Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO93/10777 PCT/US92/09587
21~120 '
COMPOSITION AND METHOD FOR REDUC~G 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 t~ hydrogen peroxide
by CuZn-superoxide dismutases (CuZnSOD) in the cells. In
a properly functioning system the hydrogen peroxide is
WO93/10777 PCT/US92/09587
ft~ 2 ~ ~ 2
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
0 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 metalloenzyme 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 th~ 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 c~ll to maintain SOD
activity.
~atalase is a large molecular weight enzyme that
contains four heme 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
W093~10777 2 1 2 i~ 1 PCT/US92/09587
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
lo 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 oxyqen 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
enzymés, their functioning and purposes are taught and
summarized by Coffey, "Catalase, CulZn-Superoxid~
Dismutase, Glutathio~e Peroxida~: Their Relationship to
O~ygen ~tilization in Cellular Physiolo~y, Clinic~l ~nd
8ubclinical Dise~se, Nutrition a~d Trace Element
~tilization in ~ivestock", The BQvine
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.
WO93/10777 PCT/U~92/09587
212~2Sli
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 membraneu 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 i5 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
- 25 alleviating free radic.al 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.
WO93/10777 2 1 2 ~ 2 ~ ~ PCT/~S92/~95~7
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, B63, 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
-15 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 S~MMARY OF T~E 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
provid~ a method of strengthening and maintaining the
functioning of enzymes in warm-blooded animals which
relate to oxidative bursts in neutrophils and
W ~ 93/10777 ~c~r/US92/09~87
212~2~4
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
S administered. These enzymes function to adequately
remove the superoxides, peroxides and hydroxides that
are formed in the cellsr
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 ~howing 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 sig~ificant and can ~ary depending upon
the species of animal, its vulnerability to free radical
toxicity or the symptoms of free radical toxicity which
are manifest due to inade~uate 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 10l6.
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
WO93/10777 PCT/US9~/09~87
~12 ~2~1
which issued September 5, 1989 and also in Ashmead et
al., Inte~tinal Ab~orptio~ of ~etal Io~ and ChQl~tes,
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 INVEN~ION
As documented by the ~shmead 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
~5 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 i5 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 zincl 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
W0~3/10777 PCT/US9Z/09587
2124204 8
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
l:l and preferably 2:l or higher, a molecular weight of
no more than 1500 and preferably not more than l000 and
a stability constant of between about l06 and l016. 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 bP 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
W093/10777 2 ~ 2 ~1 2 1~ ~I PCT/US92/~95~7
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 absorptiQn.
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 pres nt 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.
3C 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 bac~ground section above,
that catalase, CuZnSOD and glutathione peroxidase play
W093/1~777 PCT/US92/09587
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 formatlon and
~ioacti~ity 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:
~Able
8pecles CatalasQ Cu~nSOD GS~J-PX~GSH-P~
- k Vn ~ t~/Gm Units/mg Unit~/Gm
_________________________________________ ______________________
~ovll)e MO-llO 90-140 260-310
Swine 110-17U 40- 6~ 220-2~0
Sheep 17-Z4 60- 8~ 350-420
Equlne 110-150 60- 90 200-260
________________________ ________________________________________
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
W093/~0777 2 1 2 ~ 2 0 ~1 PCT/US92/09~87
11
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 con~inuous
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 u~ilization and immunity without resorting to
the use pf unproven drugs and/or chemicals which may
have a detrimental impact on the animalD
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 metho~ of treatment. For ex~mple, the
W O 93/10777 PC~r/US92/09587
2 1 2 ~ 2 ~ ~. 12
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 sérum 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
WO93/10777 2 ~ 2 4 2 , ~ PCT~US92/09587
13
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 iEon 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 l, 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
anothex which had been determined to be required ts 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
2S without undue experimentation what an "effective amount"
of a composition is and how to administer it
accoxdingly. It is not possible to categorically state
that "x" mg of trace mineral per kg of animal body
W093~l0777 PCT/US92/09587
21242U~ 14
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'l 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'l 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 l.
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 h~s been found that the optimal actiYity is obtained
when the mole ratio of Cu:Zn:Mn is about 1:1:0.5.
WO93/10777 2 1 ~ ~ 2 ~11 PCT/~S92/09587
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 1o6 and 1olÇ.
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 productior. within sperm is
highly damaging. After the first test the bull was
placed on a ration which included zinc, manganese and
W093tlO777 PCT/US92/09587
21242~3'~ 16
copper amino acid chelates. These minerals were ~helated
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 10~6. 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:
WO 93~10777 PCT/US92/09587
2l2~2a~l
17
~able 2
_____________ ______________________--______ ________ .______~___
Test Item Initial Third ~Sixth Ninth
Week Week ` Week
_____________________________~______________ .________________ _
Blood Hemoglobin
(Gm/dl) 12.02 13.54 12.57 13.05
CuZnSOD-l
(units/mg) 41.0 52.0 46.5 35.9
CuZnSOD(R)
(Unit61mg) 62.0 55.0 62~S 58.8
% Normal SOD Activity
(SOD-l/SOD-2) 66.S 94.7 74.4 60.8
Catalase
~k Units/gm) 29.7 58.0 44.0 82.0
GPx (Se Dependent)
(Units/gm) 101 197 231 310
GSH-P(Total)
(Un~t8/gm) ~ __ 246 310
_________._______________________________-____________________.._
3~
oxidative enzyme activity. The glutathione (GPx) and
catalase activities almost doubled. At the time the
sixth weeX test blood sample~ were ~aken, the bull was
producing viable sperm. The GPX level had continued to
elevate ~ut 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
- 40 had dropped from the third week test with a lowering of
SOD activity indicating a lack of copper or insufficient
copper utilization.
WO93/10777 PCT/US92/09587
2 12425' ~ ~
1~ ,
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 ~1~ 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.
Suqaested Alternate Therapies: To bolster the
CuZnSOD activity of this bull, it was suggested that
alternative therapies be adopted, both of which would
utilize copper, æinc 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
W O 93/10777 2 1 2 ~ 2 ~ 1 P ~ /US92/09587
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.
Exa~pl- 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 hyperthermia 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
W093/10777 PCT/US92/09587
21242{~ -
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 succes~.
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 completeIy and mortality fell to the anticipated
rate of O . 5 to 1. 0% . When the chelated supplement was
withdrawn the deaths increased again within 10-14 days.
Ex~mpl~ 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
WO ~3~10777 2 1 ~ ~ 2 ~3 ~ PCT/US92/09587
21
was analyzed for RBC oxidative enzyme content and ~erum
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
thP cows which is exemplary of the herd. Cow One was
dry, C~w Two had been lactating for about 40 days and
Cow Three for about 165 days.
- 10
Table 3
_________________i__________ __ ___________ _____________________
Test Item Normal Cow No. Cow No. Cow No.
Range One Two Three
________ _______________________________________________________
Blood Hemoglobin
(Gm/dl) 11.5-13.09.59 8.71 10.55
CuZnSOD-1
(Unlts/mg) 90-14049~41 38.42 41.43
CuZnSOD~R)
(Units/mg) 90-14071.43 60.61 S8.89
% Normal SOD. Activity
(SOD-1/SOD-Z) 90+ 69.17 63.39 70.35
Catalase
(k Units/gm) 30-110117.37 112.53 104.54
GPx fSe Dependent)
(Vnltslgm) 260-310210.29 Z10.93 230.~7
GSH-P(Total)
(Vnit6/gm) 260-310227.33 220.26 245.98
_______________________..________________________________________
Serum Mineral Con~ent:
Selenium (ppm) 0.07~0.30.031 Q.110 0.085
Manganese ~ppm~ 0.01-0.030.034 0.032 0.049
Copper (ppm) 0.70-1.000.43 0.52 0.51
Zinc (ppm) 1.50-1.800.63 1.50 3.81
Iron (ppm) 0.90-2.500.97 0.82 2.11
__________O_________________._.._______.__________________________
~5
WO93/10777 PCT/~S~2/09587
2~ 2al i
22
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 notresponded 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 ~ineral
supplement being mixed with the feed ration. Su~ficient
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
WO93/10777 2 1 ~ 1 2 ~ ~ PCT/US92/09587
23
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 0.2~ 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 ~0 days on the above ration the
oxidative Pnzyme and serum copper levels of the above
tested cows were all within normal ranges and each cow
had exhibited a strong heat.
E~ampl~ 4
A beef cattle study was made of forty two and three
year old first-calf purebred hei~ers to determine the
effects that amino acid ch lates of copper, manganese
and zinc would haYe, (when compared to the same amount
of minerals in inorg~nic form), on cycling activity as
determined by visual standing heats, first service
conception rate, weaning weights and superoxide
dismu~ase activity in the red blood cells. The herd
selected for the study, tconsisting of 15 Angus, 12
Horned Herefords, 5 Polled Herefords, 5 Brangus and 3
w093/~0777 PCT/US92/09587
21212~31
24
Simmental heifers), had been experiencing fertility
problems despite the fact that the cattle had been fed
ratios containing protein and energy levels in ex~ess 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
somé -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
WO93/10777 2 1 ~ 3 ~ PCT/US92/09587
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 ~CaPO4], 8% phosphorus
[CaPO4], 3% potassium [XCl 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 [~DDI] at
0.001% and 0.002% selenium [SeSO3] along with adeguate
amounts of Vitamins A, D3 and E rounded out the test
mineral supplementation. Subse~uent 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.
Th~ heifers received a base nutritional program of 20
lbs native grass hay and 5 lbs of a 20% crude protein
WO93/10777 PCT/USg2/~9587
2I2~2~4
26
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 a~ the rate of 1 lb of
fortified supplement per heifer per day. In addition to
these handfed ratios, a free choice supplement
containing 66~ dical~ium phosphate, 29% sodium chloride,
and 5% cottonseed meal was provided. Including the basal
nutritional program, the diets were formulated to supply
lS 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 chalate was
utilized.
Twelve days after the trial b~gan, a breeder
supplement containing additional minerals was utilized
for two week~ 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.
WO 93/10777 2 1 2 4 2 ~ ~ PCTtUS92/09S87
27
T~ble 4
________________________________________________________________
Milligrams Mineral In
1 lb 20~ 2 oz Total
Mineral Mineral Breeder Mineral
Form Supplement Supplement Amounts
----________
CuSO~ 17~ 4~ 226
Cu Amino Acid Chelate 178 48 226
MnO 275 80 335
Mn Amino Acid Chelate 275 80 335
ZnSO4 563 258 821
Zn Amino Acid Chelate 563 258 821
_______________ _____________________________ _ _______________
At the time-the breeder supplement was
added, each heifer was injected with 2 cc of Synchro-
Mate B ~Norgestomet plus estradiol valerate3 andimplanted with a Synchro-Mate B implant. The implant was
remo~ed 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 ~rtificially
insemïnated about 12 hours after the obser~ed 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 remo~ed from
the study. Two heifers from the amino acid chelate group
were removed, one due to lameness and one because the
WO 93/10777 PCT/US92/09~87
:' ..
212~2~l~ 28
Syncro-Mate B implant fell out. One heifer receiving the
inorganic mineral w~s removed after her calf died.
The results of the remaining 47 heifers are s~own in
Table 5:
~ablu 5
_________________________ __~______________ _ _____~_________ __
Estr~s ~ctiv~ty, Fir~t Service conception and SOD Levels
of lieiers; ~djusted Wea~ing Weight of Calves
___________________________________ _______________~__ _________
Mineral Supplement Group
S _________________________
Ch~lat~d Inorqanic
___.____ _.________
Heifer~ exhibit~ng s~tru~ 14 ;19
% Heifer~ exh~biting estrus 78 42
First ~ervice conc~ptlon 10 2
% ~eifers exhibiting estrus that
conceived on first ~ervice 71 25
% Total llelfers conceiving .
on first service 56 11
Body cond~t~on ~core 5.0 5;3
~verage weanlng welght (205 day)
lbs/calf born to heifer6~75 527
CuZnSOD activity (u~its/mg) .
1st Bleeding (average) 52.9 59.0
3rd Bleeding (average) 59.3 56.8
________________________________________________________________
~0 .
The amino acid chelate supplemented heifers
exhi~ited 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
WO93/10777 2 1 ~. ~ 2 ~3 '~ PCT/US92/095~7
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 inorgani~ 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
~o 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
'5 copper in the serum. The cell activity of the CuZnSO~,
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, Der se, may not be as significant
as the determination of symptoms of oxidative stress in
the system and then correcting that stress through the
W O 93/~0777 PC~r/US92/09587 ~ 2420~ ~ `
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 ~est mode of practicing it to the
extent that it has been developed, the invention is not
to ~e 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
W093/10777 2 1 2 ~ 2 0 ~ P~T/US92/09587
3~
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 oin~ment.
Therefore, the invention is to be limited in scope only
by the following claims and their functional
equivalents.
s
