U.S. patent application number 11/035109 was filed with the patent office on 2006-02-09 for methods and compositions for improved chromium complexes.
Invention is credited to Jovan Hranisavljevic, Dusan Miljkovic, Zbigniew Pietrzkowski.
Application Number | 20060029642 11/035109 |
Document ID | / |
Family ID | 35757663 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029642 |
Kind Code |
A1 |
Miljkovic; Dusan ; et
al. |
February 9, 2006 |
Methods and compositions for improved chromium complexes
Abstract
Complex metal-containing matrices, and especially
chromium-containing matrices are produced from a water soluble
preparation that is derived from an item suitable for animal (and
most typically human) consumption. In particularly contemplated
aspects, the water soluble preparation is an extract or filtrate of
disintegrated brewer's yeast, and the so prepared complex mixture
is combined with a chromium-3.sup.+ ions.
Inventors: |
Miljkovic; Dusan; (San
Diego, CA) ; Hranisavljevic; Jovan; (Belgrade,
YU) ; Pietrzkowski; Zbigniew; (San Diego,
CA) |
Correspondence
Address: |
ROBERT D. FISH;RUTAN & TUCKER LLP
611 ANTON BLVD 14TH FLOOR
COSTA MESA
CA
92626-1931
US
|
Family ID: |
35757663 |
Appl. No.: |
11/035109 |
Filed: |
January 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US04/25026 |
Aug 3, 2004 |
|
|
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11035109 |
Jan 12, 2005 |
|
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Current U.S.
Class: |
424/439 ;
514/185 |
Current CPC
Class: |
A61K 31/555 20130101;
A61P 9/00 20180101; A61K 47/46 20130101; A61P 21/00 20180101; A61P
3/08 20180101; A61K 9/1664 20130101 |
Class at
Publication: |
424/439 ;
514/185 |
International
Class: |
A61K 47/00 20060101
A61K047/00; A61K 31/555 20060101 A61K031/555 |
Claims
1. A metal-fortified substantially completely water soluble complex
matrix formulated for mammalian consumption.
2. The metal-fortified complex matrix of claim 1 wherein the matrix
comprises a water soluble fraction of a cellular lysate.
3. The metal-fortified complex matrix of claim 2 wherein the
cellular lysate is a brewer's yeast extract.
4. The metal-fortified complex matrix of claim 2 wherein the
cellular lysate is dehydrated.
5. The metal-fortified complex matrix of claim 1 wherein the metal
is a chromium-3.sup.+ ion.
6. The metal-fortified complex matrix of claim 5 wherein the matrix
has an absorption maximum at a wavelength between 550 nm and 570
nm.
7. The metal-fortified complex matrix of claim 6 wherein the matrix
comprises a freeze-dried filtrate of a brewer's yeast lysate.
8. The metal-fortified complex matrix of claim 1 formulated as a
nutritional item selected from the group consisting of a beverage,
a bar, a cereal, and a pill.
9. A method of preparing a chromium-containing product comprising:
preparing a water soluble complex preparation from an edible
material; and combining the preparation with a trivalent chromium
ion under conditions effective to form a complex between a
component of the preparation and the chromium ion.
10. The method of claim 9 wherein the step of preparing comprises
disintegrating a cellular edible material and a step of removing at
least part of an undissolved material from the disintegrated
material.
11. The method of claim 10 wherein the step of removing the
undissolved material comprises at least one of filtration and
centrifugation.
12. The method of claim 10 wherein the trivalent chromium is added
to the disintegrated material before the step of removing the
undissolved material.
13. The method of claim 10 further comprising a step of removing
water from the complex between the component of the preparation and
the chromium ion.
14. The method of claim 12 wherein the edible material comprises at
least one of a fruit, a vegetable, and a grain.
15. The method of claim 12 wherein the edible material comprises
brewer's yeast.
16. The method of claim 9 wherein the complex between the component
of the preparation and the chromium ion has an absorption maximum
at a wavelength between 545 nm and 565 nm.
17. A food item comprising a chromium-containing complex having an
absorption maximum at a wavelength between 545 nm and 575 nm, and
wherein the complex is present in an amount effective to reduce
fasting blood glucose in a mammal ingesting the food item.
18. The food item of claim 17 wherein the complex is substantially
completely water soluble.
19. The food item of claim 17 wherein the complex comprises a water
soluble extract from a cellular edible item.
20. The food item of claim 19 wherein the complex comprises a water
soluble extract from brewer's yeast.
Description
[0001] This application is a continuation-in-part application of
our co-pending international patent application with the serial
number PCT/US04/25026, which designates the U.S., filed Aug. 3,
2004.
FIELD OF THE INVENTION
[0002] The field of the invention is chromium-containing
nutritional compositions and methods, and particularly those with
enhanced biological activity and/or absorption.
BACKGROUND OF THE INVENTION
[0003] Numerous chromium-containing compositions are well known in
the art, however, all or almost all of them suffer from one or more
disadvantages. Most significantly, while some of the
chromium-containing supplements are embroiled in toxicity issues
(e.g., Cr-picolinate), others have only relatively low solubility
and/or bioavailability (e.g., chromium yeast), or are expensive
(e.g., chromium lactoferrin) in their production.
[0004] For example, WO 03/101436 and U.S. Pat. No. 3,925,433
describe various alpha amino acid complexes with Cr3.sup.+ in
nutritional supplements. Similarly, pure alpha amino acid chromium
complexes are taught in U.S. 2003/0228394 as animal feed additive.
To increase bioavailability of chromium from amino acid complexes,
histidine or threonine may be employed as main ligand as reported
in U.S. Pat. Nos. 6,689,383, and 6,548,687, respectively. Where
desirable, non-proteinogenic amino acid complexes with chromium may
be prepared as disclosed in U.S. Pat. No. 6,071,545 and U.S.
2004/0106591. Amino acid containing mixed complexes are taught in
WO 02/056889, where an amino acid and nicotinic acid act as
ligands. While amino acid ligands are typically considered
nutritionally safe, amino acid complexes formed with chromium tend
to pose several drawbacks. Among other things, bioavailability of
the chromium from the complex is often relatively low. Furthermore,
at least some of such complexes have displayed toxicity to some
degree.
[0005] To overcome at least some of the problems associated with
amino acid complexes, non-amino acid ligands (e.g., nicotinate and
picolinic acid) are relatively common and often exhibit improved
bioavailability, and may further be employed in mixed complexes
and/or combination formulations. For example, known non-amino acid
chromium complexes include polynicotinate chromium complexes as
described in U.S. Pat. No. 6,323,192. Similarly, complexes in which
niacin binds chromium were reported as reducing blood glucose in
U.S. 2003/0133992. In yet another example, chromium arginate or
chromium chalidamate were used as defined and water soluble
chromium complexes that were administered in combination with an
oxygen uptake enhancer as described in U.S. 2004/00053688. Still
further known preparations include those in which Cr-picolinate or
Cr-polynicotinate are combined with a cyclooxygenase inhibitor as
described in U.S. Pat. No. 6,713,469, or with conjugated linoleic
acid or conjugated linoleic alcohol as taught in U.S. Pat. No.
6,809,115.
[0006] Other isolated and defined chromium ligands include sucrose
as taught in U.S. Pat. No. 3,914,410, acetylacetonate as taught in
U.S. Pat. No. 4,571,391, short chain carboxylic acids as described
in U.S. Pat. No. 5,846,581 and U.S. Pat. No. 6,303,158, or selected
synthetic peptide-like ligands as described in U.S. Pat. No.
5,266,560. Similarly, chromium camitine complexes in combination
with vanadyl sulfate, lipoic acid and other ingredients were
reported in U.S. Pat. No. 6,733,793, while EP 0 037 144 describes a
negatively charged C3-type ligand (e.g., optionally substituted
malonaldehyde complexes). In U.S. Pat. No. 6,149,948, a complex of
the formula
[Cr.sub.3O(O.sub.2CCH.sub.2CH.sub.3).sub.6(H.sub.2O).sub.3].sup.+
is used as a chromium carrier. While such defined complexes tend to
overcome at least some of the difficulties associated with amino
acid ligands, toxicity issues frequently remain, particularly at
relatively high dosages, and/or where the compounds are
administered over a relatively long period.
[0007] Toxicity may be reduced to at least some degree where
specific naturally occurring ligands are selected. For example,
isolated bovine chromium-binding protein (e.g., Biochemistry. 1996
Oct. 1; 35(39):12963-9, or Eur J Biochem. 1987 Jun. 15;
165(3):627-31) was described as a source of chromium
supplementation as taught in U.S. Pat. No. 5,872,102. Similarly,
lactoferrin was reported as a chromium ligand in U.S. Pat. No.
6,379,693, and in yet another example (see e.g., WO 04/022083),
proteolysis-derived low-molecular weight peptides are employed as
ligands, that preferably have a proline terminus. Alternatively,
oxidized leather scrap hydrolysate from leather tanning refuse was
reported as a carrier for chromium in the preparation of animal
feed as reported in U.S. Pat. No. 6,352,714. While many hydrolyzed
protein preparations are often low- or even non-toxic, various
difficulties nevertheless remain. Among other problems, crude
hydrolysate generally has an off-taste that is hard to mask, and
where the hydrolysate is purified or otherwise processed,
production costs often significantly increase. Still further, the
use of hydrolytic enzymes may pose a health concern where the
enzymes are not properly inactivated.
[0008] In yet another known approach of preparing
chromium-containing supplements, yeast is cultivated in a medium
that includes a chromium source, which provides the chromium to the
yeast cell that is subsequently harvested, pasteurized, and
optionally dried and/or pulverized. Such products are typically
known as chromium yeast products. For example, a common preparation
of a chromium yeast product is described in U.S. Pat. Nos.
4,348,483 and 4,343,905 in which the yeast is incubated with a
non-toxic chromium compound to form a chromium-enriched yeast cell
preparation. The so fermented yeast is then isolated and/or
powderized to provide the chromium supplement. To improve the
chromium content, selected yeast strains for cultivation of yeast
in a metal-containing medium are described in U.S. Pat. No.
6,140,107. Alternatively, or additionally, mixed amino acid
nicotinate chromium complexes can be used in the fermentation
medium to boost the chromium content of a yeast as described in
U.S. Pat. No. 6,248,323. While such preparations are often well
tolerated, the low solubility of the chromium yeast product
frequently poses a significant hurdle to incorporate such products
into a food or beverage. Moreover, and at least in part due to the
relatively poor solubility, bioavailability of chromium from such
yeast products is typically low.
[0009] Yeast has also been used as starting material to purify
defined and metabolically active substances as described in U.S.
Pat. No. 6,261,606. Similarly, EP 0 248 057 describes isolation of
a glucose tolerance factor from yeast. Surprisingly, such isolated
factor was free of chromium and was identified as a quinoline
compound. However, isolation of such substances is typically labor
intensive and therefore often not economic.
[0010] Thus, while there are numerous chromium complexes known in
the art, toxicity, low bioavailability, and/or low water solubility
limit the usefulness of these complexes. Therefore, there is a
constant need to find new chromium compounds/complexes that have
higher biological activity/bioavailability, higher safety/less
toxicity, sufficient chemical stability and high water
solubility.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to compositions and
methods for water soluble, highly bioavailable chromium-containing
complex matrices. Preferably, contemplated matrices are prepared
from a filtered or otherwise clarified solution of a disintegrate
of an edible (and most typically cellular) material, wherein the
clarified solution is combined with a metal (preferably Cr3.sup.+)
to form a complex between the metal and one or more components in
the matrix.
[0012] Contemplated complex matrices are most preferably formulated
for human consumption. For example, the complex matrices may be
incorporated into a food item (e.g., snack bar, cereal, etc.), a
beverage (e.g., sports drink, liquid diet formulation, etc.), or
formulated as a pill or other orally administered dosage form,
optionally in combination with other nutritionally relevant
compounds (e.g., metabolic modulators, conjugated linoleic acid,
etc.).
[0013] Therefore, in one aspect of the inventive subject matter,
the inventors contemplate a metal-containing substantially
completely water soluble complex matrix that is formulated for
mammalian consumption. Preferably, the matrix comprises a water
soluble fraction of a cellular lysate (most preferably brewer's
yeast lysate). The cellular lysate may further be dehydrated (e.g.,
freeze-dried or spray dried) before or after combination with the
meta, which is most preferably chromium-3.sup.+. It is still
further contemplated that such complex matrices are prepared under
conditions that will provide a matrix with an absorption maximum at
a wavelength between 550 nm and 570 nm.
[0014] In another aspect of the inventive subject matter, a method
of preparing a chromium-containing product includes a step in which
a water soluble complex preparation is prepared from an edible
material. In another step, the preparation is combined with a
trivalent chromium ion under conditions effective to form a complex
between a component of the preparation and the chromium ion. Most
preferably, the conditions are selected such that the complex
between the component of the preparation and the chromium ion has
an absorption maximum at a wavelength between 545 nm and 565 nm. It
is further preferred that the step of preparing includes a step of
disintegrating a cellular edible material, and an optional step of
removing (e.g., via filtration or centrifugation) at least part of
undissolved materials from the disintegrated material.
[0015] The chromium is preferably added to the disintegrated
material while the material is in a liquid form. However,
alternative addition protocols are also contemplated. In yet
further preferred examples, the so formed chromium-containing
product may be used in a liquid form or in an at least partially
dehydrated form (e.g., freeze-dried, or gelled). Especially
contemplated edible materials include cellular materials from
plants (e.g., fruits or portion of fruits, leaves, seeds,
vegetables, etc.), fingi (e.g., brewer's yeast), and animals (e.g.,
beef, poultry, etc.).
[0016] Consequently, in a still further aspect of the inventive
subject matter, a food item includes a chromium-containing complex
having an absorption maximum at a wavelength between 545 nm and 565
nm, wherein the complex is present in an amount effective to reduce
fasting blood glucose in a mammal ingesting the food item. Most
preferably, the complex in such food items is substantially
completely water soluble, and/or comprises a water soluble extract
from a cellular edible item (e.g., brewer's yeast).
[0017] Various objects, features, aspects and advantages of the
present invention will become more apparent from the following
detailed description of preferred embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWING
[0018] FIG. 1 is an exemplary graph depicting the correlation
between biological activity and spectral characteristics (here:
absorption between 500 nm and 600 nm) of selected
chromium-containing compositions.
DETAILED DESCRIPTION
[0019] The inventors have unexpectedly discovered that water
soluble metal complexes can be prepared from an edible complex
matrix, wherein the metal has a high bioavailability. Moreover,
with particular relevance to chromium, the inventors discovered
that the bioavailability and/or biological effect of chromium
appears to be a function of the spectral characteristics.
[0020] As used herein, the term "metal-fortified" in conjunction
with a composition means that the composition has a metal content
that is intentionally raised above a natural metal content of that
composition. As also used herein, the term "substantially
completely water soluble" refers to a solubility of at least 90 wt
%, more typically of at least 95 wt %, and most typically of at
least 98 wt %. As further used herein, the term "complex matrix"
refers to a composition of matter that comprises at least one
hundred, more typically at least 1000, and most typically at least
5000 chemically distinct components, which may or may not be known
in at least one of their identity and quantity. As still further
used herein, the term "mammalian consumption" refers to oral
administration to a mammal, and most typically in solid and/or
liquid form to a human.
[0021] In one preferred exemplary aspect of the inventive subject
matter, a chromium-fortified and substantially completely water
soluble complex matrix is prepared by a reaction of water soluble
brewer's yeast extract and chromium chloride hexahydrate. Most
typically, baker's or brewer's yeast is grown to a desired density,
optionally washed and harvested. The cells are then suspended in an
aqueous solution and disintegrated (e.g., via French press,
ultrasound, shear-homogenizer, etc.). Preferably, the so prepared
lysate is cleared to produce an aqueous yeast extract. Typically,
clearing is done using centrifugation or filtration.
[0022] It should be recognized that the extract may further be
processed to achieve one or more desired properties. For example,
nucleic acids may e removed to reduce the purine content, or
protein may be removed or added to achieve an especially desirable
nutritional profile. In other contemplated examples, the aqueous
extract may be chemically and/or physically fractionated (e.g., via
solvent extraction or size exclusion chromatography) to enrich the
matrix in one or more beneficial or otherwise desirable components.
After the matrix material is prepared, chromium is typically added
in the form of a trivalent chromium ion or salt in a predetermined
quantity (e.g., to a final concentration of between about 0.1
mcg/ml to 10 mg/ml). It should be particularly recognized that the
chromium ions will form numerous chemically distinct complexes with
one or more components in the matrix (e.g., with a peptide, a
polysaccharide, a lipoprotein, a nucleic acid, etc.), and that such
complexes are sufficiently stable and water soluble. In most
typical examples, such prepared compositions are substantially
completely water soluble.
[0023] Thus, one significant consequence of using aqueous matrices
is that the chromium is complexed or otherwise bound to the complex
matrix by a material that is a priori water soluble. Based on the
assumed molar excess of potential binding/complexing sites for the
chromium ions, complexation of the chromium ions will not (or only
to a negligible extent) alter the water solubility of the complex
matrix. Such approach is clearly superior to conventionally
produced chromium yeast as a substantial portion of chromium yeast
in insoluble, and with that not available for release of the
chromium. For example, where chromium is bound to a membrane or
lipopolysaccharide, chromium is not available for hydrophilic
exchange.
[0024] It is generally contemplated that the chromium fortified and
substantially completely water soluble complex matrices will then
be formulated for mammalian consumption, which may be in numerous
forms. For example, the fortified matrices may be prepared in
dehydrated form (e.g., spray-dried, freeze-dried, triple point
dried, etc.) as a powder, pill, or otherwise dried form.
Alternatively, aqueous or gel forms are also deemed suitable,
especially when such forms are combined with a fluid or gel-like
nutrient (e.g., sports drink, syrup, preserve, etc.). It should
further be appreciated that contemplated fortified matrices may
also be combined with any other nutrient for human (or other
mammalian) consumption, and all known nutrients are deemed suitable
for combination with fortified matrices presented herein.
Typically, chromium will be present in the edible formulation such
that one serving will provide the recommended daily amount for a
human (e.g., between 10 and 100 mcg/serving), or even more.
[0025] In alternative aspects of the inventive subject matter, it
is contemplated that the matrix need not be limited to an aqueous
extract of brewer's yeast, but that numerous alternative matrices
may also be suitable, including (typically aqueous) extracts from
various unprocessed and/or processed edible foodstuff. For example,
fruit and/or vegetables may be pressed or otherwise disintegrated,
optionally clarified (e.g., via filtration) to provide a liquid
matrix that can be further processed (e.g., concentration, physical
or chemical separation, etc). prior to addition of the chromium.
Where desirable, multiple and different matrices may be combined
(e.g., yeast matrix and fruit matrix, or water soluble extracts of
one or more edible plant portions with filtered yeast
disintegrate). Similarly, suitable matrices may also be produced
from animal material.
[0026] Additionally, contemplated metal-fortified matrices may also
be prepared from a complex matrix that is combined with one or more
known and defined ligands for chromium or another metal, including
nicotinic acid, polynicotinic acid, picolinate, various amino acids
(proteinogenic and/or non-proteinogenic), carnosine, carnitine,
citrate, etc. Thus, it should be appreciated that mixed complexes
between a component of the matrix, a known ligand, and the metal
are also deemed suitable.
[0027] Depending on the specific composition and/or further
processing, it is generally preferred that the matrix is water
soluble to a significant degree (i.e., at least 50 wt %, and more
typically at least 70 wt %), and it is particularly preferred that
the chromium-fortified matrix is substantially completely water
soluble. To that end, processing steps in the preparation of the
matrix may be included to remove water insoluble material, and
particularly preferred processing steps include physical separation
(e.g., sedimentation, centrifugation, filtration, etc.), chemical
separation (e.g., phase separation with hydrophobic solvent,
adsorption on hydrophobic matrix, etc.), and all reasonable
combinations thereof. Such processing steps may be performed
before, concurrent with, or after addition of the chromium. It
should be noted that chromium picolinate, chromium niacin and
heretofore known chromium yeast are all water insoluble.
Consequently, it can be expected that the bioavailability is
accordingly diminished. In contrast, all or almost all of the
compounds contemplated herein are substantially completely water
soluble, sufficiently stable, and bioavailable to a relatively
large degree.
[0028] It should still further be recognized that numerous metals
other than chromium are also contemplated, and all metals and metal
ions suitable for human and mammalian ingestion are deemed
appropriate. For example, alternative metals include ionic forms of
Na, Mg, Ca, Zn, I, Co, Cu, V, Fe, Ni, and all reasonable
combinations thereof etc. Most preferably, metal-fortified matrices
are at least partially dehydrated (typically freeze-dried,
evaporated, etc), and may then be packaged into a bulk preparation,
individual dosage forms, or incorporated into another edible
product. In still further contemplated aspects, numerous non-metal
elements (e.g., group 13-16 elements) are also contemplated, and
especially include those of nutritional significance. For example,
suitable non-metal elements include B, Si, and Ge. Regardless of
their chemical nature, it is generally preferred (but not
necessary) that the element that is bound to the matrix is in ionic
form. Furthermore, it should be noted that the absorption maximum
of alternative metal or non-metal complex matrices will vary, and
the exact position of the maximum will predominantly depend on the
ligand and the synthetic protocol. Nevertheless, it is contemplated
that the optimum biological activity will be correlated with
spectral characteristics, and particularly with an absorbance
maximum in the UV and/or VIS range.
[0029] Based on various experiments (see below) and further
observations (data not shown), the inventors discovered that the
biological activity of known and contemplated Cr-3.sup.+ complexes
is correlated with their spectral characteristics. More
specifically, the position of an absorption maximum in the range of
between 500 nm and 600 nm correlated with the vigor of the
biological response. While not limiting to the inventive subject
matter, the inventors contemplate that the chemical stability of
the chromium complexes with different ligands is directly
associated with the position of a maximum in the visible spectrum,
wherein the most suitable stability (i.e., stability that provides
highest bioavailability) of a chromium complex is that of a complex
having an absorption maximum at about 560 nm.
[0030] Viewed from a different perspective, the stability of a
chromium complex is a predictor for its biological activity. Thus,
if complexes are too stable (e.g., Cr-picolinate) biological
activity is low, and the same is true if the complexes are very
unstable. For example, relatively stable complexes appear to be
comparably bioavailable, but once they reach the target cells they
do not readily transfer the chromium atom to a Cr-specific binding
protein(s). On the other hand, relatively week complexes are
typically unstable and are therefore not sufficiently bioavailable
as insoluble chromium hydroxide is formed in the digestive tract
upon dissociation of the chromium ion from the complex. Therefore,
only chromium complexes of an intermediate stability are both
bioavailable and active in target cells since they transfer their
Cr-ion faster and easier than strong complexes would do.
Consequently, the inventors contemplate chromium complexes, and
especially water soluble complex chromium-containing matrices that
have an absorption maximum at between 530 nm to 580 nm, more
preferably between 545 nm to 575 nm, and most preferably between
550 nm to 570 nm.
[0031] It should be noted that the association between the
wavelength of absorption in the range between 500 nm and 600 nm and
the biological activity may be used to select for, design, and/or
modify one or more ligands for a chromium-3.sup.+ ion. Similarly,
selected products may be advertised as having an increased
biological activity (e.g., reduce fasting blood glucose, improve
glucose tolerance, reduce LDL cholesterol, etc.) based on the
confirmed position of an absorption maximum (e.g., between 530 nm
to 580 nm, more preferably between 545 nm to 575 nm, and most
preferably between 550 nm to 570 nm).
[0032] In still further contemplated aspects, compositions
according to the inventive subject matter may be further combined
with a variety of other nutritional components, wherein such
combinations exhibit additive, or even synergistic, effect with
respect to their intended effect. Especially preferred combinations
of chromium-containing water-soluble matrices include those in
which the combinations are intended to positively affect the
metabolism of a person. For example, it is known that chromium
positively influences glucose utilization. Therefore, all known
nutritional supplements for improving glucose utilization are
especially contemplated herein. Similarly, chromium is also known
to improve insulin sensitivity. Therefore, all known nutritional
supplements for treatment or prevention of diabetes are especially
contemplated suitable for use herein. Furthermore, chromium has
also been implicated in prevention and/or improvement of elevated
cholesterol. Consequently, all known nutritional supplements for
treatment or prevention of heart disease or elevated cholesterol
are especially contemplated appropriate. Still further, it is known
in the art that chromium may also have an anabolic effect.
Therefore, all known dietary supplements for increase of muscle
mass are considered suitable for combination with contemplated
chromium-containing matrices. Further particularly preferred
combinations include those in which chromium-containing matrices
are combined with metabolic enhancers, and especially conjugated
linoleic acid and/or conjugated linoleic alcohol. Yet other
preferred combinations include those in which an anabolic agent
(e.g., DHEA) is combined with contemplated chromium-containing
matrices.
EXAMPLES
[0033] The following abbreviations are used: CROA-1C
(Chromium-Citrate-Aminooxyacetate); CROA-1
(Chromium-mono-Aminooxyacetate)--1 mmol sodium bicarbonate; CROA-2
(Chromium-bis-Aminooxyacetate)-2 mmol sodium bicarbonate; CROA-3
(Chromium-tris-Aminooxyacetate)--3 mmol sodium bicarbonate; CROX-1C
(Chromium-Citrate-Oxamate); CROX-1 (Chromium-mono-oxamate) 1 mmol
sodium bicarbonate; CROX-2 (Chromium-bis-oxamate) 2 mmol sodium
bicarbonate; CROX-3 (Chromium-tris-oxamate) 3 mmol sodium
bicarbonate; CROC-1 (Chromium-mono-Citrate); CROC-2
(Chromium-di-Citrate); HEX (ChromEx) (Chromium-chloride in YEX);
YEX yeast extract;
Defined Ligands for Chromium-3+ Ions
[0034] Particularly striking examples include certain reference
complexes (chromium complexes with oligopeptides [sequence data not
shown]), which were labeled CPC5 (chromium-penta-oligopeptide),
CPC3R (chromium-tri-oligopeptide-red), and CPC3V (chromium
tri-oligopeptide-violet). Each of these reference complexes were
prepared by reacting one mole of Cr3.sup.+ with five or three moles
of the same oligopeptide under different experimental conditions.
Depending on the reaction conditions (typically reaction time and
temperature, as well as slightly acidic, neutral, or slightly
alkaline medium), products were obtained with distinct spectral
characteristics. Thus, it is especially noted that the reaction
conditions (here: control of temperature and pH) may significantly
affect the spectral properties of an otherwise chemically identical
composition.
[0035] For example, CPC3 compounds are prepared by dissolving CC-hh
in 3 ml water; the oligopeptide is dissolved in 3 ml water and
quickly mixed with the CC-hh solution. A precipitate forms; the
product is heated for a short time and 252 mg SB are slowly added.
Heat 2 hours at boiling water bath. The final pH of this
preparation is basic. In contrast, CPC3R is prepared by dissolving
CC-hh in 6 ml water. Heat at the boiling water bath for a short
time. Add slowly to hot solution solid oligopeptide. No precipitate
forms. Heat for another 10 minutes and then add slowly and
cautiously 160 mg SB. No precipitate forms. On prolonged heating
(two to three hours) the solution stays clear. The final color is
in between red and violet, closer to red. The final pH is close to
neutral. In further contrast, CPC3V is produced by dissolving CC-hh
in water (6 ml). Heat and add slowly oligopeptide. No precipitate
forms, and no SB is added. Heat for 2 hours at a boiling water
bath. The product solution stays clear and has a distinct violet
color. The final pH is acidic.
[0036] Exemplary synthesis of other defined complexes: 1 mmol (266
mg) of CrCl.sub.3 hexahydrate (CC-hh) in all cases, and optionally
1 mmol (210 mg) of citric acid mono-hydrate (CA-mh) where indicated
are combined with 1 mmol of ligand. The so prepared mixture is
heated and sodium bicarbonate (for amounts see above) is carefully
added. Heating is continued (typically in boiling water) bath for
another 2 hours, and the resultant solution is then diluted or
dehydrated as desired.
Yeast Matrix as Ligand for Chromium-3+ Ions
[0037] A solution of 400 mg water soluble yeast extract
(Commercially available under the trade name AMBEREX) in 10 mL
water is prepared and filtered if needed. 266 mg CC-hh are added
and the resultant solution is kept for 2 hours at room temperature.
If desired, the so prepared chromium-fortified matrix is filtered
and dehydrated. It contemplated that similar to the CPC protocol
given above, (a) a change in the pH of the resultant solution
and/or (b) moderate temperature modifications will provide modified
products in which the position of the absorbance maximum will vary.
For example, it is contemplated that acidification of the yeast
extract may provide a hypochromatic shift of the absorbance maximum
("ChromexA" in FIG. 1), while alkalinization may provide a
hyperchromatic shift ("ChromexB" in FIG. 1). FIG. 1 depicts a graph
in which the position of the absorbance maximum of various
compounds is correlated with the biological activity (reduction of
blood glucose increase, see below) of the compounds.
Test Results
[0038] In in vitro experiments (on total glucose uptake into L6
muscle cells at 100 nmol concentration), CPC5 increases on average
3.57, CPC-3R 4.00 and CPC-3V 4.45 fold over control. In vivo
experiments gave much more dramatic differences, which are
summarized in Table 1 below in which the increase in fasting blood
glucose was measured after four weeks as compared to untreated
animals: TABLE-US-00001 TABLE 1 DIABETIC RATS BLOOD GLUCOSE TREATED
WITH LAMDA MAX. INCREASE Cr-picolinate 504 nm 2.37 (95.56%) Chromex
569 nm 1.88 (75.80% CPC 5 531.5 nm 2.15 (86.70%) CPC3R 540.5 nm
1.74 (70.16%) CPC3V 558 nm 1.09 (43.95%) Untreated 2.48 (100%)
[0039] Based on the data from Table 1, it can be seen that there is
a clear relationship between the position of the absorption maximum
(lambda max) of the chromium complex (i.e., between the strength of
the Cr-Ligand Coordination Bond) and its biological activity. The
optimal range for an absorption maximum is around 560 nm.
In Vitro Glucose Uptake in L6 Muscle Cells Induced By Various
Chromium Compounds
[0040] Total glucose uptake was measure using fluorescent analog of
glucose, 2-NBDG from Molecular Probes Inc. L6 myoblastic cells were
treated for 2 hrs with tested compounds in culture medium SkBM from
Clonetics. After washing, cells were transferred to HBSA
(Hepes-buffered Saline), pH 7.0 with 50 mcM of 2-NBDG without
glucose. One minute later, cells were washed with ice-cold PBS, and
fixed in -20 C 70% ethanol. Fluorescence was measured at 480/530
(excitation/emission). Table 2 below lists exemplary results.
TABLE-US-00002 TABLE 2 Com- Conc. Aver- pounds nM Fold over Control
Range age CrCl3 10 1.44, 1.23, 0.95, 3.38, 0.94, 1.50, 0.94-3.38
1.55 1.44, 2.1, 1.3, 1.34, 1.46 100 1.46, 1.33, 4.05, 2.0, 2.44,
1.56, 1.33-4.05 2.05 2.30, 1.52, 1.93 1000 1.64, 1.24, 3.05, 2.83,
2.76. 1.68, 1.24-3.05 2.08 1.60, 1.63, 2.42 Chromex 10 4.44, 1.66,
3, 1.70, 2.40, 1.50, 1.40-4.44 2.15 1.40, 1.51 100 4.88, 3.94,
2.75, 2.01, 4.50, 2.40, 1.84-4.88 3.07 1.84, 2.29 1000 3.83, 4.26,
4.10, 2.44, 3.90, 3.3 1.94-4.26 3.26 2.39, 1.94 CPC3R 10 3.5, 3.0,
2.5, 1.6 1.6-3.5 2.65 100 4.5, 4.0, 4.5, 3.0, 3.0-4.5 4.00 1000
6.0, 2.60, 3.20, 2.0, 2.0-6.0 3.45 CPC3V 10 4.70, 1.88, 2.0, 3.0,
1.71 1.71-4.70 2.65 100 8.50, 2.20, 4.9, 2.2, 2.20-8.50 4.45 1000
4.10, 2.90, 3.00, 3.40, 2.10 2.10-4.10 3.10 CPC5 10 2.50, 1.77,
1.60, 1.30 1.30-2.50 1.73 100 3.17, 6.10, 3.2, 2.10, 2.10-6.10 3.57
1000 3.20, 4.50, 2.60, 2.60 2.60-4.50 3.22 CROA-1 10 1.41, 1.66,
2.10, 1.41-2.10 1.72 100 2.12, 2.16, 2.80, 2.12-2.80 2.36 1000
2.58, 2.10, 2.50 2.10-2.58 2.39 CROA-1C 10 1.08, 1.07, 1.44,
1.07-1.44 1.19 100 1.32, 1.35, 1.90 1.32-1.90 1.52 1000 1.48, 1.96,
2.06 1.48-2.06 1.83
In Vivo Activity of Selected Chromium Compounds
[0041] Streptozocin-induced insulin deficient rats were used to
evaluate the insulin potentiating activity of several compounds.
Steptozocin causes damage of pancreas resulting in drastically
reduced secretion of insulin. As consequence, these rats develop
severe hyperglycemia. So far, certain known chromium compounds were
known to potentiate action of insulin, however, the actual
mechanism was not clearly understood. Only recently, chromium was
found to stimulate AKT, thus possibly inducing glucose uptake to
muscle cells also in a insulin-independent way. In our studies in
vivo, chromium compounds were provided in drinking water for four
weeks at a concentration of about 42 .mu.g/kg. Vein blood was
collected following four hrs fasting and used for fasted blood
glucose level test. The study results for two tests are given below
in Table 3 in which the increase in blood glucose in given as a
fold increase over four weeks. TABLE-US-00003 TABLE 3 Compound
Fasted Blood Glucose STUDY 1 Untreated 1.95 CrCl3 1.32 CPC3V 0.80
CPC5 1.02 CrPic 2.02 Chromex 1.55 CrNiacin 1.91 Metformin 0.96
STUDY 2 Untreated 2.48 CrPic 2.37 Chromex 1.88 CPC3R 1.74 CPC3V
1.09 CPC5 2.15
[0042] These results show quite dramatic improvement of glucose
transport in insulin-deficient rats. These rats are hypoinsulinemic
and hyperglycemic due to severe pancreatitis conditions.
Improvement under such conditions indicate that the treatment
overpass insulin-deficiency and stimulate glucose utilization.
[0043] Thus, specific embodiments and applications of compositions
and methods for improved chromium complexes have been disclosed. It
should be apparent, however, to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted except in the
spirit of the appended claims. Moreover, in interpreting both the
specification and the claims, all terms should be interpreted in
the broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps may be present, or utilized, or combined with
other elements, components, or steps that are not expressly
referenced. Furthermore, where a definition or use of a term in a
reference, which is incorporated by reference herein is
inconsistent or contrary to the definition of that term provided
herein, the definition of that term provided herein applies and the
definition of that term in the reference does not apply.
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