Some Humifulvate® Science - Chapter 2: Components of Humic Substances
2. THE COMPONENTS IF HUMIC SUBSTANCES
2.1. Humic acid
Structural and chemical properties
One could assume that due to the ubiquitous nature and structural and chemical properties of humic substances the environment is dependent on them. It is known that the specific functional groups of humic acids are responsible for chelating various compounds in the environment, thereby improving nutrient utilisation and preventing metal toxicity in waters, soils, and thus possibly in plants, animals and humans as well.
Humic acids from peats show significant levels of phenolic carbons (C6) and methoxyl carbons (-OCH3), associated with the presence of lignin-like materials [2]. Lignin, being the starting material of humic and fulvic acid, and various phenolic compounds such as vanillin, vanillin acid, resorcinol, ferulic acid, protochatechuic acid and benzoic acid are the degradation products of these lignins [15, 20]. It is apparent that humic substances consist of a heterogeneous mixture of compounds for which no single structural formula will suffice. Nevertheless, humic acids are thought to be complex aromatic macromolecules with amino acids, amino sugars, peptides and aliphatic compounds involved in linkages between the aromatic groups. The hypothetical structure for humic acid is shown in Figure 1 below. It contains free and bound phenolic OH groups, quinone structures, nitrogen and oxygen as bridge units and carboxylic acid groups variously placed on aromatic rings.
Figure 1.
Because of the variable molecular composition of humic acids, a wide range of dissociation constants exists for the metals that are chelated by humic acids [1]. In addition, different metals are bound to humic acids with varying strength, and this would mean that a particular chelate-bond cation will modify the binding stability of the other metal linkages. This peculiar metal binding capacity of humic acids is exemplified by the fact that when some alkali metals, such as K and Na, are bound by previously empty functional groups, then the chelated bonds of Fe and Al may rupture easier than if the humic acid molecule contains an alkali earth metal, such as Ca [1]. This is why vegetation suffers from microelement deficiency in the presence of Ca-humate in the soils, although the needed elements abound in the humus. This peculiar metal binding capacity also protects plants by the ability of water-soluble fractions of humic substances (humic and fulvic acids) to form precipitates with a number of metals (Ca, Cd, Hg, Pb, Ba), forming insoluble complexes. The complexes formed are not available to plants and the concentration of toxications in the soil solution is reduced [2]. This may also partly explain the role of humic substances in alleviating heavy metal toxicity in humans.
Conformational changes also occur when metals bind to different sites of the humic acid. This can affect the binding stability of various metals [13, 21]. Different metals have different affinities for different binding sites of the humic acid. The pH, ionic strength, molecular weight and functional group content are all factors influencing the quantity of metal ions bound by humic substances [6]. But most importantly, due to the heterogeneous molecular composition of humic acids, a given metal may bind very strongly, while another humic acid may affix or release the same metal much easier [1].
In regards to the metal ion exchange processes, it is thought that the humic acids with smaller molecular mass bind metals 2 to 6 times better than larger molecules from the same sample. Additionally, bivalent ions (e.g. Zn2+, Cu2+, Ca2+, Mn2+, Cd2+) are more likely to become bound as compared to trivalent ions (e.g. Cr3+, Al3+). The bivalent ions that have been characterised based on their ability for cation exchange are in the following order:
Pb2+>Cd2+>Cu2+>Ni2+>Fe2+>Co2+>Mn2+>Zn2+>VO22+ [1].
Biological role
Although the physical and chemical properties of humic acids and their biological role in bacteria, fungi, viruses and plants have been well documented, it is not yet clear exactly how humic acids affect mammalian cells. Research indicates that humic acid is absorbed in vivo, and can act as an active agent modifying biochemical reactions. Its effects on cell metabolism, enzymes, free radicals and minerals have been documented in the literature. In addition, due to the ability of humic acids to form bonds with metal ions, they are also responsible for forming complexes with amino acids, peptides, carbohydrates and steroids. These physicochemical properties of humic acids may also be responsible for some of the effects occurring in tissues; including the elimination of heavy metals, desmutagenic effects, antioxidant and anticoagulant activity.
Absorption and bioavailability
Studies involving rats demonstrated that the liver is the organ in which humic acids are absorbed and broken down. The breakdown of humic acids may take place also in the gut, as some microorganisms are known to utilise them as their source of organic carbon [2]. Other research reports that after a single oral dose of humic substances given to rats, the bioavailability was determined to be 0.1% of the administered dose, indicating that only a small fraction of the humic substances was absorbed through the intestinal tract [22].
Metabolic effects
Humic acids seem to accelerate cell metabolism, the rate of breakdown of glucose, leucine and uridine. Humic acids seem to retard the rate of incorporation of these organic molecules into the liver, but once they are absorbed, humic acids appear to accelerate their metabolism. [7].
There are other data also that support the indirect influence of humic acids in enhancing the utilisation of nutrients. Humic acids are known to bind inorganic ions and thus facilitate the transport of these minerals through the intestinal membrane of rats [7]. The amount of transfer across the intestinal membrane was found to increase in the following order: alkali metals (Na, K) by 1 - 16%; alkali earth metals (Mg, Ca) by 50% and heavier metals (Mn, Fe, Zn) 80%. Elements such as Mn, Fe, Cd and Zn are known to be able to participate actively in ligand formation with organic compounds and therefore, the ability of humic acids to act as ligand formers, may explain their facilitator action of transporting inorganic ions through biological membranes.
It has also been suggested that humic acids promote the restoration of energy levels by stimulating increased oxygen uptake resulting in the generation of energy-rich molecules necessary for metabolic processes. Humic acids can apparently stimulate respiration and increase the efficiency of oxidative phosphorylation in rat liver mitochondria. [3]. Cellular respiration, occurring only in the presence of oxygen, results in the breakdown of nutrient molecules to generate ATP. Cells, such as in the liver and muscle, use this ATP for energy to fuel various processes like stimulating the uptake of nutrients [23]. As a result, more energy is generated for the individual for sustaining his normal functioning and for compensating for extra energy requirement caused by illness or some other stress.
Removal of heavy metals
It appears that the cation exchange capacity and ligand formation ability of humic substances may partially explain why humic acids can bind and release ions of lower atomic mass while binding heavier ions with a higher atomic mass. It is known that lead and cadmium are among those bivalent ions that are most likely to be bound to the humic acid molecule. This is of great significance since both metals are considered toxic when accumulated in biological systems.
Cadmium, lead and mercury are among the most toxic and ubiquitous environmental metallic contaminants. to which the population is exposed. Cadmium is known to be toxic to every body system, whether ingested or inhaled, and tends to accumulate in body tissues. The half-life of cadmium is 20 to 30 years. Cadmium is a known central nervous system neurotoxin and carcinogen. Daily intakes of 25 to 60 mcg of cadmium for a 70 kg individual have been estimated for typical diets in Europe and the United States [9].
Long-term exposures to cadmium can result in renal damage [9]. Cadmium toxicity is manifested in a variety of syndromes, including kidney dysfunction [9, 10], hypertension, hepatic injury and lung damage. Furthermore, cadmium has an affinity to bind the transport proteins for minerals and displaces the ability of Cu and Zn to bind and be utilised effectively in the body [19].
The nervous system of new-borns and infants is highly sensitive to lead exposure. The crucial effect of lead exposure on adults is hypertension: a significant direct correlation has been shown between blood lead levels and high blood pressure. This was manifested most explicitly in the blood pressure of men of 40 to 59 years of age.
The toxicity of mercury greatly depends on the form in which it appears: the elementary form and the inorganic and organic mercury compounds show different toxicity properties. The neurotoxicity of mercury poses the highest risk for the adult population, while methyl mercury has been shown to affect the development of the foetus in pregnant women.
Several studies were conducted in order to describe more clearly the role and mechanism of humic acids in alleviating heavy metal toxicity. These studies have produced conflicting results. In one study to determine the effect of humic acid on the absorption of cadmium in rat intestine, researchers found that an increased distribution of cadmium to the metallothionein fraction may contribute to a lower absorption of cadmium in the intestine.
It was concluded that the complexation of cadmium to the humic acid does not happen in the intestinal lumen, but rather humic acid may be responsible for influencing the metabolism of cadmium inside the cells (as reflected by the increased distribution of cadmium to the transport proteins), instead of affecting the uptake of cadmium into the intestinal cells [18].
Another study was designed to determine if the formation of cadmium complexes with humic acid would occur in the intestinal lumen. The observed effects on intestinal absorption and tissue accumulation of this complex were also studied [22]. In contrast to the decreased absorption of cadmium in the presence of humic acid in the intestines of rats in the previous study [18], the absorption of cadmium in mice was not affected by humic compounds in this study [22]. However, the organ distribution of cadmium was affected after absorption as indicated by the decreased fractional retention in the kidneys at the highest humic acid exposure level.
The authors explained the difference in results in the two former studies. In the first study the intestinal lumen was carefully rinsed and competing complexing agents normally present were removed. However, in the latter study this was not the case and therefore, a dissociation of the cadmium humic complex could occur in the presence of other binding ligands.
Furthermore, the heterogeneous nature of humic substances and varying functional group capacity could also be responsible for different results. Further studies are warranted to clarify the role that humic substances play in the speciation of cadmium in the intestines and their role in the distribution of this metal in the body.
Apart from these studies conducted with HFC (Humifulvate multimineral concentrate) there are only sporadic references to the relationship of lead and mercury with humin substances.
Desmutagenic effects
Due to the chelating properties of humic acids, emphasis has been placed on the possible role of these substances in preventing mutagenesis. A number of medicines, chemicals and physical agents such as ionising radiation and ultraviolet light have the ability to act as mutagens and cause genetic mutations. Some natural plant-derived materials (e.g. humic acids, gycyrrhiza glabra extract, glutathione and bioflavinoids) have been classified as desmutagens or antimutagens based on their ability to react with or bind to formed mutagens, or break down a mutagen or promutagen, thereby providing a means of defence against mutagenesis [26].
Research suggests that the desmutagenic activity of humic acids is characterised by their ability to adsorb mutagens rather then decompose them [27]. By adsorption or through the formation of humic acid-mutagen complexes, humic acids may act extracellularly by preventing the formation of genotoxic compounds that would affect the DNA of the cell [8]. This is more characteristic of humic acids than the direct protection of the DNA from damage at the intracellular level.
Thus, the mechanism of action of humic acid is not by inhibition of the metabolism of the mutagen, but the humic acid is instead binding to and inactivating the mutagen. It has been found that the ability of humic acid to adsorb mutagens increases with the molecular weight of humic acid. [27].
Cardioprotective effects
The leading health care problem and cause of death in the United States is cardiovascular disease, with 733,834 deaths in 1996. In 1994 22.3 million Americans were reported to be suffering from heart disease [29]. Risk factors associated with the development of this disease include obesity, high blood pressure, diabetes, smoking and decreased antioxidant protection. Atherosclerosis, or the narrowing of the blood vessels, may lead to heart attacks. During an ischaemic insult, alleviating arrythmias upon the return of blood flow to the heart muscle (reperfusion) are important in protecting the cardiac muscle. Additional damage upon reperfusion includes increased free radical production, histological damage to the heart tissue and increased blood-clotting activity, which can further exacerbate the narrowing of blood vessels and ischaemia. It is thought that humic acid may scavenge free radicals and thereby decrease blood-clotting activity. Studies have documented the potential cardioprotective role of humic acid [30, 31].
In an experiment, in which rats were subjected to ischaemic insult, the administration of standardised Humifulvate concentrate (HFC) with microelements known as the Humet®-R product manifested beneficial effects. Coronary blood flow, aortic blood flow and left ventricular and diastolic pressure were improved in the hearts of the rats [30]. More data are needed to substantiate the mechanism by which the humic acid is beneficial as a cardioprotective agent.
It has been proposed that humic acid could play a protective role during myocardial reperfusion by exhibiting antioxidant activity. Humic acid may have the ability, as an antioxidant, to limit the potential formation of oxyradicals produced during tissue injury that occurs with ischaemia and reperfusion.
Another potential role for humic acids as cardioprotective agents has been exhibited in an in vivo study examining the anticoagulant effects of these humates [31]. Although a less potent anticoagulant effect was seen with the natural humic acids as compared to the synthetic, the use of the natural substances may still be effective in the treatment of thrombotic disorders.
Many of the biological actions of humic acid are thought to occur because of their complex chemical structure, consisting of numerous phenol and quinone rings [6], held together through epsilon donor acceptor complexes [32]. These epsilon donor acceptor complexes contain molecules that have electrons to donate as well as molecules, like molecular oxygen, which are considered epsilon acceptors because they accept an electron. A reactive free radical is formed during the transfer of an electron to molecular oxygen leaving the other molecule with a single unpaired electron, which will, in turn, react with other molecules within the compound. This resulting epsilon transfer brings about molecular bonds between the individual molecules that are further stabilised producing intermolecular mesomery. These complexes may then form covalent hydrogen and epsilon bonds with macromolecules [1, 32], inorganic compounds and exogenous species (viruses, mutagens etc.). Therefore, it is thought that humic substances interact with a wide array of reactants in the environment, such as carbohydrates, amino acids, phenols, enzymes, minerals, free radicals, viruses and mutagens. It is then possible to assume that these interactions also occur in vivo and in humans with the potential for modulating biochemical functions; therefore, establishing a role for them in the health of these biological systems.
Safety and toxicology
Contrary to a large body of research confirming that peat derived humic substances are safe, some research indicates that humic substances in well water may be a potential cause in the development of an endemic peripheral vascular disease known as "Blackfoot disease". Furthermore, the potential mutagenic and prooxidant effects of particular humic acids have been documented in vitro and in vivo. Because humic acids occur ubiquitously in our environment and researchers are considering supplementing the diet with these substances, information concerning the safety of humic acid should be considered.
Blackfoot disease found in the south-western coast of Taiwan is a chronic disease of infarction (death of tissue following cessation of blood supply) in blood vessel terminals [33]. Clinically, it is characterised by numbness, black discoloration, ulceration or gangrenous changes in the extremities. A high concentration of humic acid (200 ppm) was found in well water from the areas where Blackfoot disease is endemic, as well as a high arsenic content [34]. Inhabitants of the endemic areas are very prone to chronic arsenism [35]. Both Blackfoot disease and arsenism are limited to people drinking artesian well water with a variable but high concentration of arsenic (0.10 - 1.81 ppm).
Chinese researchers [33, 35, 36] hypothesised that the combined effects of the arsenic and humic acid content of the well water may cause this endemic disease. It appears that arsenic alone does not have an effect on blood coagulation; therefore, arsenic may act as an auxiliary agent when combined with high amounts of humic acid to increase blood clotting in vitro.
Additional in vitro research indicates that fluorescent humic acid is a potent inhibitor of protein C activity, when in the presence of arsenic, which enhances protein C activity [34]. Protein C is responsible for the prevention of blood coagulation or clotting and it also acts indirectly as a promoter of plasminogen activators. These results do not support the data from other research [31] that suggest that peat humic acid may be effective in promoting plasminogen activator and thus dissolving thrombi in vivo. Protein C activity is only one component of a complex system of blood clotting. Additional research [33] indicates that well water humic acid inhibits human fibrin clot formation in vitro, and therefore may be a factor in the balance of blood coagulation and anti-coagulation. The concentrations of humic acids used in the above in vitro experiments were significantly higher than the normal physiological concentrations.
Blackfoot disease is endemic to a particular area where the average daily intake of humic acid is estimated to be 400 mg [38], and high levels of arsenic intake have also been reported. As the composition and thus physicochemical properties of humic substances vary with different geographic regions, fluorescent humic acid from well water in China and humic acid derived from peat and their effects on blood clotting were compared. It is apparent that the humic acid from two different sources affected the same parameter of the blood clotting system in different ways. Therefore, it cannot be assumed that the same physiological effects, as in Blackfoot disease, would be seen if humic acids were isolated from a different source, used in much smaller doses, and given in the absence of arsenic. More research is needed to elucidate the role of natural humic acids from well water in vascular disorders.
Although there has been a great deal of attention focused on the carcinogenic nature of compounds complexed with humic acid (e.g. arsenic), only one study has found humic acid to be toxic in vivo. This study focused on the chromosomal behaviour in cells for studying any possible genotoxic effects of humic acid [39]. Using mice, the researchers were able to analyse their intestinal and bone marrow cells for numerical and structural chromosome abnormalities. Induction of aberrant cells was time dependent and reached a maximum after 24 hours with continual aberrations up until 72 hours after animal exposure to humic acid. The researchers hypothesise that the humic acid could become chlorinated in the gastrointestinal tract and the resulting compound is the main factor responsible for humic acid mutagenicity and toxic side effects in Chinese hamster ovary [28], and other in vitro and in vivo studies on chlorinated humic acid seem to confirm this [40, 41].
A researcher of the International Agency for Research on Cancer suggests that "although chlorinated humic compounds present a hypothetical risk to man, their instability in vivo suggests that they are unlikely to be carcinogens".
In addition, the amounts of humic acid used for these cytogenetic studies were extremely high in comparison to the amount of humic acid that would be ingested from the HFC product. Moreover, none of the before-mentioned studies used peat-derived humic acid for their experiments, but humic acid that was synthetically derived in the laboratory. It is premature to state that peat-derived humic acid, when ingested in reasonable physiological doses would contribute to the formation of chlorinated humic acid by-products. Even if the chlorination of humic acid did occur in vivo, the humic acid chlorinated by-products would most likely be present in insignificant amounts that are not likely to be carcinogenic. Furthermore, these compounds are very unstable in vivo, and are detoxified by inherent biological enzyme systems in the body.
Humic acid was shown to exhibit only weak mutagenicity and toxicity in human peripheral lymphocytes in vitro at a high dosage of 250 and 500 mcg/ml. Although a positive mutagenic response with humic acid is apparent, it was quite low when compared to the alachlor and maleic hydrazide, two known herbicides used to destroy unwanted weeds. It should be noted that the variable results concerning the mutagenic activity of humic substances have been attributed to the heterogeneous structures of humic acids and their reactivity with various compounds, which may produce toxic by-products. Thus interpretation of the exact biological role of humic acids in mutagenesis is difficult. Because most studies use high doses of humic acid in vitro and in vivo, it is not reasonable to assume that these same effects would be seen if animals or humans that ingested reasonable doses of humic acid. Furthermore, extrapolating in vitro and in vivo data to human safety is often misleading. The type and amount of humic acid used in these studies are very different from what is present in HFC. Peat-derived humic acid has been documented as non-mutagenic and safe based on a series of acute, cumulative and mutagenic toxicology studies of HFC that contained humic acid (see the HFC safety and toxicology chapter). These data are important to consider because they provide a safety profile of the use of humic acid in animals in both reasonable physiological doses as well as amounts far exceeding the recommended dose.
There is also some concern that humic acids, due to their potential antioxidant capacity may also exhibit pro-oxidant characteristics when ingested by animals or humans. It is known that nutrients such as carotenoids, tocopherols or ascorbate derivatives will demonstrate an antioxidant or pro-oxidant characteristic depending on the individual redox potential of the molecule, the inorganic chemistry of the cell and the quality of the nutrient available to the cells and tissues [44].
Research has documented that humic acids can cause the depletion of glutathione in human red blood cells in vitro, but with fairly high amounts of 50 - 100 mcg/ml of humic acid [38]. In the same study, humic acids were also shown to decrease other antioxidant enzymes (CuZN SOD, catalase, G6PD) when humic acid was present at high concentrations of 100 mcg/ml. Cells have enzymatic systems, which convert oxidants into non-toxic molecules, thus protecting the organism from the deleterious effects of oxidative stress. When enzyme systems (i.e. superoxide dismutase, catalase, glutathione) are depleted in the presence of the testing compound, such as with humic acid, this indicates that these enzyme systems are working to detoxify any of the reactive oxygen species that have been initiated by the particular test substance. This substance would then be termed a pro-oxidant because of its ability to initiate oxidative stress. However, the development of a beneficial or a detrimental cellular response by a nutrient will depend on the nutrient's antioxidant or pro-oxidant characteristics, which in turn are a product of the cellular oxygen environment that is influenced by normal metabolic processes as well as already existing pathologies. Furthermore, the pro-oxidant potency of various compounds is determined by several factors, including oxygen tension, concentration of the potential pro-oxidant and interactions with other antioxidants [46, 47].
When an inappropriate pro-oxidant activity develops in normal cells, the reactive oxygen metabolites generated could damage the DNA and cellular membranes. This damage to DNA is believed to be partly responsible for the process of ageing, diabetes mellitus, inflammatory diseases and liver disease [45]. Furthermore, damage to proteins may cause alterations in transport systems or enzyme activities. And the well-known event of lipid peroxidation, when reactive oxygen species damage lipids in cell membranes, is thought to be related to several pathologies such as diabetes, atherosclerosis and liver disease [45]. Although one study has documented the potential pro-oxidant activity of synthetic humic acid when used in high amounts, none of the adverse events of pro-oxidation have been documented in numerous animal and human studies using peat derived humic acid in reasonable physiological doses and amounts far exceeding the recommended dose.
Pro-oxidant activity can induce either beneficial or harmful results in biologic systems and influence the development of human chronic diseases. Most antioxidants can act as pro-oxidants under certain conditions, and more research is needed to determine the occurrence and importance of this in vivo. These pro-oxidant effects usually occur when the test substance is used in high amounts, far exceeding the recommended doses of humic acid in HFC. So far, no in vivo studies have demonstrated the pro-oxidant effects of the humic acids found in HFC.
A small body of literature points to a potential mutagenic (induction of structural changes in cells) and pro-oxidant effect (oxidative damage) of humic acid when given in high doses. However, many nutrients such as vitamin C and vitamin A are also thought to be pro-oxidants in high amounts, which in certain conditions can influence tumour growth. The mutagenic and pro-oxidant results from these studies with humic acid can, therefore, hardly be extrapolated to human consumption of HFC at the recommended dose levels. High doses of humic acid or its chlorinated by-products would not likely be found in humans given a reasonable dose of a humic acid-containing supplement.
In summary, it appears that the concerns about the safety of humic acid can be somewhat misleading if careful attention is not focused on several particular issues. The confusion arises from the fact that most studies concerning its safety have used high doses of humic acid isolated from well water or synthetically derived in the laboratory. Furthermore, different isolation techniques and experimental conditions will almost surely affect the synthesis of a complete and thorough review of the safety of humic acid. Therefore, to accurately assess the safety of humic acid, one must isolate the particular humic acid of concern from its respective environment. Its safety should then be tested based on amounts that would normally be consumed in the diet from that particular environment. These results should then be compared to a reasonable physiological amount of the humic acid to generate a pharmacodynamic profile to substantiate acceptable amounts of these substances in the body. Recent research has clarified the safety of peat-derived humic acid and fulvic acid (i.e. Humifulvate from Hungarian peat) for use as a dietary supplement in animals and humans. It is apparent that Humifulvate isolated from peats, specifically Hungarian peat, does not react under certain test conditions as does well water, soil or synthetic humic acid.
2.2. Fulvic acid
Structural and chemical properties
Fulvic acid is considered a macromolecular polymer with a structure and characteristics that change along with its origins and humification processes [6]. Fulvic acids, like humic acid, occur naturally in water, soil and peat. They are produced by the chemical and microbial decomposition of plants, also known as humification. It is thought that fulvic acid may be formed after the formation of humic acid. However, two different laboratory analyses have confirmed that a complex mixture of humic and fulvic acid exists in the same Hungarian peat deposit.
For the most part, fulvic acids and humic acids have been thought of as two distinct entities and their characteristics have been described in this manner. Fulvic acids are generally known to be more oxygen-rich and carbon-poor than humic acids. Similarly to humic acids, fulvic acid contains many reactive functional groups, including carboxyls, hydroxyls, carbonyls, phenols, quinones and semiquinones. These reactive groups make fulvic acid a candidate for both metal chelating and antioxidant activity. The molecular weights of fulvic acids are thought to be less than those of humic acids. Peat fulvic acids contain significant levels of carbohydrate-like materials, derived from decomposing plant polysaccharides. Figure 2 shows a model structure of fulvic acid, depicting its numerous functional groups.
Figure 2.
Biological role
Just like humic acids, fulvic acids have also been shown to be effective chelators of both mineral ions and heavy metals, as well as stimulators of oxidative phosphorylation and the energy production. They have been found to favourably affect seed germination and plant growth, as well as increase the number and length of roots of plants [48]. Fulvic acid from peat has also been used for the clinical treatment of diseases induced by damage of oxygenated free radicals, such as arthritis, cancer, ulcers and rheumatism disease [49].
The structure and chemical properties of fulvic acids are thought to be responsible for chelating mineral ions, and therefore indirectly affecting nutrient uptake and utilisation of these minerals in plants. That is why fulvic acids are commonly used for enhancing seed germination and plant growth [6]. Fulvic acids may directly influence plant growth by stimulating oxidative phosphorylation, the process in plant and animal cells that generates energy. The quinol and phenol functional groups of fulvic acid may influence the respiratory processes of plants [48]. Fulvic acid has been shown to increase oxidative phosphorylation in vitro, however, the results with fulvic acid do not appear as strong as those for humic acid [3]. Fulvic acid was found to affect the uptake of cadmium in the intestinal segment of rats [18]. This is very important due to the carcinogenic and toxic nature of cadmium. Fulvic acids, like humic acids, have the ability to chelate heavy metals based on their cation exchange ability and reactive functional groups.
In summary, it appears that fulvic acids may act similarly to humic acids. This may be due to their acidic functional groups, primarily carboxylic acid and phenolic hydroxyl groups, which give them the capacity to react with various species such as free radicals, minerals and biological enzyme systems [2, 6, 9]. However, due to the complexity of the structure and functions of fulvic acid, it is difficult to determine the exact mechanisms responsible for the effects seen in vivo. Further research may help to explain the way these substances interact with biological systems.
Safety and toxicology
Information on the safety and toxicology of isolated fulvic acid is minimal. However, fulvic acid is present in the standardised Humifulvate concentrate known as Humet®-R, which has been subjected to toxicology and mutagenicity studies that have justified the safety of fulvic acid also. Some human studies have reported a limited number of adverse effects when using HFC containing fulvic acid. The data on aquatic fulvic acid and its relationship to an endemic degenerative joint disease in China further exemplify the case with well water humic acid and the endemic vascular disease. Fulvic acid and humic acid that occur in terrestrial and aquatic environments are different in many ways. Furthermore, the fulvic acid and humic acid found in these endemic regions in China are very different from peat fulvic and humic acids. In fact, several other factors are present in determining the aetiology of both endemic diseases. The following description of fulvic acid and its proposed role in the Keshan Beck disease will provide further support for the essentiality of distinguishing the humic substances in endemic regions of China from those found in peat. It will also provide additional documentation that many other factors play a role in the development of these endemic diseases.
Much research has focused on implicating isolated fulvic acid from well water in the generation of an endemic degenerative joint disorder in China. Kashin-Beck disease (KBD) is characterised by shortened stature and deformities of various joints in individuals residing in certain regions of China. Although the exact aetiology of this disease has not been identified, three hypotheses for its development have been proposed: 1) organic substances (i.e. fulvic acids) in potable water, 2) mycotoxin polluted cereals, and 3) low environmental selenium levels. It is thought that removing any one of the three causes (adding extra selenium to food, reducing fulvic acid in drinking water and providing toxin free grain) can reduce the incidence of the disease [50].
KBD has always occurred in low selenium areas [51], but it appears that selenium deficiency alone is not sufficient to be the cause of the disease. The pathology is quite complex, but in general terms the initial lesion is thought to occur as a selective necrosis of the cartilage cells, when there is a deficiency of the required nutrient, selenium. Selenium has been shown to prevent the cartilage cells from damage in the presence of fulvic acid from water or fusarium oxysporum from grain. In addition, high amounts of fulvic acid supplementation combined with selenium deficiency induced degeneration of the articular cartilage in the knee joints of mice [52]. It should be noted that the fulvic acid concentration in local drinking water in the endemic KBD areas is higher than in other areas, and it has some unique chemical properties.
It was also documented that peat fulvic acid differs from the fulvic acid from soil and drinking water in terms of their composition and content of elements, and content of their functional groups [49]. Peat fulvic acid has been compared with other sources of fulvic acid, specifically from areas that fulvic acids are believed to play a part in the development of Keshan Beck disease. The results indicated that peat fulvic acid could scavenge free radicals produced by biological and non-biological systems in vitro, while fulvic acid from other sources (KBD and non-KBD regions) accelerated the generation of hydroxyl radicals in a dose dependent manner. In non- KBD regions, the toxicity of water fulvic acid is inhibited because selenium is a scavenging agent to reactive oxygen species, such as superoxide and hydroxyl radicals. The differences of fulvic acid from KBD regions, non-KBD regions and peat on free radical production and scavenging abilities indicate that the fulvic acids of different origins cannot be confused or replaced by each other in the etiologic study of KBD.
2.3 The Role of Metals
Cobalt is known to influence iron metabolism and increase the haemoglobin concentration in red blood cells. Daily doses of 25 - 40 mg of cobalt have been used in blood disorders such as the anaemia in renal failure and thalassemia [81]. Cobalt is necessary for the biological activity of vitamin B12 and it is also a component of several enzymes necessary for amino acid metabolism [9, 19]. The addition of cobalt to feedstuff of cattle and sheep has improved the digestibility of nutrients and the utilisation of food. Growing cattle can consume up to 50 mg cobalt per 45 kg body weight without ill effects, but higher doses are thought to be injurious [82]. Cobalt has been also observed to increase the reproduction capacity and growth of poultry.
There is no established recommended daily allowance (RDA) or intake (RDI) for cobalt for humans from the Hungarian Academy of Sciences. Reports have noted various concentrations of cobalt in the diet, but the average daily intake can range from 25 mcg up to 600 mcg without resulting side effects [81, 83, 84, 85]. The oral intake necessary to induce the toxic effects are equivalent to a dietary cobalt concentration of 250-300 mg/kg, which is approximately a thousand times the concentration of cobalt in most normal diets [85]. Cobalt is excreted from the body by the kidneys; therefore any risk of toxic effects of the amount ingested as a dietary supplement (approx. 200 mcg) would be minimal.
Copper has a significant role in the formation of red blood cells. It is required to absorb, utilise and synthesise haemoglobin, which is necessary for red blood cell formation. Copper helps to maintain the integrity of the outer covering of the nerves, metabolise vitamin C and utilise fatty acids for energy. It influences iron and zinc balances and when copper is in proper balance with zinc, the two elements act as antioxidants, i.e. they remove damaging free radicals. Its deficiency may cause and increase in cadmium toxicity [19]. The estimated safe and adequate daily dietary intake for copper is 1.5 - 3.0 mg/day [86]. The optimal intake is considered to be 1.5 - 4 mg/day. Diets provide about 0.9 - 1.0 mg/day for women and approximately 1.2 mg/day for men, which is below the estimated safe and adequate intake range of 1.5 to 3.0 mg/day. Therefore, a supplement containing 2 mg of copper would assure that an individual obtains an adequate and possibly and optimal amount of copper for health maintenance.
Iron is the basic component of haemoglobin, the oxygen-carrying protein found in red blood cells. It is also an essential component in the electron transporting cytochromes, which are found in the mitochondrial membrane, the site of energy synthesis. Therefore, its deficiency results in fatigue, headache, loss of appetite and resulting iron deficiency anaemia. Iron intake potentiates the elimination of toxic lead [87, 19]. The RDA for iron varies with gender and age [86]. It is 15 mg/day for women of all ages (optimal intake 20 mg/day for age 11-18 and over age 51 and 22 mg for ages 19-50). Men require 12 mg/day for ages 11-18 and 10 mg/day after age 18 (optimal intakes for males are 15 and 20 mg, respectively).
Iron deficiency anaemia is one of the most common deficiency diseases in the world. Even in the United States, dietary surveys indicate iron intake to be inadequate to meet the RDA [19]. The most common cause of this is inadequate absorption of iron due to poor iron intake and reduced bioavailability. Iron loss resulting from pregnancy, internal bleeding, infections and low stomach acid are also important factors contributing to iron deficiency. Iron can be a pro-oxidant in high doses and therefore iron supplementation should be restricted to cases of iron deficiency and anaemia, vegetarians, pregnant or lactating women, or individuals with inadequate dietary intake, malabsorption or lack of stomach acid. The deleterious effects of daily intakes between 25 and 75 mg are unlikely in healthy persons [86]. The average daily intake of iron is about 10 mg/day, and the recommended dietary allowance (RDA) is 15 mg/day, therefore an intake of 14 mg/day with a dietary supplement is still safe unless the individual has an iron storage problem.
Magnesium is involved in more than 3oo enzyme reactions in the body. One of its most important roles is maintaining the function of the nervous system and neuromuscular transmission and activity. Magnesium is also involved in glucose and protein metabolism and influences the metabolism of other minerals, such as calcium, phosphorus, and sodium, thereby affecting cardiac function and muscle tone of blood vessels. Magnesium is thought to help protect against the toxic effects of excess aluminium intake [19]. The RDA for magnesium is 270, 400, 350 mg/day for ages 11-14, 15-18, and 19-51+, for males, respectively. For women, 280, 300, 280 mg/day is required for the respective age groups [86]. Optimal intakes range from 300 to 500 mg/day [19].
Magnesium concentrations have been observed to decrease in individuals with chronic alcoholism, diabetes and renal and intestinal disorders, hyperaldosteronism, inadequate nutritional intake and drug therapy (i.e. thiazide treatment) [19]. Average intakes for women, children and men have been reported as 207 mg, 193 mg, and 343 mg, respectively, which fall below safe and adequate daily dietary intake for magnesium. Therefore, a supplement containing 15 mg of magnesium sulphate would not only be considered safe, but would also help individuals meet the recommended dietary allowance as established by the Hungarian Academy of Sciences.
Manganese is involved in protein, fat, and energy metabolism. It is also required for bone growth and development, and reproduction. Its deficiency can cause dermatitis, pigment disturbances of hair, growth problems, and infertility. The estimated safe and adequate daily dietary intake for manganese is 2-5 mg/day for all adult age groups [86]. Optimal intakes are 5 mg/day and 10 mg/day in individuals over age 51 [19]. Diets high in refined carbohydrates and low in plant foods may result in an inadequate intake of this essential nutrient. Furthermore, due to the addition of supplemental iron in the diet and its effects on manganese retention time, it is imperative to also include manganese as a supplement with iron [86]. Research also documents that manganese competes with iron and cobalt for common binding sites during absorption [9]. Thus, any of these metals, can exert an inhibitory effect on the absorption of others. Furthermore, a high fibre diet and supplementation with various nutrients (i.e. calcium iron, phosphorus, magnesium, copper, vitamin E, D, and certain B vitamins) are thought to reduce the absorption of manganese [9, 88].
The Total Diet Study conducted in the United States between 1982 and 1986 indicated that the mean daily dietary manganese intake was 2.7 mg and 2.2 mg for adult men and women, respectively [86]. In humans, toxicity has not been observed as a consequence of dietary intake as much as 8 to 9 mg of manganese per day in their food [86]. Furthermore, due to the low toxicity of manganese, an intake up to 10 mg/day by adults can be considered safe and some researchers feel that increasing the upper value to 10 mg per day should be considered. The Hungarian Academy of Sciences recommends an upper limit of 5 mg/day for adults, which provides an extra margin of safety. Unfortunately, few data are available to support this estimate. The consumption of 3 mg of manganese sulphate per day in supplement form would be considered safe and may help individuals achieve optimal intakes for health maintenance.
Molybdenum functions as an enzyme cofactor in many biochemical reactions. It also acts as an electron transfer agent in oxidation-reduction reactions. The current estimated safe and adequate daily dietary intake for molybdenum in adults is 75 to 250 mcg per day, however, some sources indicate that a range of 150-500 mcg per day is safe and adequate for adults [90]. Although daily intake of molybdenum ranges between 50 and 350 mcg, most diets are thought to supply only about 50-100 mcg per day [9, 86], Furthermore, a diet high in protein, copper, or sulphate can decrease molybdenum availability from the diet [91, 92]. Thus, many diets do not meet the minimum level of the suggested safe and adequate intake for molybdenum [9]. In addition, the percent of absorption of ingested molybdenum falls within the range of 25 and 80% and urinary excretion is 17-80% of the total dose [90, 9]. Therefore, molybdenum is considered a relatively non-toxic element since large oral doses are necessary to overcome the homeostatic control of molybdenum.
In non-ruminants, an intake of molybdenum of 100 to 5000 mg/kg of food or water is required to produce clinical symptoms of toxicity. When researchers apply uncertainty factors of 10 for intraspecies and 10 for interspecies differences to "no observable adverse effect levels" in animals, a tolerable daily intake (TDI) can be derived for humans when human safety studies are limited. The most recent TDI has been given a medium confidence rating and it is more than double the upper limit of adequate intake for adolescents and adults that was derived from the average molybdenum levels in the diet in the United States [90]. Estimated based on the molybdenum levels of the human diet, the molybdenum variable absorption rate, its status as a relatively non-toxic element, and recent TDI estimates, a dietary supplement containing 175 mcg per day would not only help an individual meet the recommended adequate intakes of molybdenum, but also appears to be safe for human consumption.
Potassium is an essential element in maintaining fluid balance in our cells, contributing to the transmission of nerve impulses, the control of skeletal muscle contractility, and the maintenance of normal blood pressure. There is no RDA for potassium; however, research indicates that the minimum requirement should be between 1,600 to 2,000 milligrams a day [86]. Toxicity of potassium only results from sudden enteral or parenteral increases in potassium intake to levels of about 18 grams. A high sodium diet and the use of diuretics often administered in hypertensive patients promote potassium excretion. In addition, potassium is found primarily in fruits and vegetables, which are lacking in the US diet. Therefore, supplementation with potassium may not only be necessary in some individuals, but it is also extremely safe at a level of 37 mg a day.
Selenium is a trace element with a number of biological effects, although it is best known as an antioxidant because of its relationship with vitamin E. There is evidence that selenium may be protective against certain cancers. Selenium is also thought to be protective against the affects of toxic elements, such as arsenic, mercury, and cadmium by binding these metals [19, 93]. The RDA for selenium is 40, 50, and 70 mcg/day for men aged 11-14, 15-18, and 19-51+, respectively. Females require 45, 50 and 55 mcg/day for the respective age groups. Optimal intakes range from 60-250 mcg/day depending on age and condition.
An estimated average dietary intake of 108 mcg/day between 1924 and 1982 has been noted in the literature [86]. However, dietary selenium intakes are difficult to estimate because of the variation of the selenium content of the soil in which the vegetables for food are grown. Furthermore, some minerals (e.g. zinc and copper) are antagonistic to selenium, thereby affecting its absorption [94]. Based on the available research indicating it is safe in usual and therapeutic doses, and because only intakes over 750 micrograms [19, 95] over an extended period are harmful, its use in a dietary supplement in the amount of 175 mcg per day seems reliably safe.
Vanadium has been shown to be an essential trace element in the growth of animals [9, 96]. More recent research has indicated its use in the treatment of diabetes, hypertension, and lowering of serum cholesterol [96, 97, 98, 99, 100]. Vanadium is relatively abundant in nature and is found in a variety of foods; however, there is no RDA for vanadium from the Hungarian Academy of Sciences. Most diets are thought to supply between 6 and 20 mcg daily [9]. However, other sources have indicated daily dietary intake in amounts up to 2 mg [96, 98]. The preferred range for vanadium intake in man may be 0.0007 to 2.0 mg/kg/day (or 52.5 mcg to 150,000 mcg for a 75 kg man) to show therapeutic benefits. In fact, during the age of metallotherapy, metavanadate was given in amounts of 1-8 mg (1,000 to 8,000 mcg) by mouth without any resulting signs of toxicity [96]. In addition, it is not uncommon to see dietary supplements on the market containing vanadium in the range of 500-150,000 mcg.
Recently, research has documented the use of sodium metavanadate in amounts of 125 mg (125,000mcg)/day for two weeks in insulin dependent and non-insulin dependent diabetics. The data not only suggests that vanadium may have a potential role as adjunctive therapy in these patients [100], but that side effects using higher amounts of vanadium were mild (i.e. gastrointestinal intolerance). Biochemical evidence of vanadium treatment revealed no sign of toxicity based on assessment of blood electrolytes, blood urea nitrogen, creatinine, liver function studies, thyroid functions, urinalysis, and complete blood count. Furthermore, another study has documented no significant side effects observed in non-insulin dependent diabetics who were given daily doses of 100 mg (100,000 mcg)/day of vanadyl sulphate for three weeks [99].
It seems reasonable to examine some explanations for its use in these amounts. Due to the fact that certain dietary components (i.e. ascorbic acid, chromium, protein, ferrous iron, chloride, and aluminium hydroxide) affect the speed at which vanadium is transformed into a useable form, the percentage of ingested vanadium absorbed is effected [9]. Furthermore, little absorbed vanadium (less than 5%) is retained under normal conditions in the body due to homeostatic regulation [9, 98, 101]. Therefore, due to the variability of vanadium concentrations in the diet, the factors affecting its complete absorption, its homeostatic regulation by controlled accumulation and its low toxicity upon oral intake in humans, dosages of 500 mcg (or 0.5 mg) in a dietary supplement appears safe.
Zinc is essential for the functioning of over 200 enzymes in biologic systems. A critical function of zinc is its role in the structure and function of biomembranes. It is also responsible for the synthesis of DNA and RNA. Furthermore, zinc is involved in immunity, wound healing, and the functioning of the central nervous system. The presence of zinc is especially important in preventing toxicity of metal ions, such as lead, arsenic, and cadmium [19]. The RDA for zinc is 15 mg/day for males and 12 mg/day for females [86]. Optimal intakes range from 15-20 mg/day, males requiring 20 mg/day as they get older (19-51+), and the same is the case with females, but in amounts of 17 mg/day [19].
The average dietary intake for zinc in the United States has been reported as 8.6 to 14 mg per day [9, 86]. However, absorption of zinc is largely dependent upon the presence of substances in the food that alter solubility or availability of zinc at the absorption sites [9]. Plant foods contain phytic acid, which explain, in part, the lower availability of zinc from these foods. In addition, several elements with similar physicochemical characteristics as zinc compete for common pathways. For example, zinc competes with copper, cadmium and iron for binding to the same carrier protein. Thus metal competition exists, which can result in reduced bioavailability of one or more of these nutrients.
Acute or chronic toxicity of zinc is very rare. Endogenous faecal zinc losses can be increased several fold in order to maintain zinc homeostasis when there are high intakes of zinc. In addition, there is no specific zinc "store", thus it is difficult to accumulate zinc in excessive amounts in tissues. Zinc absorption decreases as an individual ages, and older adults average less than 2/3 of the RDA for zinc. Levels of zinc supplements as low as 25 mg per day have been reported to induce copper deficiency, thus it is recommended to consume supplements containing less than this amount of zinc. Due to the fact that several factors affect zinc absorption and its toxicity is rare, a dietary supplement of zinc sulphate containing 66% of the RDA (10 mg) is considered safe [9].
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