Biological assimilation of metals - Patent Review 4167564

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BACKGROUND

The rate of growth and well-being of a living organism is dependent upon the ability and rate at which that organism is able to assimilate life's building blocks, i.e., proteins, carbohydrates, lipids, minerals and vitamins and convert or metabolize them into useful products for that organism. It is well known that many metals are essential to the proper functioning of a living organism. It is also well known that some metals are very difficult to assimilate into the cells of a living organism and are, for that reason, available in various forms, i.e., usually various organic and inorganic salts.

DESCRIPTION OF THE INVENTION

It has now been found that metabolically essential metals and trace elements such as iron, copper, zinc, manganese, cobalt, chromium, calcium, magnesium, vanadium and the like can be assimilated into a living organism if they are made available to that organism in the form of chelated coordination complexes with amino acids and other hydrolysis products of proteins in a buffered state at a relatively constant pH. For purposes of this disclosure, a chelate is defined to mean a coordination complex between the metal ion and an organic compound which is an amino acid or other organic compound derived from hydrolyzed protein and in which atoms within the organic compound are coordinated with the metal ion. Since proteins are made up of amino acids, the chelated coordination complex thus formed will hereinafter be referred to as a proteinate of the metal ion or a metal proteinate. Such terms shall be inclusive of coordination complexes with amino acids, proteins and proteins in any state of hydrolysis.

Any of the amino acids produced from proteins by hydrolysis are applicable to this invention. Most proteins yield about twenty different amino acids. These are all alpha amino acids and are listed as follows: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, cystine, cysteine, methionine, proline and hydroxyproline. Preferred of the above amino acids are methionine, glycine, aspartic acid and glutamic acid. Especially preferred is methionine.

The buffer system functions to control the pH of the metal proteinate and its surrounding media and maintain it at a relatively constant state. By thus creating optimum conditions, the uptake or assimilation of metal proteinate into the surrounding tissues is hastened. For purposes of this application, it is irrelevant at which portion along the digestive tract the assimilation of the metal proteinate occurs. As will be seen in the following examples, the essential metal is rapidly distributed throughout the body tissues. The buffer system may change according to the metal proteinate being administered, and one or more metal proteinates may be administered at one time.

The choice of buffer system will depend upon the pH desired. The amino acids alone will interact with both acidic and basic solutions such as hydrocholoric acid (HCl) and sodium hydroxide (NaOH) to form buffered systems. Typical buffer solutions include acid phthalates, neutralized phthalates, phosphates, carbonates and bicarbonates and combinations thereof. Either alkali or alkaline earth metals may be utilized in the buffer system. Examples of buffering systems in the pH range of 3 to 11 are set forth below:

__________________________________________________________________________ BUFFERS pH __________________________________________________________________________ 3.0 50 ml M/5 KHPhthalate + 22.3 ml M/5 HCl diluted to 200 ml 4.0 50 ml M/5 KHPhthalate + 0.1 ml M/5 HCl diluted to 200 ml 5.0 50 ml M/5 KHPhthalate + 22.6 ml M/5 NaOH diluted to 200 ml 6.0 50 ml M/5 KH.sub.2 PO.sub.4 + 5.6 ml M/5 NaOH diluted to 200 ml 7.0 50 ml M/5 KH.sub.2 PO.sub.4 + 22.4 ml M/5 NaOH diluted to 200 ml 8.0 50 ml M/5 KH.sub.2 PO.sub.4 + 46.1 ml M/5 NaOH diluted to 200 ml 6.0 23.2g KH.sub.2 PO.sub.4 + 4.3g Na.sub.2 HPO.sub.4 per liter 7.0 9.1g KH.sub.2 PO.sub.4 + 19.7g Na.sub.2 HPO.sub.4 per liter 10.0 6.5g NaHCO.sub.3 + 13.2g Na.sub.2 CO.sub.3 per liter 11.0 11.4g Na.sub.2 HPO.sub.4 + 19.7g Na.sub.3 PO.sub.4 per __________________________________________________________________________ liter

Other systems which have been successfully used are the combination of proteins which have a tendency to keep solutions at pH 7 with metal chelates and vitamins including ascorbic acid (Vitamin C). The present invention encompasses any combination of organic or inorganic substituents which will buffer or maintain a system at a pH range of from about 3 to 11 with acidic ranges of from about 3 to 6.9 and basic range of about 7 to 11. There are numerous other buffering systems readily available to one with ordinary skill in the art and mere enumeration of them at this point would be meaningless. What is important to the invention is that a buffering system be selected which will not only stabilize a metal proteinate solution but be non toxic and assist in the assimilation of such metal proteinate into the living organism.

While the term "living organism" has been referred to throughout this disclosure, the invention is primarily intended for the treatment of warm-blooded animals. The dosage to be administered will, of course, be dependent upon the type of animal, i.e., species, size, weight, age, sex, etc. and the metal proteinate being administered. Dosage is not a critical function of this invention as it may be empirically determined by one with ordinary skill in the art once the invention is defined. The method of dosage may also vary. The metal proteinate may be administered orally by ingestion or by means of a stomach tube. It may also be given by injection intramuscularly, intravenously or subcutaneously. If desired, the proteinate may be injected directly into any portion of the gastrointestinal tract by any appropriate means. When given to pregnant animals it has also been found that the buffered proteinates are sufficiently stable that they are transported across the placental barrier to the fetus. What is critical is the discovery that the uptake of essential metal can be enhanced by the use of the stabilized buffered metal proteinate complexes of this invention.

In order to demonstrate the improved assimilation of metal ions as metal proteinates into living tissue, several studies were undertaken utilizing radio isotopes. These metals were so tagged in order to quantitatively measure their uptake into the living organism.

The following examples are illustrative of the invention and are not to be construed as limitations thereon.

EXAMPLE I

Traces of Zn.sup.65 Cl.sub.2 were mixed with non radioactive zinc chloride and complexed with methionine as an amino acid. To demonstrate structure and how tightly the methionine was bound to the zinc, a polarographic study was made. A solution was prepared containing 0.0001 moles of zinc per 100 mls of solution of ZnCl.sub.2, and there was added thereto sufficient 0.2 M methionine to produce a solution having a molar ratio of amino acid to zinc as follows:

______________________________________ Mls of 0.2M Moles Ratio Solution Methionine Methionine Zinc ______________________________________ 1 0 ml 0 2 2.5 ml 0.5 3 5.0 ml 1.0 4 10.0 ml 2.0 5 20.0 ml 4.0 6 40.0 ml 8.0 7 80.0 ml 16.0 ______________________________________

To each of the above solutions was added 10 mls of 1 M potassium nitrate (KNO.sub.3) as an electrolyte and 10 mls of a 0.1% gelatin solution. Each solution was corrected to a pH of 7 by the addition of a few drops of concentrated (6 N) sodium hydroxide (NaOH) solution.

Using a Metrohm E 261 polarograph with a silver/silver chloride (Ag/AgCl) reference electrode, the following E.sub.1/2's were recorded:

______________________________________ Solution No. E.sub.1/2 ______________________________________ 1 -1.008 2 -1.033 3 -1.057 4 -1.079 5 -1.090 6 -1.110 7 -1.129 ______________________________________

A plot of the log of the proteinate ligand concentration against the E.sub.1/2 gives a sloped line which is indicative of the number of ligands in the complex. It was found that the Zn.sup.++ ion complexes with two molecules of methionine.

While not wishing to be bound by any specific theory, it is believed that at higher ligand concentrations and at a higher pH (more basic) the complex is probably a bicyclic complex.

By knowing the number of ligands, the stability constant at different concentrations and pH's can be determined. It has been found that the logarithm of the stability constant equals: ##EQU1## where p=the number of ligands and [ligand] refers to the concentration of the ligand.

Solution Number 7 (16 moles of methionine per mole of zinc) was found to have a stability constant equal to 4.94.times.10.sup.7 at pH 7. The same solution was adjusted to a pH of 9 and the stability constant was found to be 4.41.times.10.sup.12. In other words, by changing the pH of a zinc methionate solution of the same concentration from 7 to 9 an increase in stability of 10.sup.5 or 100,000 was obtained. Similar results can be demonstrated with copper, iron, chromium, calcium, manganese, magnesium, vanadium and other essential metals.

EXAMPLE II

The following study was done to correlate the results obtained in Example I with the way animals absorb the buffered metal proteinate complexes. White laboratory rats were used as experimental animals, and each rat received the same amount of tagged zinc chloride by dosing with a pipette directly into the rat's stomach. The molar ratio of zinc to methionine was one to two for Rat II and III, and the pH was adjusted according to the following table:

__________________________________________________________________________ Rat I Rat II Rat III __________________________________________________________________________ 24 microliters Zn.sup.65 Cl.sub.2 24 microliters Zn.sup.65 Cl.sub.2 24 microliters Zn.sup.65 Cl.sub.2 75 microliters H.sub.2 O 25 microliters H.sub.2 O con- 25 microliters H.sub.2 O con- taining NaHCO.sub.3 /Na.sub.2 CO.sub.3 taining NaOH to pH 7 to pH 10 50 microliters methionine- 50 microliters methionine- solition 2:1 molar solution 2:1 molar ratio with Zn.sup.++ ratio with Zn.sup.++ __________________________________________________________________________

The rats were placed in metabolic cages on a normal diet and were observed for one week during which time the feces were collected. At the end of the week, the rats were sacrificed, and the total excreta measured by scintillation count for radioactivity as compared to a blank. The following amounts of Zn.sup.++65 were excreted by each of the rats as measured by the collected feces for the week:

______________________________________ % of Total Dose Excreted ______________________________________ Rat I 52% Rat II 12% Rat III 36% ______________________________________

More than half of the Zn.sup.65 Cl.sub.2 in the control animal was lost. The Zn.sup.65 methionate retention in Rat II administered at pH 10 was significantly better than the Zn.sup.65 methionate retention in Rat III administered at pH 7. However, both showed marked improvement in Zn.sup.++ retention over Rat I.

EXAMPLE III

Example II was essentially repeated using Fe.sup.59 SO.sub.4 as the control. The solution was orally administered by pipette into the stomach. Each rat received 36.7 micrograms of Fe.sup.59 in 20 microliters of sollution. Rat II was administered a methionine solution and Rat III a glycine solution, both buffered to a pH of 10 in a molar ratio of two to one metal to amino acid. At the end of a week, the rats were sacrificed and parts of various organs analyzed for Fe.sup.++59 by scintillation count.

The following results were obtained:

______________________________________ CORRECTED COUNTS PER MINUTE PER GRAM Rat I Rat II Rat III Tissue FeSO.sub.4 FeMet FeGly ______________________________________ Heart 63. 151. 83. Liver 136. 243. 83. Gastroc 2. 54. 83. Masseter 14. 138. 65. Brain 31. 130. 142. Kidney 2. 327. 150. Testes 20. 109. 75. Serum 700. 1,797. 840. Cells 742. 2,076. 773. Blood 1,335. 4,215 1,602. Feces 302,400. 314,000. 205,800. Urine 490. 370. 690. Rat I Rat II Rat III Feces 45.8% Lost 32.4% Lost 31.2% Lost ______________________________________

The results reported above are very dramatic. The amounts of Fe.sup.++59 retained by Rats II and III administered the buffered Fe.sup.59 proteinate were significantly higher than in Rat I as demonstrated by the feces analysis. The amounts of metal retained in the tissues were also significantly higher in almost every instance. However, detailed results were not computable because the complete organ was not removed for analysis.

Buffering systems which were used in the above examples include amino acid-NaOH solutions and a solution of 6.5 grams of sodium bicarbonate (NaHCO.sub.3) and 13.2 grams of sodium carbonate (Na.sub.2 CO.sub.3) per liter of solution which will produce a buffered solution of about pH 10.

EXAMPLE IV

To further substantiate the effects of buffering metal proteinates, the following tests were conducted on rats which had been fasted over night. Each rat was given the following dosage of radioactive calcium by injection into the duodenum:

Rat I

250 microliters of CaCl.sub.2 solution (1 mg. Ca) is distilled H.sub.2 O

40 microliters of distilled H.sub.2 O (40 mcC Ca.sup.45)* as Ca.sup.45 Cl.sub.2

*mcC=micro curies

Rat II

250 microliters of CaCl.sub.2 solution (1 mg Ca) in distilled H.sub.2 O buffered to pH 7 with NaOH and the amino acids and containing in molar ratio with calcium 2 moles of aspartic acid, 2 moles of glycine and 1 mole of methionine

40 microliters of distilled H.sub.2 O (40 mcC Ca.sup.45) as Ca.sup.45 Cl.sub.2

Rat III

Same as Rat II except buffered to Ph 10 with NaHCO.sub.3 /Na.sub.2 CO.sub.3.

The rats were fed a normal diet for one week, and the feces were collected. At the end of one week the rats were sacrificed and the total feces and portions of the tissues were analyzed by scintillation count. The results obtained are as follow:

______________________________________ CORRECTED COUNTS PER MINUTE PER GRAM Tissues Rat I Rat II Rat III ______________________________________ Frontal Bone 3682 5878 5772 Massater 602 844 904 Gastroc (muscle) 614 620 1206 Heart 642 598 932 Liver 664 546 742 R. Cerebrum 698 726 804 Kidney 686 656 730 Lung 676 672 648 Serum 8.4 39.6 31.0 100 microliters Cells Blood 18.6 0 13.2 Total Blood 27.0 39.6 44.2 ______________________________________

It is evident from the tissue counts that much more of the calcium proteinate was assimilated into the tissues at the buffered Ph of 10 than at pH 7. However, it is also evident that more of the calcium proteinate was absorbed at the buffered pH 7 than was the calcium salt control.

Insofar as the feces is concerned, it can readily be seen that about four times as much calcium was excreted in the simple organic salt control (Rat I) than in the buffered (pH 10) calcium proteinate. Moreover, the pH 10 proteinate was approximately twice as effective in retaining calcium than was the buffered (pH 7) proteinate. It would thus appear that buffering undoubtedly assists in both promoting stability of the metal proteinate solution and in improving its assimilation into a host of various tissues.

EXAMPLE V

The above examples tend to show that a buffer system at about pH 10 will improve certain metal assimilation into living tissues. This pH however is not optimum for all metals. Some metals actually are better absorbed at a lower (more acidic) buffered range. The object is to find and maintain the optimum pH range for the metal to be administered. This may be empirically established for each metal proteinate.

Manganese, for example, is absorbed better as a proteinate at lower pH's than in more basic systems where it tends to form Mn (OH).sub.2.

Manganese, calcium also do not function well with carbonate buffered solutions in that they tend to form insoluble carbonates.

The absorptive capacities of manganese proteinates at a buffered pH of 7 are demonstrated below. Two solutions utilizing Mn.sup.54 were made up as follows:

Solution I

250 microliters distilled H.sub.2 O containing 100 mg of Mn as MnCl.sub.2

20 microliters Mn.sup.54 solution (14.3 mcC) (Slightly acidic pH) as Mn.sup.54 Cl.sub.2

Solution II

250 microliters distilled H.sub.2 O containing 100 mg of Mn as MnCl.sub.2

20 microliters Mn.sup.54 solution (14.3 mcC) as Mn.sup.54 Cl.sub.2

Based on a molar ratio of total manganese, the solution contained per mole of manganese, 2 moles each of the amino acids--methionine, glycine, aspartic acid and glutamic acid. The solution was buffered to a pH 7 with NaOH interacting with the amino acids.

The solutions prepared were injected into the duodenum of laboratory rats (labeled Rat I and Rat II according to solution given) which were fed a normal diet for one week and then sacrificed. The tissues were then measured by scintillation count as an indication of manganese proteinate uptake. The results are as follows:

______________________________________ CORRECTED COUNTS PER MINUTE PER GRAM cc/min/gm dry wt I II ______________________________________ Heart 370 1190 Kidney 470 600 Brain 620 1170 Gastroc 800 660 Masseter 270 310 Liver 760 1070 Lung 720 330 Frontal Bone 350 780 Duodenum 170 480 ______________________________________

As will be noted from the above table, almost all counts were higher in Rat II administered the manganese proteinate than in control Rat I. Counts in urine and feces from these animals were not obtained.

EXAMPLE VI

The above examples illustrate the assimilation of buffered metal chelate complexes with isolated amino acids or limited combinations of acids. This example demonstrates that hydrolized protein (containing essentially all the amino acids) may be used as effectively. This example further demonstrates the placental transfer of stabilized metal proteinates from the mother to the unborn fetus. Mink were chosen for these tests and iron was chosen for the metal proteinate. This was done because many authorities in mink production believe that mink have difficulty in placental transfer of iron from mother to young.

Two pregnant mink individually housed were fasted for twenty to twenty-four hours and were then given 24.17 milligrams of iron containing 5 mcC of Fe.sup.59 ratio active isotope. Mink No. 1 was given the iron in the form of Fe.sup.59 SO.sub.4 which had been chelated into hydrolyzed protein and buffered with a NaHCO.sub.3 /Na.sub.2 CO.sub.3 solution to a pH of 10. Mink No. 2 was given the same amount of iron as Fe.sup.59 SO.sub.4. In each case, the isotopes were mixed with 25 grams of food which was consumed by the mink by ingestion. The iron was administered to each mink 15 days before whelping. Faces and urine from each mink were collected to determine the amount of Fe.sup.++ excreted. Measurements were recorded 4 days after dosing and at the time of sacrifice. Fifteen days after dosing each mink was sacrificed and the various biological tissues measured for radio active iron by scintillation count. Measurements were also made of the hemoglobin and hematocrit of the mother and the kits. The data obtained are given in the two following tables.

__________________________________________________________________________ GENERAL DATA Mink No. 1 Mink No. 2 __________________________________________________________________________ Total % Fe.sup.++ Retained 70.4 42.7 Total % Fe.sup.++ Excreted in feces 24.4 29.6 Total % Fe.sup.++ Excreted in urine 5.17 27.7 % Fe.sup.++ Excreted in Feces 4 days after dosing 21.5 23.8 % Fe.sup.++ Passed on to young (kits) 0.03 (7.3 micrograms) 0 Hemoglobin Mother-gm % 20.5 20.0 Hematocrit % 45 44 Average Hemoglobin of young (kits) 19.5 19 gm/% Average Hematocrit of young (kits) 53 50 Whole Body Counts Without Organs 112.4 68.1 Mother (corrected counts/minute) Average Body Counts Per Kit 42.3 1 (Corrected Counts/minute) __________________________________________________________________________

______________________________________ CORRECTED COUNTS PER MINUTE PER GRAM Tissue Mink No. 1 Mink No. 2 ______________________________________ Masseter 7.81 12.00 Pectoralis Major 1.22 5.02 Spleen 15.3 10.60 Brain 9.7 7.57 Lung 6.4 4.05 Heart 2.7 5.12 Liver 4.98 4.73 Neck Fur and Skin 6.24 3.82 Scalp 5.74 6.33 ______________________________________

From the above data several conclusions can be drawn. It is at once evident that the amount of Fe.sup.++ retained in Mink No. 1, dosed with the buffered iron proteinate was 65% greater than the amount retained in Mink No. 2 dosed with Fe.sub.2 SO.sub.4. Stating it another way about 70% of the iron in the buffered iron proteinate was metabolized whereas only 42.7% was retained in the mink treated with Fe.sub.2 SO.sub.4.

Comparing the amounts of iron excreted after 4 days with the final analysis, it is evident that in Mink No. 2 33.5% of the iron initially dosed was absorbed but not metabolized, and was eventually eliminated between the fourth and fifteenth day after dosing. In Mink No. 1 only about 8% of the absorbed iron proteinate was later eliminated and not metabolized. The data show that a measurable amount of Fe.sup.++ as iron proteinate was carried to the kits from Mink No. 1 by placental transfer (42.3 cc/min) whereas the Fe.sup.++59 in the kits from Mink No. 2 was barely evident (1 cc/min). The hemoglobin and hematocrit measurements were higher from the iron proteinate dosed mink than from the control. The iron proteinate is utilized in the blood, skin and organs more readily as shown by tissue counts. The spleen, which is 90% blood, contains about 50% more iron from the buffered iron proteinate than from the Fe.sub.2 SO.sub.4 control. This is important as it demonstrates that the iron proteinate is better for building hemoglobin than the corresponding Fe.sub.2 SO.sub.4. Finally, the data show that hydrolyzed protein is as effective a ligand for complexing with buffered metals to form metal proteinates for transport of metal into the blood stream from the intestinal tract as the individual amino acids.

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