made to make metabolite detection and quantification technology more robust, automated and comprehensive. While promising advances have been made, current techniques are only capable of detecting perhaps 1/10th of the relevant metabolome. This expanded breadth and depth of coverage is particularly important in food and nutrition studies.
Many more reference spectral or chromatographic databases on metabolites, food components and phytochemicals need to be developed and made public. It is only through these databases that nutritionally relevant compounds can be routinely identified or quantified. Indeed a comprehensive effort, similar to that undertaken to annotate the human metabolome, needs to be made to complete and annotate the “food metabolome”. Similar efforts also need to be directed towards creating publicly accessible, comprehensive nutritional phenotype databases that include quantitative metabolomic (and other omic) data collected from diet-challenge or food intervention experiments. While these kinds of endeavours may take years to complete and cost millions of dollars, hopefully the food science community (and its funding agencies) will find a way of coordinating its activities to complete these efforts. Indeed, having public resource like a food metabolome database or a nutritional phenotype database could be as valuable to food scientists as GenBank has been to molecular biologists.
Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com
With the growing worldwide obesity epidemic, including huge populations in developing countries, such as China, India, Mexico and Brazil, the causes of this health and economic catastrophe have been increasingly studied. It is well known that metabolic syndrome and obesity exhibit a high correlation with low or absent physical exercise practices and the consumption of calorie-rich diets in developing countries; however, although the inhabitants may actually experience a nutrition transition, high levels of overweight and obese individuals could not be justified solely by diet and physical inactivity, other hallmarks, such as metabolic programming by the under nutrition early in life and epigenetic modification could also be underlining the obesity onset.
In addition to the pathophysiological aspects that have emerged from studies on metabolic programming caused by environmental insults during fetal life, another interesting point that is relevant to this issue is the role of epigenetic changes in the increased risk of developing metabolic diseases, such as type 2 diabetes and obesity, later in life. Epigenetic mechanisms, such as DNA methylation and/or nucleoprotein acetylation/methylation, are crucial to the normal/physiological development of several tissues in mammals, and they involve several mechanisms to guarantee fluctuations of enzymes and other proteins that regulate the metabolism. As previously reviewed, the intrauterine phase of development is particularly important for the genomic processes related to genes associated with metabolic pathways. Therefore, this phase of life may be particularly important for nutritional disturbance. In humans who experienced the Dutch famine Winter in 1944–1945 and in rats that were deprived of food in utero, epigenetic modifications were detected in the insulin-like growth factor 2 (IGF2) and pancreatic and duodenal home box 1 (Pdx1), which are the major factors involved in pancreas development and pancreatic β-cell maturation. Although it is known that the pancreas and the pancreatic β-cells develop/maturate during the embryonic phase, the postnatal life is also crucial for the maintenance processes that control the β-cell mass, such as proliferation, neogenesis and apoptosis. Nevertheless, no data on metabolic programming as the result of protein-restricted diet during lactation only have yet been reported, and no direct association with epigenetic modifications has been observed; on the other hand, because stressor insults during the milk suckling phase can lead to disturbances in glucose metabolism, hypothalamic neurons, ANS activity and β-cell mass/function of the pancreatic β-cells in rodents, further studies are needed on this topic.
Two decades ago, it was observed that low birth weight was related to adult chronic, non-transmissible diseases, such as type 2 diabetes, cardiovascular disease and obesity. It has been speculated that a nutritional injury during perinatal growth, including uterine and early postnatal life, may contribute to adapting the adult metabolism toward nutritional restriction. However, if an abundant diet is offered to people who have been undernourished during the perinatal life, this opportunity induces a metabolic shift toward the storage of energy and high fat tissue accumulation, thus leading to high risks of the onset of metabolic/coronary diseases onset. These observations led to the introduction of the term DOHaD (Developmental Origins of Health and Disease) previously known as the Barker thrifty phenotype hypothesis. Currently, the concept of DOHaD is extended to any other insults during perinatal life, pregnancy and/or lactation, such as underweight, overweight, diabetic or hyperplasic mothers. This concept also includes any type of stressful situations that may predispose babies or pups to develop metabolic disorders when they reach adulthood.
A large part of the world’s population is undernourished by the standards of Western Europe and North America. Scientists and nonscientists alike recognize as one of the major challenges of our time the problem of how to ensure that the production and distribution of food keep pace with the increasing number of mouths to be fed. In the world as a whole the most widespread and serious dietary deficiency is that of protein. This fact emerges clearly from the reports of the expert committees of WHO and FAO (World Health Organization, 1951, 1953). Nevertheless, many protein chemists, even those associated with medical research, may not realize the extent and severity of protein malnutrition, because it occurs chiefly in the technically underdeveloped countries far from where they work.
Dietary histories and response to treatment point to deficiency of total protein as the primary cause of the clinical syndrome kwashiorkor. The level of calorie intake has an important influence on the pattern of the disease. Deficiency of one or more specific amino acids, or amino acid imbalances in the diet, may perhaps be responsible for some of the symptoms and signs, particularly those whose incidence varies from one part of the world to another. All these variations on a theme are covered by the general term protein malnutrition. The onset is often precipitated by the added burden of diarrhea, infection, and parasitic infestation. The nutritional state influences the resistance to infection, and conversely the presence of an infection affects the state of nutrition. A further contributory factor may be the psychological upheaval in the child when the next baby in the family is born. At the root of all these causes lie poverty, ignorance, and disruption of the family life.
The planning of preventive measures cannot be effective unless it is based on some knowledge of the magnitude of the problem to be tackled. At a very rough estimate, in some countries perhaps 10% of the children suffer from severe protein malnutrition at some age between birth and 4 years. The marginal deficiency states must be much more common, Clinical signs and biochemical changes are of little value in diagnosing the early case; a deficit in body weight still seems to be the best criterion. Prevention ideally would be by greater production and consumption of animal protein, and by the increased use of skim milk and of surplus fish at present often wasted. However, animal protein is likely to remain scarce and expensive. Plant sources are being investigated with a view to encouraging not only domestic production, but also the production on an industrial scale of cheap foodstuffs rich in protein. A preventive program that is nutritionally sound may fail if account is not taken of local food habits, traditions, and customs. Protein requirements are affected by the quality of protein, the intake of calories, and by the state of the body (growth, the presence of disease, etc.). The maintenance requirement and the amount required for growth in children can be estimated, but the requirement for health is still unknown. For the time being, the allowances of protein recommended for people in the world as a whole are based empirically on the known physiological requirement with an arbitrarily added wide margin of safety.
The absorption of nitrogen is remarkably efficient even in severely malnourished infants. In general the nitrogen of plant proteins is less well absorbed than that of milk. When a baby receives a diet in which the protein is derived entirely from vegetabIe sources, incomplete absorption of nitrogen may play a significant part in the production of protein malnutrition. The malnourished baby who responds to treatment is able to retain and utilize nitrogen very efficiently; there is no evidence of any impairment in the mechanisms of protein synthesis. It is possible, however, that these mechanisms may be irreversibly damaged in babies who die, and that this may be the cause of death. The level of calorie intake has an important influence on the efficiency of utilization of nitrogen. An adequate calorie intake promotes conservation of nitrogen in the body as a whole when supplies of protein are short, but this protective effect may not be exerted equally in all organs. In this way the level of calorie intake may modify the pattern of protein depletion. A greater than normal calorie intake is needed for the restoration of depleted protein stores.
The discussion of protein metabolism in protein malnutrition has been purposely limited to a narrow field-to studies made on man, and to the few animal experiments that have a direct bearing on those studies. For technical reasons most of the work discussed relates to plasma proteins. There is a conflict of evidence between results obtained in man and animals about the effect of protein depletion or a low protein diet on the rate of catabolism of plasma albumin. It is of great importance to settle this point. A priori there seems no reason why the rate of protein catabolism should be affected by nutritional state. Preliminary studies with radioactive methionine in infants suggest, as working hypotheses, that in protein malnutrition there may be an increase in the reutilization of amino acids liberated by tissue catabolism, and an apparent concentration of protein synthesis in the more essential organs at the expense of the less essential. There is some experimental support for both these ideas, but further work is badly needed. The concept of protein stores or reserve protein is based entirely on dynamic and not on chemical considerations. It is suggested that the essential difference between a “labile” and a “fixed” protein is a difference in turnover rate. An attempt is made to show that the changes produced by protein depletion in the protein content of organs such as liver and muscle are a necessary consequence of the metabolic characteristics of proteins in those organs. There may be no need to invoke the help of homeostatic or compensatory regulations to explain the changes found in protein depletion.
Aging and growth are processes during which some metabolic adjustments must take place. It is believed that it may be better to regard the changes which are found in protein malnutrition in a similar light: as evidence of an alteration in functional pattern, rather than of damage or disease. Protein malnutrition in man has two aspects-a practical and a theoretical one. From the practical point of view it is an extremely common disease with a high mortality, and there is every reason to believe that it will become more common unless urgent preventive measures are taken. Theoretically it raises many questions that are of interest in relation to other branches of medicine and biochemistry. It is believed that the two aspects are linked, and that progress towards prevention is still impeded by our lack of basic knowledge as well as by our failure to apply what is already known. In protein malnutrition there is no sharp line between health and disease. The simple concept of specific deficiency diseases that grew from the discovery of vitamins is not applicable. We have to go back instead to the ideas of an earlier era, when nutrition was regarded as a branch of physiology, concerned with the functions, fate, and metabolic interrelationships of the major nutrients.
It is a characteristic of protein metabolism that nitrogen balance can be maintained at many different levels of protein intake. These different steady states are achieved by adjustments of the amount and distribution of proteins in the body as a whole, in organs, and in cells. It is believed that these changes in amount and distribution of proteins must result in alterations of metabolic pattern, with a gradation of change from an optimum, which cannot be defined, to a state of irreversible breakdown incompatible with life. In the intermediate stages function is modified and efficiency perhaps impaired. It seems possible that variations in diet, and particularly in the amount and quality of the protein, may underlie many of the differences in incidence and symptomatology of disease which are gradually being uncovered in different parts of the world.
Benefits of Functional Foods in Nutrient Imbalance of Vulnerable Populations
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
There are clear distinctions between a food and a drug. Nutraceuticals, however, occupy a place between the two. Nutraceuticals are naturally derived phytochemicals with potential health benefits and without the characteristics of being essential nutrients. Foods that contain these non-essential substances with potential health benefits may qualify as “functional foods.” As defined by the Food and Nutrition Board of the National Academy of Sciences, the term functional food refers to foods that provide health benefits beyond basic nutrition. Examples of these are
may be increased in or added to traditional foods. An example is a genetically modified tomato high in lycopene, which has potent antioxidant capabilities.
The risk of nutrient imbalance is highest in vulnerable populations unable to access essential or conditionally essential nutrients. To a large extent, the
very young and the
frail elderly
are the select groups who might benefit most from alleviating this risk. The lack of adequate nutrition may be due to seasonal and unexpected losses of agricultural produce; however, poverty is a factor on a global scale as a result of growing economic disparities. The question then becomes what role functional foods offer to improve recognized population nutritional deficiencies. The range of work being done on functional foods is impressive, from
modified oils that contain heart-healthy ω-3 fatty acids to
cassava plants developed with an increased protein content to help counter malnutrition in developing nations.
However, the nutraceutical industry has responded to and relies on the untested expectations of the healthiest members of the world’s population rather than its more vulnerable ones. Due largely to economic causes, those in need are less likely to receive the benefits of nutraceuticals from whole foods or from manufactured foods or supplements. This is particularly striking where the source is locally available and extracted for commerce but is unaffordable or unavailable to the native population.
The rapid advances in biotechnology and functional foods confront us with a need to address the benefits of these with regard to improving health and managing or decreasing disease risks.Conventional dietary recommendations have focused on the consumption of fruits, vegetables, legumes, and whole grains, a decreased sugar intake, and an emphasis on plant oils, recommendations that have unproved benefits for the prevention of chronic diseases and that have complexities involving individual, environmental, and genetic influences.
Although the potential benefits of phytochemicals could have an impact on health status for vulnerable populations, the recommendations focused on plant foods do not address the primary concerns of the undernutrition associated with a poor quality of protein intake. Taken individually, plant sources do not provide a balanced amino acid profile necessary for protein synthesis, being deficient in lysine and/or methionine. Animal sources of protein, specifically meat and fish, also provide essential fatty acids not found in plant sources of protein and that may be otherwise limited. In addition, plants may contain antinutritional factors (wheat, cassava roots, cabbages, soy beans), and plant-based diets may be deficient in important essential nutrients.
Programs must focus on the sustainable production and local processing of indigenous products that can be used by needy populations to improve their nutritional intake and enhance economic stability. In addition, dietary recommendations must not exclude important sources of nutrition for more vulnerable populations by focusing primarily on plant-based sources of food, decreasing saturated fat, and de-emphasizing the importance of high-value biologic protein. The global economic crisis has touched the lives of 80% of the population in most developing countries with a threat to the development of a generation of children (approximately 250 million) who are most vulnerable in the first 2 years of life. An investment in nutrition in this circumstance has a high value, and the use of complementary food supplements to increase a meal’s nutrient content is warranted.
A recent proposal has concluded there are health benefits for foods and food constituents put together in a synergic diet pattern, suggesting that the interrelation between constituents within whole foods is significant, and has recommended dietary variety and the selection of nutrient-rich foods. Providing vulnerable populations with an adequate supply of whole foods should take precedence over the recommendation of food products in supplying not only essential macro- and micronutrients and energy but also phytochemicals whose value to the human diet is still to be determined.
Author and Curator: Larry H. Bernstein,MD, FACP, Clinical Pathologist, Biochemist, and Transfusion Physician
Brief introduction
Transthyretin (also known as prealbumin) has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompted a review of the benefit of using this test in acute and chronic care. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu. A much better understanding of the significance of this program has emerged from studies of nitrogen and sulfur in health and disease.
Transthyretin protein structure (Photo credit: Wikipedia)
Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)
Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively.
There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR and in the context of an ICU setting, the contribution of TTR is essential. The only consideration is the timing of initiation since the metabolic burden is sufficiently high that a substantial elevation is expected in the first 3 days post admission, although the level of this biomarker is related to the severity of injury. Despite the complexity of the situation, TTR is not to be considered a test “for all seasons”. In the context of age, prolonged poor meal intake, chronic or acute illness, TTR needs to be viewed in a multivariable lens, along with estimated lean body mass, C-reactive protein, the absolute lymphocyte count, presence of neutrophilia, and perhaps procalcitonin if there is remaining uncertainty. Furthermore, the reduction of risk of associated complication requires a systematized approach to timely identification, communication, and implementation of a suitable treatment plan.
The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.
Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis
We have proposed an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability. The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. These are not easily performed or adequately expedited. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.
The laboratory role in nutritional management of the patient has seen remarkable growth while there have been dramatic changes in technology over the last 25 years, and it is bound to be transformative in the near term. This editorial is an overview of the importance of the laboratory as an active participant in nutritional care.
The discipline emerged divergently along separate paths with unrelated knowledge domains in physiological chemistry, pathology, microbiology, immunology and blood cell recognition, and then cross-linked emerging into clinical biochemistry, hematology-oncology, infectious diseases, toxicology and therapeutics, genetics, pharmacogenomics, translational genomics and clinical diagnostics.
In reality, the more we learn about nutrition, the more we uncover of metabolic diversity of individuals, the family, and societies in adapting and living in many unique environments and the basic reactions, controls, and responses to illness. This course links metabolism to genomics and individual diversity through metabolomics, which will be enlightened by chemical and bioenergetic insights into biology and translated into laboratory profiling.
Vitamin deficiencies were discovered as clinical entities with observed features as a result of industrialization (rickets and vitamin D deficiency) and mercantile trade (scurvy and vitamin C)[2]. Advances in chemistry led to the isolation of each deficient “substance”. In some cases, a deficiency of a vitamin and what is later known as an “endocrine hormone” later have confusing distinctions (vitamin D, and islet cell insulin).
The accurate measurement and roles of trace elements, enzymes, and pharmacologic agents was to follow within the next two decades with introduction of atomic absorption, kinetic spectrophotometers, column chromatography and gel electrophoresis. We had fully automated laboratories by the late 1960s, and over the next ten years basic organ panels became routine. This was a game changer.
Today child malnutrition prevalence is 7 percent of children under the age of 5 in China, 28 percent in sub-Saharan African, and 43 percent in India, while under-nutrition is found mostly in rural areas with 10 percent of villages and districts accounting for 27-28 percent of all Indian underweight children. This may not be surprising, but it is associated with stunting and wasting, and it has not receded with India’s economic growth. It might go unnoticed viewed alongside a growing concurrent problem of worldwide obesity.
The post WWII images of holocaust survivors awakened sensitivity to nutritional deprivation.
In the medical literature, Studley [HO Studley. Percentage of weight loss. Basic Indicator of surgical risk in patients with chronic peptic ulcer. JAMA 1936; 106(6):458-460. doi:10.1001/jama.1936.02770060032009] reported the association between weight loss and poor surgical outcomes in 1936. Ingenbleek et al [Y Ingenbleek, M De Vissher, PH De Nayer. Measurement of prealbumin as index of protein-calorie malnutrition. Lancet 1972; 300[7768]: 106-109] first reported that prealbumin (transthyretin, TTR) is a biomarker for malnutrition after finding very low TTR levels in African children with Kwashiorkor in 1972, which went unnoticed for years. This coincided with the demonstration by Stanley Dudrick [JA Sanchez, JM Daly.Stanley Dudrick, MD. A Paradigm Shift. Arch Surg. 2010; 145(6):512-514] that beagle puppies fed totally through a catheter inserted into the superior vena cava grew, which method was then extended to feeding children with short gut. Soon after Bistrian and Blackburn [BR Bistrian, GL Blackburn, E Hallowell, et al. Protein status of general surgical patients. JAMA 1974; 230:858; BR Bistrian, GL Blackburn, J Vitale, et al. Prevalence of malnutrition in general medicine patients, JAMA, 1976, 235:1567] showed that malnourished hospitalized medical and surgical patients have increased length of stay, increased morbidity, such as wound dehiscence and wound infection, and increased postoperative mortality, later supported by many studies.
Michael Meguid,MD, PhD, founding editor of Nutrition [Elsevier] held a nutrition conference “Skeleton in the Closet – 20 years later” in Los Angeles in 1995, at which a Beckman Prealbumin Roundtable was held, with Thomas Baumgartner and Michael M Meguid as key participants. A key finding was that to realize the expected benefits of a nutritional screening and monitoring program requires laboratory support. A Ross Roundtable, chaired by Dr. Lawrence Kaplan, resulted in the first Standard of Laboratory Practice Document of the National Academy of Clinical Biochemists on the use of the clinical laboratory in nutritional support and monitoring. Mears then showed a real benefit to a laboratory interactive program in nutrition screening based on TTR [E Mears. Outcomes of continuous process improvement of a nutritional care program incorporating serum prealbumin measurements. Nutrition 1996; 12 (7/8): 479-484].
A later Ross Roundtable on Quality in Nutritional Care included a study of nutrition screening and time to dietitian intervention organized by Brugler and Di Prinzio that showed a decreased length of hospital stay with $1 million savings in the first year (which repeated), which included reduced cost for dietitian evaluations and lower complication rates.
Presentations were made at the 1st International Transthyretin Congress in Strasbourg, France by Mears [E Mears. The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 2002; 40:1360-1369] on the impact of TTR in screening for PEM in a public hospital in Louisiana, and by Potter [MA Potter, G Luxton. Prealbumin measurement as a screening tool for patients with protein calorie malnutrition in emergency hospital admissions: a pilot study.Clin Invest Med. 1999; 22(2):44-52] that indicated a 17% in-hospital mortality rate in a Canadian hospital for patients with PCM compared with 4% without PCM (p < 0.02), while only 42% of patients with PCM received nutritional supplementation. Cost analysis of screening with prealbumin level projected a saving of $414 per patient screened. Ingenbleek and Young [Y Ingenbleek, VR Young. Significance of transthyretin in protein metabolism. Clin Chem Lab Med. 2002; 40(12):1281–1291. ISSN (Print) 1434-6621, DOI: 10.1515/ CCLM.2002.222, December 2002. published online: 01/06/2005] tied the TTR to basic effects reflected in protein metabolism.
TTR levels correlate with patient outcomes and are an accurate predictor of patient recovery in non-critically ill patients, but it is uncertain whether or not TTR level correlates with level of nutrition support and outcome in critically ill patients. This issue has been addressed only in critically ill patients on total parenteral nutrition and there was no association reported with standard outcome measures. We revisit this in all patients admitted to a medical intensive care unit.
Serum TTR was measured on the day of admission, day 3 and day 7 of their ICU stay. APACHE II and SOFA score was assessed on the day of admission. A registered dietician for their entire ICU stay assessed the nutritional status and nutritional requirement. Patients were divided into three groups based on initial TTR level and the outcome analysis was performed for APACHE II score, SOFA score, ICU length of stay, hospital length of stay, and mortality.
TTR showed excellent concordance with the univariate or multivariate classification of patients with PEM or at high malnutrition risk, and followed for seven days in the ICU, it is a measure of the metabolic burden. TTR levels decline from day 1 to day 7 in spite of providing nutritional support. Twenty-five patients had an initial TTR serum concentration more than 17 mg/dL (group 1), forty-eight patients had mild malnutrition with a concentration between 10 and 17 mg/dL (group 2), Forty-five patients had severe malnutrition with a concentration less than 10 mg/dL (group 3). Initial TTR level had inverse correlation with ICU length of stay, hospital length of stay, and APACHE II score, SOFA score; and predicted mortality, especially in group 3.
The acute reaction to stress is characterized by major metabolic, endocrine and immune alterations. According to classical descriptions, these changes clinically present as a succession of 3 adaptive steps – ebb phase, catabolic flow phase, and anabolic flow phase. The ebb phase, shock and resuscitation, is immediate, lasts several hours, and is characterized by hypokinesis, hypothermia, hemodynamic instability and reduced basal metabolic rate. The catabolic flow phase, beginning within 24 hours and lasting several days, is characterized by catabolism with the flow of gluconeogenic substrates and ketone bodies in response to the acute injury. The magnitude of the response depends on the acuity and the severity of the stress. The last, a reparative anabolic flow phase, lasts weeks and is characterized by the accretion of amino acids (AAs) to rebuilding lean body mass.
The current opinion is that the body economy is reset during the course of stress at novel thresholds of metabolic priorities. This is exemplified mainly by proteolysis of muscle, by an effect on proliferating gut mucosa and lymphoid tissue as substrates are channeled to support wound healing, by altered syntheses of liver proteins with preferential production of acute phase proteins (APPs) and local repair in inflamed tissues (3). The first two stages demonstrate body protein breakdown exceeding the rate of protein synthesis, resulting in a negative nitrogen (N) balance, muscle wasting and weight loss. In contrast, the last stage displays reversed patterns, implying progressive recovery of endogenous N pools and body weight.
These adaptive alterations undergo continuing elucidation. The identification of cytokines, secreted by activated macrophages/monocytes or other reacting cells, has provided further insights into the molecular mechanisms controlling energy expenditure, redistribution of protein pools, reprioritization of syntheses and secretory processes.
The free fraction of hormones bound to specific binding-protein(s) [BP(s)] manifests biological activities, and any change in the BP blood level modifies the effect of the hormone on the end target organ. The efficacy of these adaptive responses may be severely impaired in protein-energy malnourished (PEM) patients. This is especially critical with respect to changes of the circulating levels of transthyretin (TTR), retinol-binding protein (RBP) and corticosteroid-binding globulin (CBG) conveying thyroid hormones (TH), retinol and cortisol, respectively. This reaction is characterized by cytokine mediated autocrine, paracrine and endocrine changes. Among the many inducing molecules identified, interleukins 1 and 6 (Il-1, Il-6) and tumor necrosis factor a (TNF) are associated with enhanced production of 3 counterregulatory hormonal families (cortisol, catecholamines and glucagon). Growth hormone (GH) and TH also have roles in these metabolic adjustments.
There is overproduction of cortisol mediated by several cytokines acting on both the adrenal cortex (10) and on the pituitary through hypothalamic CRH with loss of feedback regulation of ACTH production (11). Hypercortisolemia is a major finding observed after surgery (12), sepsis (13), and medical insults, usually correlated with severity of insult and of complications. Rising cortisol values parallel hyperglycemic trends, as an effect of both gluconeogenesis and insulin resistance. Working in concert with TNF, glucocorticoids govern the breakdown of muscle mass, which is regarded as the main factor responsible for the negative N balance.
Under normal conditions, GH exerts both lipolytic and anabolic influences in the whole body economy under the dual control of the hypothalamic hormones somatocrinin (GHRH) and somatostatin (SRIH). GH secretion is usually depressed by rising blood concentrations of glucose and free fatty acids (FFAs) but is paradoxicaly elevated despite hyperglycemia in stressed patients.
The oversecretion of counterregulatory hormones working in concert generates subtle equilibria between glycogenolytic/glycolytic/gluconeogenic adaptive processes. The net result is the neutralization of the main hypoglycemic and anabolic activities of insulin and the development of a persisting and controlled hyperglycemic tone in the stressed body. The molecular mechanisms whereby insulin resistance occurs in the course of stress refer to
cytokine- and hormone-induced phosphorylation abnormalities affecting receptor signaling. The insulin-like anabolic processes of GH are mediated by IGF1 working as relay agent. The expected high IGF1 surge associated with GH oversecretion is not observed in severe stress as plasma values are usually found at the lower limit of normal or even in the subnormal range. The end result of this dissociation between high GH and low IGF1 levels is to favor the proteolysis of muscle mass to release AAs for gluconeogenesis and the breakdown of adipose tissue to provide ketogenic substrates.
The acute stage of stress is associated with the onset of a low T3 syndrome typically delineated by the drop of both total (TT3) and free (FT3) triiodothyronine plasma levels in the subnormal range. In contrast, both total (TT4) and free (FT4) thyroxine values usually remain within normal ranges with declining trends observed for TT4 and rising tendencies for FT4 (44). This last free compound is regarded as the sensor reflecting the actual thyroid status and governing the release of TSH whereas FT3 works as the active hormonal mediator at nuclear receptor level. The maintenance of an euthyroid sick syndrome is compatible with the down-regulation of most metabolic and energetic processes in healthy tissues. These inhibitory effects , negatively affecting all functional steps of the hypothalamo-pituitary-thyroid axis concern TSH production, iodide uptake, transport and organification into iodotyrosyl residues, peroxidase coupling activity as well as thyroglobulin synthesis and TH leakage. Taken together, the above-mentioned data indicate that the development of hyperglycemia and of insulin-resistance in healthy tissues – mainly in the muscle mass – are hallmarks resulting from the coordinated activities of the counterregulatory hormones.
A growing body of recent data suggest that the stressed territory, whatever the causal agent – bacterial or viral sepsis, auto-immune disorder, traumatic or toxic shock, burns, cancer – manifest differentiated metabolic and immune reactions. The amplitude, duration and efficacy of these responses are reportedly impaired along several ways in PEM patients. These last detrimental effects are accompanied by a number of medical, social and economical consequences, such as extended length of hospital stay and increased complication / mortality rates. It is therefore mandatory to correctly identify and follow up the nutritional status of hospitalized patients. Such approaches are prerequisite to timely and scientifically grounded nutritional and pharmacological mediated interventions.
Contrary to the rest of the body, energy requirements of the inflamed territory are primarily fulfilled by anaerobic glycolysis, an effect triggered by the inhibition of key-enzymes of carbohydrate metabolism, notably pyruvate-dehydrogenase. This non-oxidative combustion of glucose reveals low conversion efficiency but offers the major advantage to maintain, in the context of hyperglycemia, fuel provision to poorly irrigated and/or edematous tissues. The depression of the 5’-monodeiodinating activity (5’-DA) plays a pivotal role in these adaptive changes, yielding inactive reverse T3 (rT3) as index of impaired T4 to T3 conversion rates, but at the same time there is an augmented supply of bioactive T3 molecules and local overstimulation of thyro-dependent processes characterized by thyroid down-regulation. The same differentiated evolutionary pattern applies to IGF1. In spite of lowered plasma total concentrations, the proportion of IGF1 released in free form may be substantially increased owing to the proteolytic degradation of IGFBP-3 in the intravascular compartment. The digestion of BP-3 results from the surge of several proteases occurring the course of stress, yielding biologically active IGF1 molecules available for the repair of damaged tissues. In contrast, healthy receptors oppose a strong resistance to IGF1 ligands freed in the general circulation, likely induced by an acquired phosphorylation defect very similar in nature to that for the insulin transduction pathway.
PEM is the generic denomination of a broad spectrum of nutritional disorders, commonly found in hospital settings, and whose extreme poles are identified as marasmus and kwashiorkor. The former condition is usually regarded as the result of long-lasting starvation leading to the loss of lean body mass and fat reserves but relatively well-preserved liver function and immune capacities. The latter condition is typically the consequence of (sub)acute deprivation predominantly affecting the protein content of staplefood, an imbalance causing hepatic steatosis, fall of visceral proteins, edema and increased vulnerability to most stressful factors. PEM may be hypometabolic or hypermetabolic, usually coexists with other diseased states and is frequently associated with complications. Identification of PEM calls upon a large set of clinical and analytical disciplines comprising anthropometry, immunology, hematology and biochemistry.
CBG, TTR and RBP share in common the transport of specific ligands exerting their metabolic effects at nuclear receptor level. Released from their specific BPs in free form, cortisol, FT4 and retinol immediately participe to the strenghtening of the positive and negative responses to stressful stimuli. CBG is a relatively weak responder to short-term nutritional influences (73) although long-lasting PEM is reportedly capable of causing its significant diminution (74). The dramatic drop of CBG in the course of stress appears as the combined effect of Il-6-induced posttranscriptional blockade of its liver synthesis (75) and peripheral overconsumption by activated neutrophils (61). The divergent alterations outlined by CBG and total cortisolemia result in an increased disposal of free ligand reaching proportions considerably higher than the 4 % recorded under physiological conditions.
The appellation of negativeAPPs that was once given to the visceral group of carrier-proteins. The NDAD concept takes the opposite view, defending the opinion that their suppressed synthesis releases free ligands which positively contribute to strengthen all aspects of the stress reaction, justifying the ABR denomination. This implies that the role played by ABRs should no longer be interpreted in terms of concentrations but in terms of functionality.
Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.
Metabolic pathways: Met molecules supplied by dietary proteins are submitted to TM processes allowing to release Hcy which may in turn either undergo Hcy – Met RM pathways or be irreversibly committed into TS decay. Impairment of CbS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. This last beneficial effect is counteracted by decreased concentration of most components generated downstream to CbS, explaining the depressed CbS- and CbL-mediated enzymatic production of H2S along the TS cascade. The restricted dietary intake of elemental S further operates as a limiting factor for its non-enzymatic reduction to H2S which contributes to downsizing a common body pool. Combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.
Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.
Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting
FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg. Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content.
SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues. The LBM of the reference man contains 98% of total
body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass). The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells.
During ageing, all the protein components of the human body decrease regularly. This shrinking tendency is especially well documented for SM whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure, intracellular K content and working capacit. TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline
with an accelerated decrease after 65 years. The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction.
The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth consistent with superimposable N accretion and protein synthesis rates. Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males. The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age. The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research. TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.
TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs. The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise.
LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability. In kidney patients, LBM is proposed as an excellent predictor of outcome working in the same direction as TTR plasma levels. High N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome. It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.
Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12).
Significant positive correlations are found between tHcy and plasma urea and plasma creatinine, indicating that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. In healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein
components which are downregulated in protein-depleted states, tHcy values are upregulated. Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking.
Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance. Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation. This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004).
Aminoacidemia studies performed in PEM children, adult patients and elderly subjects have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the above described evolutionary profiles, showing unusual stability in chronically protein depleted states.
Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEMadults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children. In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione, taurine, and sulfaturia. Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli, thereby promoting the upstream retention of tHcy in biological fluids. It
was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity has recently provided. The tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction. In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. This is consistent with the unique role played by Met in the preservation of N body stores.
The classical interpretation that strict vegans, who consume plenty of folates in their diet and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts, needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide, being often incriminated as deficient in vegan subjects. It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy
variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies.
A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products,
vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma
concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship that was established in Western Africa from comparable field studies involving country dwellers living on plant products.
The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokine induced proteolysis. This has been documented in coronary infarction and in acute pancreatitis where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation. tHcy is considered stable in plasma and the two investigations report unaltered folate and cobalamin plasma concentrations.
The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition.
The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations.
Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996).
1INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse F-31300, France.
2CNRS, U5282, Toulouse F-31300, France.
3CPTP, Université de Toulouse, Université Paul Sabatier (UPS), Toulouse F-31300, France.
4Institut National de la Recherche Agronomique (INRA), UMR 1319 Micalis, Commensal and Probiotics-Host Interactions Laboratory, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
5Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
6Pôle Digestif, CHU Purpan, Toulouse F-31059, France.
7Division of Gastroenterology, Hepatology and Nutrition, Children’s Hospital, Toulouse F-31059, France.
8Institut Pasteur, Unité de Défense Innée et Inflammation, Paris F-75015, France.
9INSERM U874, Paris F-75724, France.
10Universite Paris Diderot, Sorbonne Paris Cite, Cellule Pasteur F-75013, France.
ABSTRACT
Elafin, a natural protease inhibitor expressed in healthy intestinal mucosa, has pleiotropic anti-inflammatory properties in vitro and in animal models. We found that mucosal expression of Elafin is diminished in patients with inflammatory bowel disease (IBD). This defect is associated with increased elastolytic activity (elastase-like proteolysis) in colon tissue. We engineered two food-grade strains of lactic acid bacteria (LAB) to express and deliver Elafin to the site of inflammation in the colon to assess the potential therapeutic benefits of the Elafin-expressing LAB. In mouse models of acute and chronic colitis, oral administration of Elafin-expressing LAB decreased elastolytic activity and inflammation and restored intestinal homeostasis. Furthermore, when cultures of human intestinal epithelial cells were treated with LAB secreting Elafin, the inflamed epithelium was protected from increased intestinal permeability and from the release of cytokines and chemokines, both of which are characteristic of intestinal dysfunction associated with IBD. Together, these results suggest that oral delivery of LAB secreting Elafin may be useful for treating IBD in humans.
Cytokines involved in IBD (Photo credit: Wikipedia)
Metabolism
Front. Physio., 10 October 2012 | doi: 10.3389/fphys.2012.00401
Outlook: membrane junctions enable the metabolic trapping of fatty acids by intracellular acyl-CoA synthetases
Joachim Füllekrug*, Robert Ehehalt and Margarete Poppelreuther
Molecular Cell Biology Laboratory, Internal Medicine IV, University of Heidelberg, Heidelberg, Germany
The mechanism of fatty acid uptake is of high interest for basic research and clinical interventions. Recently, we showed that mammalian long chain fatty acyl-CoA synthetases (ACS) are not only essential enzymes for lipid metabolism but are also involved in cellular fatty acid uptake. Overexpression, RNAi depletion or hormonal stimulation of ACS enzymes lead to corresponding changes of fatty acid uptake. Remarkably, ACS are not localized to the plasma membrane where fatty acids are entering the cell, but are found instead at the endoplasmic reticulum (ER) or other intracellular organelles like mitochondria and lipid droplets. This is in contrast to current models suggesting that ACS enzymes function in complex with transporters at the cell surface. Drawing on recent insights into non-vesicular lipid transport, we suggest a revised model for the cellular fatty acid uptake of mammalian cells which incorporates trafficking of fatty acids across membrane junctions. Intracellular ACS enzymes are then metabolically trapping fatty acids as acyl-CoA derivatives. These local decreases in fatty acid concentration will unbalance the equilibrium of fatty acids across the plasma membrane, and thus provide a driving force for fatty acid uptake.
English: Acyl-CoA from the cytosol to the mitochondrial matrix. Français : Transport de l’Acyl-CoA du Cytosol jusqu’à la matrice mitochondriale. (Photo credit: Wikipedia)
English: The mechanism for Long Chain Fatty Acyl-CoA Synthetase (Photo credit: Wikipedia)
One of the latest posts address to issue of immunoreactivity and nanotechnology and I wanted to take advantage of this stage to address this topic again. On the many, potentially good effects and goals of nanotechnology, we have emerging side effects and human health issues that needs to be addressed.
It is estimated that the average person in a developed country consumes between 10xE12 and 10xE14 man-made fine (diameter, 0.1–1 mm) to ultrafine (diameter, ,100 nm) particles every day. These dietary particles are mainly TiO2, silicates and aluminosilicates derived from food additives such as stabilizers and anticaking agents . Because most of these micro- and nanoparticles have negatively charged surfaces, they can bind to biomolecules in the gut lumen, absorb across the gastrointestinal tract and accumulate at the base of Peyer’s patches, where a large concentration of M cells are found. M cells transport microorganisms and particles from the gut lumen to immune cells across the intestinal epithelium, and are important for defending the body against ingested toxic substances and stimulating mucosal immunity.
In a research collaboration led by Michael Shuler, the Samuel B. Eckert Professor of Chemical Engineering and the James and Marsha McCormick Chair of Biomedical Engineering, studied how large doses of polystyrene nanoparticles — a common, FDA-approved material found in substances from food additives to vitamins — affected how well chickens absorbed iron, an essential nutrient, into their cells (http://www.nature.com/nnano/journal/v7/n4/full/nnano.2012.3.html).
The researchers tested both acute and chronic nanoparticle exposure using human gut cells in petri dishes as well as live chickens and reported matching results. They chose chickens because these animals absorb iron into their bodies similarly to humans, and they are also similarly sensitive to micronutrient deficiencies.
More so, the authors chose iron absorption as a subject because iron is an example of an essential nutrient that is transported across the intestinal epithelium by means of complex, highly regulated, protein-assisted vesicular and non-vesicular mechanisms.
The researchers used commercially available, 50-nanometer polystyrene carboxylated particles that are generally considered safe for human consumption. They found that following acute exposure, a few minutes to a few hours after consumption, both the absorption of iron in the in vitro cells and the chickens decreased. But following exposure of 2 milligrams per kilogram for two weeks — a slower, more chronic intake — the structure of the intestinal villi began to change and increase in surface area. This was an effective physiological remodeling that led to increased iron absorption.
The increased iron uptake by monolayers exposed to +50 nm particles is probably due to the increased tight junction permeability, as increased transcytosis of luminal material often accompanies tight junction dysfunction.
The in vivo experiments indicate that nanoparticle exposure causes a disruption in iron transport and that the intestinal villi remodel to increase the surface area available for absorption. This increased area compensates for the disruption in iron transport caused by the nanoparticles.
Ferritin levels were analysed in all samples to exclude pre-existing differences in iron status as a cause for differences in iron transport or uptake. Ferritin levels in all nanoparticle-exposed and control cultures were not significantly different.
The authors concluded that The intestinal epithelial layer represents the initial gate that ingested nanoparticles must pass to reach the body. The polystyrene particles used in these experiments are generally considered non-toxic, but their interaction with a normal physiological process suggests a potential mechanism for a chronic, harmful, but subtle response.
Similar disruptions in nutrient absorption could be possible in relation to other inorganic elements such as calcium, copper and zinc, which require passive or active transport systems for them to be absorbed through the intestinal epithelium. Fat-soluble vitamins such as vitamins A, D, E and K are absorbed only after micellization by pancreatic lipase.
oral exposure to polystyrene nanoparticles can disrupt iron transport and chronic exposure can cause remodelling of the intestinal villi. Remodelling of the villi increases the surface area available for iron absorption. Nanoparticle size, concentration and charge can influence iron uptake and iron transport at doses that represent potential human exposure.
Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP
Article 1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
This discussion is a continuation of an earlier piece on the technologic framework for , proteomics, nutrigenomics, and translational medicine. The last decade has seen the emergence of a genomic science that is changing the trajectory of biological sciences and medicine. It has not resolved all of our problems by any means, but it has begun to redraw the map, which began with the elucidation of major metabolic pathways in the first half of the 20th century, was then captured by the transformation of genetics with the discovery of the “Watson-Crick Model”, and then later was recharged with the discovery of the Toll-like receptor and the drawing of “signaling pathways”. What we have seen in an unraveling of protein-genome interactions, small peptide regulators, and dynamic changes in pathway dominance, bloackage, and reentry, depending on genetic, dietary, and environmental conditions, mostly expressed in what we refer to as “oxidative stress”.
Unraveling the multitude of nutrigenomic, proteomic, and metabolomic patterns that arise from the ingestion of foods or their bioactive food components will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as microarrays, RNA interference, and nanotechnologies, will provide needed insights into molecular targets for specific bioactive food components and how they harmonize to influence individual phenotypes. A challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems.
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. The new era of nutrition research translates empirical knowledge to evidence-based molecular science. Following this trend, Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that aims to elucidate how diet can influence human health. It is already well known that bioactive food compounds can interact with genes affecting transcription factors, protein expression and metabolite production. The study of these complex interactions requires the development of advanced analytical approaches combined with bioinformatics. The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy. Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of
genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,
individualized medical nutrition therapy for disease management, and
better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that maximize benefit and minimize adverse outcomes within genetically diverse human populations.
For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with high sample numbers in reliable measurement times with respect to both technical accuracy and the identification and quantitation of small-molecular-weight metabolites. This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology. The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand the impact of food on defined health outcomes. Both strongly depend on appropriate analytical tools to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and to monitor molecular changes in the body in a highly specific and sensitive manner. Based on these requirements, mass spectrometry has become the analytical method of choice with broad applications throughout all areas of nutrition research.
Dynamic Construct of the –Omics
Metabolomics is a term that encompasses several types of analyses, including
metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;
metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and
target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing only a few selected metabolites that comprise a specific biochemical pathway.
Any unifying concept of the metabolome was incomplete or debatable in the first 30 years of the 20th century. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 28-32 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.
For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP. The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:
Pyruvate Carboxylase (Gluconeogenesis) catalyzes pyruvate + HCO3- + ATP — oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP —- phosphoenolpyruvate + GDP + CO2
Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids.
An important and revolutionary aspect of ‘The 2010 Project’ is that it implicitly endorses the allocation of resources to attempts to assign function to genes that have no known function. This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that for the first time it is feasible to envision a whole-systems approach to gene and protein function. I shall not discuss the emerging field of bioinformatics that makes this possible. In this review, the end-of-the line “detector will be considered having been covered. The entire focus proceeds to a discussion of separation methods. Separation methods have always been tricky, time consuming, and a multiple step process that depended on using anionic and cationic resins as intermediate steps in bulk separation, and then molecular size separation. Therapeutic Targets will be identified as they are seen.
Affinity Chromatography The rapid development of biotechnology and biomedicine requires more reliable and efficient separation technologies for the isolation and purification of biopolymers such as therapeutic proteins, antibodies, enzymes and nucleic acids. In particular, monoclonal antibodies are centrally important as therapeutics for the treatment of cancer and other diseases, leading to recombinant monoclonal antibodies that dominate today’s biopharmaceutical pipeline. The large-scale production of therapeutic biopolymers requires
a manufacturing process that delivers reliability and in high-yield, as well as
an effective purification process affording extremely pure products.
Because of its high selectivity, affinity chromatography has been used extensively to isolate a variety of biopolymers. The retention of solutes is based on specific, reversible interactions found in biological systems, such as the binding of an enzyme with an inhibitor or an antibody with an antigen. These interactions are exploited in affinity chromatography by immobilizing an affinity ligand onto a support, and using this as a stationary phase. Non-porous particles having an average diameter of 2.1 mm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S–MMA–GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode.
The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S–MMA–GMA) particles were slurry packed into a 1.0 cm30.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 mg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level. CH Chen, WC Lee. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate. Journal of Chromatography A 2001; 921: 31–37.
Affinity separation membranes, consisting of electrospun nanofibers, have been developed recently. Affinity ligands are attached to the surface of the constituent fibers, offering a potential solution to some of the problems of traditional, column-based, affinity chromatography. Electrospun fibers are good candidates for use in affinity separation because of their
unique characteristics of high surface area to volume ratio, resulting in
high ligand loading, and
their large porosity, resulting in
high throughput operation.
A number of polymers have been used for electrospun fiber mesh-based affinity membrane separations including poly (ether-urethane-urea), cellulose, poly(ethylene terephthalate, polysulphone, and polyacrlonitrile. Typically, very thin electrospun fiber meshes are produced by electrostatically collecting negatively charged fibers on a collector electrode. These very thin 2D electrospun fiber mesh mats provide excellent solution permeability as compared to 3D column packed with affinity beads. M Miyauchi, J Miao, TJ Simmons, JS Dordick and RJ Linhardt. Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin. J Chromatograph Separat Techniq 2011; 2:2 http://dx.doi.org/10.4172/2157-7064.1000110
Dye Affinity Chromatography Biomimetic Dyes Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins.
Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation,
the model of the protein and also
a number of suitable synthetic ligands which mimic natural biological ligands of the protein.
There are several examples of enzyme purifications
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-b-cyclodextrin (HP-b-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-b-CD revealed only two absorbing species, indicating a host–guest ratio of 1:1. The dissociation constant for this HP-b-CD–CB F3GA complex, K , was found d to be 0.43 mM. The data for HP-b-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific. JA Lopez-Mas, SA Streitenberger, F Garcıa-Carmona, AA Sanchez-Ferrer. Cyclodextrin biospecific-like displacement in dye-affinity chromatography. Journal of Chromatography A 2001; 911: 47–53.
Affinity chromatography uses biospecific binding usually between an antibody and an antigen, an enzyme and a substrate or other pairs of key-lock type of matching molecules. Due to its high selectivity, it is able to purify proteins and other macromolecules from very dilute solutions. In this work, a general rate model for affinity chromatography was used for scale-up studies. Parameters for the model were estimated from existing correlations, or from experimental results obtained on a small column with the same packing material. As anexample, Affi-Gel with 4.5mol cm−3 Cibacron Blue F-3GA as immobilized ligands covalently attached to cross-linked 6% agarose was used for column packing. Cibacron Blue F-3GA was also used as a soluble ligand in the elution stage. Satisfactory scale-up predictions were obtained for a 98.2 ml column and a 501 ml column based on a few experimental data obtained on a 7.85 ml small column. T. Gu, K.-H. Hsu and M.-J. Syu, “Scale-Up of Affinity Chromatography for Purification of Enzymes and Other Proteins.” Enzyme and Microbial Technology 2003; 33:433-437.
Affinity Column with AAAA as a Model Sense Ligand The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. R Zhao, X Yu, H Liu, L Zhai, S Xiong, et al. Study on the degeneracy of antisense peptides using affinity chromatography. Journal of Chromatography A 2001; 913: 421–428.
Frontal AC for Biomolecular Interactions Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 mm diameter), and packed into a miniature column (e.g., 1034.0 mm, 0.126 ml). The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V ); and the dissociation constant, K , was calculated according to the literature. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/ cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 mg/ml for protein) analyses of lectin–carbohydrate interactions. J Hirabayashi, Y Arata, K Kasai. Reinforcement of frontal affinity chromatography for effective analysis of lectin–oligosaccharide interactions. Journal of Chromatography A 2000; 890:261–271.
Immobilized Metal Ion Affinity New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant b-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media.
IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins. For example, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole).
The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions. However, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface, but the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. C Mateo , G Fernandez-Lorente , BCC Pessela , A Vian, et al. Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A 2001; 915:97–106. The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail. The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner. Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-
thioredoxin
ubiquitin
calmodulin
lysozyme
cytochrome c
myoglobin
on immobilized transition metal ions
Co2+
Ni2+
Cu2+
Zn2
-allows establishment of the following facets of the histidyl side chain distribution:
either interior or surface;
when localized on the surface, accessible or unaccessible for coordination;
single or multiple;
When multiple, either distant or vicinal.
Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution). We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique. IMAC can be used to ascertain several facets of the status of a histidyl residue(s) in a protein molecule:
localization (interior vs. surface)
coordination potential as defined by the steric accessibility and the state of protonation
A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to .99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate–polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.
Adenosine Affinity Ligand for Glutamine Synthase Glutamine synthetase has been purified from both procaryotic and eucaryotic sources using various types of affinity chromatography. For example, ADP-agarose has been used to purify glutamine synthetase from photosynthetic bacteria, while the related “Blue” chromatography media (e.g. Affigel Blue) have been used to purify glutamine synthetases from a variety of sources. In addition, 2’,5’-ADPSepharose 4B has been used to purify glutamine synthetase from procaryotes, plants and insects. However, this latter affinity ligand resembles NADP more than ADP, particularly with respect to the position of the phosphate moieties. This is reflected in the more general use of this affinity ligand in the purification of NADPH-dependent enzymes. In the present report, we characterize the ability of glutamine synthetase to be purified by three different adenosine-affinity ligands: 5’-ADP-agarose (an ADP analogue), 2’,5’-ADP-Sepharose 4B (an NADP analogue) and 3’,5’-ADP-agarose (a cyclic AMP analogue). We report conditions for the successful purification of insect flight muscle glutamine synthetase using each of these three different affinity ligands. The enzyme bound most strongly to the
ADP analogue (S-ADP-agarose),
followed by the NADPH analogue (2’,5’-ADP-Sepharose 4B), and least strongly to
the cyclic AMP analogue (3’J’-ADP-agarose).
In all cases, binding was strongest in the presence of Mn2+ when compared to Mg”. These results suggest that the binding of glutamine synthetase to adenosine-affinity media is related to the participation of Mn. ADP in the y-glutamyl transferase reaction that is catalyzed by glutamine synthetase. M Dowton, IR Kennedy. Purification of glutamine synthetase by adenosine-affinity chromatography. Journal of Chromatography A 1994; 664: 280-283
Aptamer Based Stationary Phase An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an 21 aqueous mobile phase containing 20 mM Mg , adenosine was strongly retained on the column. A gradient of increasing 21 Ni (to 18 mM), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. The adenosine assay, which required no sample preparation, was used on microdialysis samples. Total analysis times were short so samples could be injected every 5 min. Q Deng, CJ Watson, RT Kennedy. Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo. Journal of Chromatography A 2003; 1005:123–130.
We will realize the full power of proteomics only when we can measure and compare the proteomes of many individuals to identify biomarkers of human health and disease and track the blood-based proteome of an individual over time. Because the human proteome contains an estimated 20,000 proteins – plus splicing and post-translational variants – that span a concentration range of ,12 logs, identifying and quantifying valid biomarkers is a great technical challenge. Proteomic measurements demand
extreme sensitivity
specificity
dynamic range
accurate quantification.
We describe a new class of DNA-based aptamers enabled by a versatile chemistry technology that endows nucleotides with protein-like functional groups. These modifications greatly expand the repertoire of targets accessible to aptamers. The resulting technology provides efficient, large-scale selection of exquisite protein-binding reagents selected specifically for use in highly multiplexed proteomics arrays. Aptamers are a class of nucleic acid-based molecules discovered twenty years ago, and have since been employed in diverse applications including
therapeutics
catalysis
proteomics
Aptamers are short single-stranded oligonucleotides, which fold into diverse and intricate molecular structures that bind with high affinity and specificity to
proteins
peptides
small molecules.
Aptamers are selected in vitro from enormously large libraries of randomized sequences by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX). A SELEX library with 40 random sequence positions has 440 (,1024) possible combinations and a typical selection screens 1014–1015 unique molecules. This is on the order of 105 times larger than standard peptide or protein combinatorial molecular libraries.
The interrogation of proteomes (‘‘proteomics’’) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 mL of serum or plasma).
Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (,100 fM–1 mM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray.
Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes.
This is a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. L Gold, D Ayers, J Bertino, Christopher Bock, et al. Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PlosONE 2010; 5 (12): e15004
Biomarker Discovery, Diagnostics, and Therapeutics Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to
the discovery of new biomarkers for diagnostics
new targets for therapeutics
can identify patients most likely to benefit from treatment.
Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of “real-time” health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as
both folded binding entities with defined shapes and
unique nucleic acid sequences recognizable by specific hybridization probes.
Aptamers and Smart Drug delivery Targeting In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained, as their has been very recent progress in controlled drug release based on molecular gating achieved with aptamers. Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas.
Nucleic acids are recognized as attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. An active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles.
Nanoparticle-encapsulated drug delivery aims to deliver the active therapeutic ingredients to the disease site in stable compartments in order to reduce premature release. This ensures that the effects of drug are maximized and the side effects are reduced. An encapsulated nanoparticle system requires a specific targeting mechanism and at the same time the retention of drugs inside the container should be high. The balance between specificity of targeting and the extent of premature leakage determines the success of a given delivery system.
Nanotechnology research approaches in drug delivery include a wide variety of nanomaterials ranging from soft hydrogels to solid polymeric particles. Large surface area, high drug loading efficiency and potential combination with other organic/inorganic materials are the main properties of hollow nanostructures that are attractive for biomedical applications.
Packaging of small-molecule drugs
improves their availability
compatibility
reduces toxicity
Controlling the drug release profile is the main challenge in drug delivery development when the drug is to be successfully targeted to a specific site. Stimuli-responsive materials have been created by using biological, physical and chemical properties of materials for heat-activated, light-activated or pH-activated delivery. Nucleic acids are utilized to construct rationally designed nanostructures at molecular levels for nanotechnology applications. Integration of the properties of nucleic acids can offer many opportunities for drug delivery systems, including stimuli-responsive nanogates for nanocarriers and molecular sensors. Favorable drug release kinetics can be achieved at the target sites by aptamer-based capping systems.
Activity Based Profiling Powerful strategies for the gel-free analysis of proteomes have emerged, including isotope-coded affinity tagging (ICAT) for quantitative proteomics and multidimensional protein identification technology (MudPIT) for comprehensive proteomics, both of which utilize liquid chromatography (LC) and MS for protein separation and detection, respectively. Nonetheless, these methods, like 2DE-MS, still focus on measuring changes in protein abundance and, therefore, provide only an indirect estimate of dynamics in protein function. Indeed, several important forms of post-translational regulation, including protein–protein and protein–small-molecule interactions, may elude detection by abundance-based proteomic methods. To facilitate the analysis of protein function, several proteomic methods have been introduced to characterize the activity of proteins on a global scale. These include large-scale yeast two-hybrid screens and epitope tagging immunoprecipitation experiments, which aim to construct comprehensive maps of protein–protein interactions, and protein microarrays, which aim to provide an assay platform for the rapid assessment of protein activities. A chemical proteomic strategy referred to as activity-based protein profiling (ABPP) has emerged that utilizes active site-directed probes to profile the functional state of enzyme families directly in complex proteomes.
Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.
Cells with fundamental metabolic alterations commonly arise during tumorigenesis, and it is these types of changes that help to establish a biochemical foundation for disease progression and malignancy. A seminal example of this was discovered in the 1920s when Otto Warburg found that cancer cells consume higher levels of glucose and secrete most of the glucose carbon as lactate rather than oxidizing it completely. Since then, studies by multiple groups have uncovered a diverse array of metabolic changes in cancer, including alterations in
glycolytic pathways
the citric acid cycle
glutaminolysis
lipogenesis
lipolysis
proteolysis
These in turn modulate the levels of cellular building blocks
lipids, nucleic acids and amino acids,
cellular energetics,
oncogenic signaling molecules
the extracellular environment to confer protumorigenic and malignant properties.
Despite these advances, our current understanding of cancer metabolism is far from complete and would probably benefit from experimental strategies that are capable of profiling enzymatic pathways on a global scale. To this end, conventional genomic and proteomic methods, which comparatively quantify the expression levels of transcripts and proteins, respectively, have yielded many useful insights. These platforms are, however, limited in their capacity to identify changes in protein activity that are caused by posttranslational mechanisms.
Annotating biochemical pathways in cancer is further complicated by the potential for enzymes to carry out distinct metabolic activities in tumor cells that might not be mirrored in normal physiology. In addition, a substantial proportion of the human proteome remains functionally uncharacterized, and it is likely that at least some of these poorly understood proteins also have roles in tumorigenesis. These challenges require new proteomic technologies that can accelerate the assignment of protein function in complex biological systems, such as cancer cells and tumors.
Metabolomics has emerged as a powerful approach for investigating enzyme function in living systems. Metabolomic experiments in the context of enzyme studies typically start with
the extraction of metabolites from control and enzyme-disrupted biological systems,
followed by metabolite detection and comparative data analysis.
For example, lipophilic metabolites can be enriched from cells or tissues by organic extraction. Mass spectrometry (MS) has become a primary analytical method for surveying metabolites in complex biological samples, with upfront separation accomplished by liquid chromatography (LC–MS) or gas chromatography (GC–MS). MS experiments can be carried out using
targeted or untargeted approaches,
depending on whether the objective is
to profile and quantitate known metabolites or
to broadly scan for metabolites across a large mass range, respectively.
As metabolomic experiments generate a large amount of data, powerful software tools are needed for identification and quantitation of ions in LC–MS data sets (see the figure; the mass to charge ratio (m/z) is indicated). One such program is XCMS95, which
aligns,
quantifies and
statistically ranks ions that are altered between two sets of metabolomic data.
This program can be used to rapidly identify metabolomic signatures of various disease states or to assess metabolic networks that are regulated by an enzyme using pharmacological or genetic tools that modulate enzyme function. Additional databases assist in metabolite structural characterization, such as HMDB96,97, METLIN98,99 and LIPID MAPS100. In this Review, we discuss one such proteomic platform, termed activity based protein profiling (ABPP), and its implementation in the discovery and functional characterization of deregulated enzymatic pathways in cancer. We discuss the evidence that, when coupled with other large scale profiling methods, such as metabolomics and proteomics, ABPP can provide a compelling, systems level understanding of biochemical networks that are important for the development and progression of cancer.
Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signaling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.
Representative activity-based probes and their application to cancer research
enzyme class applications in cancer
Serine hydrolases increased KIAA1363 and MAGL
aggressive human cancer lines
uPA and tPA serine protease aggressive cancers
RBBP9 activity in pancreatic carcinoma
Metalloproteinases neprilysin activity in melanoma cell lines
Cysteine proteases cathepsin cysteine protease in pancreatic islet tumours
Kinases Inhibitor selectivity profiling of kinase inhibitors
Caspases visualization of apoptosis in colon tumour-bearing mice treated with Apomab
Deubiquitylases Identified increased carboxy-terminal hydrolase UCHL3 and UCH37 activity in HPV cervical carcinomas
Cytochrome P450s Identified the aromatase inhibitor anastrazole as an inducer of CYP1A2
Serine hydrolases KIaa1363 and MaGL regulate lipid metabolic pathways that support cancer pathogenesis. Activity-based protein profiling (ABPP) identified
KIAA1363 and
monoacylglycerol (MAG) lipase (MAGL)
as being increased in aggressive human cancer cells from multiple tumour types. Pharmacological and/or RNA interference ablation of KIAA1363 and MAGL coupled with metabolomic analysis revealed specific roles for KIAA1363 and MAGL in cancer metabolism. Disruption of KIAA1363 by the small-molecule inhibitor AS115 lowered monoalkylglycerol ether (MAGE), alkyl lysophosphatidic acid (alkyl LPA) and alkyl lysophosphatidyl choline (alkyl LPC) levels in cancer cells. Disruption of MAGL by the small-molecule inhibitor JZL184 raised MAG levels and reduced free fatty acid, lysophosphatidic acid (LPA) and prostaglandin E2 (PGE2) levels in cancer cells. Disruption of KIAA1363 and MAGL leads to impairments in cancer cell aggressiveness and tumour growth, PAF, platelet-activating factor.
At a glance
• Activity-based protein profiling (ABPP) facilitates the discovery of deregulated enzymes in cancer. • Competitive ABPP yields selective inhibitors for functional characterization of cancer enzymes. • ABPP can be integrated with metabolomics to map deregulated enzymatic pathways in cancer. • ABPP can be integrated with other proteomic methods to map proteolytic pathways in cancer. • ABPP probes can be used to image tumour development in living animals.
Activity-based protein profiling (ABPP), the use of active site-directed chemical probes to monitor enzyme function in complex biological systems, is emerging as a powerful post-genomic technology. ABPP probes have been developed for several enzyme classes and have been used to inventory enzyme activities en masse for a range of (patho)physiological processes.
ABPP uses active site–directed, small molecule–based covalent probes to report on the functional state of enzyme activities directly in native biological systems. ABPP probes are designed or selected to target a subset of the proteome based on shared principles of binding and/or reactivity and have been successfully developed for many enzyme classes, including
serine
cysteine,
aspartyl
metallo hydrolases
kinases
glycosidases
histone deacetylases and
oxidoreductases.
These probes have been shown to selectively label active enzymes but not their inactive precursor (zymogen) or inhibitor-bound forms, thus allowing researchers to capture functional information that is beyond the scope of standard proteomic methods. By presenting specific examples, we show here that ABPP provides researchers with a distinctive set of chemical tools to embark on the assignment of functions to many of the uncharacterized enzymes that populate eukaryotic and prokaryotic proteomes.
Vinyl-methylester UL from HSV-1 Deubiquitinating enzyme (DUB)
Aryl 2-deoxy-2-fluoro glycoside Cfx from C. fimi Glycosidase (β-1-4-glycanase) Fluorophosphonate SAE Serine hydrolase
Examples of enzymes assigned to specific mechanistic classes by ABPP
ABPP can also be implemented as a direct assay for inhibitor discovery, allowing researchers to develop potent and selective pharmacological probes for uncharacterized enzymes.
Examples of enzymes assigned to specific mechanistic classes by ABPP.
Probe Leu-Asp-αCA probe selectively labeled Upβ
Substrate the endogenous Upβ substrate, N-carbamoyl-β-alanine
Substrate mimicry of an ABPP probe.
Multidimensional profiling strategy for the annotation of the cancer-related enzyme KIAA1363. ABPP using fluorophosphonate probes identified KIAA1363 as a highly elevated enzyme activity in aggressive cancer cells. Competitive ABPP was then used to develop a selective KIAA1363 inhibitor (AS115). Metabolomic analysis of cancer cells treated with AS115 determined a role for this enzyme in the regulation of MAGE lipids in cancer cells. Biochemical studies confirmed that KIAA1363 acts as 2-acetyl MAGE hydrolase in a metabolic network that bridges the platelet activating factor and lysophosphatidic acid classes of signaling lipids. Assignment of enzyme mechanism by ABPP
There are multiple levels of annotation for enzymes. The most basic level is assignment to a specific mechanistic class based on the general chemical reaction catalyzed by the enzyme (for example, hydrolase, kinase, oxidoreductase and others). Additional annotation involves determining the endogenous substrates and products for the enzyme. Finally, complete annotation requires an understanding of how the specific chemical transformation(s) catalyzed by an enzyme integrate into larger metabolic and signaling pathways to influence cell physiology and behavior.
Many of the predicted enzymes uncovered by genome sequencing projects can be assigned to a mechanistic class or ascribed a putative biochemical function based on sequence homology to well-characterized enzymes. But some enzymes have insufficient sequence relatedness for class assignment or have a function different from that predicted by sequence comparisons. ABPP has facilitated class annotation for several such uncharacterized enzymes.
A principal goal of modern biomedical research is to discover, assemble, and experimentally manipulate molecular pathways in cells and organisms to reveal new disease mechanisms.
Toward this end, complete genome sequences for numerous bacteria and higher organisms, including humans, have laid the fundamental groundwork for understanding the molecular basis of life in its many forms. However, the information content of DNA sequences is limited and, on its own, cannot describe most physiological and pathological processes.
Unlike oligonucleotides, proteins are a very diverse group of biomolecules that display a wide range of chemical and biophysical features, including
membrane-binding,
hetero/homo-oligomerization, and
posttranslational modification.
The biochemical complexity intrinsic to protein science intimates that several complementary analytical strategies will be needed to achieve the ultimate goal of proteomics – a comprehensive characterization of the expression, modification state, interaction map, and activity of all proteins in cells and tissues.
A powerful LC-MS strategy for proteomics involves the use of isotope-coded affinity tags (ICAT). This approach enables the comparison of protein expression in proteomes by treating samples with isotopically distinct forms of a chemical labeling reagent. ICAT methods provide superior resolving power compared to gel-based methods and improve access to membrane-associated proteins. More recently, isotope-free MS methods for quantitative proteomics have emerged.
Reverse protein microarrays have also been described in which proteomes themselves are arrayed and the antibodies used for detection in a format analogous to Western blotting. In addition to increasing the throughput of proteomic experiments by integrating the protein separation and detection steps, microarrays consume much less material than conventional proteomic methods. Still, the general application of microarrays for proteomics is currently limited by the availability of high-quality capture reagents (e.g., antibodies, aptamers, etc).
These approaches, by measuring protein abundance provide, like genomics, only an indirect assessment of protein activity and may fail to detect important posttranslational events that regulate protein function, such as protein–protein or protein–small-molecule interactions. To address these limitations, complementary strategies for the functional analysis of proteins have been introduced. Prominent among these functional proteomic efforts is the use of chemistry for the design of active site-directed probes that measure enzyme activity in samples of high biological complexity.
Many post-translational modes of enzyme regulation share a common mechanistic foundation – they perturb the active site such that catalytic power and/or substrate recognition is impaired. Accordingly, it was hypothesized that chemical probes capable of reporting on the integrity of enzyme active sites directly in cells and tissues might serve as effective functional proteomic tools. These activity based protein profiling (ABPP) probes consist of at least two general elements:
a reactive group for binding and covalently modifying the active sites of many members of a given enzyme class or classes
a reporter tag for the detection, enrichment, and identification of probe-labeled proteins
ABPP probes have been successfully developed for more than a dozen enzyme classes, including
all major classes of proteases
kinases
phosphatases
glycosidases
GSTs
oxidoreductases.
Post-translational regulation of enzyme activity. Many enzymes are produced as inactive precursors, or zymogens, which require proteolytic processing for activation. Enzyme activity can be further regulated by interactions with endogenous protein inhibitors. The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.
Advanced analytical platforms utilize a range of separation and detection strategies, including LC-MS, CELIF, and antibody microarrays, to achieve an unprecedented breadth and depth of proteome coverage in ABPP investigations. The complementary strengths and weaknesses of each of these methods suggest that the selection of an appropriate analytical platform should be guided by the specific experimental question being addressed. SA Sieber and BF Cravatt. Analytical platforms for activity-based protein profiling – exploiting the versatility of chemistry for functional proteomics. Chem. Commun. 2006, 2311–2319. http://www.rsc.org/chemcomm
Diagnostic Therapeutics in Activity Based Probes Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features;
a ‘warhead’ (binds irreversibly but selectively to the active site),
a ‘tag’ (allowing enzyme ‘handling’, with a combination of fluorescent, affinity and/or radio labels),
a linker region between warhead and tag.
From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. Heal WP, Wickramasinghe SR, Tate EW. Activity based chemical proteomics: profiling proteases as drug targets. Curr Drug Discov Technol 2008; 5(3):200-12. PMID: 18690889
The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them. The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states.
In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity. Berger AB, Vitorino PM, Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am J Pharmacogenomics. 2004;4(6):371-81.
Recent advances in global genomic and proteomic methods have led to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.
Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes.
As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.
Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials.
Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex.
Critical clues to the identity of γ-secretase included: (1) Genes encoding the multi-pass membrane proteins presenilin-1 and presenilin-2 are, like APP, associated with familial, early-onset Alzheimer’s disease. The disease-causing missense mutations were found to alter how γ-secretase cuts APP, leading to increased proportions of longer, more aggregation-prone forms of Aβ. (2) Knockout of presenilin genes eliminates γ-secretase cleavage of APP. (3) Peptidomimetics that inhibit γ-secretase contain moieties typically found in aspartyl protease inhibitors. These findings led to the identification of two conserved transmembrane aspartates in the multi-pass presenilins that are critical for γ-secretase cleavage of APP, evidence that presenilins are aspartyl proteases. Presenilin is endoproteolytically cleaved into two polypeptides, an N-terminal fragment (NTF) and a C-terminal fragment (CTF), the formation of which is
regulated
metabolically stable
part of a high-molecular weight complex
suggesting that the NTF-CTF heterodimer is the biologically active form. NTF and CTF each contribute one of the essential and conserved aspartates, and transition-state analogue inhibitors of γ-secretase, compounds designed to interact with the active site of the protease, bind directly to presenilin NTF and CTF. Presenilins are also required for Notch signaling (Levitan and Greenwald, 1995), a pathway essential for cell differentiation during development and beyond.
The highly conserved role of γ-secretase in Notch signalling and its importance in development led to genetic screens in Caenorhabditis elegans that identified three other integral membrane proteins besides presenilin that modify Notch signaling. Designed inhibitors have proven to be useful tools in understanding the mechanism of γ-secretase and substrate recognition – affinity labelling with transition-state analogue inhibitors showed binding at the interface between the presenilin NTF and CTF subunits, consistent with the active site residing at this interface, with each presenilin subunit contributing one of the essential aspartates. The concept of presenilin as the catalytic component for γ-secretase was considerably strengthened when
signal peptide peptidase (SPP) was found to be a similar intramembrane aspartyl protease
SPP is exploited by the hepatitis C virus for the maturation of its core protein, suggesting that this protease may be a suitable target for antiviral therapy
SPP was identified by affinity labeling with a peptidomimetic inhibitor, and the protein sequence displayed similarities with presenilin.
SPP contains two conserved aspartates, each predicted to lie in the middle of a transmembrane domain, and the aspartate-containing sequences resemble those found in presenilins.
SPP appears to be less complicated than γ-secretase.
Expression of human SPP in yeast reconstituted the protease activity, suggesting that the protein has activity on its own and does not require other mammalian protein cofactors.
Aspartyl I-CLiPs are found in all forms of life and play essential roles in biology and disease. How these enzymes carry out hydrolysis in the membrane is a fascinating question that is not entirely resolved, but evidence suggests an initial substrate docking site and a lateral gate into a pore where water and the active site aspartates reside. Designed inhibitors have been critical in elucidating these mechanisms, but inhibitors targeting γ-secretase for the treatment of Alzheimer’s disease must avoid interfering with Notch signaling.
MS Wolfe. Structure, Mechanism and Inhibition of γ-Secretase and Presenilin-Like Proteases. Biol Chem. 2010 August; 391(8): 839–847. doi: 10.1515/BC.2010.086. PMCID: PMC2997569. NIHMSID: NIHMS254540 Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought Genomics Monday, June 4, 2012 A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought.
An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms. The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. “We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, principal developer of the new DNA bases.
Adding to the DNA Alphabet Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA. But these bases’ lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.
An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase.” Yet in test after test, the NaM-5SICS pair was efficiently replicable.
Edge to Edge The NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. “The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair.” NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair.”
An Arbitrary Choice? The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems.” Source: The Scripps Research Institute
DNA damage response (DDR) network
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR.
We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Mitochondria have various essential functions in metabolism and in determining cell fate during apoptosis. In addition, mitochondria are also important nodes in a number of signaling pathways. For example, mitochondria can modulate signals transmitted by second messengers such as calcium. Because mitochondria are also major sources of reactive oxygen species (ROS), they can contribute to redox signaling—for example, by the production of ROS such as hydrogen peroxide that can reversibly modify cysteine residues and thus the activity of target proteins. Mitochondrial ROS production is thought to play a role in hypoxia signaling by stabilizing the oxygen-sensitive transcription factor hypoxia-inducible factor–1α. New evidence has extended the mechanism of mitochondrial redox signaling in cellular responses to hypoxia in interesting and unexpected ways. Hypoxia altered the microtubule-dependent transport of mitochondria so that the organelles accumulated in the perinuclear region, where they increased the intranuclear concentration of ROS. The increased ROS in turn enhanced the expression of hypoxia-sensitive genes such as VEGF (vascular endothelial growth factor) not by reversibly oxidizing a protein, but by oxidizing DNA sequences in the hypoxia response element of the VEGF promoter. This paper and other recent work suggest a new twist on mitochondrial signaling: that the redistribution of mitochondria within the cell can be a component of regulatory pathways.
A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ.
2012pharmaceutical
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