Posts Tagged ‘methionine’

The relationship of S amino acids to marasmic and kwashiorkor PEM

Larry H. Bernstein, MD, FCAP, Curator


Sulfur is perhaps the most abundant element in the human body.  It is found in most proteins in the sulfur-containing amino acids, along with phosphorus and nitrogen.  These three elements form a triad of important elements needed as building blocks or structural components of all animal tissues.  The sulfur gives animal tissues their strength, and their resiliency.

Sulfur and nutritional balancing.  Suflur is not supplemented in pill form because it is plentiful in foods.  However, nutiritonal balancing emphasizes it and we find if one wishes to be healthy then one must eat meat, eggs and cooked vegetables and then your sulfur needs will be taken care of.

In Metabolic Reactions in the Nervous System, 1970; pp 225-287

Medical Research Council, Neuropsychiatric Research Unit, Carshalton, Surrey, England

Several sulfur amino acids and sulfur compounds are found in mammalian tissues. While some find their origin in the diet, other sulfur amino acids are formed in vivo from methionine in the tissues. Thus it is known that methionine is converted into homocysteine, cystathionine, cysteine, hypotaurine, and taurine. These metabolites are formed in the course of transferring a methyl group to other compounds. The mechanism of demethylation and the subsequent metabolism of the demethylated product, homocysteine, is now well established. The enzyme systems in most cases were first studied in liver preparations. The demonstration that 35S-methionine is converted into 35S-cysteine and 35S-taurine by rat brain in vitro and in vivo gave evidence that the sulfur amino acids are metabolized also in the mammalian brain. Several subsequent studies have shown similarities between the metabolism of methionine in liver and in brain, but they have also revealed some characteristic differences in the metabolism of sulfur amino acids in the brain: (1) the cystathionine and taurine concentrations are much higher in the brain than in the liver, (2) the enzyme cysteine sulfinic acid decarboxylase is predominantly a particulate deaminated to form isethionic acid by rat brain and heart and not by liver. An interesting feature of sulfur amino acid metabolism is that many of the enzyme systems involved in the conversion of methionine into its several metabolites require pyridoxal phosphate (vitamin B6) as a cofactor. Whereas in liver this cofactor is tightly bound to some of these enzymes, the corresponding enzymes in the brain are bound loosely to this cofactor, and their activity in the brain can be demonstrated in vitro only by adding the cofactor.

Sulfur-Containing Amino Acids

The sulfur-containing amino acids (cysteine and methionine) are generally considered to be nonpolar and hydrophobic. In fact, methionine is one of the most hydrophobic amino acids and is almost always found on the interior of proteins. Cysteine on the other hand does ionize to yield the thiolate anion. Even so, it is uncommon to find cysteine on the surface of a protein. There are several reasons. First, sulfur has a low propensity to hydrogen bond, unlike oxygen. A consequence of this fact is that H2S is a gas under conditions that H2O is a liquid. Second, the thiol group of cysteine can react with other thiol groups in an oxidation reaction that yields a disulfide bond. Perhaps as a consequence, cysteine residues are most frequently buried inside proteins.

When in its natural L-form, methionine is a proteinogen amino acid. It is classed as an essential amino acid and cannot be synthesized by the body itself. This means that a sufficient supply of methionine in the diet or as a dietary supplement is of particular importance.

Sulphur compounds occur in all living creatures and have a multitude of functions. Besides cysteine, methionine is the only sulphur-containing amino acid. Furthermore methionine plays an important role in the synthesis of other proteins, such as carnitine or melatonine. Methionine has a fat-dissolving effect and reduces the depositing of fat in the liver.

Methionine is an important cartilage-forming substance

The cartilage in the joints requires sulphur for its production. If there is not enough sulphur available in the body, this can have negative effects for the healthy individual over the long term. People who suffer from arthritis can experience negative effects such as a prolonged healing process for the damaged tissue, if there is a sulphur deficiency at the beginning of the illness.

Studies have shown that the cartilage from healthy people contains approximately three times more suphur than in arthritis patients.To make things more complicated, various arthritis medications connect sulphur, which are the salts in the sulphuric acid. The demand for sulphur is increasing to more than average levels.

J Nutr. 2006 Jun;136(6 Suppl):1636S-1640S.

The sulfur-containing amino acids: an overview.

Brosnan JT1Brosnan ME.

Author information


Methionine, cysteine, homocysteine, and taurine are the 4 common sulfur-containing amino acids, but only the first 2 are incorporated into proteins. Sulfur belongs to the same group in the periodic table as oxygen but is much less electronegative. This difference accounts for some of the distinctive properties of the sulfur-containing amino acids. Methionine is the initiating amino acid in the synthesis of virtually all eukaryotic proteins; N-formylmethionine serves the same function in prokaryotes. Within proteins, many of the methionine residues are buried in the hydrophobic core, but some, which are exposed, are susceptible to oxidative damage. Cysteine, by virtue of its ability to form disulfide bonds, plays a crucial role in protein structure and in protein-folding pathways. Methionine metabolism begins with its activation to S-adenosylmethionine. This is a cofactor of extraordinary versatility, playing roles in methyl group transfer, 5′-deoxyadenosyl group transfer, polyamine synthesis, ethylene synthesis in plants, and many others. In animals, the great bulk of S-adenosylmethionine is used in methylation reactions. S-Adenosylhomocysteine, which is a product of these methyltransferases, gives rise to homocysteine. Homocysteine may be remethylated to methionine or converted to cysteine by the transsulfuration pathway. Methionine may also be metabolized by a transamination pathway. This pathway, which is significant only at high methionine concentrations, produces a number of toxic endproducts. Cysteine may be converted to such important products as glutathione and taurine. Taurine is present in many tissues at higher concentrations than any of the other amino acids. It is an essential nutrient for cats.


Sulfur is an essential nutrient (micro-mineral). It is a nonmetallic element that is essential for life. In most animals it represents about 0.25% of the body weight. However, sulfur is normally present as part of larger compounds, and the requirement for pure sulfur has not been determined for most species. In recent years, sulfur toxicity has become more common because of its high concentration in many byproduct feeds. The use of these feeds in ruminant diets is increasing, which in turn may increase the trace mineral requirement. The purpose of this article is to review our current understanding of sulfur nutrition and to look at how sulfur level in the diet may influence the copper requirement.

Essential Functions

Compounds containing sulfur play a variety of essential functions in the body. They act as structural entities (collagen), as catalysts (enzymes), as oxygen carriers (hemoglobin), as hormones (insulin), and as vitamins (thiamine and biotin). Sulfur is present in four amino acids: methionine, cystine, cysteine and taurine. The secondary structure of many proteins is determined by the cross linkage or folding due to covalent disulfide bonds between amino acids.

Sulfur is the element that gives many key compounds their unique functional properties. For example, acetate is linked to coenzyme A by a thioester linkage to form acetyl coenzyme A. This compound is required for the formation of key metabolic intermediates such as citrate, acetoacetate and malonate. The sulfur in thiamine allows it to serve as a molecule which transfers carbonyl groups. Thiamine plays a key role in the formation of pentose sugars which are required for ribonucleic acids synthesis and photosynthesis. Biotin, another sulfur-containing B vitamin, acts a carrier for carbon dioxide in carboxylation reactions.

Inorganic vs. Organic Sulfur

Despite the fact that sulfur is a key mineral in many compounds essential for life, dietary inorganic sulfur is not necessary for the health of most animals. Pigs and poultry can do quite well with only organic sulfur (sulfur amino acids, thiamine, biotin, etc.) sources in their diets. The total absence of inorganic sulfur from the diet may increase the sulfur-amino acid requirement, which suggest that sulfur from the amino acids is used to synthesize other organic compounds containing sulfur.

In contrast, ruminants may respond to inorganic sulfur supplementation, especially if the diet is high in nonprotein nitrogen. Block et al., (1951) showed that ruminal microorganism are capable of synthesizing all organic sulfur containing compounds essential for life from inorganic sulfur. When urea or other nonprotein nitrogen sources are fed, the diet may become deficient in sulfur. Goodrich et. al., (1978) reported that the nitrogen to sulfur ratio in rumen microbial protein averages 14.5:1. The common recommendation for the nitrogen:sulfur ratio is 10:1 in diets containing high levels of urea.

Sulfur Source

The source of sulfur can influence its bioavailability. Goodrich et. al,. (1978) gave the following rankings from the most available to the least available: L-methionine> calcium sulfate >ammonium sulfate> sodium sulfate>molasses sulfur>sodium sulfide>lignin sulfonate>elemental sulfur. The recommend concentration of sulfur in beef cattle diets is 0.15% (NRC, 1996). However, this assumes the sulfur source is highly bioavailable.

The type of forage in the diet may also influence sulfur requirement. For example, Archer and Wheeler, (1978) showed that increasing the sulfur concentration from 0.08% to 0.12% in cattle grazing sorghum sudangrass increased weight gains by 12%. Sulfur requirements may be higher for cattle grazing sorghum sudangrass because sulfur is required in the detoxification of the cyanogenic glucosides found in most sorghum forages. Sulfur bioavailability varies with the type of forage; fescue has a lower sulfur availability than other grasses. Cattle consuming fescue hay will often respond with improved intake and fiber digestion following sulfur supplementation. Forages usually contain between 0.1-0.3% sulfur, except for corn silage which is often lower.

Zinn et al., (1997) reported that when ammonium sulfate was used to produce diets containing 0.15, 0.20 and 0.25% sulfur (DM basis), feedlot performance was reduced with the higher sulfur concentration. The diets were based on steam-flaked corn and fed to heifers weighing 845 pounds initially. Increasing dietary sulfur above 0.20%, caused a strong trend (P <.10) for decreased gains, feed intake and gain per unit of feed intake. The excess sulfur also caused a reduction (P <.05) in the ribeye area which is an important factor determining the yield grade of the carcass. Sulfur intake from the drinking water was not reported.


Another problem that can occur when high dietary sulfur leads to the production of excess sulfides in the rumen is polioencephalomalacia, or PEM (Gould et al., 1991; Lowe et al., 1996). The most defining sign of PEM is the necrosis of the cerebrocortical region of the brain. Animals with PEM will often press their head against a wall or post. In some instances they become “star gazers,” where they stand with their head back over their shoulders looking up at the sky. If not treated with thiamine, most animals with PEM will die within 48 hours.

A thiamine deficiency has been considered the most common cause of PEM in ruminants. However, recent research suggests that sulfur may play a key role in many instances of PEM. PEM has often been seen in animals that have had access to plants containing high amounts of thiaminase such as bracken fern (Merck, 1991). Thiamine is a B vitamin that plays a key role in the tri-carboxcylic acid cycle and pentose shunt. When thiamine is deficient, key tissues that require large amounts of thiamine, such as the brain and heart, are the first to show lesions.

The exact interaction between dietary sulfur, thiaminase production, and PEM is not understood. Kung et al. (1998) postulated that sulfates in the feed or water are converted to hydrogen sulfide in the rumen. When the hydrogen sulfide is eructated with the other rumen gases, it is inhaled and can damage lung and brain tissues. Several researchers (Oliveria et al., 1996; Brent and Bartley, 1994 and Olkowski, et al., 1992) have suggested that high sulfide levels could cause the brain lesions associated with PEM.

Kung et al., (1998) summarized six different reports in the literature where high sulfur intakes were associated with PEM. In these studies thiamine status was within normal ranges and giving thiamine did not prevent the signs in all cases. In these cases sulfur intakes from feed and water would have ranged from 0.40 to over 0.80% of the diet dry matter.

Drinking Water

Sulfates in the water can be a major source of sulfur intake. For example, in one of the cases cited by Kung et al., (1998), sulfates in the drinking water ranged from 2,200 to 2,800 ppm. When the water sulfur intake was expressed as a percent of the dry matter consumed, it averaged 0.67%. Digesti and Weeth (1976) proposed that the maximum safe concentration of sulfates in drinking water for cattle was 2,500 ppm. Water sulfate concentrations as high as 5,000 ppm have been reported (Veenhuizen et al., 1992).

Accurately estimating water intake in these situations can also be a challenge. Water meters can be used for confined livestock to estimate the average intake, but with grazing animals drinking from ponds or streams, one can only estimate the intake. Usually water consumption will be 3-5 times the dry matter intake. Dry matter intake for grazing beef cattle and sheep will normally be between 1.5 and 2.5% of their body weight. Lactating dairy cows may consume over 3.5% of their body weight when grazing high quality forage. Although this is not a precise means of measuring water sulfur intake, it does allow one to estimate the relative contributions of the feed and water.

Excess Sulfur

Excess sulfur can also impair animal performance by reducing the availability of other minerals. For example, hydrogen sulfide in the rumen binds with molybdenum to form thiomolybdates. Thiomolybdates bind with copper in the rumen to form an insoluble complex. Some thiomolybdates are absorbed and impair the metabolism of copper in the body. For example, Gooneratne et al. (1989) reported that certain thiomolybdates cause copper to be bound to blood albumins which renders the copper unavailable for biochemical reactions in the body. Price et al., (1987) showed that tri- and tetrathiomolybdates were the sulfur-molybdenum complexes responsible for reducing copper absorption, while the di- and trithiomolybdates had the greatest effect on copper metabolism in the body.

Sulfur also reduces copper absorption by the formation of insoluble copper sulfide in the rumen, independent of the formation of thiomolybdates. Rumen protozoa degrade sulfur amino acids to sulfide which binds to copper to form an insoluble complex. Smart et al., (1986) reported that decreasing the sulfate concentration of drinking water from 500 to 42 ppm, improved the copper status of cattle. These same researchers reported that the 10 ppm copper recommended by the Beef NRC (1996) was not adequate when cattle drank high-sulfur water, which resulted in a total dietary sulfur intake of 0.35%.

Copper Supplement

The optimum level of copper supplementation required to combat high sulfur intakes has not been determined. The maximum tolerable level of copper for cattle has been estimated at 100 ppm (NRC 1980). Although this level is being fed in diets that are high in sulfur, certain breeds of dairy cattle such as the Jersey and Guernsey are susceptible to copper toxicity at concentrations below 100 ppm.

In these situations, the source of copper is also important. Although copper sulfate is a common copper source, it would not be recommended if the diet is already high in sulfur. Copper oxide would not contribute to the sulfur problem, but because of its poor availability is not recommended. Copper carbonate is probably the best copper source for this situation. It has a bioavailability similar to copper sulfate, with out increasing sulfur intake.

The Importance of Macro Minerals: Sulfur

K.E. Lanka, Ph.D., P.A.S.

Sulfur (S) is one of seven generally recognized macro minerals needed in the diets of dairy cattle and other animals. Sulfur is a mineral that is found in the amino acids methionine, cysteine (cystine), homocysteine and in taurine. It is also in the B-vitamins, thiamin and biotin. It is an important component of healthy cartilage. As a part of the specified amino acids, it is key to the structure of proteins. Heating protein s u p p l e m e n t s can rearrange the structures of proteins, due to the sulfurcontaining amino acids, which can determine whether these nutrients are soluble and rumen degradable or if they will resist rumen degradation in cattle. Heating also affects the essential amino acid, lysine, when carbohydrates are present in a supplement. An example of this change by heating can be observed when an egg is boiled.

Animals have a need for essential sulfur-containing nutrients, such as methionine and cysteine. However, the microbes in the rumens of cattle and other ruminants can use mineral sources of sulfur to produce some of these important nutrients for dairy and beef cattle. Thus, it is important to feed sulfur at recommended dietary levels to meet the needs of the microbes, as well as the animals. In dairy cattle, it is needed in the diet at the level of 0.20%. For beef cattle, the recommended concentration is a minimum of 0.15% of dietary dry matter (DM). Since about 0.15% of the body weight is sulfur, commercial concentrations in typical beef cattle rations range from 0.18 to 0.24%. Sulfur is essential when a nonprotein nitrogen source, such as urea, is fed. The total Nitrogen:Sulfur (N:S) ratio in a diet should range from 10:1 to 12:1, and the rumen soluble N:S ratio should be 4.0:1 to 5.5:1. Common sources of sulfur for livestock include:

• potassium sulfate

• magnesium sulfate

• sodium sulfate

• ammonium sulfate

• calcium sulfate

• corn gluten feed, distillers grains and other corn coproducts.

Sulfate forms of macro and trace minerals are among the most digestible and easily absorbed forms in the digestive tract. Elemental sulfur in water and feed is not a readily available source for animals. A deficiency of sulfur in the diets of animals can have detrimental effects on their performance. Marginal deficiency symptoms include:

• reduced microbial synthesis

• reduced fiber digestion due to slow microbial growth in ruminants

• slow growth

• reduced milk production

• reduced feed efficiency

• reduced intakes

Severe deficiencies can cause the following symptoms:

• unwillingness to eat

• weight loss

• dullness and slow movement

• excessive salivation

• death

For ruminants, the maximum tolerable level of sulfur in diets is .40% of their dry matter intakes. Excess sulfur will interfere with the digestion and absorption of other minerals, particularly the trace minerals, copper and selenium. Even though these minerals may be adequate in the diet, secondary deficiency symptoms can be observed, simply because the trace minerals were made unavailable, due to too much sulfur in the feed. Other toxicity symptoms or problems that can occur from high levels of sulfur include:

• reduced intakes

• overloading the urinary system, leading to kidney failure

• interference with nerve impulses, including blindness, coma, muscle twitches and intestinal inflammation or bleeding

• The breath of cattle may smell like “rotten eggs,” due to the toxic form of sulfur, hydrogen sulfide.

• polioencephalomalacia (PEM)

With recent increased usage of distillers grains in dairy and feedlot diets, the association between sulfur and PEM has been noted and documented. One of the causes of PEM in ruminants is the interference by sulfur with the B-vitamin, thiamin. Supplementation with thiamine may help to alleviate PEM. This is one reason why thiamin is included in Agri-King base mineral products. The symptoms of PEM include: • excessive salivation • nervousness and twitching (hypersensitivity) • poor muscle coordination and dullness • tilting the head to the side and walking in circles (star gazing) • head pressing • blindness • death Sulfur is an important element in the pH balance of the blood of animals. Sulfates are some of the anionic salts that are used to adjust PCI (Pre-Fresh Cow Index) that affects calcium utilization in cows prior to calving. This can be a key factor in the prevention of milk fevers and retained placentae in fresh cows. In summary, sulfur is needed in dairy rations at a minimum level of .20% of dry matter for a TMR. It is a key macro mineral in maintaining life and production in animals, and it is an essential component of some amino acids, vitamins and other nutrients needed by all animals. Like all required nutrients, too much S can become toxic. The maximum level of sulfur is .40% of the dry matter intake for cattle. Agri-King, Inc is a leader and innovator in animal nutrition. AgriKing rations are balanced to meet the nutrient needs of the animals that are fed by clients. For more information about Agri-King nutrition, contact a representative near you or visit the website at

Protein Malnutrition

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

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.

Source References:

Voluntary and Involuntary S- Insufficiency

Writer and Curator: Larry H Bernstein, MD, FCAP

Transthyretin and the Stressful Condition


This article is written among a series of articles concerned with stress, obesity, diet and exercise, as well as altitude and deep water diving for extended periods, and their effects.  There is a reason that I focus on transthyretin (TTR), although much can be said about micronutients and vitamins, and fat soluble vitamins in particular, and iron intake during pregnancy.    While the importance of vitamins and iron are well accepted, the metabolic basis for their activities is not fully understood.  In the case of a single amino acid, methionine, it is hugely important because of the role it plays in sulfur metabolism, the sulfhydryl group being essential for coenzyme A, cytochrome c, and for disulfide bonds.  The distribution of sulfur, like the distribution of iodine, is not uniform across geographic regions.  In addition, the content of sulfur found in plant sources is not comparable to that in animal protein.  There have been previous articles at this site on TTR, amyloid and sepsis.

Transthyretin and Lean Body Mass in Stable and Stressed State

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Stabilizers that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity

Thyroid Function and Disorders

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal

Malnutrition in India, high newborn death rate and stunting of children age under five years

Vegan Diet is Sulfur Deficient and Heart Unhealthy

How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Amyloidosis with Cardiomyopathy

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

Automated Inferential Diagnosis of SIRS, sepsis, septic shock

Transthyretin and the Systemic Inflammatory Response 

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 syndrom (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 will affect TTR and will be put in context in this review. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  copyright @ Bentham Publishers Ltd. 2009.

Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H. Bernstein.
Clin Biochem Oct 2008; 41(14-15): 1126-1130

A determination of TTR level is an objective method od measuring protein catabolic loss of severly ill patients and numerous studies show that TTR levels correlate with patient outcomes of non-critically ill patients. We evaluated whether TTR level correlates with the prevalence of PEM in the ICUand evaluated serum TTR level as an indicator of the effectiveness of nutrition support and the prognosis in critically ill patients.

TTR showed excellent concordance with patients classified with PEM or at high malnutrition risk, and followed for 7 days, it is a measure of the metabolic burden. TTR levels did not respond early to nutrition support because of the delayed return to anabolic status. It is particularly helpful in removing interpretation bias, and it is an excellent measure of the systemic inflammatory response concurrent with a preexisting state of chronic inanition.

 The Stressful Condition as a Nutritionally Dependent Adaptive Dichotomy

Yves Ingenbleek and Larry Bernstein
Nutrition 1999;15(4):305-320 PII S0899-9007(99)00009-X

The injured body manifests a cascade of cytokine-induced metabolic events aimed at developing defense mechanisms and tissue repair. Rising concentrations of counterregulatory hormones work in concert with cytokines to generate overall insulin and insulin-like growth factor 1 (IGF-1), postreceptor resistance and energy requirements grounded on lipid dependency. Dalient features are self-sustained hypercortisolemia persisting as long as cytokines are oversecreted and down-regulation of the hypothalamo-pituitary-thyroid axis stabilized at low basal levels. Inhibition of thyroxine 5’deiodinating activity (5’DA) accounts for the depressed T3 values associated with the sparing of both N and energy-consuming processes. Both the liver and damaged territories adapt to stressful signals along up-regulated pathways disconnected from the central and peripheral control systems. Cytokines stimulate 5’DA and suppress the synthesis of TTR, causing the drop of retinol-binding protein (RBP) and the leakage of increased amounts of T4 and retinol in free form. TTR and RBP thus work as prohormonal reservoirs of precursor molecules which need to be converted into bioactive derivatives (T3 and retinoic acids) to reach transcriptional efficiency. The converting steps (5’DA and cellular retinol-binding protein-1) are activated to T4 and retinol, themselves operating as limiting factors to positive feedback loops. …The suicidal behavior of TBG, CBG, and IGFBP-3 allows the occurrence of peak endocrine and mitogenic influences at the site of inflammation. The production rate of TTR by the liver is the main determinant of both the hepatic release and blood transport of holoRBP, which explains why poor nutritional status concomitantly impairs thyroid- and retinoid-dependent acute phase responses, hindering the stressed body to appropriately face the survival crisis.  …
abbreviations: TBG, thyroxine-binding globulain; CBG, cortisol-binding globulin; IGFBP-3, insulin growth factor binding protein-3; TTR, transthyretin; RBP, retionol-binding protein.

Why Should Plasma Transthyretin Become a Routine Screening Tool in Elderly Persons? 

Yves Ingenbleek.
J Nutrition, Health & Aging 2009.

The homotetrameric TTR molecule (55 kDa as MM) was first identified in cerebrospinal fluid (CSF).  The initial name of prealbumin (PA)  was assigned based on the electrophoretic migration anodal to albumin. PA was soon recognized as a specific binding protein for thyroid hormone. and also of plasma retinol through the mediation of the small retinol-binding protein (RBP, 21 kDa as MM), which has a circulating half-life half that of TTR (24 h vs 48 h).

There exist at least 3 goos reasons why TTR should become a routine medical screening test in elderly persons.  The first id grounded on the assessment of protein nutritional status that is frequently compromized and may become a life threatening condition.  TTR was proposed as a marker of protein-energy malnutrition (PEM) in 1972. As a result of protein and energy deprivation, TTR hepatic synthesis is suppressed whereas all plasma indispensable amino acids (IAAs) manifest declining trends with the sole exception of methionine (Met) whose concentration usually remains unmodified. By comparison with ALB and transferrin (TF) plasma values, TTR did reveal a much higher degree of reactivity to changes in protein status that has been attributed to its shorter biological half-life and to its unusual tryptophan richness. The predictive ability of outcome offered by TTR is independent of that provided by ALB and TF. Uncomplicated PEM primarily affects the size of body nitrogen (N) pools, allowing reduced protein syntheses to levels compatible with survival.  These adaptiver changes are faithfully identified by the serial measurement of TTR whose reliability has never been disputed in protein-depleted states. On the contrary, the nutritional relevance of TTR has been controverted in acute and chronic inflammatory conditions due to the cytokine-induced transcriptional blockade of liver synthesis which is an obligatory step occurring independently from the prevailing nutritional status. Although PEM and stress ful disorders refer to distinct pathogenic mechanisms, their combined inhibitory effects on TTR liber production fueled a long-lasting strife regarding a poor specificity.  Recent body compositional studies have contributed to disentagling these intermingled morbidities, showing that evolutionary patterns displayed by plasma TTR are closely correlated with the fluctuations of lean body mass (LBM).

The second reason follows from advances describing the unexpected relationship established between TTR and homocysteine (Hcy), a S-containing AA not found in customary diets but resulting from the endogenous transmethylation of dietary methionine.  Hcy may be recycled to Met along a remethylation pathway (RM) or irreversibly degraded throughout the transsulfuration (TS) cascade to relase sulfaturia as end-product. Hcy is thus situated at the crossrad of RM and TS pathways which are in equilibrium keeping plasma Met values unaltered.  Three dietary water soluble B viatamins are implicated in the regulation of the Hcy-Met cycle. Folates (vit B9) are the most powerful agent, working as a supplier of the methyl group required for the RM process whereas cobalamines (vit B12) and pyridoxine (vit B6) operate as cofactors of Met-synthase and cystathionine-β-synthase.  Met synthase promotes the RM pathway whereas the rate-limiting CβS governs the TS degradative cascade. Dietary deficiency in any of the 3 vitamins may upregulate Hcy plasma values, an acquied biochemiucal anomaly increasingly encountered in aged populations.

The third reason refers to recent and fascinating data recorded in neurobiology and emphasizing the specific properties of TTR in the prevention of brain deterioration. TTR participates directly in the maintenance of memory and normal cognitive processes during the aging process by acting on the retinoid signaling pathway.  Moreover, TTR may bind amyloid β peptide in vitro, preventing its transformation into toxic amyloid fibrils and amyloid plaques.  TTR works as a limiting factor for the plasma transport of retinoid, which in turn operates as a limiting determinant of both physiologically active retinoic acid (RA) derivatives, implying that any fluctuation in protein status might well entail corresponding  alterations in cellular bioavailability of retinoid compounds.  Under normal aging circumstances, the concentration of retinoid compounds declines in cerebral tissues together with the downregulation of RA receptor expression. In animal models, depletion of RAs causes the deposition of amyloid-β peptides, favoring the formation of amyloid plaques.

Prealbumin and Nutritional Evaluation

Larry Bernstein, Walter Pleban
Nutrition Apr 1996; 12(4):255-259.

We compressed 16-test-pattern classes of albumin (ALB), cholesterol (CHOL), and total protein (TPR) in 545 chemistry profiles to 4 classes by conveerting decision values to a number code to separate malnourished (1 or 2) from nonmalnourished (NM)(0) patients using as cutoff values for NM (0), mild (1), and moderate (2): ALB 35, 27 g/L; TPR 63, 53 g/L; CHOL 3.9, 2.8 mmol/L; and BUN 9.3, 3.6 mmol/L. The BUN was found to have  to have too low an S-value to make a contribution to the compressed classification. The cutoff values for classifying the data were assigned prior to statistical analysis, after examining information in the structured data. The data was obtained by a natural experiment in which the test profiles routinely done by the laboratory were randomly extracted. The analysis identifies the values used that best classify the data and are not dependent on distributional assumptions. The data were converted to 0, 1, or 2 as outcomes, to create a ternary truth table (eaxch row in nnn, the n value is 0 to 2). This allows for 3(81) possible patterns, without the inclusion of prealbumin (TTR). The emerging system has much fewer patterns in the information-rich truth table formed (a purposeful, far from random event). We added TTR, coded, and examined the data from 129 patients. The classes are a compressed truth table of n-coded patterns with outcomes of 0, 1, or 2 with protein-energy malnutrition (PEM) increasing from an all-0 to all-2 pattern.  Pattern class (F=154), PAB (F=35), ALB (F=56), and CHOL (F=18) were different across PEM class and predicted PEM class (R-sq. = 0.7864, F=119, p < E-5). Kruskall-Wallis analysis of class by ranks was significant for pattern class E-18), TTR (6.1E-15) ALB (E-16), CHOL (9E-10), and TPR (5E-13). The medians and standard error (SEM) for TTR, ALB, and CHOL of four TTR classes (NM, mild, mod, severe) are: TTR = 209, 8.7; 159, 9.3; 137, 10.4; 72, 11.1 mg/L. ALB – 36, 0.7; 30.5, 0.8; 25.0, 0.8; 24.5, 0.8 g/L. CHOL = 4.43, 0.17; 4.04, 0.20; 3.11, 0.21; 2.54, 0.22 mmol/L. TTR and CHOL values show the effect of nutrition support on TTR and CHOL in PEM. Moderately malnourished patients receiving nutrition support have TTR values in the normal range at 137 mg/L and at 159 mg/L when the ALB is at 25 g/L or at 30.5 g/L.

An Informational Approach to Likelihood of Malnutrition 

Larry Bernstein, Thomas Shaw-Stiffel, Lisa Zarney, Walter Pleban.
Nutrition Nov 1996;12(11):772-776.  PII: S0899-9007(96)00222-5.

Unidentified protein-energy malnutrition (PEM) is associated with comorbidities and increased hospital length of stay. We developed a model for identifying severe metabolic stress and likelihood of malnutrition using test patterns of albumin (ALB), cholesterol (CHOL), and total protein (TP) in 545 chemistry profiles…They were compressed to four pattern classes. ALB (F=170), CHOL (F = 21), and TP (F = 5.6) predicted PEM class (R-SQ = 0.806, F= 214; p < E^-6), but pattern class was the best predictor (R-SQ = 0.900, F= 1200, p< E^-10). Ktuskal-Wallis analysis of class by ranks was significant for pattern class (E^18), ALB (E^-18), CHOL (E^-14), TP (@E^-16). The means and SEM for tests in the three PEM classes (mild, mod, severe) were; ALB – 35.7, 0.8; 30.9, 0.5; 24.2, 0.5 g/L. CHOL – 3.93, 0.26; 3.98, 0.16; 3.03, 0.18 µmol/L, and TP – 68.8, 1.7; 60.0, 1.0; 50.6, 1.1 g/L. We classified patients at risk of malnutrition using truth table comprehension.

Downsizing of Lean Body Mass is a Key Determinant of Alzheimer’s Disease

Yves Ingenbleek, Larry Bernstein
J Alzheimer’s Dis 2015; 44: 745-754.

Lean body mass (LBM) encompasses all metabolically active organs distributed into visceral and structural tissue compartments and collecting the bulk of N and K stores of the human body. Transthyretin (TTR)  is a plasma protein mainly secreted by the liver within a trimolecular TTR-RBP-retinol complex revealing from birth to old age strikingly similar evolutionary patterns with LBM in health and disease. TTR is also synthesized by the choroid plexus along distinct regulatory pathways. Chronic dietary methionine (Met) deprivation or cytokine-induced inflammatory disorders generates LBM downsizing following differentiated physiopathological processes. Met-restricted regimens downregulate the transsulfuration cascade causing upstream elevation of homocysteine (Hcy) safeguarding Met homeostasis and downstream drop of hydrogen sulfide (H2S) impairing anti-oxidative capacities. Elderly persons constitute a vulnerable population group exposed to increasing Hcy burden and declining H2S protection, notably in plant-eating communities or in the course of inflammatory illnesses. Appropriate correction of defective protein status and eradication of inflammatory processes may restore an appropriate LBM size allowing the hepatic production of the retinol circulating complex to resume, in contrast with the refractory choroidal TTR secretory process. As a result of improved health status, augmented concentrations of plasma-derived TTR and retinol may reach the cerebrospinal fluid and dismantle senile amyloid plaques, contributing to the prevention or the delay of the onset of neurodegenerative events in elderly subjects at risk of Alzheimer’s disease.

Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

Pallavi Manral and Natalia Reixach

TTR (transthyretin) amyloidosis are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membraneprotein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a KD near 650nM. Only amyloidogenic V122I TTR compete with fluorescent V122I force ll-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.

Emerging roles for retinoids in regeneration and differentiation in normal and disease states

Lorraine J. Gudas
Biochimica et Biophysica Acta 1821 (2012) 213–221

The vitamin (retinol) metabolite, all-transretinoic acid (RA), is a signaling molecule that plays key roles in the development of the body plan and induces the differentiation of many types of cells. In this review the physiological and pathophysiological roles of retinoids (retinol and related metabolites) in mature animals are discussed. Both in the developing embryo and in the adult, RA signaling via combinatorial Hoxgene expression is important for cell positional memory. The genes that require RA for the maturation/differentiation of T cells are only beginning to be cataloged, but it is clear that retinoids play a major role in expression of key genes in the immune system. An exciting, recent publication in regeneration research shows that ALDH1a2(RALDH2), which is the rate-limiting enzyme in the production of RA from retinaldehyde, is highly induced shortly after amputation in the regenerating heart, adult fin, and larval fin in zebrafish. Thus, local generation of RA presumably plays a key role in fin formation during both embryogenesis and in fin regeneration. HIV transgenic mice and human patients with HIV-associated kidney disease exhibit a profound reduction in the level of RARβ protein in the glomeruli, and HIV transgenic mice show reduced retinol dehydrogenase levels, concomitant with a greater than 3-fold reduction in endogenous RA levels in the glomeruli. Levels of endogenous retinoids (those synthesized from retinol within cells) are altered in many different diseases in the lung, kidney, and central nervous system, contributing to pathophysiology.

The Membrane Receptor for Plasma Retinol-Binding Protein, A New Type of Cell-Surface Receptor

Hui Sun and Riki Kawaguchi
Intl Review Cell and Molec Biol, 2011; 288:Chap 1. Pp 1:34

Vitamin A is essential for diverse aspects of life ranging from embryogenesis to the proper functioning of most adul torgans. Its derivatives (retinoids) have potent biological activities such as regulating cell growth and differentiation. Plasma retinol-binding protein (RBP) is the specific vitamin A carrier protein in the blood that binds to vitamin A with high affinity and delivers it to target organs. A large amount of evidence has accumulated over the past decades supporting the existence of a cell-surface receptor for RBP that mediates cellular vitamin A uptake. Using an unbiased strategy, this specific cell-surface RBP receptor has been identified as STRA6, a multi-transmembrane domain protein with previously unknown function. STRA6 is not homologous to any protein of known function and represents a new type of cell-surface receptor. Consistent with the diverse functions of vitamin A, STRA6 is widely expressed in embryonic development and in adult organ systems. Mutations in human STRA6 are associated with severe pathological phenotypes in many organs
such as the eye, brain, heart, and lung. STRA6 binds to RBP with high affinity and mediates vitamin A uptake into cells. This review summarizes the history of the RBP receptor research, its expression in the context of known functions of vitamin A in distinct human organs, structure/function analysis of this new type of membrane receptor, pertinent questions regarding its very existence, and its potential implication in treating human diseases.

Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer’s disease

Ibrahim González-Marrero, Lydia Giménez-Llort, Conrad E. Johanson, et al.
Front Cell Neurosc  Feb2015; 9(17): 1-10

Compromised secretory function of choroid plexus (CP) and defective cerebrospinal fluid (CSF) production, along with accumulation of beta-amyloid (Aβ) peptides at the blood-CSF barrier (BCSFB), contribute to complications of Alzheimer’s disease (AD). The AD triple transgenic mouse model (3xTg-AD) at 16 month-old mimics critical hallmarks of the human disease: β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) with a temporal-and regional-specific profile. Currently, little is known about transport and metabolic responses by CP to the disrupted homeostasis of CNS Aβ in AD. This study analyzed the effects of highly-expressed AD-linked human transgenes (APP, PS1 and tau) on lateral ventricle CP function. Confocal imaging and immunohistochemistry revealed an increase only of Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries; this buildup may reflect insufficient clearance transport from CSF to blood. Still, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: the low density lipoprotein receptor-related protein1 (LRP1) and the receptor for advanced glycation end product (RAGE). A thickening of the epithelial basal membrane and greater collagen-IV deposition occurred around capillaries in CP, probably curtailing solute exchanges. Moreover, there was attenuated expression of epithelial aquaporin-1 and transthyretin(TTR) protein compared to Non-Tg mice. Collectively these findings indicate CP dysfunction hypothetically linked to increasing Aβ burden resulting in less efficient ion transport, concurrently with reduced production of CSF (less sink action on brain Aβ) and diminished secretion of TTR (less neuroprotection against cortical Aβ toxicity). The putative effects of a disabled CP-CSF system on CNS functions are discussed in the context of AD.

Endoplasmic reticulum: The unfolded protein response is tangled In neurodegeneration

Jeroen J.M. Hoozemans, Wiep Scheper
Intl J Biochem & Cell Biology 44 (2012) 1295–1298

Organelle facts•The ER is involved in the folding and maturation ofmembrane-bound and secreted proteins.•The ER exerts protein quality control to ensure correct folding and to detect and remove misfolded proteins.•Disturbance of ER homeostasis leads to protein misfolding and induces the UPR.•Activation of the UPR is aimed to restore proteostasis via an intricate transcriptional and (post)translational signaling network.•In neurodegenerative diseases classified as tauopathies the activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau.•The involvement of the UPR in tauopathies makes it a potential therapeutic target.

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ERand tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.

Chemical and/or biological therapeutic strategies to ameliorate protein misfolding diseases

Derrick Sek Tong Ong and Jeffery W Kelly
Current Opin Cell Biol 2011; 23:231–238

Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation.

Vegan Diet is Sulfur Deficient and Heart Unhealthy

Larry H. Bernstein, MD, FCAP, Curator Diet is Sulfur Deficient and Heart Unhealthy

The following is a reblog of “Heart of the Matter: Plant-Based Diets Lead to High Homocysteine, Low Sulfur and Marginal B12 Status”
Posted on September 26, 2011 by Dr Kaayla Daniel in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Yves Ingenbleek

It is a report of a scientific study carried out by Kilmer S. Cully and Yves Ingenbleek, Harvard Pathology and Univ Louis Pasteur.  I have previously written about the conundrum of transthyretin as an accurate marker of malnutrition, but also being lowered by the septic state.  This is accounted for by the catabolic state that sets off autocannabalization of skeletal muscle and lean body mass to provide gluconeogenic precursors to sustain life.  While serum albumin and transthyretin both decline, the former has a half-life of 20 days, while the latter is 48 hours.  Much work has been done to gain a better understand this rapid turnover protein that transports thyroxine, and the immediate result of the decline in concentration is a shift the the hormone protein binding equilibrium increasing the free thyroxine, a euthyroid hyperthyroid effect.  However, much work by Prof. Inglenbleek, some ion collaboration with Vernon Young, at MIT, showed that transthyretin reflects the sulfur stores of animals.  The sulfur to nitrogen ratio of plants is 1:20, but it is 1:12 in man, so the dietary intake would affect an omnivorous animal.  Recall that S is carried on amino acids that take part in disulfide linkage.  A deficiency in S containing amino acids would have a negative health effect.  The story is presented here.

The World Health Organization (WHO) reports that 16.7 million deaths occur worldwide each year due to cardiovascular disease, and more than half of those deaths occur in developing countries where plant-based diets high in legumes and starches are eaten by the vast majority of the people.

Yet “everyone knows” plant-based diets prevent heart disease.  Indeed this myth  is repeated so often that massive numbers of educated, health-conscious individuals in first world countries are consciously adopting third world style diets in the hope of preventing disease, optimizing health and maximizing longevity.   But if the WHO statistics are correct, plant-based diets might not be protective at all.   And today’s fashionable experiment in veganism could end very badly indeed.

A study out August 26 in the journal Nutrition makes a strong case against plant-based diets for prevention of heart disease.  The title alone  –  “Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis” — sounds a significant warning.   The article establishes  why subjects who eat mostly vegetarian diets develop morbidity and mortality from cardiovascular disease unrelated to vitamin B status and Framingham criteria.

Co-author Kilmer S. McCully, MD, “Father of the Homocysteine Theory of Heart Disease,” is familiar to WAPF members as winner of the Linus Pauling Award, WAPF’s Integrity in Science Award, and author of numerous articles published in peer-reviewed journals as well as the popular books The Homocysteine Revolution and The Heart Revolution.   In 2009 Dr. McCully was one of the signers of the Weston A. Price Foundation’s petition to the FDA in which we asked the agency to retract its unwarranted 1999 soy/heart disease health claim.  (

Dr. McCully teamed up with Yves Ingenbleek, MD, of the University Louis Pasteur in Strasbourg, France, which funded the research.   Dr. Ingenbleek is well known for his work on malnutrition, the essential role of sulfur to nitrogen, and sulfur deficiency as a cause of  hyperhomocysteinemia.

The study took place in Chad, and involved 24 rural male subjects age 18 to 30, and 15 urban male controls, age 18-29.   (Women in this region of Chad could not be studied because of their animistic beliefs and proscriptions against collecting their urine.)

The rural men were apparently healthy, physically active farmers with good lipid profiles.  Their staple foods included cassava, sweet potatoes, beans, millet and ground nuts.   Cassava leaves, cabbages and carrots provided good levels of carotenes, folates and pyridoxine (B6).  The diet is plant-based there because of a shortage of grazing lands and livestock, but subjects occasionally consume  some B12-containing foods, mostly poultry and eggs, though very little dairy or meat.   Their diet could be described as high carb, high fiber,  low in both protein and fat, and low in the sulfur containing amino acids.    In brief, the very diet recommended by many of today’s nutritional “experts” for overall good health and heart disease prevention.

The urban controls were likewise healthy and ate a similar diet, but with beef, smoked fish and canned or powdered milk regularly on the menus.  Their diet was thus higher in protein, fat and the sulfur-containing amino acids though roughly equivalent in calories.

Dr. McCully’s research over the past 40 years on the pathogenesis of atherosclerosis has shown the role of homocysteine in free radical damage and the protective effect of  vitamins B6, B12 and folate.   Indeed, many doctors today recommend taking this trio of B vitamins as an inexpensive heart disease “insurance policy.”

In Chad, both groups showed adequate levels of B6 and folate.  The B12 levels of the vegetarian group were lower, but the difference was only of “borderline significance.”   However, as the researchers point out, ”A previous study undertaken in the same Chadian area in a larger group of 60 rural participants did demonstrate a weak inverse correlation between B12 and homocysteine concentrations in the 20 subjects most severely protein depleted .  .  .  It is therefore likely that the hyperhomocysteinemia status of some of our rural subjects in the present survey might have resulted from combined B12 and protein deficiencies.   The correlation of B12 deficiency with hyperhomocysteinemia could well reach statistical significance if a larger groups of subjects were studied.”

Clearly it’s wise for people on plant-based diets to supplement their diets with B12, but protein malnutrition must also be addressed.   And the issue is not just getting enough protein to eat, but the right kind.   Quality, not just quantity.   The bottom line is we must eat  protein rich in bioavailable, sulfur-containing amino acids — and that means animal products.   (Vegans at this point will surely claim the issue is insufficient protein and trot out soy as the solution.   Soy is indeed a  complete plant based protein, but notoriously low in methionine.  It does contain decent levels of cysteine, but the cysteine is bound up in protease inhibitors, making it largely  biounavailable. (For more information, read  my book The Whole Soy Story: The Dark Side of America’s Favorite Health Food, endorsed by Dr. McCully, as well as our petition to the FDA noted above.)

So what did  Drs. Ingenbleek and  McCully find among the study group of protein-deficient people?   Higher levels of homocysteine, of course.  Also significant alterations in body composition,  lean body mass, body mass index and plasma transthyretin levels.  In plain English, the near-vegetarian subjects were thinner, with poorer muscle tone and showed subclinical signs of protein malnutrition.   (So much for popular ideas of extreme thinness being healthy. )

The plant-based diet of the study group was low in all of the sulfur-containing amino acids.   As would be expected, labwork on these men showed lower plasma cysteine and glutathione levels compared to the controls.  Methionine levels, however,  tested comparably.   The explanation for this is  “adaptive response.”   In brief, mammals trying to function with insufficient sulfur-containing amino acids will do whatever’s necessary to survive.   Given the essential role of methionine in metabolic processes, that means deregulating the transsulfuration pathway, increasing homocysteine levels, and methylating homocysteine to make methionine.

Ultimately, it all boils down to our need for sulfur.   As Stephanie Seneff, PhD, and many others have written in Wise Traditions and on this website, sulfur is vital for disease prevention and maintenance of good health.   In terms of heart disease, Drs. Ingenbleek and McCully have shown sulfur deficiency not only leads to high homocysteine levels, but is the likeliest reason some clinical trials using B6, B12 and folate interventions have proved ineffective for the prevention of cardiovascular and cerebrovascular diseases.    Over the past few years, headlines from such studies have led to widespread dismissal of Dr. McCully’s  “Homocysteine Theory of Heart Disease” and renewed media focus on cholesterol, c-reactive protein and other possible culprits that can be treated by statins and other profitable drugs.   In contrast, Drs. McCully and Ingenbleek research suggests we can better prevent heart disease with three inexpensive B vitamins and traditional diets rich in the sulfur-containing amino acids found in animal foods.

In the blaze of publicity surrounding Forks Over Knives and other blasts of vegan propaganda, few people are likely to hear about this study.   That’s sad, for it provides an important missing piece in our knowledge of heart disease development, a strong argument against the plant-based fad, and a bright new chapter in what the New York Times has called “The Fall and Rise of Kilmer McCully.”

*  *  *  *  *

Thanks to Sylvia Onusic PhD who was able to access a full text copy of this article to share with  me.

This entry was posted in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Naughty Nutritionist, soy, sulfur, Yves Ingenbleek. Bookmark the permalink.

Food Insecurity in Africa and GMOs

Reporter and Curator: Larry H. Bernstein, MD, FCAP

In the GMO-free future, farming still looks pretty much the same. Without insect-resistant crops, farmers spray more broad-spectrum insecticides, which do some collateral damage to surrounding food webs. Without herbicide-resistant crops, farmers spray less glyphosate, which slows the spread of glyphosate-resistant weeds and perhaps leads to healthier soil biota. Farmers also till their fields more often, which kills soil biota, and releases a lot more greenhouse gases.

The banning of GMOs hasn’t led to a transformation of agriculture because GM seed was never a linchpin supporting the conventional food system: Farmers could always do fine without it. Eaters no longer worry about the small potential threat of GMO health hazards, but they are subject to new risks: GMOs were neither the first, nor have they been the last, agricultural innovation, and each of these technologies comes with its own potential hazards. Plant scientists will have increased their use of mutagenesis and epigenetic manipulation, perhaps. We no longer have biotech patents, but we still have traditional seed-breeding patents. Life goes on.

In the other alternate future, where the pro-GMO side wins, we see less insecticide, more herbicide, and less tillage. In this world, with regulations lifted, a surge of small business and garage-biotechnologists got to work on creative solutions for the problems of agriculture.

Genetic engineering is just one tool in the tinkerer’s belt. Newer tools are already available, and scientists continue to make breakthroughs with traditional breeding. So in this future, a few more genetically engineered plants and animals get their chance to compete. Some make the world a little better, while others cause unexpected problems. But the science has moved beyond basic genetic engineering, and most of the risks and benefits of progress are coming from other technologies. Life goes on.

In many ways he’s right. GMOs on the market today – and most of the ones planned – are about making agriculture more efficient and profitable for farmers and seed providers. This is not a trivial thing, but would global agriculture collapse without these GMOs? Of course not.

We rarely see transformative technologies coming. And remember that we are still in the very early days of genetic engineering of crops and animals. I suspect that you could go back and look at the early days of almost any new technology and convincingly downplay its transformative potential.

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer, Curator: Larry H. Bernstein, MD, FCAP

The new era of nutrition research translates empirical knowledge to evidence-based molecular science (9). Modern nutrition research focuses on

  • promoting health,
  • preventing or delaying the onset of disease,
  • optimizing performance, and
  • assessing risk.

Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics

  • build the science foundation for understanding human variability in
  • preferences, requirements, and responses to diet and
  • may become the future tools for consumer assessment

motivated by personalized nutritional counseling for health maintenance and disease prevention.

The primary aim of ―omic‖ technologies is

  • the non-targeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample.

By their nature, these technologies reveal unexpected properties of biological systems.

A second and more challenging aspect of ―omic‖ technologies is

  • the refined analysis of quantitative dynamics in biological systems (10).

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.

In modern nutrition research, mass spectrometry has developed into a tool

  • to assess health, sensory as well as quality and safety aspects of food.

In this review, we focus on health-related benefits of food components and, accordingly,

  • on biomarkers of exposure (bioavailability) and bioefficacy.

Current nutrition research focuses on unraveling the link between

  • dietary patterns,
  • individual foods or
  • food constituents and

the physiological effects at cellular, tissue and whole body level

  • after acute and chronic uptake.

The bioavailability of bioactive food constituents as well as dose-effectcorrelations 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 (11).

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Metabolomic analysis of two leukemia cell lines. II.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

Leaders in Pharmaceutical Intelligence


In Part I of metabolomics of two leukemia cell lines, we have established a major premise for the study, an insight into the use of an experimental model, and some insight into questions raised.

I here return to examine these before pursuing more detail in the study.

Q1. What strong metabolic pathways come into focus in this study?

Answer – The aerobic and anaerobic glycolytic pathways, with a difference measured in the extent of participation of mitochondrial oxidative phosphorylation.

Q2. Would we expect to also gain insight into the effect, on balance, played by a suppressed ubiquitin pathway?

Answer – lets look into this in Part II.

Q3. Would the synthesis of phospholipid and the maintenance of membrane structures requires availability of NADPH, which would be a reversal of the TCA cycle at the cost of delta G in catabolic energy, be consistent with increased dependence of anaerobic glycolysis  with unchecked replication?

Answer: Part II might show this, as the direction and the difference between the cell lines is consistent with a Warburg (Pasteur) effect.

Recall the observation that the model is based on experimental results from  lymphocytic leukemia cell lines in cell culture.  The internal metabolic state is inferred from measurement of external metabolites.

The classification of the lymphocytic leukemias in humans is based on T-cell and B-cell lineages, but actually uses cell differentiation (CD) markers on the cytoskeleton for recognition.  It is only a conjecture that if the cells line were highly anaplastic, they might not be sustainable in cell culture in perpetuity.
The analogue of these cells to what I would expect to see in humans is the SLL having the characteristic marking: CD5, see

Micro description

● Effacement of nodal architecture by pale staining pseudofollicles or proliferation centers with ill-defined borders, containing small round mature lymphocytes, prolymphocytes (larger than small lymphocytes, abundant basophilic cytoplasm, prominent nucleoli), paraimmunoblasts (larger cells with distinct nucleoli) and many smudge cells
● Pseudofollicular centers are highlighted by decreasing light through the condenser at low power; cells have pale cytoplasm but resemble soccer balls or smudge cells on peripheral smear (cytoplasm is bubbly in mantle cell lymphoma); may have plasmacytoid features
● May have marginal zone, perifollicular or interfollicular patterns, but these cases also have proliferation centers (Mod Pathol 2000;13:1161)
● Interfollicular pattern: large, reactive germinal centers; resembles follicular lymphoma but germinal centers are bcl2 negative and tumor cells resemble SLL by morphology and immunostains
(Am J Clin Path 2000;114:41)
● Paraimmunoblastic variant: diffuse proliferation of paraimmunoblasts (normally just in pseudoproliferation centers); rare, <30 reported cases; usually multiple lymphadenopathies and rapid disease progression; case report in 69 year old man (Hum Pathol 2002;33:1145); consider as mantile cell lymphoma if t(11;14)(q13;q32) is present; may also represent CD5+ diffuse large B cell lymphoma
Bone marrow: small focal aggregates of variable size with irregular, poorly circumscribed outlines; lymphocytes are well differentiated, small, round with minimal atypia; may have foci of transformation; rarely has granulomas (J Clin Pathol 2005;58:815)
● Marrow infiltrative patterns are also described as diffuse (unmutated IgH genes, ZAP-70+, more aggressive), nodular (associated with IgH hypermutation, ZAP-70 negative) or mixed (variable mutation of IgH, variable ZAP-70, Hum Pathol 2006;37:1153)


Positive stains

● CD5, CD19, CD20 (dim), CD23, surface Ig light chain, surface IgM (dim)
● Also CD43, CD79a, CD79b (dim in 20%, Arch Pathol Lab Med 2003;127:561), bcl2
● Variable CD11c, FMC7 (42%)
Negative stains

● CD10, cyclin D1

● Trisomy 12 (30%, associated with atypical CLL and CD79b), deletion 13q14 (25-50%),
deletion of 11q23 (worse prognosis, 10-20%)



We set up a pipeline that could be used to

  • infer intracellular metabolic states from semi-quantitative data
  • regarding metabolites exchanged between cells and their environment.

Our pipeline combined the following four steps:

  1. data acquisition,
  2. data analysis,
  3. metabolic modeling and
  4.  experimental validation of
  • the model predictions (Fig. 1A).

We demonstrated the pipeline and the predictive potential

  • to predict metabolic alternations in diseases such as cancer
  • based on two lymphoblastic leukemia cell lines.

The resulting Molt-4 and CCRF-CEM condition-specific cell line models were able

  • to explain metabolite uptake and secretion
  •  by predicting the distinct utilization of central metabolic pathways by the two cell lines.

Whereas the CCRF-CEM model

  • resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype,
  • our predictions suggested  a more respiratory phenotype for the Molt-4  model.

We found these predictions to be in agreement with measured gene expression differences

  • at key regulatory steps in the central metabolic pathways, and
  • they were also consistent with  data regarding the energy and redox states of the cells.

After a brief discussion of the data generation and analysis steps, the results

  • derived from model generation and analysis will be described in detail.


2.1 Pipeline for generation of condition-specific metabolic cell line models

2.1.1 Generation of experimental data

We monitored the growth and viability of lymphoblastic leukemia cell lines in
serum- free medium (File S2, Fig. S1). Multiple omics  data sets  were derived  from these cells.

Extracellular metabolomics (exo-metabolomic) data,

  • comprising measurements of the metabolites in the spent medium of the cell cultures
    (Paglia et al. 2012a),
  • were collected along with transcriptomic data, and
  • these data sets were used to construct the models.


2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells

To determine whether we had obtained two distinct models,

  • we evaluated the reactions, metabolites, and genes of the two models.

Both the Molt-4 and CCRF-CEM models contained approximately

  • half of the reactions and metabolites present in the global model (Fig. 1C).

They were very similar to each other in terms of their

  • reactions,
  • metabolites, and
  • genes (File S1, Table S5A–C).

The Molt– 4 model contained

  • seven reactions that were not present in the CCRF-CEM model
    (Co-A biosynthesis pathway and exchange reactions).

In contrast, the CCRF-CEM  contained

31 unique reactions

  • arginine and proline metabolism,
  • vitamin B6  metabolism,
  • fatty acid activation,
  • transport, and exchange reaction.
  • There  were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models,  respectively
    (File S1, Table S5B).
    Approximately three quarters of the global  model  genesremained in the condition-specific cell line models  (Fig. 1C).

The Molt-4 model contained

  • 15 unique genes, and

the CCRF-CEM model had

  • 4 unique genes (File S1, Table S5C).

Both models lacked NADH dehydrogenase
(complex I of the electron transport chain—ETC),

  •  determined by  the  absence of expression of a mandatory subunit
    (NDUFB3, Entrez gene ID 4709).

The ETC was fueled by FADH2 originating from

  1. succinate dehydrogenase and
  2. from fatty acid oxidation, which
  • through flavoprotein electron transfer
  • could contribute to the same ubiquinone pool as
  • complex I and complex II (succinate dehydrogenase).

Despite their different in vitro growth rates
(which differed by 11 %, see File S2, Fig. S1) and

  • differences in exo-metabolomic data (Fig. 1B) and
  • transcriptomic data,
  • the internal networks were largely conserved
  • in the two condition-specific cell line models.


2.1.5 Condition-specific cell line models predict distinct metabolic strategies

Despite the overall similarity of the metabolic models,

  • differences in their cellular uptake and secretion patterns suggested
  • distinct metabolic states in the two cell lines
    (Fig. 1B and see “Materials and methods” section for more detail).

To interrogate the metabolic differences, we sampled the solution space

  • of each model  using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005).

For this  analysis, additional constraints were applied, emphasizing

  • the  quantitative differences in commonly uptaken and secreted metabolites.

The  maximum possible uptake and maximum possible secretion flux rates were

  • reduced according to the measured relative differences between the cell lines
    (Fig. 1D, see “Materials and methods” section).

We plotted the number of sample points containing a particular flux rate for each reaction. The resulting

  • binned histograms can be understood as representing the probability that
  • a particular reaction can have a certain flux value.

A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed

  • a  considerable shift in the distributions, suggesting
  • a higher utilization of  glycolysis by the CCRF-CEM model (File S2, Fig. S2).

This result  was further  supported by differences

  • in medians calculated from sampling points (File S1,  Table S6).

The shift persisted throughout all reactions of the pathway and

  • was  induced by the higher glucose uptake (35 %) from
  • the extracellular medium in CCRF-CEM cells.

The sampling median for glucose uptake was 34 % higher

  • in the  CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).

The usage of the  TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2).

  • the models used succinate dehydrogenase differently (Figs. 23).

The Molt-4 model utilized an associated reaction to generate FADH2, whereas

  • in  the CCRF-CEM model, the histogram was shifted in the opposite direction,
  • toward  the generation of succinate.

Additionally, there was a higher efflux of  citrate toward

  • amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2).

There was higher flux through anaplerotic and cataplerotic reactions

  • in the CCRF-CEM model than in the Molt-4 model (Fig. 2);
  • these reactions include the efflux  of citrate through


  1. ATP-citrate lyase,
  2. uptake of glutamine,
  3. generation of  glutamate from glutamine,
  4. transamination of pyruvate and
  5.  glutamate to alanine  and to 2-oxoglutarate,
  6. secretion of nitrogen, and
  7. secretion of alanine.

The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3),

  • supported by elevated median flux through ATP synthase (36 %) and other  enzymes,
  • which contributed to higher oxidative metabolism.

The sampling  analysis therefore revealed different usage of

  • central metabolic pathways by the condition-specific models.


Fig. 2

Differences in the use of the TCA cycle by the CCRF-CEM

Differences in the use of the TCA cycle by the CCRF-CEM

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).
The table provides the median values of the sampling results. Negative values in histograms and Table

  • describe reversible  reactions with flux in the reverse direction.

There are multiple reversible  reactions for the transformation of

  1. isocitrate and α-ketoglutarate,
  2. malate and  fumarate, and
  3. succinyl-CoA and succinate.

These reactions are  unbounded,  and therefore histograms are not shown.
The details of participating cofactors  have been removed.

Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoAcoa coenzyme-A,
icit isocitrate, αkg α-ketoglutarate, succcoa succinyl-CoAsucc succinate, fumfumarate, mal malate,
oxa oxaloacetate,  pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport  chain.


Electronic supplementary material The online version of this article 
contains supplementary material,  which  is available to authorized users.

  1.  K. Aurich _ G. Paglia _ O ´ . Rolfsson _ S. Hrafnsdo´ ttir _
  2. Magnu´sdo´ ttir _ B. Ø. Palsson _ R. M. T. Fleming _ I. Thiele. Center for Systems Biology,
    University of Iceland, Reykjavik, Iceland
  3.  K. Aurich _ R. M. T. Fleming _ I. Thiele (&). Luxembourg Centre for Systems Biomedicine,
    University of Luxembourg, Campus Belval, Esch-Sur-Alzette, Luxembourg
  4. M. Stefaniak. School of Health Science, Faculty of Food Science and Nutrition,
    University of Iceland, Reykjavik, Iceland
  5. Ø. Palsson. Department of Bioengineering, University of California San Diego, La Jolla, CA, USA


Fig. 3

Fatty acid oxidation and ETC _Fig3

Fatty acid oxidation and ETC _Fig3


Sampling reveals different utilization of oxidative phosphorylation by the

  • generated models.

Different distributions are observed for the CCRF-CEM model (red) and the Molt-4 model (blue).

  • Molt-4 has higher  median  flux through ETC reactions II–IV.

The table provides the median values  of the sampling results. Negative values in the histograms and in the table describe

  • reversible reactions with flux in the reverse direction.

Both models lack Complex I of the ETC because of constraints

  • arising from the mapping of transcriptomic data.

Electron transfer flavoprotein and

  • electron transfer flavoprotein–ubiquinone oxidoreductase
  •  both also carry higher flux in the Molt-4 model


2.1.6 Experimental validation of energy and redox status of CCRF-CEM and Molt-4 cells

Cancer cells have to balance their needs

  •  for energy and biosynthetic precursors, and they have
  • to maintain redox homeostasis to proliferate (Cairns et al. 2011).

We conducted enzymatic assays of cell lysates to measure levels and/or ratios of

  • ATP,
  • NADH + NAD, and
  • glutathione.

These measurements were used to provide support for

  • the in silico predicted metabolic differences (Fig. 4).

Additionally, an Oxygen Radical Absorbance Capacity (ORAC) assay was used

  • to evaluate the cellular antioxidant status (Fig. 4B).

Total concentrations of NADH + NAD, GSH + GSSG, NADPH + NADP and ATP, were higher in Molt-4 cells  (Fig. 4A).

The higher ATP concentration in Molt-4 cells could either result from

  • high production rates, or intracellular  accumulation connected to high or
  • low reactions fluxes (Fig. 4A).

Our simplified view that oxidative Molt-4 produces less ATP and was contradicted by

  • the higher ATP concentrations measured (Fig. 4L).

Yet we want to emphasize that concentrations

  • cannot be compared to flux values,
  • since we are modeling at steady-state.

NADH/NAD+ ratios for both cell lines were shifted toward NADH (Fig. 4D, E), but

  • the shift toward NADH was more pronounced in CCRF-CEM (Fig. 4E),
  • which matched  our expectation based on the higher utilization of
  • glycolysis and 2-oxoglutarate  dehydrogenase in the CCRF-CEM model (Fig. 4L).


Fig. 4 (not shown)

A–K  Experimentally determined ATP, NADH + NAD, NADPH + NADP, and GSH + GSSG concentrations, and ROS detoxification in the CCRF-CEM and Molt-4 cells.

L Expectations for cellular energy and redox states. Expectations are based on predicted metabolic differences of the Molt-4 and CCRF-CEM models

2.1.7 Comparison of network utilization and alteration in gene expression

With the assumption that

  • differential expression of particular genes would cause reaction flux changes,

we determined how the differences in gene expression (between CCRF-CEM and Molt-4)

  • compared to the flux differences observed in the  models.

Specifically, we checked whether the reactions associated with genes upregulated
(significantly more expressed in CCRF-CEM cells compared to Molt-4  cells)

  • were indeed more utilized by the CCRF-CEM model,

and we  checked  whether downregulated genes

  • were associated with reactions more utilized by the Molt-4 model.

The set of downregulated genes was associated with 15 reactions, and

  • the set of 49 upregulated genes was associated with 113 reactions in the models.

Reactions were defined as differently utilized

  • if the difference in flux exceeded 10 % (considering only non-loop reactions).

Of the reactions associated with upregulated genes,

  • 72.57 % were more utilized by the CCRF-CEM model, and
  • 2.65 % were more utilized by the Molt-4 model (File S1, Table S7).

In contrast, all 15 reactions associated with the 12 downregulated genes

  • were more utilized in the CCRF-CEM model (File S1, Table S8).

After this initial analysis, we approached the question from a different angle, asking

  • whether the majority of the reactions associated with each individual gene
  • upregulated in CCRF-CEM were more utilized by the CCRF-CEM model.
  •  this was the case for 77.55 % of the upregulated genes.

The majority of reactions associated with two (16.67 %) downregulated genes

  • were more utilized by the Molt-4 model.

Taken together, our comparisons of the

  • direction of gene expression with the fluxes of the two cancer cell-line models
  • confirmed that reactions associated with upregulated genes in the CCRF-CEM
    cells were generally more utilized by the CCRF-CEM model.

2.1.8 Accumulation of DEGs and AS genes at key metabolic steps

After we confirmed that most reactions associated with upregulated genes

  • were more utilized by the CCRF-CEM model,

we checked the locations of DEGs within the network. In this analysis, we paid special attention to

  • the central metabolic pathways that we had found
  • to be distinctively utilized by the two models.

Several DEGs and AS events were associated with

  • glycolysis,
  • the ETC,
  • pyruvate metabolism, and
  • the PPP (Table 1).


Table 1

DEGs and AS events of central metabolic and cancer-related pathways

Full lists of DEGs and AS are provided in the supplementary material.

Upregulated significantly more expressed in CCRF-CEM compared to Molt-4 cells

PPP pentose phosphate pathway, OxPhos oxidative phosphorylation, Glycolysis/gluconglycolysis/gluconeogenesis, Pyruvate met. pyruvate metabolism

Moreover, in glycolysis, the DEGs and/or AS genes

  • were associated with all three rate-limiting steps, i.e., the steps mediated by
  1. hexokinase,
  2. pyruvate kinase, and
  3. phosphofructokinase.

Of these key enzymes,

  • hexokinase 1 (Entrez Gene ID: 3098) was alternatively spliced,
  • pyruvate kinase (PKM, Entrez gene ID: 5315) was significantly more
    expressed in the CCRF-CEM cells (Table 1),

in agreement with the higher in silico predicted flux.

However, in contrast to the observed

  • higher utilization of glycolysis in the CCRF-CEM model,
  • the gene associated with the rate-limiting glycolysis step, phosphofructokinase (Entrez Gene ID: 5213),
  • was significantly upregulated in Molt-4 cells relative to CCRF-CEM cells.

This higher expression was detected for only a single isozyme, however. Two of
the three genes associated with phosphofructokinase were also subject to
alternative splicing (Table 1). In addition to the key enzymes, fructose
bisphosphate aldolase (Entrez Gene ID: 230) was also significantly

  • upregulated in Molt-4 cells relative to CCRF-CEM cells,
  • in contrast to the predicted higher utilization of glycolysis in the CCRF-CEM model.

Additionally, glucose-6P-dehydrogenase (G6PD), which catalyzes

  • the first reaction and committed step of the PPP,
  • was an AS gene (Table 1).

A second AS gene associated with

  •  the PPP reaction of the deoxyribokinase
  • was RBKS (Entrez Gene ID: 64080).

This gene is also associated with ribokinase, but ribokinase was removed

  • because of the lack of ribose uptake or secretion.

Single AS genes were associated with different complexes of the ETC (Table 1).

Literature query revealed that at least 13 genes associated with alternative

  • splicing events were mentioned previously in connection with both alternative
    splicing and cancer (File S1, Table S14), and
  • 37 genes were associated with cancer, e.g., upregulated, downregulated at the
    level of mRNA or protein, or otherwise
  • connected to cancer metabolism and signaling.

One general observation was that there was a surprising

  • accumulation of metabolite transporters among the AS.

Overall, the high incidence of

  • differential gene expression events at metabolic control points
  • increases the plausibility of the in silico predictions.


2.1.9 Single gene deletion

Analyses of essential genes in metabolic models have been used

  • to predict candidate drug targets for cancer cells (Folger et al. 2011).

Here, we conducted an in silico gene deletion study for all model genes to identify

  • a unique set of knock-out (KO) genes
  • for each condition-specific cell line model.

The analysis yielded 63 shared lethal KO genes and

  • distinct sets of KO genes for the CCRF-CEM model (11 genes) and the Molt-4 model (3 genes).

For three of the unique CCRF-CEM KO genes,

  • the genes were only present in the CCRF-CEM model (File S1, Table S9).


The essential genes for both models were then

  • related to the cell-line-specific differences in metabolite uptake and secretion (Fig. 1B).

The CCRF-CEM model

  1. needed to generate putrescine from ornithine
    (ORNDC, Entrez Gene ID: 4953)
  2. to subsequently produce 5-methylthioadenosine for secretion (Fig. 1B).
  3. S-adenosylmethioninamine produced by adenosylmethionine decarboxylase
    (arginine and proline metabolism, associated with Entrez Gene ID: 262)
  • is a substrate required for generation of 5-methylthioadenosine.

Another example of a KO gene connected to an enforced exchange reaction was

  • glutamic-oxaloacetic transaminase 1 (GOT1, Entrez Gene ID: 2805).

Without GOT1, the CCRF-CEM model was forced to secrete

  • 4-hydroxyphenylpyruvate (Fig. 1B),
  • the second product of tyrosine transaminase,
  • which is produced only by that enzyme.


One KO gene in the Molt-4 model (Entrez Gene ID: 26227) was associated with

  • phosphoglycerate dehydrogenase (PGDH),
  • which catalyzes the conversion of 3-phospho-d-glycerate to 3-phosphohydroxypyruvate
  • while generating NADH from NAD+.

This KO gene is particularly interesting, given

  • the involvement of this reaction in a novel pathway for ATP generation in rapidly proliferating cells
    (Locasale et al. 2011; Vander Heiden 2011; Vazquez et al. 2011).

Reactions associated with unique KO genes were in many cases utilized more by the model, in which

  • the gene KO was lethal,
  • underlining the potential importance of these reactions for the models.

Thus, single gene deletion provided unique sets of lethal genes that could be

  • specifically targeted to kill these cells.


3 Discussion

In the current study, we explored the possibility of

  • semi-quantitatively integrating metabolomic data with
  • the human genome-scale reconstruction to facilitate analysis.

By constructing condition-specific cell line models

  • to provide a structured framework,
  • we derived insights that could not have been obtained from data analysis alone.

We derived condition-specific cell line models

  • for CCRF-CEM and
  • Molt-4 cells

that were able to explain the observed exo-metabolomic differences (Fig. 1B).

Despite the overall similarities between the models, the analysis revealed

  • distinct usage of central metabolic pathways (Figs. 234),
  • which we validated based on experimental data and
  • differential gene expression.

The additional data sufficiently supported

  • metabolic differences in the cell lines,
  • providing confidence in the generated models and the model-based predictions.

We used the validated models

  • to predict unique sets of lethal genes
  • to identify weak links in each model.

These weak links may represent potential drug targets.

Integrating omics data with the human genome-scale reconstruction

  • provides a structured framework (i.e., pathways)
  • that is based on careful consideration of the available biochemical literature
    (Thiele and Palsson2010).

This network context can simplify omics data analysis, and

  • it allows even non-biochemical experts
  • to gain fast and comprehensive insights
  • into the metabolic aspects of omics data sets.

Compared to transcriptomic data,

  • methods for the integration and analysis of metabolomic data
  • in the context of metabolic models are less well established,

although it is an active field of research (Li et al. 2013; Paglia et al. 2012b).
In contrast to other studies, our approach emphasizes

  • the representation of experimental conditions rather than
  • the reconstruction of a generic, cell-line-specific network,
  • which would require the combination of data sets from
  • many experimental conditions and extensive manual curation.

Rather, our way of model construction allowed us to efficiently

  • assess the metabolic characteristics of cells.

Despite the fact, that only a limited number of exchanged metabolites can be

  • measured by available metabolomics platforms and
  • at reasonable time-scale,

and that pathways of measured metabolites might still be unknown to date
(File S1, Tables S2–S3), our methods have the potential

  • to reveal metabolic characteristics of cells
  • which could be useful for biomedicine and personalized health.

The reasons why some cancers respond to certain treatments and not others
remain unclear, and choosing a treatment for a specific patient is often difficult
(Vander Heiden 2011). One potential application of our approach could be the
characterization of cancer phenotypes to explore how cancer cells or other cell

  • with particular metabolic characteristics respond to drugs.

The generation of our condition-specific cell line models involved

  • only limited manual curation,
  • making this approach a fast way to place metabolomic data
  • into a network context.

Model building mainly involves

  • the rigid reduction of metabolite exchanges
  • to match the observed metabolite exchange pattern
  • with as few additional metabolite exchanges as possible.

It should be noted that this reduction determines,

  • which pathways can be utilized by the model.

Our approach mostly conserved the internal network redundancy. However, a

  • more significant reduction may be achieved using different data.

Generally, a trade-off exists between the reduction of the internal network and

  • the increasing number of network gaps that need to be curated
  • by using additional omics data, such as transcriptomics and proteomics.

One way to prevent the emergence of network gaps would be

  • to use mapping algorithms that conserve network functionality,
    such as GIMME (Becker and Palsson 2008).

However, several additional methods exist for the integration of
transcriptomic data (Blazier and Papin 2012), and

  • which model-building method is best depends on the available data.

Interestingly, the lack of a significant contribution of our

  • gene expression data to the reduction of network size
  • suggests that the use of transcriptomic data is not necessary
  • to identify distinct metabolic strategies;
  • rather, the integration of exo-metabolomic data alone
    may provide sufficient insight.

However, sampling of the cell line models constrained

  • according to the exo-metabolomic profiles only, or
  • increasing the cutoff for the generation of absent and present calls (p < 0.01),
  • did not yield the same insights as presented herein (File S1, Table S18).

Only recently Gene Inactivation Moderated by Metabolism, Metabolomics and
Expression (GIM(3)E) became available, which

  • enforces minimum turnover of detected metabolites
  • based on intracellular metabolomics data as well as
  • gene expression microarray data (Schmidt et al. 2013).

In contrast to this approach, we emphasized our analysis on the

  • relative differences in the exo-metabolomic data of two cell lines.

GIM(3)E constitutes another integration method when the analysis should be

  • emphasized on intracellular metabolomics data (Schmidt et al. 2013).

The metabolic differences predicted by the models are generally plausible.
Cancers are known to be heterogeneous (Cairns et al. 2011), and

  • the contribution of oxidative phosphorylation to cellular ATP production
    may vary (Zu and Guppy 2004).

Moreover, leukemia cell lines have been shown

  • to depend on glucose, glutamine, and fatty acids to varying extents
  • to support proliferation.

Such dependence may cause the cells to adapt their metabolism

  • to the environmental conditions (Suganuma et al. 2010).

In addition to identifying supporting data in the literature, we performed

  • several analyses to validate the models and model predictions.

Our expectations regarding the levels and ratios of metabolites

  • relevant to energy and redox state were largely met (Fig. 4L).

The more pronounced shift of the NADH/NAD+ ratio

  • toward NADH in the CCRF-CEM cells
  • was in agreement with the predicted Warburg phenotype (Fig. 4),
  • and the higher lactate secretion in the CCRF-CEM cells (File S2, Fig. S2)
  • implies an increase in NADH relative to NAD+
    (Chiarugi et al. 2012; Nikiforov et al. 2011), again
  • matching the known Warburg phenotype.

ROS production is enhanced in certain types of cancer (Droge 2002; Ha et al. 2000), and

  • the generation of ROS is thought to contribute to
  1. mutagenesis,
  2. tumor promotion, and
  3. tumor progression (Dreher and Junod1996; Ha et al. 2000).

However, decreased mitochondrial glucose oxidation and

  • a transition to aerobic glycolysis
  • protect cells against ROS damage during biosynthesis and cell division
    (Brand and Hermfisse1997).

The higher ROS detoxification capability in Molt-4 cells, in combination with

  • higher spermidine dismutase utilization by the Molt-4 model (Fig. 4),
  • provided a consistent picture of the predicted respiratory phenotype (Fig. 4L).

Control of NADPH maintains the redox potential through GSH and

  • protects against oxidative stress, yet
  • changes in the NADPH ratio in response to oxidative damage
  • are not well understood (Ogasawara et al.2009).

Under stress conditions, as assumed for Molt-4 cells,

  • the NADPH/NADP+ ratio is expected to decrease because of
  • the continuous reduction of GSSG (Fig. 4L), and
  • this was confirmed in the Molt-4 cells (Fig. 4).

The higher amounts of GSH found in Molt-4 cells in vitro may demonstrate

  • an additional need for ROS scavengers because of
  • a greater reliance on oxidative metabolism.

Cancer is related to metabolic reprogramming, which results from

  • alterations of gene expression and
  • the expression of specific isoforms or
  • splice forms to support proliferation
    (Cortes-Cros et al. 2013; Marin-Hernandez et al. 2009).

The gene expression differences detected between the two cell lines in this study
supported the existence of

  • metabolic differences in these cell lines, particularly because
  • key steps of the metabolic pathways central to cancer metabolism
  • seemed to be differentially regulated (Table 1).

The detailed analysis of the respective

  • differences on the pathway fluxes exceeds the scope of this study, which was to
  • demonstrate the potential of the integration of exo-metabolomic data into the network context.

We found discrepancies between differential gene regulation and

  • the flux differences between the two models as well as
  • the utilization AS gene-associated reaction.

This is not surprising, since analysis of the detailed system is required

  • to make any further assumptions on the impact that
  • the differential regulation or splicing might have on the reaction flux,
  • given that for many of the concerned enzymes isozymes exist, or
  • only one of multiple subunits of a protein complex was concerned.

Additionally, reaction fluxes are regulated by numerous post-translational factors, e.g.,

  • protein modification,
  • inhibition through proteins or metabolites,
  • alter reaction fluxes (Lenzen 2014),

which are out of the scope of constraint-based steady-state modeling.

Rather, the results of the presented  approach

  • demonstrate how the models can be used to generate
  • informed hypothesis that can guide experimental work.

The combination of our tailored metabolic models and

  • differential gene expression analysis seems well-suited
  • to determine the potential drivers
  • involved in metabolic differences between cells.

Such information could be valuable for drug discovery, especially when more

  • peripheral metabolic pathways are considered.

Statistical comparisons of gene expression data with sampling-derived flux data

  • could be useful in future studies (Mardinoglu et al. 2013).

A single-gene-deletion analysis revealed that PGDH was

  • a lethal KO gene for the Molt-4 model only.

Differences in PGDH protein levels

  • correspond to the amount of glycolytic carbon
  • diverted into glycine biosynthesis.

Rapidly proliferating cells may use an

  • alternative glycolytic pathway for ATP generation,
  • which may provide an advantage in the case of
  • extensive oxidative phosphorylation and proliferation
    (Locasale et al.2011; Vander Heiden 2011; Vazquez et al. 2011).

For breast cancer cell lines, variable dependency on

  • the expression of PGDH has already been demonstrated
    (Locasale et al. 2011).

This example of a unique KO gene demonstrates how

  • in silico gene deletion in metabolomics-driven models
  • can identify the metabolic pathways used by cancer cells.

This approach can provide valuable information for drug discovery.

In conclusion, our contextualization method produced

  • metabolic models that agreed in many ways with the validation data sets.

The analyses described in this study have great potential to reveal

  • the mechanisms of metabolic reprogramming,
  • not only in cancer cells but also in other cells affected by diseases, and
  • for drug discovery in general.


4.3 Analysis of the extracellular metabolome

Mass spectrometry analysis of the exo-metabolome was performed by
Metabolon®, Inc. (Durham, NC, USA) using a standardized analytical platform.
In total, 75 extracellular metabolites were detected in the initial data set for at
least 1 of the 2 cell lines (Paglia et al. 2012a). Of these metabolites, 15 were not
part of our global model and were discarded. Apart from being absent in our
global model, an independent search in HMDB (Wishart et al. 2013) revealed no
pathway information was available for most of these metabolites (File S1, Tables S2–S3).
It should be noted that metabolites e.g.,

  • N-acetylisoleucine,
  • N-acetyl-methionine or pseudouridine,

constitute protein and RNA degradation products, which were out of the scope
of the metabolic network.

Thiamin (Vitamin B1) was part of the minimal medium of essential compounds
supplied to both models.Riboflavin (Vitamin B2) and Trehalose were excluded
since these compounds cannot be produced by human cells. Erythrose and
fructose were also excluded. In contrast 46 metabolites that were part of the
global model. The data set included two different time points, which allowed us
to treat the increase/decrease of a metabolite signal between time points as

  • evidence for uptake or secretion when the change was greater than 5 %
    from what was observed in the control (File S1, Tables S2–S3).

We found 12 metabolites that were taken up by both cell lines and
10 metabolites that were commonly secreted by both cell lines over
the course of the experiment.

Molt-4 cells took up three metabolites not taken up by CCRF-CEM cells, and
secreted one metabolite not secreted by CCRF-CEM cells. Two of the three
uniquely uptaken metabolites were essential amino acids:

  1. valine and
  2. methionine.

It is unlikely that these metabolites were not taken up by the CCRF-CEM cells,
and the CCRF-CEM model was allowed to take up this metabolite. Therefore,
no quantitative constraints were applied for the sampling analysis either.
CCRF-CEM cells had

  • four unique uptaken
  • and seven unique secreted metabolites
    (exchange not detected in Molt-4 cells).


4.4 Network refinement based on exo-metabolic data

Despite its comprehensiveness, the human metabolic reconstruction is

  • not complete with respect to extracellular metabolite transporters
    (Sahoo et al. 2014; Thiele et al. 2013).

Accordingly, we identified metabolite transport systems

  • from the literature for metabolites that were already part of the global model,
  • but whose extracellular transport was not yet accounted for.

Diffusion reactions were included whenever a respective transporter could not be identified.

In total, 34 reactions [11 exchange reactions, 16 transport reactions and 7 demand reactions
(File S1, Table S11)] were added to Recon 2 (Thiele et al. 2013), and 2 additional reactions
were added to the global model (File S1, Table S10).

4.5 Expression profiling

Molt-4 and CCRF-CEM cells were grown in advanced RPMI 1640 and 2 mM
GlutaMax, and the cells were resuspended in medium containing DMSO
(0.67 %) at a concentration of 5 × 105 cells/mL. The cell suspension (2 mL)
was seeded in 12-well plates in triplicate. After 48 h of growth, the cells
were collected by centrifugation at 201×g for 5 min. Cell pellets were snap-frozen
in liquid N2 and kept frozen until RNA extraction and analysis by Aros
(Aarhus, Denmark).

4.6 Analysis of transcriptomic data

We used the Affymetrix GeneChip Human Exon 1.0 ST Array to measure whole
genome exon expression. We generated detection above background (DABG) calls
using ROOT (version 22) and the XPS package for R (version 11.1), with Robust
Multi-array Analysis summarization. Calls for data mapping were assigned based
on p < 0.05 as the cutoff probability to distinguish presence versus absence for
the 1,278 model genes (File S1, Table S12).

Differential gene expression and alternative splicing analyses were performed by
using AltAnalyse software (v2.02beta) with default options on the raw data files
(CEL files). The Homo sapiens Ensemble 65 database was used, probe set filtering
was kept as DABG p < 0.05, and non-log expression < 70 was used for
constitutive probe sets to determine gene expression levels. For the comparison,
CCRF-CEM was the experimental group and Molt-4 was the baseline group. The
set of DEGs between cell lines was identified based on a p < 0.05 FDR cutoff
(File S1, Table S13A–B). Alternative splicing analysis was performed on core probe sets
with a minimum alternative exon score of 2 and a maximum absolute gene
expression change of 3 because alternative splicing is a less critical factor among
highly DEGs (File S1, Table S14). Gene expression data, complete lists of DABG p-values,
DEGs and alternative splicing events have been deposited in the Gene
Expression Omnibus
 (GEO) database (Accession number: GSE53123).


4.7 Deriving cell-type-specific subnetworks

Transcriptomic data were mapped to the model in a manual fashion (COBRA
function: deleteModelGenes). Specifically, reactions dependent on gene products
that were called as “absent” were constrained to zero, such that fluxes through
these reactions were disabled. Submodels were extracted based on the set of
reactions carrying flux (network pruning) by running fastFVA
(Gudmundsson and Thiele 2010) after mapping the metabolomic and
transcriptomic data using the COBRA toolbox (Schellenberger et al. 2011).




Electronic supplementary material

Below is the link to the electronic supplementary material.

File S1. Supplementary material 1 (XLSX 915 kb)

File S2. Supplementary material 2 (DOCX 448 kb)


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Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP


We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:


This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)


“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.    …/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.


Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960, 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.


Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx.

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.


Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.


Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14. 


Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor


3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.

Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109. 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.




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A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Subtitle: Transthyretin and the Systemic Inflammatory Response


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

Transthyretin protein structure (Photo credit: Wikipedia)

Age-standardised disability-adjusted life year...

Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)


The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum.
Larry H Bernstein
Clin Chem Lab Med 2007; 45(11):0
ICID: 939932
Article type: Editorial

The Transthyretin Inflammatory State Conundrum
Larry H. Bernstein
Current Nutrition & Food Science, 2012, 8, 00-00

Keywords: Tranthyretin (TTR), systemic inflammatory response syndrome (SIRS), protein-energy malnutrition (PEM), C- reactive protein, cytokines, hypermetabolism, catabolism, repair.

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.


Title: The Automated Malnutrition Assessment
Accepted 29 April 2012. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.
Authors: Gil David, PhD; Larry Howard Bernstein, MD; Ronald R Coifman, PhD
Article Type: Original Article

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.


Title: The Increasing Role for the Laboratory in Nutritional Assessment
Article Type: Editorial
Section/Category: Clinical Investigation
Accepted 22 May 2012.
Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.05.024
Keywords: Protein Energy Malnutrition; Nutritional Screening; Laboratory Testing
Author: Dr. Larry Howard Bernstein, MD

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 ShiftArch 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.


Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H Bernstein
Clin Biochem 2008; 41(14-15):1126-1130
ICID: 939927
Article type: Original article

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.


A simplified nutrition screen for hospitalized patients using readily available laboratory and patient
Linda Brugler, Ana K Stankovic, Madeleine Schlefer, Larry Bernstein
Nutrition 2005; 21(6):650-658
ICID: 825623
Article type: Review article
The role of visceral protein markers in protein calorie malnutrition.
Linda Brugler, Ana Stankovic, Larry Bernstein, Frederick Scott, Julie O’Sullivan-Maillet
Clin Chem Lab Med 2002; 40(12):1360-1369
ICID: 636207
Article type: Original article

The Automated Nutrition Score is a data-driven extension of continuous quality improvement.

Larry H Bernstein
Nutrition 2009; 25(3):316-317
ICID: 939934

Transthyretin: its response to malnutrition and stress injury. clinical usefulness and economic implications.
LH Bernstein, Y Ingenbleek
Clin Chem Lab Med 2002; 40(12):1344-1348
ICID: 636205
Article type: Original article


Yves Ingenbleek  MD  PhD  and  Larry Bernstein MD
J CLIN LIGAND ASSAY  (out of print)

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 negative APPs 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.


Yves Ingenbleek. The Open Clinical Chemistry Journal, 2011, 4, 34-44.

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.

see also …

McCully, K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am. J. Pathol., 1996, 56, 111-128.

Cheng, Z.; Yang, X.; Wang, H. Hyperhomocysteinemia and endothelial dysfunction. Curr. Hypertens. Rev., 2009, 5,158-165.

Loscalzo, J. The oxidant stress of hyperhomocyst(e)inemia. J. Clin.Invest., 1996, 98, 5-7.

Ingenbleek, Y.; Hardillier, E.; Jung, L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition, 2002, 18, 40-46.

Ingenbleek, Y.; Young, V.R. The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinemia. Nutr. Res. Rev., 2004, 17, 135-153.

Hosoki, R.; Matsuki, N.; Kimura, H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 1997, 237, 527-531.

Tang, B.; Mustafa, A.; Gupta, S.; Melnyk, S.; James S.J.; Kruger, W.D. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine-􀀁-synthase. Nutrition, 2010, 26, 1170-1175.

Elshorbagy, A.K.; Valdivia-Garcia, M.; Refsum, H.; Smith, A.D.; Mattocks, D.A.; Perrone, C.E. Sulfur amino acids in methioninerestricted rats: Hyperhomocysteinemia. Nutrition, 2010, 26, 1201- 1204.


Yves Ingenbleek. Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease. Chapter 20. In S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009.

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).

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Pharma Intell Links

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I 
Mitochondrial dynamics and cardiovascular diseases 
“Seductive Nutrition”: Making Popular Dishes a Bit Healthier – Culinary Institute of America
Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model
Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
Nitric Oxide and Immune Responses: Part 2
Mitochondrial Damage and Repair under Oxidative Stress
Endothelial Function and Cardiovascular Disease
Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options
Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control
Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?
Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

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Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Author: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN

This is the second of a two part discussion of viscosity, hemostasis, and vascular risk

This is Part II of a series on blood flow and shear stress effects on hemostasis and vascular disease.

See Part I on viscosity, triglycerides and LDL, and thrombotic risk.


Hemostatic Factors in Thrombophilia

Objectives.—To review the state of the art relating to elevated hemostatic factor levels as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of specific measurements of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

Data Sources.—Review of the medical literature, primarily from the last 10 years.

Data Extraction and Synthesis.—After an initial assessment of the literature, key points were identified. Experts were assigned to do an in-depth review of the literature and to prepare a summary of their findings and recommendations.

A draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia prior to the conference. Each of the key points and associated recommendations was then presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form.

Consensus was reached on 8 recommendations concerning the use of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

The underlying premise for measuring elevated coagulation factor levels is that if the average level of the factor is increased in the patient long-term, then the patient may be at increased risk of thrombosis long-term. Both risk of thrombosis and certain factors increase with age (eg, fibrinogen, factor VII, factor VIII, factor IX, and von Willebrand factor). Are these effects linked or do we need age specific ranges? Do acquired effects like other diseases or medications affect factor levels, and do the same risk thresholds apply in these instances? How do we assure that the level we are measuring is a true indication of the patient’s average baseline level and not a transient change? Fibrinogen, factor VIII, and von Willebrand factor are all strong acute-phase reactants.

Risk of bleeding associated with coagulation factor levels increases with roughly log decreases in factor levels. Compared to normal (100%), 60% to 90% decreases in a coagulation factor may be associated with excess bleeding with major trauma, 95% to 98% decreases with minor trauma, and .99% decrease with spontaneous hemorrhage. In contrast, the difference between low risk and high risk for thrombosis may be separated by as little as 15% above normal.

It may be possible to define relative cutoffs for specific factors, for example, 50% above the mean level determined locally in healthy subjects for a certain factor. Before coagulation factor levels can be routinely used to assess individual risk, work must be done to better standardize and calibrate the assays used.

Detailed discussion of the rationale for each of these recommendations is presented in the article. This is an evolving area of research. While routine use of factor level measurements is not recommended, improvements in assay methodology and further clinical studies may change these recommendations in the future.

Chandler WL, Rodgers GM, Sprouse JT, Thompson AR.  Elevated Hemostatic Factor Levels as Potential Risk Factors for Thrombosis.  Arch Pathol Lab Med. 2002;126:1405–1414

Model System for Hemostatic Behavior

This study explores the behavior of a model system in response to perturbations in

  • tissue factor
  • thrombomodulin surface densities
  • tissue factor site dimensions
  • wall shear rate.

The classic time course is characterized by

  • initiation and
  • amplification of thrombin generation
  • the existence of threshold-like responses

This author defines a new parameter, the „effective prothrombotic zone‟,  and its dependence on model parameters. It was found that prothrombotic effects may extend significantly beyond the dimensions of the spatially discrete site of tissue factor expression in both axial and radial directions. Furthermore, he takes advantage of the finite element modeling approach to explore the behavior of systems containing multiple spatially distinct sites of TF expression in a physiologic model. The computational model is applied to assess individualized thrombotic risk from clinical data of plasma coagulation factor levels. He proposes a systems-based parameter with deep venous thrombosis using computational methods in combination with biochemical panels to predict hypercoagulability for high risk populations.


The Vascular Surface

The ‘resting’ endothelium synthesizes and presents a number of antithrombogenic molecules including

  • heparan sulfate proteoglycans
  • ecto-adenosine diphosphatase
  • prostacyclin
  • nitric oxide
  • thrombomodulin.

In response to various stimuli

  • inflammatory mediators
  • hypoxia
  • oxidative stress
  • fluid shear stress

the cell surface becomes ‘activated’ and serves to organize membrane-associated enzyme complexes of coagulation.

Fluid Phase Models of Coagulation

Leipold et al. developed a model of the tissue factor pathway as a design aid for the development of exogenous serine protease inhibitors. In contrast, Guo et al. focused on the reactions of the contact, or intrinsic pathway, to study parameters relevant to material-induced thrombosis, including procoagulant surface area.

Alternative approaches to modeling the coagulation cascade have been pursued including the use of stochastic activity networks to represent the intrinsic, extrinsic, and common pathways through fibrin formation and a kinetic Monte Carlo simulation of TF-initiated thrombin generation. Generally, fluid phase models of the kinetics of coagulation are both computationally and experimentally less complex. As such, the computational models are able to incorporate a large number of species and their reactions, and empirical data is often available for regression analysis and model validation. The range of complexity and motivations for these models is wide, and the models have been used to describe various phenomena including the ‘all-or-none’ threshold behavior of thrombin generation. However, the role of blood flow in coagulation is well recognized in promoting the delivery of substrates to the vessel wall and in regulating the thrombin response by removing activated clotting factors.

Flow Based Models of Coagulation

In 1990, Basmadjian presented a mathematical analysis of the effect of flow and mass transport on a single reactive event at the vessel wall and consequently laid the foundation for the first flow-based models of coagulation. It was proposed that for vessels greater than 0.1 mm in diameter, reactive events at the vessel wall could be adequately described by the assumption of a concentration boundary layer very close to the reactive surface, within which the majority of concentration changes took place. The height of the boundary layer and the mass transfer coefficient that described transport to and from the vessel wall were shown to stabilize on a time scale much shorter than the time scale over which concentration changes were empirically observed. Thus, the vascular space could be divided into two compartments, a boundary volume and a bulk volume, and furthermore, changes within the bulk phase could be considered negligible, thereby reducing the previously intractable problem to a pseudo-one compartment model described by a system of ordinary differential equations.

Basmadjian et al. subsequently published a limited model of six reactions, including two positive feedback reactions and two inhibitory reactions, of the common pathway of coagulation triggered by exogenous factor IXa under flow. As a consequence of the definition of the mass transfer coefficient, the kinetic parameters were dependent on the boundary layer height. Furthermore, the model did not explicitly account for intrinsic tenase or prothrombinase formation, but rather derived a rate expression for reaction in the presence of a cofactor. The major finding of the study was the predicted effect of increased mass transport to enhance thrombin generation by decreasing the induction time up to a critical mass transfer rate, beyond which transport significantly decreased peak thrombin levels thereby reducing overall thrombin production.

Kuharsky and Fogelson formulated a more comprehensive, pseudo-one compartment model of tissue factor-initiated coagulation under flow, which included the description of 59 distinct fluid- and surface-bound species. In contrast to the Baldwin-Basmadjian model, which defined a mass transfer coefficient as a rate of transport to the vessel surface, the Kuharsky-Fogelson model defined the mass transfer coefficient as a rate of transport into the boundary volume, thus eliminating the dependence of kinetic parameters on transport parameters. The computational study focused on the threshold response of thrombin generation to the availability of membrane binding sites. Additionally, the model suggested that adhered platelets may play a role in blocking the activity of the TF/ VIIa complex. Fogelson and Tania later expanded the model to include the protein C and TFPI pathways.

Modeling surface-associated reactions under flow uses finite element method (FEM), which is a technique for solving partial differential equations by dividing the vascular space into a finite number of discrete elements. Hall et al. used FEM to simulate factor X activation over a surface presenting TF in a parallel plate flow reactor. The steady state model was defined by the convection-diffusion equation and Michaelis-Menten reaction kinetics at the surface. The computational results were compared to experimental data for the generation of factor Xa by cultured rat vascular smooth muscle cells expressing TF.

Based on discrepancies between numerical and experimental studies, the catalytic activity of the TF/ VIIa complex may be shear-dependent. Towards the overall objective of developing an antithrombogenic biomaterial, Tummala and Hall studied the kinetics of factor Xa inhibition by surface-immobilized recombinant TFPI under unsteady flow conditions. Similarly, Byun et al. investigated the association and dissociation kinetics of ATIII inactivation of thrombin accelerated by surface-immobilized heparin under steady flow conditions. To date, finite element models that detail surface-bound reactions under flow have been restricted to no more than a single reaction catalyzed by a single surface-immobilized species.


Models of Coagulation Incorporating Spatial Parameter

Major findings include the roles of these specific coagulation pathways in the

  • initiation
  • amplification
  • termination phases of coagulation.

Coagulation near the activating surface was determined by TF/VIIa catalyzed factor Xa production, which was rapidly inhibited close to the wall. In contrast, factor IXa diffused farther from the surface, and thus factor Xa generation and clot formation away from the reactive wall was dependent on intrinsic tenase (IXa/ VIIIa) activity. Additionally, the concentration wave of thrombin propagated away from the activation zone at a rate which was dependent on the efficiency of inhibitory mechanisms.

Experimental and ‘virtual’ addition of plasma-phase thrombomodulin resulted in dose-dependent termination of thrombin generation and provided evidence of spatial localization of clot formation by TM with final clot lengths of 0.2-2 mm under diffusive conditions.

These studies provide an interesting analysis of the roles of specific factors in relation to space due to diffusive effects, but neglect the essential role of blood flow in the transport analysis. Additionally, the spatial dynamics of clot localization by thrombomodulin would likely be affected by restricting the inhibitor to its physiologic site on the vessel surface.

Finite Element Modeling

Finite element method (FEM) is a numerical technique for solving partial differential equations. Originally proposed in the 1940s to approach structural analysis problems in civil engineering, FEM now finds application in a wide variety of disciplines. The computational method relies on mesh discretization of a continuous domain which subdivides the space into a finite number of ‘elements’. The physics of each element are defined by its own set of physical properties and boundary conditions, and the simultaneous solution of the equations describing the individual elements approximate the behavior of the overall domain.

Sumanas W. Jordan, PhD Thesis. A Mathematical Model of Tissue Factor-Induced Blood Coagulation: Discrete Sites of Initiation and Regulation under Conditions of Flow.

Doctor of Philosophy in Biomedical Engineering. Emory University, Georgia Institute of Technology. May 2010.  Under supervision of: Dr. Elliot L. Chaikof, Departments of Surgery and Biomedical Engineering.

Blood Coagulation (Thrombin) and Protein C Pat...

Blood Coagulation (Thrombin) and Protein C Pathways (Blood_Coagulation_and_Protein_C_Pathways.jpg) (Photo credit: Wikipedia)

Coagulation cascade

Coagulation cascade (Photo credit: Wikipedia)


Cardiovascular Physiology: Modeling, Estimation and Signal Processing

With cardiovascular diseases being among the main causes of death in the world, quantitative modeling, assessment and monitoring of cardiovascular dynamics, and functioning play a critical role in bringing important breakthroughs to cardiovascular care. Quantification of cardiovascular physiology and its control mechanisms from physiological recordings, by use of mathematical models and algorithms, has been proved to be of important value in understanding the causes of cardiovascular diseases and assisting the diagnostic and prognostic process. This E-Book is derived from the Frontiers in Computational Physiology and Medicine Research Topic entitled “Engineering Approaches to Study Cardiovascular Physiology: Modeling, Estimation and Signal Processing.”

There are two review articles. The first review article by Chen et al. (2012) presents a unified point process probabilistic framework to assess heart beat dynamics and autonomic cardiovascular control. Using clinical recordings of healthy subjects during Propofol anesthesia, the authors demonstrate the effectiveness of their approach by applying the proposed paradigm to estimate

  • instantaneous heart rate (HR),
  • heart rate variability (HRV),
  • respiratory sinus arrhythmia (RSA)
  • baroreflex sensitivity (BRS).

The second review article, contributed by Zhang et al. (2011), provides a comprehensive overview of tube-load model parameter estimation for monitoring arterial hemodynamics.

The remaining eight original research articles can be mainly classified into two categories. The two articles from the first category emphasize modeling and estimation methods. In particular, the paper “Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study” by Cheng and Khoo (2012), combines computational modeling and simulations to study the autonomic and metabolic effects of obstructive sleep apnea (OSA).

The second paper, “Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal” by Fazeli and Hahn (2012), presents a model-based approach to estimate cardiac output (CO) and total peripheral resistance (TPR), and validates the proposed approach via in vivo experimental data from animal subjects.

The six articles in the second category focus on application of signal processing techniques and statistical tools to analyze cardiovascular or physiological signals in practical applications. the paper “Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration” by Cabiddu et al. (2012), uses spectral and cross-spectral analysis of heartbeat and respiration signals to assess autonomic cardiac regulation and cardiopulmonary coupling variations during different sleep stages in healthy subjects.

The paper “increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction” by Hayano et al. (2011) uses a new non-gaussian index to assess the HRV of cardiac mortality using 670 post-acute myocardial infarction (AMI) patients. the paper “non-gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and parkinson disease” by Kiyono et al. (2012), applies a non-gaussian index to assess HRV in patients with multiple system atrophy (MSA) and parkinson diseases and reports the relation between the non-gaussian intermittency of the heartbeat and increased sympathetic activity. The paper “Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings” by Faes et al. (2011), proposes an information domain approach to evaluate nonlinear causality among heartbeat, arterial pressure, and respiration measures during tilt testing and paced breathing protocols. The paper “integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans” by Lin and Sharif (2012), uses a relative multifractal complexity measure to assess HRV in healthy humans and discusses the related implications in central autonomic interactions. Lastly, the paper “Time scales of autonomic information flow in near-term fetal sheep” by Frasch et al. (2012), analyzes the autonomic information flow (AIF) with kullback–leibler entropy in fetal sheep as a function of vagal and sympathetic modulation of fetal HRV during atropine and propranolol blockade.

In summary, this Research Topic attempts to give a general panorama of the possible state-of-the-art modeling methodologies, practical tools in signal processing and estimation, as well as several important clinical applications, which can altogether help deepen our understanding about heart physiology and pathology and further lead to new scientific findings. We hope that the readership of Frontiers will appreciate this collected volume and enjoy reading the presented contributions. Finally, we are grateful to all contributed authors, reviewers, and editorial staffs who had all put tremendous effort to make this E-Book a reality.

Cabiddu, R., Cerutti, S., Viardot, G., Werner, S., and Bianchi, A. M. (2012). Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration. Front. Physio. 3:45. doi: 10.3389/fphys.2012.00045

Chen, Z., Purdon, P. L., Brown, E. N., and Barbieri, R. (2012). A unified point process probabilistic framework to assess heartbeat dynamics and autonomic cardiovascular control. Front. Physio. 3:4. doi: 10.3389/fphys.2012.00004

Cheng, L., and Khoo, M. C. K. (2012). Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study. Front. Physio. 2:111. doi: 10.3389/fphys.2011.00111

Faes, L., Nollo, G., and Porta, A. (2011). Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings. Front. Physio. 2:80. doi: 10.3389/fphys.2011.00080

Fazeli, N., and Hahn, J.-O. (2012). Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal. Front. Physio. 3:298. doi: 10.3389/fphys.2012.00298

Frasch, M. G., Frank, B., Last, M., and Müller, T. (2012). Time scales of autonomic information flow in near-term fetal sheep. Front. Physio. 3:378. doi: 10.3389/fphys.2012.00378

Hayano, J., Kiyono, K., Struzik, Z. R., Yamamoto, Y., Watanabe, E., Stein, P. K., et al. (2011). Increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction. Front. Physio. 2:65. doi: 10.3389/fphys.2011.00065

Kiyono, K., Hayano, J., Kwak, S., Watanabe, E., and Yamamoto, Y. (2012). Non-Gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and Parkinson disease. Front. Physio. 3:34. doi: 10.3389/fphys.2012.00034

Lin, D. C., and Sharif, A. (2012). Integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans. Front. Physio. 2:123. doi: 10.3389/fphys.2011.00123

Zhang, G., Hahn, J., and Mukkamala, R. (2011). Tube-load model parameter estimation for monitoring arterial hemodynamics. Front. Physio. 2:72. doi: 10.3389/fphys.2011.00072

Citation: Chen Z and Barbieri R (2012) Editorial: engineering approaches to study cardiovascular physiology: modeling, estimation, and signal processing. Front. Physio. 3:425. doi: 10.3389/fphys.2012.00425

fluctuations of cerebral blood flow and metabolic demand following hypoxia in neonatal brain

Most of the research investigating the pathogenesis of perinatal brain injury following hypoxia-ischemia has focused on excitotoxicity, oxidative stress and an inflammatory response, with the response of the developing cerebrovasculature receiving less attention. This is surprising as the presentation of devastating and permanent injury such as germinal matrix-intraventricular haemorrhage (GM-IVH) and perinatal stroke are of vascular origin, and the origin of periventricular leukomalacia (PVL) may also arise from poor perfusion of the white matter. This highlights that cerebrovasculature injury following hypoxia could primarily be responsible for the injury seen in the brain of many infants diagnosed with hypoxic-ischemic encephalopathy (HIE).

The highly dynamic nature of the cerebral blood vessels in the fetus, and the fluctuations of cerebral blood flow and metabolic demand that occur following hypoxia suggest that the response of blood vessels could explain both regional protection and vulnerability in the developing brain.

This review discusses the current concepts on the pathogenesis of perinatal brain injury, the development of the fetal cerebrovasculature and the blood brain barrier (BBB), and key mediators involved with the response of cerebral blood vessels to hypoxia.

Baburamani AA, Ek CJ, Walker DW and Castillo-Melendez M. Vulnerability of the developing brain to hypoxic-ischemic damage: contribution of the cerebral vasculature to injury and repair? Front. Physio. 2012;  3:424. doi: 10.3389/fphys.2012.00424

remodeling of coronary and cerebral arteries and arterioles 

Effects of hypertension on arteries and arterioles often manifest first as a thickened wall, with associated changes in passive material properties (e.g., stiffness) or function (e.g., cellular phenotype, synthesis and removal rates, and vasomotor responsiveness). Less is known, however, regarding the relative evolution of such changes in vessels from different vascular beds.

We used an aortic coarctation model of hypertension in the mini-pig to elucidate spatiotemporal changes in geometry and wall composition (including layer-specific thicknesses as well as presence of collagen, elastin, smooth muscle, endothelial, macrophage, and hematopoietic cells) in three different arterial beds, specifically aortic, cerebral, and coronary, and vasodilator function in two different arteriolar beds, the cerebral and coronary.

Marked geometric and structural changes occurred in the thoracic aorta and left anterior descending coronary artery within 2 weeks of the establishment of hypertension and continued to increase over the 8-week study period. In contrast, no significant changes were observed in the middle cerebral arteries from the same animals. Consistent with these differential findings at the arterial level, we also found a diminished nitric oxide-mediated dilation to adenosine at 8 weeks of hypertension in coronary arterioles, but not cerebral arterioles.

These findings, coupled with the observation that temporal changes in wall constituents and the presence of macrophages differed significantly between the thoracic aorta and coronary arteries, confirm a strong differential progressive remodeling within different vascular beds.

These results suggest a spatiotemporal progression of vascular remodeling, beginning first in large elastic arteries and delayed in distal vessels.

Hayenga HN, Hu J-J, Meyer CA, Wilson E, Hein TW, Kuo L and Humphrey JD  Differential progressive remodeling of coronary and cerebral arteries and arterioles in an aortic coarctation model of hypertension. Front. Physio. 2012; 3:420. doi: 10.3389/fphys.2012.00420

C-reactive protein oxidant-mediated release of pro-thrombotic  factor

Inflammation and the generation of reactive oxygen species (ROS) have been implicated in the initiation and progression of atherosclerosis. Although C-reactive protein (CRP) has traditionally been considered to be a biomarker of inflammation, recent in vitro and in vivo studies have provided evidence that CRP, itself, exerts pro-thrombotic effects on vascular cells and may thus play a critical role in the development of atherothrombosis. Of particular importance is that CRP interacts with Fcγ receptors on cells of the vascular wall giving rise to the release of pro-thrombotic factors. The present review focuses on distinct sources of CRP-mediated ROS generation as well as the pivotal role of ROS in CRP-induced tissue factor expression. These studies provide considerable insight into the role of the oxidative mechanisms in CRP-mediated stimulation of pro-thrombotic factors and activation of platelets. Collectively, the available data provide strong support for ROS playing an important intermediary role in the relationship between CRP and atherothrombosis.

Zhang Z, Yang Y, Hill MA and Wu J.  Does C-reactive protein contribute to atherothrombosis via oxidant-mediated release of pro-thrombotic factors and activation of platelets? Front. Physio.  2012; 3:433. doi: 10.3389/fphys.2012.00433

CRP association with Peripheral Vascular Disease

To determine whether the increase in plasma levels of C-Reactive Protein (CRP), a non-specifi c reactant in the acute-phase of systemic infl ammation, is associated with clinical severity of peripheral arterial disease (PAD).

This is a cross-sectional study at a referral hospital center of institutional practice in Madrid, Spain.  These investigators took a stratifi ed random sampling of 3370 patients with symptomatic PAD from the outpatient vascular laboratory database in 2007 in the order of their clinical severity:

  • the fi rst group of patients with mild chronological clinical severity who did not require surgical revascularization,
  • the second group consisted of patients with moderate clinical severity who had only undergone only one surgical revascularization procedure and
  • the third group consisted of patients who were severely affected and had undergone two or more surgical revascularization procedures of the lower extremities in different areas or needed late re-interventions.

The Neyman affi xation was used to calculate the sample size with a fi xed relative error of 0.1.

A homogeneity analysis between groups and a unifactorial analysis of comparison of medians for CRP was done.

The groups were homogeneous for

  • age
  • smoking status
  • Arterial Hypertension
  • diabetes mellitus
  • dyslipemia
  • homocysteinemia and
  • specifi c markers of infl ammation.

In the unifactorial analysis of multiple comparisons of medians according to Scheffé, it was observed that

the median values of CRP plasma levels were increased in association with higher clinical severity of PAD

  • 3.81 mg/L [2.14–5.48] vs.
  • 8.33 [4.38–9.19] vs.
  • 12.83 [9.5–14.16]; p  0.05

as a unique factor of tested ones.

Plasma levels of CRP are associated with not only the presence of atherosclerosis but also with its chronological clinical severity.

De Haro J, Acin F, Medina FJ, Lopez-Quintana A, and  March JR.  Relationship Between the Plasma Concentration of C-Reactive Protein and Severity of Peripheral Arterial Disease.
Clinical Medicine: Cardiology 2009;3: 1–7

Hemostasis induced by hyperhomocysteinemia

Elevated concentration of homocysteine (Hcy) in human tissues, defined as hyperhomocysteinemia has been correlated with some diseases, such as

  • cardiovascular
  • neurodegenerative
  • kidney disorders

L-Homocysteine (Hcy) is an endogenous amino acid, containing a free thiol group, which in healthy cells is involved in methionine and cysteine synthesis/resynthesis. Indirectly, Hcy participates in methyl, folate, and cellular thiol metabolism. Approximately 80% of total plasma Hcy is protein-bound, and only a small amount exists as a free reduced Hcy (about 0.1 μM). The majority of the unbound fraction of Hcy is oxidized, and forms dimers (homocystine) or mixed disulphides consisting of cysteine and Hcy.

Two main pathways of Hcy biotoxicity are discussed:

  1. Hcy-dependent oxidative stress – generated during oxidation of the free thiol group of Hcy. Hcy binds via a disulphide bridge with

—     plasma proteins

—     or with other low-molecular plasma  thiols

—     or with a second Hcy molecule.

Accumulation of oxidized biomolecules alters the biological functions of many cellular pathways.

  1. Hcy-induced protein structure modifications, named homocysteinylation.

Two main types of homocysteinylation exist: S-homocysteinylation and N-homocysteinylation; both considered as posttranslational protein modifications.

a)      S-homocysteinylation occurs when Hcy reacts, by its free thiol group, with another free thiol derived from a cysteine residue in a protein molecule.

These changes can alter the thiol-dependent redox status of proteins.

b)      N-homocysteinylation takes place after acylation of the free ε-amino lysine groups of proteins by the most reactive form of Hcy — its cyclic thioester (Hcy thiolactone — HTL), representing up to 0.29% of total plasma Hcy.

Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) — a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In human blood, N-homocysteinylated (N-Hcy-protein) and S-homocysteinylated proteins (S-Hcy-protein) such as NHcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin, and S-Hcyalbumin are known. Other pathways of Hcy biotoxicity might be apoptosis and excitotoxicity mediated through glutamate receptors. The relationship between homocysteine and risk appears to hold for total plasma concentrations of homocysteine between 10 and 30 μM.

Different forms of homocysteine present in human blood.

*Total level of homocysteine — the term “total homocysteine” describes the pool of homocysteine released by reduction of all disulphide bonds in the sample (Perla-Kajan et al., 2007; Zimny, 2008; Manolescu et al., 2010, modified).

The form of Hcy The concentration in human blood
Homocysteine thiolactone (HTL) 0–35 nM
Protein N-linked homocysteine:
N-Hcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin
about 15.5 μM: 12.7 μM, 2.8 μM
Protein S-linked homocysteine — S-Hcy-albumin about 7.3 μM*
Homocystine (Hcy-S-S-Hcy) and combined with cysteine to from mixed disulphides (Hcy-S-S-Cys) about 2 μM*
Free reduced Hcy about 0.1 μM*

As early as in the 1960s it was noted that the risk of atherosclerosis is markedly increased in patients with homocystinuria, an inherited disease resulting from homozygous CBS deficiency and characterized by episodes of

—     thromboembolism

—     mental retardation

—     lens dislocation

—     hepatic steatosis

—     osteoporosis.

—     very high concentrations of plasma homocysteine and methionine.

Patients with homocystinuria have very severe hyperhomocysteinemia, with plasma homocysteine concentration reaching even 400 μM, and represent a very small proportion of the population (approximately 1 in 200,000 individuals). Heterozygous lack of CBS, CBS mutations and polymorphism of the methylenetetrahydrofolate reductase gene are considered to be the most probable causes of hyperhomocysteinemia.

The effects of hyperhomocysteinemia include the complex process of hemostasis, which regulates the properties of blood flow. Interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis are:

  • endothelial cells
  • platelets
  • fibrinogen
  • plasminogen

Elevated plasma Hcy (>15 μM; Hcy) is associated with an increased risk of cardiovascular diseases

  • thrombosis
  • thrombosis related diseases
  • ischemic brain stroke (independent of other, conventional risk factors of this disease)

Every increase of 2.5 μM in plasma Hcy may be associated with an increase of stroke risk of about 20%.  Total plasma Hcy level above 20 μM are associated with a nine-fold increase of the myocardial infarction and stroke risk, in comparison to the concentrations below 9 μM. The increase of Hcy concentration has been also found in other human pathologies, including neurodegenerative diseases

Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities.

Hcy and HTL may act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. Therefore, we have to consider the role of phenolic antioxidants in hyperhomocysteinemia –induced changes in hemostasis.

The synthesis of homocysteine thiolactone is associated with the activation of the amino acid by aminoacyl-tRNA synthetase (AARS). Hcy may also undergo erroneous activation, e.g. by methionyl-t-RNA synthetase (MetRS). In the first step of conversion of Hcy to HTL, MetRS misactivates Hcy giving rise to homocysteinyl-adenylate. In the next phase, the homocysteine side chain thiol group reacts with the activated carboxyl group and HTL is produced. The level of HTL synthesis in cultured cells depends on Hcy and Met levels.

Hyperhomocysteinemia and Changes in Fibrinolysis and Coagulation Process

The fibrinolytic activity of blood is regulated by specific inhibitors; the inhibition of fibrinolysis takes place at the level of plasminogen activation (by PA-inhibitors: plasminogen activator inhibitor type-1, -2; PAI-1 or PAI-2) or at the level of plasmin activity (mainly by α2-antiplasmin). Hyperhomocysteinemia disturbs hemostasis and shifts the hemostatic mechanisms in favor of thrombosis. The recent reports indicate that the prothrombotic state observed in hyperhomocysteinemia may arise not only due to endothelium dysfunction or blood platelet and coagulation activation, but also due to impaired fibrinolysis. Hcy-modified fibrinogen is more resistant to the fibrinolytic action. Oral methionine load increases total Hcy, but may diminish the fibrinolytic activity of the euglobulin plasma fraction. Homocysteine-lowering therapies may increase fibrinolytic activity, thereby, prevent atherothrombotic events in patients with cardiovascular diseases after the first myocardial infarction.

Homocysteine — Fibronectin Interaction and its Consequences

Fibronectin (Fn) plays key roles in

  • cell adhesion
  • migration
  • embryogenesis
  • differentiation
  • hemostasis
  • thrombosis
  • wound healing
  • tissue remodeling

Interaction of FN with fibrin, mediated by factor XIII transglutaminase, is thought to be important for cell adhesion or cell migration into fibrin clots. After tissue injury, a blood clot formation serves the dual role of restoring vascular integrity and serving as a temporary scaffold for the wound healing process. Fibrin and plasma FN, the major protein components of blood clots, are essential to perform these functions. In the blood clotting process, after fibrin deposition, plasma FN-fibrin matrix is covalently crosslinked, and it then promotes fibroblast adhesion, spreading, and migration into the clot.

Homocysteine binds to several human plasma proteins, including fibronectin. If homocysteine binds to fibronectin via a disulphide linkage, this binding results in a functional change, namely, the inhibition of fibrin binding by fibronectin. This inhibition may lead to a prolonged recovery from a thrombotic event and contribute to vascular occlusion.

Grape seeds are one of the richest plant sources of phenolic substances, and grape seed extract reduces the toxic effect of Hcys and HTL on fibrinolysis. The grape seed extract (12.5–50 μg/ml) supported plasminogen to plasmin conversion inhibited by Hcys or HTL. In vitro experiments showed in the presence of grape seed extract (at the highest tested concentration — 50 μg/ml) the increase of about 78% (for human plasminogen-treated with Hcys) and 56% (for human plasma-treated with Hcys). Thus, in the in vitro model system, that the grape seed extract (12.5–50 μg/ml) diminished the reduction of thiol groups and of lysine ε-amino groups in plasma proteins treated with Hcys (0.1 mM) or HTL (1 μM). In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).

In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).Very similar protective effects of the grape seed extract were observed in the measurements of lysine ε-amino groups in plasma proteins treated with Hcys or HTL. These results indicated that the extract from berries of Aronia melanocarpa (a rich source of phenolic substances) reduces the toxic effects of Hcy and HTL on the hemostatic properties of fibrinogen and plasma. These findings indicate a possible protective action of the A. melanocarpa extract in hyperhomocysteinemia-induced cardiovascular disorders. Moreover, the extract from berries of A. melanocarpa, due to its antioxidant action, significantly attenuated the oxidative stress (assessed by measuring of the total antioxidant status — TAS) in plasma in a model of hyperhomocysteinemia.

Proposed model for the protective role of phenolic antioxidants on selected elements of hemostasis during hyperhomocysteinemia.

various antioxidants (present in human diet), including phenolic compounds, may reduce the toxic effects of Hcy or its derivatives on hemostasis. These findings give hope for the develop development of dietary supplements, which will be capable of preventing thrombosis which occurs under pathological conditions, observed also in hyperhomocysteinemia, such as plasma procoagulant activity and oxidative stress.

Malinowska J,  Kolodziejczyk J and Olas B. The disturbance of hemostasis induced by hyper-homocysteinemia; the role of antioxidants. Acta Biochimica Polonica 2012; 59(2): 185–194.

Lipoprotein (a)

Lipoprotein (a) (Lp(a)), for the first time described in 1963 by Berg belongs to the lipoproteins with the strongest atherogenic effect. Its importance for the development of various atherosclerotic vasculopathies (coronary heart disease, ischemic stroke, peripheral vasculopathy, abdominal aneurysm) was recognized considerably later.

Lipoprotein(a) (Lp(a)), an established risk marker of cardiovascular diseases, is independent from other risk markers. The main difference of Lp(a) compared to low density lipoprotein (LDL) is the apo(a) residue, covalently bound to apoB is covalently by a disulfide-bridge. Apo(a) synthesis is performed in the liver, probably followed by extracellular assembly to the apoB location of the LDL.


ApoB-100_______LDL¬¬___ S-S –    9

Apo(a) has been detected bound to triglyceride-rich lipoproteins (Very Low Density Lipoproteins; VLDL). Corresponding to the structural similarity to LDL, both particles are very similar to each other with regard to their composition. It is a glycoprotein which underlies a large genetic polymorphism caused by a variation of the kringle-IV-type-2 repeats of the protein, characterized by a structural homology to plasminogen. Apo(a)’s structural homology to plasminogen, shares the gene localization on chromosome 6. The kringle repeats present a particularly characteristic structure, which have a high similarity to kringle IV (K IV) of plasminogen. Apo(a) also has a kringle V structure of plasminogen and also a protease domain, which cannot be activated, as opposed to the one of plasminogen. At least 30 genetically determined apo(a) isoforms were identified in man.


  • Non covalent binding of kringle -4 types 7 and 8 of apo (a) to apo B
  • Disulfide bond at Cys4326 of ApoB (near its receptor binding domain ) and the only free cysteine group in K –IV type 9 (Cys4057) of apo(a )
  • Binding to fibrin and cell membranes
  • Enhancement by small isoforms ; high concentrations compared to plasminogen and homocysteine
  • Binding to different lysine rich components of the coagulation system (e. g. TFPI)
  • Intense homology to plasminogen but no protease activity
ApoB-100_______LDL¬¬___ S-S – 9

The synthesis of Lp(a), which thus occurs as part of an assembly, is a two-step process.

  • In a first step, which can be competitively inhibited by lysine analogues, the free sulfhydryl groups of apo(a) and apoB are brought close together.
  • The binding of apo(a) then occurs near the apoB domain which binds to the LDL receptor, resulting in a reduced affinity of Lp(a) to the LDL-receptor.

Particles that show a reduced affinity to the LDL receptor are not able to form stable compounds with apo(a). Thus the largest part of apo(a) is present as apo(a) bound to LDL. Only a small, quantitatively variable part of apo(a) remains as free apo(a) and probably plays an important role in the metabolism and physiological function of Lp(a).

The Lp(a) plasma concentration in the population is highly skewed and determined to more than 90 % by genetic factors. In healthy subjects the Lp(a)-concentration is correlated with its synthesis.

It is assumed that the kidney has a specific function in Lp(a) catabolizm, since nephrotic syndrome and terminal kidney failure are associated with an elevation of the Lp(a) plasma concentration. One consequence of the poor knowledge of the metabolic path of Lp(a) is the fact that so far pharmaceutical science has failed to develop drugs that are able to reduce elevated Lp(a) plasma concentrations to a desirable level.

Plasma concentrations of Lp(a) are affected by different diseases (e.g. diseases of liver and kidney), hormonal factors (e.g. sexual steroids, glucocorticoids, thyroid hormones), individual and environmental factors (e.g. age, cigarette smoking) as well as pharmaceuticals (e.g. derivatives of nicotinic acid) and therapeutic procedures (lipid apheresis). This review describes the physiological regulation of Lp(a) as well as factors influencing its plasma concentration.

Apart from its significance as an important agent in the development of atherosclerosis, Lp(a) has even more physiological functions, e.g. in

  • wound healing
  • angiogenesis
  • hemostasis

However, in the meaning of a pleiotropic mechanism the favorable action mechanisms are opposed by pathogenic mechanisms, whereby the importance of Lp(a) in atherogenesis is stressed.

Lp(a) in Atherosclerosis

In transgenic, hyperlipidemic and Lp(a) expressing Watanabe rabbits, Lp(a) leads to enhanced atherosclerosis. Under the influence of Lp(a), the binding of Lp(a) to glycoproteins, e.g. laminin, results – via its apo(a)-part – both in

  • an increased invasion of inflammatory cells and in
  • an activation of smooth vascular muscle cells

with subsequent calcifications in the vascular wall.

The inhibition of transforming growth factor-β1 (TGF-β1) activation is another mechanism via which Lp(a) contributes to the development of atherosclerotic vasculopathies. TGF-β1 is subject to proteolytic activation by plasmin and its active form leads to an inhibition of the proliferation and migration of smooth muscle cells, which play a central role in the formation and progression of atherosclerotic vascular diseases.

In man, Lp(a) is an important risk marker which is independent of other risk markers. Its importance, partly also under consideration of the molecular weight and other genetic polymorphisms, could be demonstrated by a high number of epidemiological and clinical studies investigating the formation and progression of atherosclerosis, myocardial infarction, and stroke.

Lp(a) in Hemostasis

Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Pleiotropic effect of Lp(a).

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Antithrombotic :

  • Inhibition of platelet activating factor acetylhydrolase (PAF -AH)
  • Inhibition of platelet activating factor
  • Inhibition of collagen dependent platelet aggregation
  • Inhibition of secretion of serotonin und thromboxane

Lp(a) in Angiogenesis

Lp(a) is also important for the process of angiogenesis and the sprouting of new vessels.

  • angiogenesis starts with the remodelling of matrix proteins and
  • activation of matrix metalloproteinases (MMP).

The latter ones are usually synthesised as

  • inactive zymogens and
  • require activation by proteases,

Recall that Apo(a) is not activated by proteases. The angiogenesis is also accomplished by plasminogen. Lp(a) and apo(a) and its fragments has an antiangiogenetic and metastasis inhibiting effect related to the structural homology with plasminogen without the protease activity.

Siekmeier R, Scharnagl H, Kostner GM, T. Grammer T, Stojakovic T and März W.  Variation of Lp(a) Plasma Concentrations in Health and Disease.  The Open Clinical Chemistry Journal, 2010; 3: 72-89.


In 1985, Brown and Goldstein were awarded the Nobel Prize for medicine for their work on the regulation of cholesterol metabolism. On the basis of numerous studies, they were able to demonstrate that circulating low-density lipoprotein (LDL) is absorbed into the cell through receptor linked endocytosis. The absorption of LDL into the cell is specific and is mediated by a LDL receptor. In patients with familial hypercholesterolemia, this receptor is changed, and the LDL particles can no longer be recognized. Their absorption can thus no longer be mediated, leading to an accumulation of LDL in blood.

Furthermore, an excess supply of cholesterol also blocks the 3-hydrox-3 methylglutaryl-Co enzyme A (HMG CoA), reductase enzyme, which otherwise inhibits the cholesterol synthesis rate. Brown and Goldstein also determined the structure of the LDL receptor. They discovered structural defects in this receptor in many patients with familial hypercholesterolemia. Thus, familial hypercholesterolemia was the first metabolic disease that could be tracked back to the mutation of a receptor gene.

Dyslipoproteinemia in combination with diabetes mellitus causes a cumulative insult to the vasculature resulting in more severe disease which occurs at an earlier age in large and small vessels as well as capillaries. The most common clinical conditions resulting from this combination are myocardial infarction and lower extremity vascular disease. Ceriello et al. show an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. The combination produces greater morbidity and mortality than either alone.

As an antiatherogenic factor, HDL cholesterol correlates inversely to the extent of postprandial lipemia. A high concentration of HDL is a sign that triglyceride-rich particles are quickly decomposed in the postprandial phase of lipemia. Conversely, with a low HDL concentration this decomposition is delayed. Thus, excessively high triglyceride concentrations are accompanied by very low HDL counts. This combination has also been associated with an increased risk of pancreatitis.

The importance of lipoprotein (a) (Lp(a)) as an atherogenic substance has also been recognized in recent years. Lp(a) is very similar to LDL. But it also contains Apo(a), which is very similar to plasminogen, enabling Lp(a) to bind to fibrin clots. Binding of plasminogen is prevented and fibrinolysis obstructed. Thrombi are integrated into the walls of the arteries and become plaque components.

Another strong risk factor for accelerated atherogenesis, which must be mentioned here, are the widespread high homocysteine levels found in dialysis patients. This risk factor is independent of classic risk factors such as high cholesterol and LDL levels, smoking, hypertension, and obesity, and much more predictive of coronary events in dialysis patients than are these better-known factors. Homocysteine is a sulfur aminoacid produced in the metabolism of methionine. Under normal conditions, about 50 percent of homocysteine is remethylated to methionine and the remaining via the transsulfuration pathway.

Defining hyperhomocysteinemia as levels greater than the 90th percentile of controls and elevated Lp(a) level as greater than 30mg/dL, the frequency of the combination increased with declining renal function. Fifty-eight percent of patients with a GFR less than 10mL/min had both hyperhomocysteinemia and elevated Lp(a) levels, and even in patients with mild renal impairment, 20 percent of patients had both risk factors present.

The prognosis of patients suffering from severe hyperlipidemia, sometimes combined with elevated lipoprotein (a) levels, and coronary heart disease refractory to diet and lipid-lowering drugs is poor. For such patients, regular treatment with low-density lipoprotein (LDL) apheresis is the therapeutic option. Today, there are five different LDL-apheresis systems available: cascade filtration or lipid filtration, immunoadsorption, heparin-induced LDL precipitation, dextran sulfate LDL adsorption, and the LDL hemoperfusion. The requirement that the original level of cholesterol is to be reduced by at least 60 percent is fulfilled by all these systems.

There is a strong correlation between hyperlipidemia and atherosclerosis. Besides the elimination of other risk factors, in severe hyperlipidemia therapeutic strategies should focus on a drastic reduction of serum lipoproteins. Despite maximum conventional therapy with a combination of different kinds of lipid-lowering drugs, sometimes the goal of therapy cannot be reached. Hence, in such patients, treatment with LDL-apheresis is indicated. Technical and clinical aspects of these five different LDL-apheresis methods are depicted. There were no significant differences with respect to or concerning all cholesterols, or triglycerides observed.

High plasma levels of Lp(a) are associated with an increased risk for atherosclerotic coronary heart       disease
(CHD) by a mechanism yet to be determined. Because of its structural properties, Lp(a) can have both atherogenic and thrombogenic potentials. The means for correcting the high plasma levels of Lp(a) are still limited in effectiveness. All drug therapies tried thus far have failed. The most effective therapeutic methods in lowering Lp(a) are the LDL-apheresismethods. Since 1993, special immunoadsorption polyclonal antibody columns (Pocard, Moscow, Russia) containing sepharose bound anti-Lp(a) have been available for the treatment of patients with elevated Lp(a) serum concentrations.

With respect to elevated lipoprotein (a) levels, however, the immunoadsorption method seems to be most effective. The different published data clearly demonstrate that treatment with LDL-apheresis in patients suffering from severe hyperlipidemia refractory to maximum conservative therapy is effective and safe in long-term application.

LDL-apheresis decreases not only LDL mass but also improves the patient’s life expectancy. LDL-apheresis performed with different techniques decreases the susceptibility of LDL to oxidation. This decrease may be related to a temporary mass imbalance between freshly produced and older LDL particles. Furthermore, the baseline fatty acid pattern influences pretreatment and postreatment susceptibility to oxidation.

Bambauer R, Bambauer C, Lehmann B, Latza R, and Ralf Schiel R. LDL-Apheresis: Technical and Clinical Aspects. The Scientific World Journal 2012; Article ID 314283, pp 1-19. doi:10.1100/2012/314283

Summary:  This discussion is a two part sequence that first establishes the known strong relationship between blood flow viscosity, shear stress, and plasma triglycerides (VLDL) as risk factors for hemostatic disorders leading to thromboembolic disease, and the association with atherosclerotic disease affecting the heart, the brain (via carotid blood flow), peripheral circulation,the kidneys, and retinopathy as well.

The second part discusses the modeling of hemostasis and takes into account the effects of plasma proteins involved with red cell and endothelial interaction, which is related to part I.  The current laboratory assessment of thrombophilias is taken from a consensus document of the American Society for Clinical Pathology.  The problems encountered are sufficient for the most common problems of coagulation testing and monitoring, but don’t address the large number of patients who are at risk for complications of accelerated vasoconstrictive systemic disease that precede serious hemostatic problems.  Special attention is given to Lp(a) and to homocysteine.  Lp(a) is a protein that has both prothrombotic and antithrombotic characteristics, and is a homologue of plasminogen and is composed of an apo(a) bound to LDL.  Unlike plasminogen, it has no protease activity.   Homocysteine elevation is a known risk factor for downstream myocardial infarct.  Homocysteine is a mirror into sulfur metabolism, so an increase is an independent predictor of risk, not fully discussed here.  The modification of risk is discussed by diet modification.  In the most serious cases of lipoprotein disorders, often including Lp(a) the long term use of LDL-apheresis is described.

see Relevent article that appears in NEJM from American College of Cardiology

Apolipoprotein(a) Genetic Sequence Variants Associated With Systemic Atherosclerosis and Coronary Atherosclerotic Burden but Not With Venous Thromboembolism

Helgadottir A, Gretarsdottir S, Thorleifsson G, et al

J Am Coll Cardiol. 2012;60:722-729

Study Summary

The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] — a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.

LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with levels of Lp(a) and, as a consequence, strongly associated with CAD. However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity), as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism). Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.

To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for 2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1] Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to widen the generalizability of their results.

Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with CAD,  peripheral arterial disease, abdominal aortic aneurysm, number of CAD vessels, age at onset of CAD diagnosis, and large-artery atherosclerosis-type stroke. They did not find an association with cardioembolic or small-vessel disease-type stroke; intracranial aneurysm; venous thrombosis; carotid intima thickness; or, in a small subset of individuals, myocardial infarction.


The main conclusion to draw from this work is that Lp(a) is probably a strong causal factor in not only CAD, but also the development of atherosclerosis in other arterial trees. Although there is no evidence from this study that Lp(a) levels contribute to venous thrombosis, the investigators do not exclude a role for Lp(a) in arterial thrombosis.

Large-artery atherosclerosis stroke is thought to involve some element of arterial thrombosis or thromboembolism, [2] and genetic substudies of randomized trials of aspirin demonstrate that individuals with LPA variants predicted to have elevated levels of Lp(a) benefit the most from antiplatelet therapy. [3] Together, these data suggest that Lp(a) probably has clinically relevant effects on the development of atherosclerosis and arterial thrombosis.

Of  note, the investigators found no association between Lp(a) and carotid intima thickness, suggesting that either intima thickness is a poor surrogate for the clinical manifestations of atherosclerosis or that Lp(a) affects a distinct step in the atherosclerotic disease process that is not demonstrable in the carotid arteries.

Although Lp(a) testing is available, these studies do not provide any evidence that testing for Lp(a) is of clinical benefit, or that screening for atherosclerosis should go beyond well-described clinical risk factors, such as low-density lipoprotein cholesterol levels, high-density lipoprotein levels, hypertension, diabetes, smoking, and family history. Until evidence demonstrates that adding information on Lp(a) levels to routine clinical practice improves the ability of physicians to identify those at highest risk for atherosclerosis, Lp(a) testing should remain a research tool. Nevertheless, these findings do suggest that therapies to lower Lp(a) may have benefits that extend to forms of atherothrombosis beyond the coronary tree.

The finding of this study is interesting:

[1] It consistent with Dr. William LaFramboise..   examination specifically at APO B100, which is part of Lp(a) with some 14 candidate predictors for a more accurate exclusion of patients who don’t need intervention.          Apo B100 was not one of 5 top candidates.

William LaFramboise • Our study ( comprised discovery research using targeted immunochemical screening of retrospective patient samples using both Luminex and Aushon platforms as opposed to shotgun proteomics. Hence the costs constrained sample numbers. Nevertheless, our ability to predict outcome substantially exceeded available methods:

The Framingham CHD scores were statistically different between groups (P <0.001, unpaired Student’s t test) but they classified only 16% of the subjects without significant CAD (10 of 63) at a 95% sensitivity for patients with CAD. In contrast, our algorithm incorporating serum values for OPN, RES, CRP, MMP7 and IFNγ identified 63% of the subjects without significant CAD (40 of 63) at 95% sensitivity for patients with CAD. Thus, our multiplex serum protein classifier correctly identified four times as many patients as the Framingham index.

This study is consistent with the concept of CAD, PVD, and atheromatous disease is a systemic vascular disease, but the point that is made is that it appears to have no relationship to venous thrombosis. The importance for predicting thrombotic events is considered serious.   The venous flow does not have the turbulence of large arteries, so the conclusion is no surprise.  The flow in capillary beds is a linear cell passage with minimal viscosity or turbulence.  The finding of no association with carotid artery disease  is interpreted to mean that the Lp(a) might be an earlier finding than carotid intimal thickness.  It is reassuring to find a recommendation for antiplatelet therapy for individuals with LPA variants based on randomized trials of aspirin substudies.

If that is the conclusion from the studies, and based on the strong association between the prothrombotic (pleiotropic) effect and the association with hyperhomocysteinemia, my own impression is that the recommendation is short-sighted.

[2]  Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Source for Lp(a)

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

References on Triglycerides and blood viscosity

Lowe GD, Lee AJ, Rumley A, et al. Blood viscosity and risk of cardiovascular events: the Edinburgh Artery Study. Br J Haematol 1997; 96:168-173.

Sloop GD. A unifying theory of atherogenesis. Med Hypotheses. 1996; 47:321-5.
Smith WC, Lowe GD, et al. Rheological determinants of blood pressure in a Scottish adult population. J Hypertens 1992; 10:467-72.

Letcher RL, Chien S, et al. Direct relationship between blood pressure and blood viscosity in normal and hypertensive subjects. Role of fibrinogen and concentration. Am J Med 1981; 70:1195-1202.

Devereux RB, Case DB, Alderman MH, et al. Possible role of increased blood viscosity in the hemodynamics of systemic hypertension. Am J Cardiol 2000; 85:1265-1268.

Levenson J, Simon AC, Cambien FA, Beretti C. Cigarette smoking and hypertension. Factors independently associated with blood hyperviscosity and arterial rigidity. Arteriosclerosis 1987; 7:572-577.

Sloop GD, Garber DW. The effects of low-density lipoprotein and high-density lipoprotein on blood viscosity correlate with their association with risk of atherosclerosis in humans. Clin Sci 1997; 92:473-479.

Lowe GD. Blood viscosity, lipoproteins, and cardiovascular risk. Circulation 1992; 85:2329-2331.

Rosenson RS, Shott S, Tangney CC. Hypertriglyceridemia is associated with an elevated blood viscosity: triglycerides and blood viscosity. Atherosclerosis 2002; 161:433-9.

Stamos TD, Rosenson RS. Low high density lipoprotein levels are associated with an elevated blood viscosity. Atherosclerosis 1999; 146:161-5.

Hoieggen A, Fossum E, Moan A, Enger E, Kjeldsen SE. Whole-blood viscosity and the insulin-resistance syndrome. J Hypertens 1998; 16:203-10.

de Simone G, Devereux RB, Chien S, et al. Relation of blood viscosity to demographic and physiologic variables and to cardiovascular risk factors in apparently normal adults. Circulation 1990; 81:107-17.

Rosenson RS, McCormick A, Uretz EF. Distribution of blood viscosity values and biochemical correlates in healthy adults. Clin Chem 1996; 42:1189-95.

Tamariz LJ, Young JH, Pankow JS, et al. Blood viscosity and hematocrit as risk factors for type 2 diabetes mellitus: The Atherosclerosis Risk in Communities (ARIC) Study. Am J Epidemiol 2008; 168:1153-60.

Jax TW, Peters AJ, Plehn G, Schoebel FC. Hemostatic risk factors in patients with coronary artery disease and type 2 diabetes – a two year follow-up of 243 patients. Cardiovasc Diabetol 2009; 8:48.

Ernst E, Weihmayr T, et al. Cardiovascular risk factors and hemorheology. Physical fitness, stress and obesity. Atherosclerosis 1986; 59:263-9.

Hoieggen A, Fossum E, et al. Whole-blood viscosity and the insulin-resistance syndrome. J Hypertens 1998; 16:203-10.

Carroll S, Cooke CB, Butterly RJ. Plasma viscosity, fibrinogen and the metabolic syndrome: effect of obesity and cardiorespiratory fitness. Blood Coagul Fibrinolysis 2000; 11:71-8.

Ernst E, Koenig W, Matrai A, et al. Blood rheology in healthy cigarette smokers. Results from the MONICA project, Augsburg. Arteriosclerosis 1988; 8:385-8.

Ernst E. Haemorheological consequences of chronic cigarette smoking. J Cardiovasc Risk 1995; 2:435-9.

Lowe GD, Drummond MM, Forbes CD, Barbenel JC. The effects of age and cigarette-smoking on blood and plasma viscosity in men. Scott Med J 1980; 25:13-7.

Kameneva MV, Watach MJ, Borovetz HS. Gender difference in rheologic properties of blood and risk of cardiovascular diseases. Clin Hemorheol Microcirc 1999; 21:357-363.

Fowkes FG, Pell JP, Donnan PT, et al. Sex differences in susceptibility to etiologic factors for peripheral atherosclerosis. Importance of plasma fibrinogen and blood viscosity. Arterioscler Thromb 1994; 14:862-8.

Coppola L, Caserta F, De Lucia D, et al. Blood viscosity and aging. Arch Gerontol Geriatr 2000; 31:35-42.


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Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

English: The citric acid cycle, also known as ...

English: The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. Produced at WikiPathways. (Photo credit: Wikipedia)

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome


Reporter& Curator:  Larry Bernstein, MD, FCAP


















Unlocking the diversity of genomic expression within tumorigenesis and “tailoring” of therapeutic options

1. Reshaping the DNA landscape between diseases and within diseases by the linking of DNA to treatments

In the NEW York Times of 9/24,2012 Gina Kolata reports on four types of breast cancer and the reshaping of breast cancer DNA treatment based on the findings of the genetically distinct types, which each have common “cluster” features that are driving many cancers.  The discoveries were published online in the journal Nature on Sunday (9/23).  The study is considered the first comprehensive genetic analysis of breast cancer and called a roadmap to future breast cancer treatments.  I consider that if this is a landmark study in cancer genomics leading to personalized drug management of patients, it is also a fitting of the treatment to measurable “combinatorial feature sets” that tie into population biodiversity with respect to known conditions.   The researchers caution that it will take years to establish transformative treatments, and this is clearly because in the genetic types, there are subsets that have a bearing on treatment “tailoring”.   In addition, there is growing evidence that the Watson-Crick model of the gene is itself being modified by an expansion of the alphabet used to construct the DNA library, which itself will open opportunities to explain some of what has been considered junk DNA, and which may carry essential information with respect to metabolic pathways and pathway regulation.  The breast cancer study is tied to the  “Cancer Genome Atlas” Project, already reported.  It is expected that this work will tie into building maps of genetic changes in common cancers, such as, breast, colon, and lung.  What is not explicit I presume is a closely related concept, that the translational challenge is closely related to the suppression of key proteomic processes tied into manipulating the metabolome.

Saha S. Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations. 9/12/2012.

Hawrylycz MJ, Lein ES, Guillozet-Bongaarts AL, Shen EH, Ng L, et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature  Sept 14-20, 2012

Sarkar A. Prediction of Nucleosome Positioning and Occupancy Using a Statistical Mechanics Model. 9/12/2012.

Heijden et al.   Connecting nucleosome positions with free energy landscapes. (Proc Natl Acad Sci U S A. 2012, Aug 20 [Epub ahead of print]).

2. Fiddling with an expanded genetic alphabet – greater flexibility in design of treatment (pharmaneogenesis?)

Diagram of DNA polymerase extending a DNA stra...

Diagram of DNA polymerase extending a DNA strand and proof-reading. (Photo credit: Wikipedia)

A clear indication of this emerging remodeling of the genetic alphabet is a new
study led by scientists at The Scripps Research Institute appeared in the
June 3, 2012 issue of Nature Chemical Biology that indicates the genetic code as
we know it may be expanded to include synthetic and unnatural sequence pairing (Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought, Genome). They infer that the genetic instructions for living organisms
that is composed of four bases (C, G, A and T)— is open to unnatural letters. 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. The implications of the application of this would further expand the translation of portions of DNA to new transciptional proteins that are heretofore unknown, but have metabolic relavence and therapeutic potential. The existence of such pairing in nature has been studied in Eukariotes for at least a decade, and may have a role in biodiversity. The investigators show how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.  This could as well be translated into human diversity, and human diseases.

The Romesberg laboratory collaborated on the new study 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). It had been clear that their chemical structures 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, but that is not the case because other bonds can be in play.

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 that resembles a ‘mispair.’ In test after test, the NaM-5SICS pair was efficiently replicable even though it appeared that the DNA polymerase didn’t recognize it. Their structural data showed that 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. NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium.

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could be used to extend the DNA alphabet and that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been developed allowing for life based on other genetic systems.

3.  Studies that consider a DNA triplet model that includes one or more NATURAL nucleosides and looks closely allied to the formation of the disulfide bond and oxidation reduction reaction.

This independent work is being conducted based on a similar concep. John Berger, founder of Triplex DNA has commented on this. He emphasizes Sulfur as the most important element for understanding evolution of metabolic pathways in the human transcriptome. It is a combination of sulfur 34 and sulphur 32 ATMU. S34 is element 16 + flourine, while S32 is element 16 + phosphorous. The cysteine-cystine bond is the bridge and controller between inorganic chemistry (flourine) and organic chemistry (phosphorous). He uses a dual spelling, using  sulfphur to combine the two referring to the master catalyst of oxidation-reduction reactions. Various isotopic alleles (please note the duality principle which is natures most important pattern). Sulfphur is Methionine, S adenosylmethionine, cysteine, cystine, taurine, gluthionine, acetyl Coenzyme A, Biotin, Linoic acid, H2S, H2SO4, HSO3-, cytochromes, thioredoxin, ferredoxins, purple sulfphur anerobic bacteria prokaroytes, hydrocarbons, green sulfphur bacteria, garlic, penicillin and many antibiotics; hundreds of CSN drugs for parasites and fungi antagonists. These are but a few names which come to mind. It is at the heart of the Krebs cycle of oxidative phosphorylation, i.e. ATP. It is also a second pathway to purine metabolism and nucleic acids. It literally is the key enzymes between RNA and DNA, ie, SH thiol bond oxidized to SS (dna) cysteine through thioredoxins, ferredoxins, and nitrogenase. The immune system is founded upon sulfphur compounds and processes. Photosynthesis Fe4S4 to Fe2S3 absorbs the entire electromagnetic spectrum which is filtered by the Allen belt some 75 miles above earth. Look up chromatium vinosum or allochromatium species.  There is reasonable evidence it is the first symbiotic species of sulfphur anerobic bacteria (Fe4S4) with high potential mvolts which drives photosynthesis while making glucose with H2S.
He envisions a sulfphur control map to automate human metabolism with exact timing sequences, at specific three dimensional coordinates on Bravais crystalline lattices. He proposes adding the inosine-xanthosine family to the current 5 nucleotide genetic code. Finally, he adds, the expanded genetic code is populated with “synthetic nucleosides and nucleotides” with all kinds of customized functional side groups, which often reshape nature’s allosteric and physiochemical properties. The inosine family is nature’s natural evolutionary partner with the adenosine and guanosine families in purine synthesis de novo, salvage, and catabolic degradation. Inosine has three major enzymes (IMPDH1,2&3 for purine ring closure, HPGRT for purine salvage, and xanthine oxidase and xanthine dehydrogenase.

English: DNA replication or DNA synthesis is t...

English: DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. (Photo credit: Wikipedia)

3. Nutritional regulation of gene expression,  an essential role of sulfur, and metabolic control 

Finally, the research carried out for decades by Yves Ingenbleek and the late Vernon Young warrants mention. According to their work, sulfur is again tagged as essential for health. Sulfur (S) is the seventh most abundant element measurable in human tissues and its provision is mainly insured by the intake of methionine (Met) found in plant and animal proteins. Met is endowed with unique functional properties as it controls the ribosomal initiation of protein syntheses, governs a myriad of major metabolic and catalytic activities and may be subjected to reversible redox processes contributing to safeguard protein integrity.

Consuming diets with inadequate amounts of methionine (Met) are characterized by overt or subclinical protein malnutrition, and it has serious morbid consequences. The result is reduction in size of their lean body mass (LBM), best identified by the serial measurement of plasma transthyretin (TTR), which is seen with unachieved replenishment (chronic malnutrition, strict veganism) or excessive losses (trauma, burns, inflammatory diseases).  This status is accompanied by a rise in homocysteine, and a concomitant fall in methionine.  The ratio of S to N is quite invariant, but dependent on source.  The S:N ratio is typical 1:20 for plant sources and 1:14.5 for animal protein sources.  The key enzyme involved with the control of Met in man is the enzyme cystathionine-b-synthase, which declines with inadequate dietary provision of S, and the loss is not compensated by cobalamine for CH3- transfer.

As a result of the disordered metabolic state from inadequate sulfur intake (the S:N ratio is lower in plants than in animals), 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. They both stimulate comparable remethylation reactions from homocysteine (Hcy), indicating that Met homeostasis benefits from high metabolic priority. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing reducing molecules implicated in the regulation of the 3 desulfuration pathways

4. The effect on accretion of LBM of protein malnutrition and/or the inflammatory state: in closer focus

Hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly and liver production of  TTR integrates the dietary and stressful components of any disease spectrum. Thus we have a depletion of visceral transport proteins made by the liver and fat-free weight loss secondary to protein catabolism. This is most accurately reflected by TTR, which is a rapid turnover protein, but it is involved in transport and is essential for thyroid function (thyroxine-binding prealbumin) and tied to retinol-binding protein. Furthermore, protein accretion is dependent on a sulfonation reaction with 2 ATP.  Consequently, Kwashiorkor is associated with thyroid goiter, as the pituitary-thyroid axis is a major sulfonation target. With this in mind, it is not surprising why TTR is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequaled 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 hyper-homocysteinemic states.

Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes. But the N balance study only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved. Using vastly improved methods the LBM has been measured in its components. 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).

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. Sulfur Methylation (SM) undergoes a 15% reduction in size per decade, an involutive process. 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 or in critically ill patients whose muscle mass undergoes proteolysis.

5.  The results of the events described are:

  • Declining generation of hydrogen sulfide (H2S) from enzymatic sources and in the non-enzymatic reduction of elemental S to H2S.
  • 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.
  • Elemental S operates as co-factor of several (apo)enzymes critically involved in the control of oxidative processes.

Combination of protein and sulfur dietary deficiencies constitute a novel clinical entity threatening plant-eating population groups. They have a defective production of Cys, GSH and H2S reductants, explaining persistence of an oxidative burden.

6. The clinical entity increases the risk of developing:

  • cardiovascular diseases (CVD) and
  • stroke

in plant-eating populations regardless of Framingham criteria and vitamin-B status.
Met molecules supplied by dietary proteins are submitted to transmethylation processes resulting in the release of Hcy which:

  • either undergoes Hcy — Met RM pathways or
  • is committed to transsulfuration decay.

Impairment of CβS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. The data show that 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.

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.

Ingenbleek Y. Hyperhomocysteinemia is a biomarker of sulfur-deficiency in human morbidities. Open Clin. Chem. J. 2009 ; 2 : 49-60.

7. The dysfunctional metabolism in transitional cell transformation

A third development is also important and possibly related. The transition a cell goes through in becoming cancerous tends to be driven by changes to the cell’s DNA. But that is not the whole story. Large-scale techniques to the study of metabolic processes going on in cancer cells is being carried out at Oxford, UK in collaboration with Japanese workers. This thread will extend our insight into the metabolome. Otto Warburg, the pioneer in respiration studies, pointed out in the early 1900s that most cancer cells get the energy they need predominantly through a high utilization of glucose with lower respiration (the metabolic process that breaks down glucose to release energy). It helps the cancer cells deal with the low oxygen levels that tend to be present in a tumor. The tissue reverts to a metabolic profile of anaerobiosis.  Studies of the genetic basis of cancer and dysfunctional metabolism in cancer cells are complementary. Tomoyoshi Soga’s large lab in Japan has been at the forefront of developing the technology for metabolomics research over the past couple of decades (metabolomics being the ugly-sounding term used to describe research that studies all metabolic processes at once, like genomics is the study of the entire genome).

Their results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer. The collaborators have published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism. The researchers summarize that shows how accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell. It shifts the balance of metabolic processes and disrupts the cell in ways that could favor development of cancer.  This is of particular interest because “fumarate” is the intermediate in the TCA cycle that is converted to malate.

Animation of the structure of a section of DNA...

Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. (Photo credit: Wikipedia)

The Keio group is able to label glucose or glutamine, basic biological sources of fuel for cells, and track the pathways cells use to burn up the fuel.  As these studies proceed, they could profile the metabolites in a cohort of tumor samples and matched normal tissue. This would produce a dataset of the concentrations of hundreds of different metabolites in each group. Statistical approaches could suggest which metabolic pathways were abnormal. These would then be the subject of experiments targeting the pathways to confirm the relationship between changed metabolism and uncontrolled growth of the cancer cells.

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