EP0614361A1

EP0614361A1 - Composition and method for reducing free radical cellular oxidative stress in warm-blooded animals

Info

Publication number: EP0614361A1
Application number: EP92924342A
Authority: EP
Inventors: H. Dewayne Ashmead; Harvey H. Ashmead; Robert B. Jeppson
Original assignee: Albion International Inc
Current assignee: Albion Laboratories Inc
Priority date: 1991-11-25
Filing date: 1992-11-05
Publication date: 1994-09-14
Also published as: WO1993010777A1; CA2124204A1; EP0614361A4

Abstract

Méthode permettant de réduire les contraintes d'oxydation cellulaire par radicaux libres chez les animaux homéothermes présentant des symptômes de toxicité causés par les radicaux libres. La méthode consiste à: confirmer la présence chez l'animal de contraintes d'oxydation cellulaire causées par des radicaux libres; déterminer quels oligoéléments minéraux sélectionnés dans le groupe constitué de cuivre, de zinc, de fer et de manganèse avec ou sans sélénium, sont nécessaires pour renforcer et maintenir le fonctionnement des enzymes liées aux accès d'oxydation des granulocytes neutrophiles et des macrophages présents dans l'animal; préparer une composition contenant des quantités efficaces d'au moins un des minéraux susmentionnés (cuivre, zinc, fer et manganèse) sous forme d'un chélate aminoacide présentant un rapport ligand/minéral d'au moins 1:1, un poids moléculaire d'au plus 1500 daltons et une constante de stabilité comprise entre 106 et 1016, avec ou sans des quantités facultatives de sélénium pouvant être présent soit sous forme d'un chélate aminoacide ou de sélénium inorganique; administrer la composition à l'animal, de préférence par voie orale. L'existence de contraintes d'oxydation cellulaires par radicaux libres est confirmée de préférence par l'analyse directe de l'activité des enzymes d'oxydation sélectionnées dans le groupe constitué de CuZnSOD, de glutathion-peroxydase et de catalase.Method for reducing the stresses of cell oxidation by free radicals in homeothermic animals showing symptoms of toxicity caused by free radicals. The method consists in: confirming the presence in the animal of cell oxidation stresses caused by free radicals; determine which mineral trace elements selected from the group consisting of copper, zinc, iron and manganese with or without selenium, are necessary to strengthen and maintain the functioning of the enzymes linked to the access of oxidation of neutrophilic granulocytes and macrophages present in the 'animal; preparing a composition containing effective amounts of at least one of the aforementioned minerals (copper, zinc, iron and manganese) in the form of an amino acid chelate having a ligand / mineral ratio of at least 1: 1, a molecular weight of at most 1500 daltons and a stability constant between 106 and 1016, with or without optional amounts of selenium which may be present either in the form of an amino acid chelate or inorganic selenium; administering the composition to the animal, preferably orally. The existence of cellular oxidation constraints by free radicals is preferably confirmed by direct analysis of the activity of the oxidation enzymes selected from the group consisting of CuZnSOD, glutathione peroxidase and catalase.

Description

COMPOSITION AND METHOD FOR REDUCING FREE

RADICAL CELLULAR OXIDATIVE STRESS IN

WARM-BLOODED ANIMALS BACKGROUND AND FIELD OF THE INVENTION

This invention relates to compositions and methods of reducing free radical cellular oxidative stress in warm-blooded animals showing symptoms of free radical toxicity. More particularly, this invention relates to amino acid chelated mineral compositions containing one or more minerals selected from the group consisting of copper, zinc, iron and manganese and to methods of administering these compositions to strengthen and maintain the functioning of enzymes which help regulate the effects of oxidative bursts for killing or deactivating pathogens and foreign matter in neutrophils and macrophages. These enzymes function to adequately remove the superoxides, peroxides and hydroxides that are formed in the cells. These enzymes are inclusive of, but not limited to, the superoxide dismutases (SOD) , catalase and glutathione peroxidase. As previously stated, they function to remove the superoxides, peroxides and hydroxides that are formed in the cells. Otherwise oxygen toxicity results. The superoxide radical is formed during various metabolic processes, many of which are considered normal. Liver cells, muscle cells, leukocytes, erythrocytes, aerobic bacteria, any cell that undergoes oxidative cellular metabolism, all form superoxide radicals during normal metabolic processes. These oxygen radicals are converted to hydrogen peroxide by CuZn-superoxide dismutases (CuZnSOD) in the cells. In a properly functioning system the hydrogen peroxide is then converted to oxygen and water by a catalase. If the hydrogen peroxide and the superoxide radical are allowed to combine, the more reactive and destructive hydroxide radical is formed. When the formation of one or more of the superoxide, hydrogen peroxide or hydroxide radicals becomes uncontrolled or the organism loses the ability to regulate these reactions, changes in cellular physiology result that become detrimental to the individual cells, organ systems, or the entire host or animal. Some of these changes include generalized tissue destruction, lameness and joint inflammation, DNA miscoding or degradation, lipid peroxidation, altered immune function and inactivation of important cellular enzymes. The primary activated superoxide dismutase (SOD) in animals is CuZnSOD. This metalloenzy e undergoes a reduction-oxidation exchange with the superoxide radical with the net result of dismutation of the superoxide radical to hydrogen peroxide and oxygen. As noted, the metals for this activity are copper and zinc. Other forms, i.e. MnSOD and FeSOD, are also known but occur primarily in bacteria and cellular mitochondria. Without the presence of copper, the eucaryotic cytocell SOD enzyme is virtually inactive in the animal. The activity of the CuZnSOD enzyme can be suppressed by the rapid accumulation of hydrogen peroxide. Therefore, it is essential that other enzymes which deplete hydrogen peroxide be functional within the cell to maintain SOD activity. Catalase is a large molecular weight enzyme that contains four he e groups per molecule. Catalase is the primary enzyme necessary for the breakdown of hydrogen peroxide in the cell to oxygen and water and is found in all cells of the body that utilize oxygen. Glutathione peroxidase (GSH-Px) has a selenium dependent form which contains four moles of selenium per mole of the enzyme. The oxidative role of this enzyme is similar to catalase in that it converts hydrogen peroxide to water and oxygen. Wherever catalase or glutathione peroxidase activity is impaired there can be a toxic build-up of peroxides. This, in turn, can lead to a build-up of the hydroxide radical. The non-selenium glutathione peroxidase (GSH-P) plays a role in controlling lipid peroxidation. The primary form of glutathione peroxidase within the red blood cell is the selenium dependent form which maintains a linear relationship to selenium status within the animal and has been used to indicate whether or not a selenium deficiency exists.

Not all aspects of oxygen radical production are detrimental. One of the most useful purposes of oxygen radical, peroxide and hydroxide radical production is the role they play in the immune response when mono- or polymorphonuclear leukocytes engulf bacteria or immune complexes and destroy them. As oxygen radicals increase systemically, a more active immune response is initiated. However, if left uncontrolled, the buildup of oxygen radicals can be devastating to the animal, causing massive cellular destruction.

The above discussion relating to the oxidative enzymes, their functioning and purposes are taught and summarized by Coffey, "Catalase, Cu/Zn-Superoxide Dismutase, Glutathione Peroxidase: Their Relationship to Oxygen Utilization in Cellular Physiology, Clinical and Subclinical Disease, Nutrition and Trace Element Utilization in Livestock", The Bovine Practitioner, (1988) 23:138.

Coffey also states that copper amino acid complexes or chelates are capable of catalyzing the dismutation of the reactive oxygen radical in a fashion similar to CuZnSOD. The superoxide dismutase activity of copper amino acid chelates has been reported by JOester, et al , FEBS Letters. (1972) 25:25, and Brigelius et al , FEBS Letters (1974) 47:72. One factor which may contribute to the inability of the body to control free radical accumulation within oxygen consuming cells is that ionic mineral absorption in the gut requires an integral protein carrier molecule embedded in and transversing the mucosal membrane. Once absorbed into the mucosal cell, the transfer of the cation from the terminal web below the microvilli to the basement membrane requires the presence of carrier proteins. For iron, apoferritin is a suitable carrier. In the case of zinc, albumin is the carrier protein. For copper the carrier is ceruloplasmin and for manganese it is transmanganin. Both protein and albumin are necessary to transport mineral ions from the gut to the plasma.

If the above oxidative enzyme systems are suppressed or do not function properly, the chemistry of the cells is altered due to buildup of free radicals and enzymes are either depleted or do not perform their tasks due to mineral deficiencies.

From the above, it is evident proper metabolic functioning of minerals such as copper, zinc, iron and manganese in addition to or independent of selenium play an important role in maintaining functioning of oxidative enzymes which relate to oxidative bursts in neutrophils and macrophages and in controlling or alleviating free radical cellular oxidative toxicity. Over a period of time, the deficiency of these minerals in the body in bioavailable form results in a compromised enzyme deactivation system and the accumulation of free radicals within oxygen consuming cells leading to free radical toxicity. It would therefore be beneficial to provide these essential minerals in a bioavailable form to warm-blooded animals exhibiting symptoms of free radical cellular oxidative toxicity in which such minerals would be readily absorbed for the repair and maintenance of the appropriate enzyme systems. Ashmead et al . , U.S. Patent 4,020,158; Ashmead, U.S. Patent No. 4,076,803; Jensen , U.S. Patent No. 4,167,564; Ashmead, U.S. Patent 4,774,089 and Ashmead, U.S. Patent 4,863,898 all teach various uses for amino acid chelates in reference to increasing absorption of essential minerals into biological tissues. Some of these patents suggest that certain mineral and ligand combinations can enhance metal uptake in specific organs or tissues where specific tissue functions are enhanced, i.e. estrus or spermatogenesis, minerals crossing the placental membranes into foeti, etc. However, it has not heretofore been taught or suggested that a biological system, as distinguished from tissues, can be affected through the proper oral in vivo administration of amino acid chelates. By definition, a system is a set or series of interconnected or interdependent parts or entities (objects, organs, fluids, organisms, etc.) that function together in a common purpose or produce results impossible of achievement by one of them acting or operating alone. Hence, there is greater complexity involved in affecting a system in order to influence or assist in the enhancement, maintaining or strengthening of such a system as compared to influencing mineral uptake or to the direction of minerals to certain tissue sites.

OBJECTS AND BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide mineral compositions of one or more minerals selected from the group consisting of copper, zinc, iron and manganese with optional amounts of selenium which, when administered, reduce free radical cellular oxidative stress in warm-blooded animals.

It is also an object of the present invention to provide a method of strengthening and maintaining the functioning of enzymes in warm-blooded animals which relate to oxidative bursts in neutrophils and macrophages through the administration of amino acid chelated mineral compositions containing one or more minerals selected from the group consisting of copper, zinc, iron and manganese. Selenium may optionally be administered. These enzymes function to adequately remove the superoxides, peroxides and hydroxides that are formed in the cells.

These and other objects may be accomplished by the proper formulation of one or more minerals selected from the group consisting of copper, zinc, iron and manganese and, optionally, selenium and the administration of one or more of these minerals to a warm-blooded animal vulnerable to or showing symptoms of free radical toxicity. By proper formulation is meant the providing of such minerals in a form which is bioavailable to the animal preferably at an intestinal absorption site other than the duodenum. Also, the ratio of one mineral to another may be significant and can vary depending upon the species of animal, its vulnerability to free radical toxicity or the symptoms of free radical toxicity which are mani est due to inadequate intestinal absorption of necessary minerals.

Bioavailable forms of copper, zinc, iron and manganese which are absorbed via the intestinal tract of a warm-blooded animal at a site other than the duodenum are those made by chelating the mineral with an amino acid or peptide ligand wherein the ligand to mineral ratio is at least 1:1 and preferably 2:1 or higher and wherein the molecular weight of the amino acid chelate formed is not greater than 1500 and preferably does not exceed 1000. Such amino acid chelates are stable and are generally absorbed intact through the intestinal tract via active dipeptide transport. Such amino acid chelates have a stability constant of between about 10⁶ and 10¹⁶. A more detailed description of such chelates and the method by which they are absorbed is found below and is also documented in Ashmead et al . , U.S. Patent 4,863,898 which issued September 5, 1989 and also in Ashmead et al ., Intestinal Absorption of Metal Ions and Chelates, Published by Charles C. Thomas, Springfield, Illinois, 1985. Selenium may also be administered along with the copper, zinc, iron and manganese amino acid chelates.

DETAILED DESCRIPTION OF THE INVENTION

As documented by the Ashmead et al . publication, referenced above, mineral absorption from the intestinal tract occurs via at least two pathways. A mineral salt, after ingestion is solubilized and ionized in the acid pH of the stomach. The metal cations passing from the stomach into the intestinal tract are absorbed, if at all, in the duodenum or upper portion of the small intestine. This requires a relatively low acid pH. It is believed that the metal cation is presented to the integral proteins in the brush border of mucosal cells of the duodenum. The transport of the metal ion across the mucosal cell membrane is accomplished by an active transport system which involves chelating or complexing the cation to complex carrier proteins. Several enzyme reactions in which the cation is moved from molecule to molecule within the system. This movement is very rapid and stops when the cation is delivered to the interior side of the mucosal membrane where the metal cation is released and rechelated by cytoplasmic proteins, such as transmanganin in the case of manganese, albumin in the case of zinc, apoferritin in the case of iron and ceruloplasmin in the case of copper. The cation chelated with cytoplasmic protein is then carried to the basement membrane of the mucosal cell. Metal ions absorbed in this manner are reacted, released, re-reacted and re- released repeatedly during this transport from the intestinal tract to the portal blood. Metal cations which are not absorbed via the duodenum descend on through the intestine and the pH is increased. As the pH increases, the metal ions react with phytates, phosphates, oxalates and other anions and form insoluble precipitates which pass through the gut and are excreted in the feces.

The Ashmead et al . publication documents that when an impermeant substance, such as a metal cation, is chemically linked to an amino acid or low molecular weight peptide, the resulting complex can be transported via a peptide transport system across the cell membrane. This has been referred to as having the impermeant substance "smuggled" across the membrane and the complex has accordingly been referred to in the literature as a "smugglin". These are the amino acid chelates, above referred to having a ligand to mineral ratio of at least 1:1 and preferably 2:1 or higher, a molecular weight of no more than 1500 and preferably not more than 1000 and a stability constant of between about 10⁶ and 10¹⁶. In the field of animal nutrition, the American Association of Feed Control Officials has issued an official definition of "amino acid chelates" as being "the product resulting from the reaction of a metal ion from a soluble salt with amino acids with a mole ratio of one mole of metal to one to three (preferably two) moles of amino acids to form coordinate covalent bonds. The average weight of the hydrolyzed amino acids must be approximately 150 and the resulting molecular weight of the chelate must not exceed 800." It is also documented that amino acid chelates can be prepared from metal ions which do not come from soluble salts. Ashmead, U.S. Patent 4,599,152 and Ashmead, U.S. Patent 4,830,716 both disclose methods of preparing pure amino acid chelates using metal sources other than soluble metal salts. However, it is not critical to the present invention in which manner the amino chelates are made.

While it is known that nutrition plays an important role in proper cellular physiology and maintenance of the appropriate oxidative enzymes, its intricacies of function in the broad aspects of cellular biology are still obscure. Researchers are just beginning to understand that trace element nutrition, or malnutrition as the case may be, is often the core of enzyme system problems and/or deficiencies. If manganese, zinc, copper, iron and selenium are deficient, the CuZnSOD, peroxidase and glutathione enzymes will be deficient. Even if these trace minerals are present in sufficient amounts in the diet, an overabundance of certain other trace minerals can interfere with their absorption. Also, as referenced above, the form of the nutrient to be absorbed is often more important than the quantity. Elemental salts are not as bioavailable has the amino acid chelates referred to, particularly when there is interference from heavy metals. However, if the enzyme and immune systems are not functioning properly, many of the drugs and or methods relied on to treat and prevent free radical toxicity are ineffectual and mortality may result.

Copper, zinc, manganese, iron and, optionally, selenium are the minerals of greatest concern which have a direct impact on maintaining and sustaining the activity and formation of catalase, CuZnSOD and GSH-Px. Besides being present in adequate quantities, the interrelationship of one mineral to another is important. Specific minerals may be present in adequate amounts according to assays of food sources. However, due to interference or competition, such minerals may not be biologically available. For example, it is known that excess molybdenum directly ties up copper. Manganese and iron compete for the same active ionic absorption sites in the small intestine. Manganese is readily excreted from the body, but there is no excretion mechanism for excess iron accumulation which also may contribute to an inhibitory affect on copper utilization.

It is known, from the background section above, that catalase, CuZnSOD and glutathione peroxidase play vital roles in controlling essential functions of cellular metabolism. It is also known that adequate manganese, iron, zinc, copper and selenium utilization and bioavailability are critical to the formation and bioactivity of these enzymes. Copper and iron are important in catalase, copper and zinc in superoxide dismutase; and selenium in glutathione peroxidase. Iron is often present in adequate amounts in food ratios or water. If sufficient iron is not present in the normal diet it may be desirable to supplement the food ration with appropriate amounts of iron amino acid chelate having the molecular weight and ligand to metal ratios mentioned above. Selenium is required in very small amounts. While requirements may vary from species to species, it has been determined from analyses of blood and serum samples that there are "normal" or "average" ranges of activity levels for each oxidative enzyme in various animal species. These are listed in the following Table 1:

-Fable 1

Species Catalase CuZnSOD GSH-Px (GSH-P; k Units/Gm Units/rag Units/Gm

The enzymes are present within the cell and are therefore reflective of the amounts and activity present at the time of cell formation. Though the erythrocytes from which measurements are made are easily accessible for analysis of these enzymes it is important to consider the average life span of the erythrocyte in each species. This is because there is continuous erythropoiesis and the blood samples collected are an average of the nutritional status over the erythrocyte life span just prior to sample collection. In the bovine species, for example, this span is about 48-63 days for ages up to 3 months, 70-126 days from 3 months to near maturity and 160 for mature animals. It is therefore important that enzyme activity levels be monitored for at least a period of time that is equivalent to the erythrocyte life in a particular species.

Therefore, under the presently disclosed invention, the correct monitoring of oxidative enzyme activity and supplementation, as necessary, with critical trace elements selected from the group consisting of manganese, zinc, iron and copper in amino acid chelated form, with or without additional selenium supplementation, should lead to an improvement in nutrient utilization and immunity without resorting to the use of unproven drugs and/or chemicals which may have a detrimental impact on the animal.

The copper, zinc, iron and manganese amino acid compositions, with or without selenium, will preferably be administered to the warm blooded animal orally. In many cases, mixing of the chelates in the food, drinking water or other ration form given to the animal may be the preferred method of treatment. For example, the chelates may be mixed with salt (sodium chloride) when being administered to animal species. In the case of humans, the chelates may be administered in the form of tablets, capsules, powders, syrups, elixir or any other suitable form. They may be mixed with fillers, excipients, vitamins and other foodstuffs.

The exact amount of mineral to be administered, and the mole ratio of one mineral to another, may depend upon the particular symptoms and level of free radical oxidative stress being treated. Often, assay results of tissue and serum samples may have to be taken before a proper formulation can be made. Then the amount of minerals and their ratios may be adjusted by modification of feed supplementation and intake. To make a determination, the correct interpretation of data may be more important than the actual numbers generated in an assay and values would additionally need to be correlated to bioavailability and antagonistic parameters of one trace element to another or from one trace element to other minerals such as copper and iron. Serum and liver assays as well as assays of the SOD, peroxidase and catalase enzymes will often serve to determine need for administration of copper, zinc, iron and manganese separately or in certain combinations and ratios. Also, the need to utilize selenium can be similarly determined. An assay of the diet may also be important to determine mineral amounts in the diet and identify deficiencies and/or antagonistic factors which may affect trace minerals when administered. For example, it will be noted from the data and tests which follow as illustrative of the invention that iron was adequately present in the food ratios administered and the separate administration of iron amino acid chelate was not indicated.

Therefore, the exact amount of amino acid chelate, which minerals to use and in what ratios, and whether to add selenium, are preferably determined on an empirical basis according to need using data, such as contained in Table 1, as a guideline. Hence, the term, "effective amount" of one or more minerals is based on both the amount of mineral and the ratio of one mineral to another which had been determined to be required to meet the needs of a particular warm-blooded animal or group of animals, including humans, which are (1) vulnerable to oxidative stress or free radical toxicity, (2) are exhibiting certain symptoms of oxidative stress or (3) are affected by free radical toxicity. In some instances, based on collected data over periods of time, it will be possible to pre-formulate compositions based on known needs of the animal species experiencing oxidative stress. However, one skilled in the art, based on the information provided herein, can determine without undue experimentation what an "effective amount" of a composition is and how to administer it accordingly. It is not possible to categorically state that "x" mg of trace mineral per kg of animal body weight is what is needed to reduce symptoms of free radical oxidative stress. Nor is it possible to state, for example, that the ratio of Cu to Zn will be "a:b" in all instances. Each animal species and form of free radical oxidative stress may require different amounts of minerals and/or ratios of minerals. For these reasons, a data bank of various trace mineral levels and ratios which are found with various symptoms of free radical oxidative stress according to animal species and a comparison these data against mineral levels and ratios found in animals of the same species not exhibiting these symptoms is being compiled. From these data the "effective amounts" of minerals to administer to a given species exhibiting identifiable symptoms will be available. For animal species in which an RDA [recommended dietary allowance], or similar nutritional guideline, has been established, that amount may be used as a minimum or threshold "effective" amount to be administered to that species. However, in some instances, it may be possible to administer even lesser amounts which are also "effective" provided the correct mineral ratios are used to bring the enzyme amounts within the ranges given in Table 1.

When utilizing copper, zinc and manganese amino acid chelates to strengthen the dismutation of the reactive oxygen radical in a similar fashion as CuZnSOD it has been found that the optimal activity is obtained when the mole ratio of Cu:Zn:Mn is about 1:1:0.5. The following examples are illustrative of the invention showing treatment of warm-blooded animals having symptoms of free radical toxicity wherein the oxidative enzyme activity in these animal species are improved by administration of copper, zinc and manganese amino acid chelates with optionally added selenium. As previously mentioned, iron was not added in these formulations because the analysis of the relevant factors, including feed and water showed that the presence of iron was adequate. Whenever the term "amino acid chelate" is used, the chelate administered has a ligand to metal ratio of 2:1 or greater. The chelate has a molecular weight not in excess of 1500 and in most cases, not in excess of 1000. The stability constant in each instance is between about 10⁶ and 10¹⁶.

Example 1 This example demonstrates the ability of chelated minerals to enhance oxidative enzyme activity to a member of the bovine species with the consequent result that reproductive ability was restored. A three year old Simmental Bull who was not producing active spermatozoa was examined for oxidative enzyme activity through blood tests. Superoxides are required by sperm to maintain cell wall integrity while in the epididymis. However, unregulated oxygen radical production within sperm is highly damaging. After the first test the bull was placed on a ration which included zinc, manganese and copper amino acid chelates. These minerals were chelated with ligands derived from hydrolyzed protein and had a ligand to mineral ratio of about 2:1, a molecular weight of under 1000 and the stability constant of each amino acid chelate fell within the range of between about 10⁶ and 10¹⁶. A mineral ration was prepared by mixing 120 lbs of an amino acid chelate mixture containing 8% zinc, 4% manganese and 1% copper with other ingredients including dicalcium phosphate, magnesium oxide, solulac, rice hulls, calcium carbonate, potassium chloride and various amounts of vitamins and other minerals, some of which were present in inorganic salt or oxide form and some of which were present as chelates or complexes. The mineral ration mixture was made up to one ton of ration with the added ingredients. The chelated mineral supplemented ration was then mixed with equal parts of salt (sodium chloride) and made available free choice to the bull. It was estimated that the average daily consumption of the mineral ration amounted to between about 2 and 3 ounces per day. At three time intervals of three weeks duration the bull was again bled and tested for oxidative enzyme activity with the results being recorded in Table 2 as follows:

oxidative enzyme activity. The glutathione (GPx) and catalase activities almost doubled. At the time the sixth week test blood samples were taken, the bull was producing viable sperm. The GPx level had continued to elevate but there was an unexplained drop in catalase activity. However, since both glutathione and catalase degrade hydrogen peroxide, the higher GPx level compensated for the lowered catalase. The CuZnSOD level had dropped from the third week test with a lowering of SOD activity indicating a lack of copper or insufficient copper utilization. At the time the ninth week samples were taken, the bull was back in fertile production. The GPx had continuously climbed to a near normal level which is reflective of improved oxidative processes. At these levels, it is optimal to have total GSH-P equal to the selenium dependent GPx in the red blood cell. Catalase activity was approaching normal (90-120 k units/gm) at the ninth week. However, the CuZnSOD activity was only about 61% of saturation. A subsequent analysis of the feed ratios of the bull showed that copper availability in the feed was only 5.7% of that present in the hay being feed. Because of that, the results showed a need to further supplement the feed with additional copper amino acid chelate to bring the CuZnSOD activity within normal levels and further enhance the reproductive capabilities of the bull.

The above data demonstrates .the effectiveness of administering essential minerals in amino acid chelated form to enhance the oxidative enzyme activity of animals.

Suggested Alternate Therapies: To bolster the CuZnSOD activity of this bull, it was suggested that alternative therapies be adopted, both of which would utilize copper, zinc and manganese amino acid chelates in a Cu:Zn:Mn mole ratio of 2:2:1. The first is a water soluble chelate consisting of 6% copper, 6% zinc and 2.5% manganese. One half pound of this mixture is dissolved in 128 gallons of drinking water and supplies about 25 PPM (106.4 mg/gallon) of copper and zinc and 10.5 PPM (44.4 mg/gallon) of manganese. As a general rule, most cattle consume about 7-15% of their body weight in water daily depending upon conditions such as temperature and amount of moisture in their feed. It is proposed to administer this amount of chelate daily from 1 to 14 days and then cut the amount in half during the remainder of the treatment period, usually an additional 14 days. As another alternative therapy, a premix consisting of 1817.6 mg/lb each of copper and zinc (as amino acid chelates) and 908.8 mg/lb of manganese (as amino acid chelate) could be fed at the rate of 1/8 to 1/4 lb/day for the first fourteen days and then cut in half for the remainder of the treatment period. Specific data on these suggested alternate therapies are not available; however, the bull continues to provide viable sperm.

Example 2 Some stress-susceptible breeds of pigs have reduced cell membrane integrity, ascribed as an antioxidant abnormality, which leads to increased damage to cell membranes by free radicals. This is referred to as porcine stress syndrome (PSS) and is manifested by rapid, fatal malignant hyperther ia leading to death. Typical clinical signs of PSS are hyperventilation, tachycardia, muscular rigidity and rapid increase in body temperature. It is brought on by stresses such as being transported, exercising and mating. PSS can also be induced in pigs (and in predisposed humans) , by exposure to halothane anesthesia. Halothane is known to produce potent free radicals which exacerbate inadequate antioxidant defense mechanisms. Financial losses attributed to PSS amount to millions of dollars each year. It is therefore of paramount importance to be able to identify susceptible animals and provide treatment to reduce and or eliminate this syndrome.

Attempts have been made to utilize antioxidants such as vitamin E and selenium salts without success. Typically, PSS mortality in susceptible pig herds ranges from about 3 to 5%.

Although data from replicated trials is not yet available, it has been found that introducing a feed supplement consisting of 450 gm/ton (1 lb/ton) of a copper, zinc and manganese amino acid chelate consisting of 6% copper, 6% zinc and 2.5% manganese [Cu:Zn:Mn mole ratio 2:2:1] into the diet of susceptible pigs solves this problem. In several herds, the stress deaths stopped completely and mortality fell to the anticipated rate of 0.5 to 1.0%. When the chelated supplement was withdrawn the deaths increased again within 10-14 days.

Example 3

This example illustrates the efficacy of amino acid chelates in a herd of dairy cows experiencing problems in reproductive efficiency. Various feed ration changes and injections of vitamins failed to rectify the problem. A cross section of blood samples from the herd was analyzed for RBC oxidative enzyme content and serum trace minerals. An extensive analysis was also done of the various feed ratios. Table 3 contains results of the oxidative enzyme and trace mineral content of three of the cows which is exemplary of the herd. Cow One was dry, Cow Two had been lactating for about 40 days and Cow Three for about 165 days.

The blood and serum profiles given above indicate that the cows were anemic. It was felt that the problem was not one of iron deficiency, one reason being that the iron content of the water was more than adequate. Rather, the problem was due to the copper deficiency that existed in the animals as confirmed by the low serum copper levels and the decreased CuZnSOD activity. The water supply had an iron content in excess of 2 ppm and was believed to play a role in the copper deficiency. Too much iron inhibits the activity of copper. The serum selenium levels were unusually low particularly since the herd had been given injections containing selenium shortly before the blood was drawn. It was believed that the above mentioned sub- clinical deficiencies were the reason the herd had not responded to the various feed ration changes and injections.

In an attempt to rectify the situation, an analysis was also made of the feed which consisted of ground ear corn, alfalfa hay, corn silage and some shelled corn. It was recommended that the only change in the feed ration being used become the addition of an amino acid chelate premix consisting of copper, zinc and manganese amino acid chelates in a 2:2:1 mole ratio to the mineral supplement being mixed with the feed ration. Sufficient amounts of an amino acid chelate containing 4% copper, 4% zinc and 2% manganese were added to the mineral supplement to make the amino acid chelate concentration between about 1.0 and 1.5% by weight. The mineral supplement also contained selenium in inorganic form. The mineral supplement containing the copper, zinc and manganese amino acid chelates and elemental selenium was then added to the feed ration to bring the overall copper, zinc and manganese contents of the ration to within a range of about 60-80, 130-160 and 90-110 ppm, respectively. The overall range of mineral supplement consumed each day will vary between about 0.25 and 0.75 lbs/animal. It becomes evident that with the amino acid chelate content being limited to about 1 and 1.5%, the total amounts of amino acid chelates consumed per animal per day can vary between about 1 and 5 grams/animal/day.

After about 30 to 60 days on the above ration the oxidative enzyme and serum copper levels of the above tested cows were all within normal ranges and each cow had exhibited a strong heat.

Example 4 A beef cattle study was made of forty two and three year old first-calf purebred heifers to determine the effects that amino acid chelates of copper, manganese and zinc would have, (when compared to the same amount of minerals in inorganic form) , on cycling activity as determined by visual standing heats, first service conception rate, weaning weights and superoxide dismutase activity in the red blood cells. The herd selected for the study, (consisting of 15 Angus, 12 Horned Herefords, 5 Polled Herefords, 5 Brangus and 3 Simmental heifers) , had been experiencing fertility problems despite the fact that the cattle had been fed ratios containing protein and energy levels in excess of NRC requirements. The fertility rate, as measured by females pregnant 90 days after the breeding season was less than 75%.

An initial blood serum analysis showed low levels of calcium, phosphorus, magnesium, selenium, copper and zinc. The Angus heifers exhibited brown pigmentation around the eyes, on the tips of ears and behind the shoulders instead of the usual black. Many heifers exhibited feet and leg problems and reduced weaning weights. General growth problems and respiratory edema were also noted. Administration of a mineral mix of 66% dicalcium phosphate, 29% trace mineralized salt and 5% cottonseed meal demonstrated some visual improvement in some of the traits but the reproductive problems remained. The mineralized salt contained about 97% sodium chloride to which had been added manganous oxide, iron oxide, ferrous carbonate, copper oxide, ethylenediamine dihydroiodide, zinc oxide, cobalt carbonate and technical white mineral oil.

In an attempt to increase the levels of trace minerals available and provide for a common, unbiased starting point, the animals were placed on a free choice mineral supplementation program consisting of a 50:50 salt:mineral supplement mixture for a one year period. All heifers were maintained in the same pasture and received the same hay and protein supplement. The base diet, excluding the mineral supplement, contained 31 ppm zinc, 81 ppm manganese and 5.6 ppm copper. The mineral supplement contained 8-9% calcium [CaP0₄] , 8% phosphorus [CaP0₄] , 3% potassium [KC1 and amino acid complex], 4.75% magnesium [MgO and amino acid chelate] . Also included were 0.7% zinc, 0.3% manganese, 0.2% copper and 0.0025% cobalt all as amino acid chelates. Iodine [EDDI] at 0.001% and 0.002% selenium [SeS0₃] along with adequate amounts of Vitamins A, D₃ and E rounded out the test mineral supplementation. Subsequent blood serum analyses showed increased serum copper levels to within the normal range.

To evaluate the effectiveness of administering trace minerals as amino acid chelates, the group was divided into two treatment groups. Utilization of the amino acid chelate supplement for a one year period prior to the beginning of this test sufficiently equalized the starting point and removed bias on the randomization of heifer selection into the groups. At the beginning of the trial, blood samples were drawn and cow-calf pairs were randomly assigned to one of the two groups by breed, calving date, and body condition score to minimize the effects across treatments. Each group was placed into adjacent 45 acre pastures divided from each other by an electric fence. The heifers received a base nutritional program of 20 lbs native grass hay and 5 lbs of a 20% crude protein range supplement. The mineral treatments consisted of administering the same amounts of copper, zinc and manganese with one group receiving the minerals in the form of amino acid chelates and the other group receiving only inorganic minerals. These minerals were incorporated into a 20% crude protein supplement fortified with 14,687 I.U. of vitamin A, 3125 I.U. of vitamin D₃, 150 I.U. of vitamin E and 2.5 mg of selenium per pound and were administered at the rate of 1 lb of fortified supplement per heifer per day. In addition to these handfed ratios, a free choice supplement containing 66% dicalcium phosphate, 29% sodium chloride, and 5% cottonseed meal was provided. Including the basal nutritional program, the diets were formulated to supply a total of 18 ppm copper, 71 ppm zinc and 89 ppm manganese. As in the previous examples, iron was deemed to be adequate so no iron amino acid chelate was utilized.

Twelve days after the trial began, a breeder supplement containing additional minerals was utilized for two weeks prior to breeding at the rate of 2 oz/heifer/day. The minerals in the breed supplement were also divided into chelated and inorganic groups. Amounts of copper, manganese and zinc in the fortified supplement and breeder supplement are given in Table 4. / / /

At the time the breeder supplement was added, each heifer was injected with 2 cc of Synchro- Mate B (Norgestomet plus estradiol valerate) and implanted with a Synchro-Mate B implant. The implant was removed on the 10th day after insertion and a blood sample was withdrawn from each heifer at this time. The heifers were observed four times daily for signs of estrus beginning on the 11th day and were artificially inseminated about 12 hours after the observed heat. The respective breeder supplements were discontinued after the 14th day of administration, but the mineral supplement was continued for a total period of 75 days, after which a third blood sample was taken. Before completion of the test, three heifers were removed from the study. Two heifers from the amino acid chelate group were removed, one due to lameness and one because the Syncro-Mate B implant fell out. One heifer receiving the inorganic mineral was removed after her calf died. The results of the remaining 47 heifers are shown in Table 5:

Table 5

_Estrus _Activity, First Service conception and SOD Levels o_f Hei_fers; Adjusted Weaning Weight of Calves

Mineral Supplement Group

Heifers

Heifers exhibiting estrus

% Heifers exhibiting estrus

First service conception

% Heifers exhibiting estrus that conceived on first service % Total Heifers conceiving on first service Body condition score Average weaning weight (205 day) lbs/calf born to heifers CuZnSOD activity (units/mg) --

1st Bleeding (average)

3rd Bleeding (average)

The amino acid chelate supplemented heifers exhibited more standing heats and had a greater percentage conceiving on first service than the inorganic mineral supplemented cows. The weaning weight of calves from heifers in the amino acid chelate supplemented group averaged 48 lbs higher than from the inorganic mineral supplemented group which translates into significantly greater income to the producer.

The amino acid chelated supplemented heifers exhibited increased SOD activity from start to finish of the trial while the inorganic trace mineral supplemented females exhibited decreased enzymatic activity during the course of the trial. It is to be remembered that the serum copper levels of all heifers were within normal range at the beginning of the study due to the fact that the chelates had been administered. The study therefore shows that serum copper levels do not necessarily correlate with SOD activity levels, i.e. "normal" serum copper levels do not mean bioavailability of all the copper in the serum. The cell activity of the CuZnSOD, which is an erythrocyte enzyme, is principally dictated by the copper status of the animal at the time of erythrocyte synthesis. Therefore the fact that the amino acid chelated supplemented heifers exhibited increased SOD activity from start to finish of the trial while the inorganic trace mineral supplemented females exhibited decreased enzymatic activity during the course of the trial is significant, particularly when considered along with the other, more visible results. This demonstrates that quantifications, per se. may not be as significant as the determination of symptoms of oxidative stress in the system and then correcting that stress through the administration of appropriate amounts of copper, zinc and manganese as amino acid chelates.

The above examples demonstrate, in cases where symptoms of free radical toxicity are present, that necessary minerals, administered in the form of amino acid chelates were able to alleviate symptoms of this toxicity whereas, administration of inorganic mineral salts could not. It is therefore believed that the ingestion or absorption of effective amounts of essential minerals as amino acid chelates by warm-blooded animal species may alleviate some or all of the symptoms of free radical toxicity by increasing the uptake of these bioavailable minerals into the enzyme system to assure that the natural enzyme system remains operable, thus controlling the pathological effects in the body brought on by uncontrolled or excessive oxidative bursts associated with neutrophils and macrophages. While the above provides a detailed description of the invention and the best mode of practicing it to the extent that it has been developed, the invention is not to be limited solely to the description and examples. There are modifications which may become apparent to one skilled in the art in view of the description contained herein. For example, in some species the minerals, in the form of amino acid chelates, may be administered transdermally, with or without the aid of penetration enhancers. Such administration for transdermal absorption could be done in the form of a patch, form- filled liquid seal or simply as a creme or ointment. Therefore, the invention is to be limited in scope only by the following claims and their functional equivalents.

Claims

1. A method for reducing free radical cellular oxidative stress in warm-blooded animals showing symptoms of free radical toxicity which comprises the steps:

(1) confirming the presence of a free radical cellular oxidative stress in said animal,

(2) determining which trace minerals selected from the group consisting of copper, zinc, iron and manganese, and optionally, selenium, are needed to strengthen and maintain the functioning of enzymes which relate to oxidative bursts in neutrophils and macrophages in said animal, (3) providing a composition containing effective amounts of said trace minerals selected from the group consisting of copper, zinc, iron and manganese from step (2) in the form of amino acid chelates having a ligand to mineral ratio of at least 1:1, a molecular weight of no more than 1500 daltons and a stability constant of between about 10⁶ and 10¹⁶, with or without optional amounts of selenium which may be present either in amino acid chelated form or as inorganic selenium, and (4) administering said composition to said warm-blooded animal.

2. A method according to Claim 1 wherein said ligand to mineral ratio in the amino acid chelates is 2:1 or greater.

3. A method according to Claim 2 wherein said chelate has a molecular weight no greater than about

1000 daltons.

4. A method according to Claim 3 wherein said composition is administered orally.

5. A method according to Claim 4 wherein said composition is administered in the food of said animal.

6. A method according to Claim 5 wherein said composition is continuously available to said animal.

7. A method according to Claim 4 wherein said composition is administered in unit dosage form.

8. A method according to Claim 3 wherein the trace minerals to administer and the effective amount of trace minerals in said composition are determined by reference to collected data of mineral amounts and ratios found in animals afflicted by free radical cellular oxidative stress and compared to mineral amounts and ratios found in animals of the same species not afflicted by free radical cellular oxidative stress.

9. A method according to Claim 8 wherein the presence of free radical cellular oxidative stress in said animal is confirmed by an assay for red blood cells for activity of oxidative enzymes selected from the group consisting of CuZnSOD, glutathione peroxidase and catalase.

10. A method according to Claim 9 wherein the composition administered contains copper, zinc and manganese.

11. A method according to Claim 10 wherein the copper, zinc and manganese are present in molar ratio ranges of about 1.5-2.5:1.5-2.5:0.5-1.5 respectively.

12. A method according to Claim 11 wherein the copper, zinc and manganese are present in molar ratios of about 2:2:1 respectively.

13. A method according to Claim 8 wherein the animal is of the porcine species and exhibits symptoms of porcine stress syndrome.

14. A method according to Claim 4 wherein the animal is of the bovine species.

15. A method according to Claim 4 wherein the effective amount of trace minerals to be administered is determined after taking into consideration the content and balance of nutrients in the food ration and water also available to said animal.