U.S. patent application number 14/234622 was filed with the patent office on 2014-06-05 for formulas comprising highly soluble elements and vitamin for the prevention and amelioration of osteoporosis.
This patent application is currently assigned to SINOVEDA CANADA, INC.. The applicant listed for this patent is Yun Kau Tam. Invention is credited to Yun Kau Tam.
Application Number | 20140154332 14/234622 |
Document ID | / |
Family ID | 40913349 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154332 |
Kind Code |
A1 |
Tam; Yun Kau |
June 5, 2014 |
FORMULAS COMPRISING HIGHLY SOLUBLE ELEMENTS AND VITAMIN FOR THE
PREVENTION AND AMELIORATION OF OSTEOPOROSIS
Abstract
The present invention provides methods of producing dosage forms
for formulas of elemental compositions encompassing acetate salts
of calcium, magnesium and zinc along with vitamin D.sub.3. The
acetate salts could be extracted from natural sources such as
pearls, coral, and oyster or compounded using synthetic materials.
The dosage and ratio of calcium to magnesium was estimated using in
vitro and in vivo estimations. The dosage for promoting bone health
and alleviation of osteoporosis is about a quarter to a third of
the conventional dose.
Inventors: |
Tam; Yun Kau; (Edmonton,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tam; Yun Kau |
Edmonton |
|
CA |
|
|
Assignee: |
SINOVEDA CANADA, INC.
Edmonton
AB
|
Family ID: |
40913349 |
Appl. No.: |
14/234622 |
Filed: |
July 27, 2012 |
PCT Filed: |
July 27, 2012 |
PCT NO: |
PCT/IB2012/053872 |
371 Date: |
January 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61512685 |
Jul 28, 2011 |
|
|
|
Current U.S.
Class: |
424/523 ;
514/167 |
Current CPC
Class: |
A23L 33/165 20160801;
A23L 33/40 20160801; A61K 9/20 20130101; A61K 9/2095 20130101; A61K
45/06 20130101; A61K 31/593 20130101; A61K 33/30 20130101; A61P
3/02 20180101; A61K 33/06 20130101; A23V 2250/1578 20130101; A61K
33/30 20130101; A23V 2200/306 20130101; A61K 2300/00 20130101; A61K
33/06 20130101; A23V 2002/00 20130101; A61P 19/08 20180101; A61K
35/60 20130101; A23L 33/155 20160801; A23L 2/02 20130101; A61K
35/02 20130101; A61P 43/00 20180101; A23V 2250/1642 20130101; A23V
2250/161 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A23V 2250/7106 20130101; A61K 2300/00 20130101; A61P 19/10
20180101; A23L 33/15 20160801; A61K 35/02 20130101; A61K 31/19
20130101; A23V 2002/00 20130101; A61K 31/593 20130101; A23L 33/16
20160801 |
Class at
Publication: |
424/523 ;
514/167 |
International
Class: |
A23L 1/29 20060101
A23L001/29; A61K 33/30 20060101 A61K033/30; A61K 31/19 20060101
A61K031/19; A61K 35/60 20060101 A61K035/60; A61K 31/593 20060101
A61K031/593 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 27, 2011 |
TW |
100126601 |
Claims
1. A method of preparing tablets comprising calcium acetate,
magnesium acetate, zinc acetate and vitamin D.sub.3, the method
comprises the steps of: (i) blending a first calcium composition
comprising calcium acetate, magnesium acetate, and zinc acetate
with a composition comprising vitamin D.sub.3; and (ii) blending
the composition obtained from (i) with a second calcium composition
comprising calcium acetate, magnesium acetate, and zinc acetate;
(iii) performing one or more rounds of blending wherein in each
blending a composition obtained from a previous step is blended
with yet another calcium composition comprising calcium acetate,
magnesium acetate, and zinc acetate, thereby obtaining composition
comprising calcium acetate, magnesium acetate, zinc acetate and
vitamin D.sub.3 for preparing tablets.
2. A tablet produced by the method of claim 1.
3. A method of preparing soft gel capsules comprising calcium
acetate, magnesium acetate, zinc acetate, oil and vitamin D.sub.3,
the method comprises the steps of: (i) dissolving vitamin D.sub.3
in oil comprising omega 3 or omega 3-6-9; and (ii) blending the
composition obtained from (i) with a calcium composition comprising
calcium acetate, magnesium acetate, and zinc acetate, thereby
obtaining composition comprising calcium acetate, magnesium
acetate, zinc acetate, oil and vitamin D.sub.3 for preparing soft
gel capsules.
4. The method of claim 1 or 3, wherein the calcium composition
comprises at least 10 percent by weight of calcium acetate, at
least 5 percent by weight of magnesium acetate, and at least 0.2
percent by weight of zinc acetate.
5. The method of claim 3, wherein the oil is fish oil or flaxseed
oil.
6. The method of claim 3, wherein the oil to calcium acetate blend
ratio is selected from the group consisting of 1:1, 1.5:1 and
2:1.
7. The method of claim 3, wherein the oil to calcium ratio is
selected from the group consisting of 1:0.14, 1.5:1 and 2:1.
8. The method of claim 3, wherein the oil to magnesium ratio is
selected from the group consisting of 1:0.07, 1.5:0.07 and
2:0.07.
9. The method of claim 3, wherein the oil to zinc ratio is selected
from the group consisting of 1:0.007, 1.5:0.007 and 2:0.007.
10. A soft gel capsule produced using the method of claim 3.
Description
[0001] This application claims the benefit of priority of Taiwanese
Application No. 100126601, filed Jul. 27, 2011, and U.S.
Application No. 61/512,685 filed Jul. 28, 2011. The entire contents
and disclosures of the preceding applications are incorporated by
reference into this application.
[0002] Throughout this application, various references are referred
to and disclosures of these publications in their entireties are
hereby incorporated by reference into this application to more
fully describe the state of the art to which this invention
pertains.
BACKGROUND OF THE INVENTION
[0003] Calcium is the major element in bones with over 99% of the
body's calcium existing in bone. Adequate intake of calcium from
the diet is necessary for bone growth and maintenance. Osteoporosis
is a disease caused by a significant loss of bone mass leading to
increased susceptibility to fracture, most often occurring in women
age 35 or above, but more frequently, occurring in postmenopausal
women (1, 2).
[0004] Dietary supplements with calcium were thought to be primary
to maintaining bone health in the past 50 years (3). However, the
benefit of increased overall calcium consumption on bone health has
not been clearly demonstrated, and there are conflicting reports in
the literature on its effectiveness. In 43 studies of calcium
supplementation reviewed by Heaney published between 1988 and 1993,
16 of the 19 placebo-controlled studies in which calcium intake was
controlled showed that the mineral prevented or slowed bone loss,
but 16 studies showed that calcium had no effect on bone loss (4,
5).
[0005] In the 12 studies that excluded women who were within 5
years of menopause, a period when estrogen deficiency overwhelms
the effect of calcium supplementation (6), all showed that calcium
had a significant beneficial effect.
[0006] In elderly women, it was shown that there was a significant
relationship between bone mineral density (BMD) and several
critical nutrients: energy, protein, calcium, magnesium, zinc and
vitamin C (1). It has also, however, been found that high levels of
calcium intake may be linked to higher incidence of cardiovascular
disease (3).
[0007] Since total calcium intake has not shown to be conclusive
with respect to bone health, other factors have also been taken
into account, such as the calcium to magnesium ratio in modern
diets, and in supplement form. The ratio of Ca/Mg in the modern
diet increased from 2/1 in the first 40 years of the 1900s to
>3/1 in the 1960s, to >6/1 in the year 2000. The daily
recommended intake (DRI) in the year 2000 of Ca/Mg was >3/1 to
>4/1. This change correlates with negative consequences with
respect to bone health as well as an increased risk of
cardiovascular disease.
[0008] It should be noted that the increase in Ca/Mg is mainly due
to the increase in calcium intake, not a change in magnesium. In
the early 2000s, daily calcium intake reached a new high of 2,500
mg (3). The daily requirement of calcium was recently re-evaluated
(7). It was found that an average intake of 749 mg of calcium is
required, an estimate lower than previously estimated.
[0009] Supporting the thesis that Ca/Mg ratio, among other factors,
is a more important factor in bone maintenance and health than
absolute calcium consumption, is one clinical trial, wherein 43
early postmenopausal women were randomly assigned to one treatment
group for administration of the following: percutaneous estradiol,
oral calcium (2000 mg/day) or placebo. Bone mineral content in the
forearm, the entire body and spine remained the same in the
estradiol group; however, there was a decline in the calcium and
placebo groups. Calcium did not show any significant effect and
calcium supplementation may have a minor effect on the loss of
cortical bone, but it had no effect on the trabecular bone (6).
[0010] In a National Health and Nutritional Examination Survey
(NHANES) conducted from 1988 to 1994, predictive models were
established to evaluate parameters such as race, body composition,
exercise, alcohol intake, smoking status and nutritional intake
(8). The nutritional intake analysis included study of elements
such as calcium, phosphorus, magnesium, iron, zinc, sodium and
potassium. Among the 7,532 women in the study who were 20 years or
older, elemental intake was not a predictor of osteoporosis.
However, the average calcium intake was 659 mg and magnesium was
241 mg--lower than that of the RDA of 1000 and 310 mg,
respectively.
[0011] Physical activity was associated with increase in vertebral
bone mineral density (9). When activity was removed, vertebral bone
mineral density was dependent on calcium intake. The relationship
disappeared when calcium intake exceeded 800 to 1000 mg/day. A
ceiling effect of calcium was also observed by Celotti and
Bignamini (10). They reported that calcium supplementation is
important for maintaining bone health. However, an excessive amount
of calcium may be useless and could cause hypercalciuria and kidney
stones. Supplementation with a small amount of magnesium was
suggested.
[0012] Other studies show not just the importance of the ratio of
Ca/Mg consumed or administered, but the importance of optimizing
zinc levels. Mutlu et al. (11) showed that magnesium and zinc
levels are the lowest in postmenopausal women, lower than
postmenopausal women with osteopenia, and lower than postmenopausal
women with normal bone density. Calcium supplementation may reduce
zinc absorption, and magnesium and zinc retention. Consequently,
calcium supplementation in the absence of the administration of
other optimized amounts of minerals may further aggravate the
severity of osteoporosis (2, 12, 13). Apart from calcium,
magnesium, zinc, manganese and copper deficiencies are linked to
osteoporosis (14).
[0013] Angus et al. (15) showed that calcium was not a predictor of
bone mineral density in pre- and post-menopausal women. Magnesium
and iron were, however, predictors of bone mineral density. In this
study, however, the test subjects ingested less than the
recommended amounts of elements. About 29% of the post-menopausal
women consumed less than 500 mg of calcium per day (16), while
other nutrients such as magnesium, etc. were also deficient.
[0014] A study emphasizing the benefit of magnesium on
postmenopausal women found that a Mg/Ca ratio of 1.2/1 was more
effective at maintaining bone health than that of a ratio of 0.4/1
(17). The study used 500 mg of calcium in the form of calcium
citrate and 200 mg of magnesium in the form of magnesium oxide for
the 0.4/1 group and 600 mg of magnesium in the form of magnesium
oxide in the 1.2/1 group. The study showed that women on the 1.2/1
diet for 6 to 12 months had an average of an 11% increase in bone
mineral density, whereas, the other group had a non-significant
increase of 0.7%.
[0015] Although bone health is dependent on a variety of factors,
there is enough evidence to show that, in the area of elemental
requirements, apart from calcium, other elements such as magnesium,
phosphorus, zinc, copper, etc. are also important for maintaining
or improving bone health. Further, due to differences in
bioavailability, it is proposed that elemental salts would be more
accurately characterized in terms of absorbability, and that
calcium formulas be optimized through the use of preferred
salts.
[0016] The selection of appropriate salts for optimized
formulations has not received appropriate attention because of
reports showing that solubility of calcium salts is not related to
the element's bioavailability. The absorption of calcium salt,
soluble or insoluble, is not affected by gastric acid secretion
(18). The hypothesis that calcium carbonate can be converted to a
more soluble calcium salt in the stomach, namely calcium chloride,
thus enhancing calcium absorption, has been tested. The results
showed that calcium carbonate absorption is not influenced by
gastric acid (18). The average amount absorbed in humans is
24%.
[0017] The bioavailability of calcium carbonate, D-calcium lactate,
L-calcium lactate and oyster shell calcium was found to be
independent of the salt's solubility (19). This study used a method
which was different from that of the balance study. It measured
changes in the pituitary thyroid hormone (PTH), etc. instead of
actual calcium absorption. However, indirect methods of
measurement, such as PTH, do not provide truly accurate comparisons
of calcium bioavailability.
[0018] Using Ca.sup.45 as a tracer, fractional absorption values of
calcium carbonate and calcium citrate were found to be
insignificantly different from each other at a low dose (300 mg
calcium); however, calcium absorption from calcium carbonate was
slightly but significantly better than calcium citrate (20). Heaney
(21) reported that the rates of urinary excretion for three
marketed calcium products (marketed calcium carbonate, encapsulated
calcium carbonate and marketed calcium citrate) were identical.
[0019] Despite these observations, there are reports showing that
not all calcium salts have the same bioavailability.
Bioavailability of calcium ascorbate is higher than that of calcium
carbonate and calcium chloride (22).
[0020] Bioavailability of calcium acetate was measured using
.sup.45Ca (23). Compared to calcium ascorbate, bioavailability of
calcium acetate was significantly lower (70% vs 45% at 25 mg
calcium load). A kinetic model consisting of 8 compartments was
used to fit the plasma calcium vs. time data. The difference was
attributed to a saturable process. It is also reasoned that the
solubility of calcium acetate may be reduced in the intestine
because calcium from the acetate salt may precipitate phosphate or
chloride ions in the intestine. Therefore, it is not surprising
that the bioavailability of calcium acetate is not different from
that of calcium chloride and calcium phosphate.
[0021] Magnesium absorption from 10 organic and inorganic salts was
tested in rats (24). The bioavailability of magnesium ranged from
50 to 66%. Magnesium gluconate provided the highest value. The
solubility of these salts in the small and large intestine and
cecum was also measured. Solubility of these salts was quite high
at the proximal section of the intestine; it dropped off very
quickly as pH increased along the intestinal tract. Differences in
absorption of these magnesium salts may not be important
considering the variability among individuals.
[0022] Zinc absorption occurs throughout the small intestine and it
is dose dependent in humans (25). With respect to zinc, there was
no difference in the bioavailability of zinc oxide and zinc sulfate
as measured using dual isotope techniques (26); both were at
approximately 24%. The bioavailability of iron was 15.9%. However,
zinc sulfate tended to reduce the bioavailability of iron to 11.5%
and this number is significant. Eight to 11 mg of zinc per day is
the recommended intake (http://ods.od.nih
gov/factsheets/Zinc-HealthProfessional/). The recommended daily
allowance of zinc was 6 mg (27).
[0023] The following are inventions and disclosures noteworthy in
the art:
[0024] U.S. Pat. No. 5,879,698 issued in 1999 for a calcium dietary
supplement comprising calcium, magnesium, zinc, etc. (28). The
calcium to magnesium ratio is high and the range of magnesium used
was between 50 to 150 mg. The salt for calcium is calcium
carbonate. The quantity of calcium and magnesium used and the type
of salts employed are different from the present invention.
[0025] U.S. Pat. No. 6,716,454, awarded to Meignant and Stenger in
2004, cites a composition which consists of calcium and a vitamin D
mixture.
[0026] U.S. Pat. No. 6,790,462, awarded to Hendricks in 2004,
describes a dietary supplement containing calcium and phosphorus.
Vitamins including vitamin D could also be included in the
supplement. Hendricks emphasized the effects of phosphorus, and
optionally vitamins B.sub.12, folate and Vitamin B.sub.6. The
present application, however, does not include phosphorus.
[0027] Mazer et al. were granted U.S. Pat. No. 5,698,222 in 1997 on
a calcium supplement in solid form which contains calcium
glycerophosphate, vitamin D and vitamin C. The present invention
does not contain calcium salt of this kind.
[0028] In another patent, U.S. Pat. No. 5,075,499, issued in 1991,
Walsdorf et al. described the synthesis of dicalcium
citrate-lactate by mixing stoichiometric mixtures of citrate and
lactate salts to produce the calcium salt (29).
[0029] Krumhar and Johnson designed a diet supplement for bone
health, disclosed in U.S. Pat. No. 7,029,703 which issued in 2006,
consisting of microcrystalline calcium hydroxyapatite, protein
(mostly collagen), phosphorus, fat, and other minerals. It also
contains vitamin D.sub.3, cholecalciferol, and a preferred
osteoblast stimulant, ipriflavone. In addition to these basic
ingredients, the composition can further include various other
minerals known to occur in bone, vitamin C, and glucosamine
sulfate, all of which have been claimed to have beneficial effects
on the growth and maintenance of healthy bone.
[0030] Sultenfuss, in U.S. Pat. No. 5,514,382, issued in 1996,
described another daily vitamin and mineral supplement for women
comprising vitamin A, beta-carotene, niacin, riboflavin,
pantothenic acid, pyridoxine, cyanocobalamin, biotin,
para-aminobenzoic acid, inositol, choline, vitamin C, vitamin D,
vitamin E, vitamin K, boron, calcium, chromium, copper, iodine,
iron, magnesium, manganese, molybdenum, selenium, zinc and
bioflavonoid. For women up to 40 years of age, iron is included.
For women over 40 years of age, iron is optionally included. The
Ca/Mg ratio is in a range of 10-15/4-6.
[0031] A dietary supplement consisting of an extensive list of
minerals and vitamins was described in U.S. Pat. No. 5,654,011
(30). The patent sets forth no quantitative description on the
contribution of each component to bone health.
SUMMARY OF THE INVENTION
[0032] The present invention describes formulations of a dietary
supplement comprising acetate salts of calcium, magnesium, zinc and
vitamin D.sub.3. These preparations are highly soluble in water,
gastric and intestinal fluids.
DETAILED DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows mean (.+-.S.D.) percentage-time profiles of
calcium of various formulas in artificial gastric juice (USP).
[0034] FIG. 2 shows the average cumulative net amount of calcium
retained (.+-.S.E.M.) in rats receiving calcium free diet over a
four day period.
[0035] FIG. 3 shows the cumulative net amount of magnesium retained
(.+-.S.E.M.) in rats receiving calcium free diet over a four day
period.
[0036] FIG. 4 shows the cumulative net amount of zinc retained
(.+-.S.E.M.) in rats receiving calcium free diet over a four day
period.
[0037] FIG. 5 shows the plasma calcium (A), magnesium (B) and zinc
(C) levels sampled from rats at the end of the treatment period
while receiving calcium free diet.
[0038] FIG. 6 shows the average cumulative net amount of calcium
retained (.+-.S.E.M.) in rats receiving normal diet over a four day
period.
[0039] FIG. 7 shows the average cumulative net amount of magnesium
retained (.+-.S.E.M.) in rats receiving normal diet over a four day
period.
[0040] FIG. 8 shows the average cumulative net amount of zinc
retained (.+-.S.E.M.) in rats receiving normal diet over a four day
period.
[0041] FIG. 9 shows the plasma calcium (A), magnesium (B) and zinc
(C) levels sampled from rats at the end of the treatment period
while receiving normal diet.
[0042] FIG. 10 shows the cumulative net amount of calcium retained
(.+-.S.E.M.) in rats receiving calcium free diet plus a daily
consumed dose of calcium over a four day period.
[0043] FIG. 11 shows the cumulative net amount of magnesium
retained (.+-.S.E.M.) in rats receiving calcium free diet plus a
daily consumed dose of calcium over a four day period.
[0044] FIG. 12 shows the cumulative net amount of zinc retained
(.+-.S.E.M.) in rats receiving calcium free diet plus a daily
consumed dose of calcium over a four day period.
[0045] FIG. 13 shows the plasma calcium (A), magnesium (B) and zinc
(C) levels sampled from rats at the end of the treatment period
while receiving calcium free diet and a normal daily dose of
calcium.
[0046] FIG. 14 is the body mass record of rats which received
individual elemental treatments.
[0047] FIG. 15 shows trabecular BMD of Distal Femur Averaged from 3
pQCT Slices. *: significantly different from OVX-control
(p<0.05).
[0048] FIG. 16 shows trabecular BMD of Proximal Tibia Averaged from
3 pQCT Slices. *: significantly different from OVX-control
(p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0049] In general, soluble calcium salts have a lower percentage of
calcium. For example, calcium ascorbate has only 9% of calcium. The
content is several folds lower than that of the insoluble calcium
carbonate (40% calcium). Among soluble calcium, calcium acetate has
the highest calcium content (25% calcium). This makes calcium
acetate a suitable candidate for making a solid dosage.
[0050] The addition of elements and vitamins to a formula lowers
the percentage of calcium. This poses a severe challenge to prepare
a dosage form that has an acceptable size to consumers.
[0051] The present invention describes methodologies for preparing
dosage forms with acceptable sizes.
[0052] The present invention also provides a method of preparing
tablets comprising calcium acetate, magnesium acetate, zinc acetate
and vitamin D.sub.3, comprising the steps of: (i) blending a
calcium composition comprising calcium acetate, magnesium acetate,
and zinc acetate with a composition comprising vitamin D.sub.3; and
(ii) blending the composition obtained from (i) with a calcium
composition comprising calcium acetate, magnesium acetate, and zinc
acetate, thereby obtaining tablets comprising calcium acetate,
magnesium acetate, zinc acetate and vitamin D.sub.3. In one
embodiment, the calcium composition comprises at least 10 percent
by weight of calcium acetate, at least 5 percent by weight of
magnesium acetate, and at least 0.2 percent by weight of zinc
acetate.
[0053] The present invention also provides a tablet produced by the
method described above.
[0054] The present invention also provides a method of preparing
soft gel capsules comprising calcium acetate, magnesium acetate,
zinc acetate and vitamin D.sub.3, comprising the steps of: (i)
dissolving vitamin D.sub.3 in fish oil, flaxseed oil, or other oils
containing either omega 3 or omega 3-6-9; (ii) mixing the
composition obtained from (i) with a calcium composition comprising
calcium acetate, magnesium acetate, and zinc acetate, thereby
obtaining soft gel capsules comprising calcium acetate, magnesium
acetate, zinc acetate. In one embodiment, the calcium composition
comprises at least 10 percent by weight of calcium acetate, at
least 5 percent by weight of magnesium acetate, and at least 0.2
percent by weight of zinc acetate.
[0055] The invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative, and are not meant to limit the invention as
described herein, which is defined by the claims which follow
thereafter.
[0056] Throughout this application, various references or
publications are cited. Disclosures of these references or
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art to which this invention pertains.
[0057] It is to be noted that the transitional term "comprising",
which is synonymous with "including", "containing" or
"characterized by", is inclusive or open-ended and does not exclude
additional, un-recited elements or method steps.
Example 1
Formulations of Pearl Extracts
[0058] A pearl extract was prepared by adapting the patented method
reported by Li and Li (31). Briefly, pearls are pulverized to a
size between 80 to 120 mesh. The powder is soaked in a mixture of
saturated sodium chloride solution with titrated amount of acetic
acid. Electrical current is applied to the mixture for several
days. After dilution with water and magnetization, the mixture was
filtered and precipitated. The precipitate, rich in calcium
acetate, is dried and ready for consumption as a dietary
supplement. A detailed list of elements present in the extract is
presented on Table 1:
TABLE-US-00001 TABLE 1 Content of Pearl Extract Element Quantity,
ppm Calcium 233,000 Magnesium 253 Zinc 3281 Potassium 1650
Manganese 1170 Sodium 680 Strontium 158 Molybdenum 55.4 Silicon
38.0 Selenium 27.9
[0059] This extract, A1, is fortified with acetate salts of
magnesium to provide Ca/Mg ratios of 0.5/1 (A6), 1/1 (A4) and 2/1
(A5). The major elemental content of the pearl extract and its
fortified mixtures are listed on Table 2:
TABLE-US-00002 TABLE 2 The Content of Each Element in Each Formula
(n = 3) The content of three elements in each formula Ca (%) Mg (%)
Zn (%) Labeled Labeled Labeled Formula No. Determined content.sup.a
Determined content.sup.a Determined content.sup.a A1 23.30 .+-.
1.26 23.4 0.0253 .+-. 0.0013 0.0012*** 0.328 .+-. 0.03 0.330 A4
7.65 .+-. 0.62 7.51 7.56 .+-. 0.32 7.50 0.372 .+-. 0.029 0.375 A5
11.5 .+-. 0.34 11.3 5.41 .+-. 0.04 5.64 0.556 .+-. 0.044 0.565 A6
4.58 .+-. 0.09 4.50 8.29 .+-. 0.15 8.99 0.256 .+-. 0.012 0.225 Data
are expressed as mean .+-. S.D. .sup.aIn-house Data. ***p <
0.001
[0060] Besides Pearl, the method described in this example can also
be used to extract multiple acetate salts of calcium, magnesium and
zinc from natural sources such as corals, oysters, mineral mines,
etc. The composition of formulas A1, A4 through A6 could also be
achieved by mixing appropriate amounts of acetates salts of
calcium, magnesium and zinc.
[0061] Experimental Data on Elemental Solubility. The
gastrointestinal tract is a complex organ. There are a number of
factors which could alter the solubility of elements including
calcium, magnesium and zinc; subsequently, their rate of absorption
and bioavailability. Examples 2-5 highlight some of the
physiological factors which have been postulated to have a
significant impact on the solubility of elements.
Example 2
Solubility of Calcium in Artificial Gastric and Intestinal
Juice
[0062] The solubility of calcium in the four formulas in an
artificial gastric (pH=1) and intestinal fluid (pH=7) was tested
using a method developed for ICP-OES (Inductively Coupled Plasma
Optical Emission Spectrometer) (PerkinElmer Optima 4300DV). Two
commercial samples, Caltrate.TM. and calcium acetate, were also
tested in parallel for comparison. The results are shown in Table
3.
[0063] Compared to Caltrate.TM., the solubility of calcium acetate
is approximately 45 times higher in the artificial gastric juice
and 26,000 times higher in the artificial intestinal juice. The
solubility of the pearl extract, A1, comprising mostly calcium
acetate, is similar to that of calcium acetate in the artificial
gastric juice and intestinal juice (p>0.05). The solubility of
calcium acetate is pH dependent; it is lower in the artificial
intestinal fluid when compared to the artificial gastric juice.
Magnesium has a tendency to lower the solubility of calcium. When
the ratio of Ca/Mg decreases, the solubility of the extract
decreases, A5>A4>A6. Nevertheless, A6, the least soluble
pearl extract formula, is .about.12 times more soluble in
artificial gastric juice and 8,500 times more soluble in artificial
intestinal juice than that of Caltrate.TM. Therefore, unlike
Caltrate.TM., solubility of acetate salts should not be an issue in
gastrointestinal tract fluids because the acetate salts will still
be in solution.
[0064] The solubility profile of magnesium salts is very similar to
that of calcium (Table 4). In general, acetate salts of magnesium
are highly soluble. They are more soluble in artificial gastric
juice than artificial intestinal juice. In contrast to magnesium
acetate, the solubility of magnesium carbonate in Caltrate.TM. is
low.
[0065] The solubility profile of zinc salts is also similar to that
of magnesium and calcium, except the magnitude of difference
between salt forms under differing pH and environmental conditions
is less drastic (Table 5).
[0066] This set of experiments thus leads to the conclusion that
acetate salts are preferred salts in the disclosed formulations for
their high solubility.
TABLE-US-00003 TABLE 3 Saturated Solubility of Calcium in
Artificial Gastric And Intestinal Fluid (n = 3) Saturated
solubility of calcium Formula No. Gastric fluid (g/L) Intestinal
fluid (g/L) A1 72.93 .+-. 4.14 64.97 .+-. 6.29 A4 33.60 .+-. 1.18
29.90 .+-. 2.14 A5 53.97 .+-. 8.34 45.50 .+-. 7.24 A6 19.87 .+-.
3.11 20.90 .+-. 2.36 Calcium 77.73 .+-. 8.13 68.43 .+-. 2.55
Acetate Caltrate .TM. 1.70 .+-. 0.24 0.00246 .+-. 0.00015 Data are
expressed as Mean .+-. S.D.
TABLE-US-00004 TABLE 4 Saturated Solubility Of Magnesium In
Artificial Gastric Fluid And Intestinal Fluid Saturated solubility
of magnesium Formula No. Gastric fluid (g/L) Intestinal fluid (g/L)
A1 0.13 .+-. 0.006 0.13 .+-. 0.04 A2 0.11 .+-. 0.01 0.10 .+-. 0.03
A3 0.12 .+-. 0.04 0.09 .+-. 0.01 A4 40.78 .+-. 2.46 26.57 .+-.
1.81*** A5 24.97 .+-. 2.95 19.03 .+-. 2.73*** A6 49.30 .+-. 2.61
38.67 .+-. 4.33*** Calcium Acetate 0.50 .+-. 0.07 0.42 .+-. 0.10
Caltrate .TM. 0.17 .+-. 0.17 0.09 .+-. 0.02 Data are expressed as
mean .+-. S.D. (n = 3). ***P < 0.001 compared with solubility in
the artificial gastric fluid.
TABLE-US-00005 TABLE 5 Saturated Solubility Of Zinc In Artificial
Gastric Fluid And Intestinal Fluid Saturated solubility of zinc
Formula No. Gastric fluid (g/L) Intestinal fluid (g/L) A1 1.04 .+-.
0.16 0.76 .+-. 0.07* A2 3.72 .+-. 0.68 2.14 .+-. 0.14* A3 3.25 .+-.
0.19 2.31 .+-. 0.08** A4 2.22 .+-. 0.17 1.19 .+-. 0.11*** A5 2.64
.+-. 0.38 1.64 .+-. 0.07* A6 1.54 .+-. 0.13 1.07 .+-. 0.11**
Calcium Acetate 0.60 .+-. 0.17 0.53 .+-. 0.14 Caltrate .TM. 0.33
.+-. 0.10 0.23 .+-. 0.08 Data are expressed as mean .+-. S.D. (n =
3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the
solubility in artificial gastric fluid.
Example 3
Effects of pH on the Solubility of Calcium in Different
Formulations
[0067] In this example, the effects of pH (ranging from 1 to 9) on
the solubility of three elements of the four pearl formulas (A1,
A4, A5 and A6), a commercial product (Caltrate.TM.) and a synthetic
compound (Calcium Acetate, Ca ACE) were investigated. Solution pH
was adjusted using appropriate amounts of acetic acid (AcOH),
nitric acid (HNO.sub.3) or ammonium hydroxide (NH.sub.4OH).
Saturated solutions were prepared by dissolving each preparation in
a solution with a final pH value ranging from 1 to 9. The resultant
mixtures were incubated in a water bath at 37.degree. C. for one
hour. Each sample was then filtered (with or without
centrifugation) immediately, and the filtrate was diluted to an
appropriate concentration for elemental analysis. The concentration
of calcium, magnesium, and zinc was measured using ICP-OES. The
results are shown in Tables 6-8. Statistical analysis was performed
using one-way ANOVA and the P value was set at 0.05.
[0068] Throughout the pH range tested, both A1 and calcium acetate
showed significantly higher calcium content in solution than the
other preparations. Caltrate.TM. had the lowest calcium content
(p<0.05). A1 and calcium acetate have the highest solubility at
pH 1 (Table 6)
[0069] Magnesium has a negative effect on the content of calcium in
solution; the rank order in terms of solubility is A5>A4>A6.
Except for Caltrate.TM., calcium acetate and A1, which are more
soluble at pH 1, pH has no effect on the solubility of magnesium in
solution (Table 7).
[0070] Similarly, the amount of zinc in solution correlated well
with the zinc content in different formulations
(A5>A4>A1>A6) (Table 8). For all four acetate formulas
tested, pH values higher than 5 were associated with higher
solubility of zinc than that at pH 2 and 3.
[0071] Since intestinal pH values are typically higher than 6, the
present formulations present advantages in terms of solubility,
when compared with the solubility of calcium carbonate in
Caltrate.TM. under such pH conditions. These results are consistent
with those reported in Table 3.
TABLE-US-00006 TABLE 6 Calcium Solubility (g/L) In Different pH
Solutions (N = 3) pH Caltrate .TM. Ca ACE A1 A4 A5 A6 1 4.60 .+-.
0.28 99.0 .+-. 19.2 101 .+-. 12.9 37.6 .+-. 2.5 48.7 .+-. 2.1 23.3
.+-. 3.2 2 4.04 .+-. 0.23 61.3 .+-. 0.97 64.6 .+-. 5.1 29.4 .+-.
2.1 43.5 .+-. 5.3 22.6 .+-. 0.54 3 0.507 .+-. 0.10 75.7 .+-. 4.8
71.4 .+-. 22.2 38.0 .+-. 4.7 48.6 .+-. 0.98 19.5 .+-. 2.5 4 0.237
.+-. 0.03 85.4 .+-. 5.7 75.3 .+-. 3.4 38.5 .+-. 2.9 48.3 .+-. 0.82
20.8 .+-. 0.98 5 0.240 .+-. 0.06 75.6 .+-. 5.5 65.4 .+-. 11.8 39.3
.+-. 4.4 47.4 .+-. 8.6 24.7 .+-. 2.5 6 0.317 .+-. 0.10 76.0 .+-.
5.9 83.8 .+-. 12.7 41.2 .+-. 1.3 52.3 .+-. 5.0 19.3 .+-. 2.7 7
0.133 .+-. 0.05 80.0 .+-. 3.5 84.2 .+-. 16.8 34.6 .+-. 3.3 49.5
.+-. 8.1 20.6 .+-. 3.8 8 0.160 .+-. 0.03 71.2 .+-. 1.6 78.3 .+-.
13.0 30.0 .+-. 3.8 55.3 .+-. 7.9 19.8 .+-. 2.0 9 0.227 .+-. 0.13
74.5 .+-. 6.8 84.8 .+-. 8.2 35.8 .+-. 3.5 50.2 .+-. 1.5 19.6 .+-.
4.2
TABLE-US-00007 TABLE 7 Magnesium Solubility (g/L) in Different pH
Solutions (n = 3) pH Caltrate .TM. Ca ACE A1 A4 A5 A6 1 0.197 .+-.
0.015 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-. 4.836 23.927
.+-. 1.747 41.797 .+-. 5.622 2 0.100 .+-. 0.000 0.360 .+-. 0.010
0.133 .+-. 0.006 29.117 .+-. 2.204 20.020 .+-. 2.174 39.957 .+-.
1.050 3 0.133 .+-. 0.006 0.413 .+-. 0.035 0.143 .+-. 0.021 32.640
.+-. 41.65 21.880 .+-. 0.849 34.100 .+-. 5.169 4 0.123 .+-. 0.012
0.490 .+-. 0.010 0.173 .+-. 0.015 33.560 .+-. 2.606 21.733 .+-.
0.248 34.153 .+-. 1.560 5 0.107 .+-. 0.012 0.500 .+-. 0.040 0.137
.+-. 0.012 34.510 .+-. 2.817 24.367 .+-. 3.916 45.353 .+-. 7.294 6
0.110 .+-. 0.010 0.473 .+-. 0.076 0.177 .+-. 0.040 35.747 .+-.
1.738 24.997 .+-. 0.817 34.477 .+-. 4.730 7 0.093 .+-. 0.006 0.460
.+-. 0.035 0.153 .+-. 0.015 30.197 .+-. 2.818 21.677 .+-. 3.127
36.983 .+-. 7.234 8 0.097 .+-. 0.006 0.433 .+-. 0.040 0.157 .+-.
0.015 31.023 .+-. 6.548 24.953 .+-. 3.410 34.480 .+-. 4.046 9 0.097
.+-. 0.006 0.433 .+-. 0.045 0.160 .+-. 0.010 33.473 .+-. 7.169
23.607 .+-. 1.055 34.410 .+-. 6.836
TABLE-US-00008 TABLE 8 Zinc Solubility (g/L) in Different pH
Solutions (n = 3) pH Caltrate .TM. Ca ACE A1 A4 A5 A6 1 0.007 .+-.
0.006 0.030 .+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637 .+-.
0.143 1.440 .+-. 0.140 2 0.003 .+-. 0.006 0.013 .+-. 0.006 0.793
.+-. 0.093 1.457 .+-. 0.032 2.220 .+-. 0.204 1.143 .+-. 0.025 3
0.000 .+-. 0.000 0.017 .+-. 0.006 0.843 .+-. 0.315 1.593 .+-. 0.216
2.103 .+-. 0.134 0.817 .+-. 0.064 4 0.000 .+-. 0.000 0.017 .+-.
0.006 1.137 .+-. 0.092 1.867 .+-. 0.078 2.383 .+-. 0.071 0.933 .+-.
0.032 5 0.003 .+-. 0.006 0.017 .+-. 0.006 0.993 .+-. 0.195 1.930
.+-. 0.164 2.790 .+-. 0.305 1.250 .+-. 0.193 6 0.000 .+-. 0.000
0.020 .+-. 0.000 1.227 .+-. 0.133 2.063 .+-. 0.059 2.837 .+-. 0.135
0.870 .+-. 0.096 7 0.007 .+-. 0.012 0.023 .+-. 0.006 1.237 .+-.
0.223 1.770 .+-. 0.132 2.493 .+-. 0.372 0.990 .+-. 0.157 8 0.003
.+-. 0.006 0.027 .+-. 0.012 1.180 .+-. 0.180 1.787 .+-. 0.306 2.903
.+-. 0.300 0.940 .+-. 0.082 9 0.007 .+-. 0.006 0.027 .+-. 0.006
1.260 .+-. 0.087 1.970 .+-. 0.364 2.753 .+-. 0.133 0.917 .+-.
0.152
Experimental Data on Solubility in the Presence of Common Gastric
and Intestinal Anions and Cations
[0072] The following analyses using anions which are present in
abundance in gastro-intestinal tract fluids were performed on the
four test formulas (A1, A4, A5 and A6), Caltrate.TM. and calcium
acetate in order to assess the solubility and subsequently, their
rate of absorption and bioavailability. The following are the
standard ranges of common anions and cations in the human
gastrointestinal fluids.
TABLE-US-00009 TABLE 9 Concentration Of Ions In Human Gastric And
Intestinal Fluids Concentration of ion (mM) Ions In Stomach/Gastric
Fluid.sup.a In Intestine/Intestinal Fluid.sup.a Na.sup.+ 0-100
(0-80) (155) K.sup.+ 0-10 (0-15) (70-150) H.sup.+ 1-140 (20-120)
(pH 7.7-8.2) Cl.sup.- 100-170 (120-160) (30-90) Phosphate ions Up
to 100** HCO.sub.3.sup.- (70-130) .sup.aValues were cited from The
Digestive System (ISBN 0443062455). The values in brackets were
cited from The Medical Physiology (ISBN 0781719364). **based on the
solubility of sodium phosphate.
Example 4
Effects of Anions on the Solubility of Calcium, Magnesium and Zinc
in the Test Preparations
[0073] The following analyses using anions which are present in
abundance in gastro-intestinal tract fluids were performed on the
four test formulas (A1, A4, A5 and A6), Caltrate.TM. and calcium
acetate in order to assess the solubility and subsequently, their
rate of absorption and bioavailability. In this example, the
effects of bicarbonate and phosphate (HCO.sub.3.sup.- and
PO.sub.4.sup.3-) on the solubility of calcium, magnesium, and zinc
were studied at pH 7. Furthermore, the effects of chloride on the
absorption of these three elements at pH 1 and pH 7 were also
studied. The procedures described in Example 3 for pH adjustment
and solubility measurements were used. ICP-OES was used to quantify
calcium, magnesium and zinc. Statistical analysis was performed
using one-way ANOVA and the level of significance was set at
p<0.05.
A. Chloride Effects at pH 1
[0074] Tables 10-12 are the results of chloride effects at pH 1.
This condition mimics that of the acidic environment in the
stomach. Chloride has the most intense effect on the solubility of
calcium, magnesium and zinc in Caltrate.TM. at pH 1 (Tables 10-12).
At a Cl.sup.- concentration of 200 mM, the solubility of calcium
was the highest. The maximum magnesium and zinc solubility was
reached at Cl.sup.- concentrations of 50 mM and 120 mM,
respectively. The fluctuations of calcium, magnesium and zinc
solubility are minimal in all the acetate formulations: calcium
acetate, A1, A4, A5 and A6. Significant differences are often
obtained at the highest Cl.sup.- concentration (p<0.05).
TABLE-US-00010 TABLE 10 The Effect of Cl.sup.- Concentration On The
Solubility Of Calcium (g/L) In Different Formulations At pH 1
Cl.sup.- Conc. Caltrate .TM. Ca ACE A1 A4 A5 A6 0 mM 4.597 .+-.
0.276 98.950 .+-. 19.224 101.353 .+-. 12.947 37.637 .+-. 2.509
48.670 .+-. 2.102 23.337 .+-. 3.162 50 mM 8.160 .+-. 0.497 80.857
.+-. 10.277 73.950 .+-. 0.987 29.950 .+-. 6.933 42.413 .+-. 12.931
22.290 .+-. 4.543 100 mM 7.333 .+-. 1.572 71.060 .+-. 1.660 85.627
.+-. 14.191 30.023 .+-. 4.042 43.853 .+-. 2.264 24.690 .+-. 0.746
120 mM 8.157 .+-. 1.210 76.453 .+-. 6.196 83.967 .+-. 0.479 36.883
.+-. 1.966 50.283 .+-. 2.977 24.850 .+-. 1.077 150 mM 5.883 .+-.
1.416 73.353 .+-. 1.037 87.340 .+-. 3.166 39.657 .+-. 4.659 44.443
.+-. 5.495 24.647 .+-. 0.775 180 mM 9.073 .+-. 0.325 80.977 .+-.
12.440 88.593 .+-. 5.579 41.710 .+-. 2.836 50.343 .+-. 1.392 26.067
.+-. 1.891 200 mM 12.123 .+-. 1.178 77.257 .+-. 12.364 97.840 .+-.
12.364 42.313 .+-. 6.119 63.027 .+-. 3.406 29.387 .+-. 4.062
TABLE-US-00011 TABLE 11 The Effect of Cl.sup.- Concentration On The
Solubility Of Magnesium (g/L) In Different Formulations At pH 1
Cl.sup.- Conc. Caltrate .TM. Ca ACE A1 A4 A5 A6 0 mM 0.197 .+-.
0.015 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-. 4.836 23.927
.+-. 1.747 41.797 .+-. 5.622 50 mM 0.357 .+-. 0.471 0.440 .+-.
0.075 0.133 .+-. 0.006 31.420 .+-. 6.649 20.547 .+-. 6.525 45.827
.+-. 6.006 100 mM 0.113 .+-. 0.012 0.380 .+-. 0.020 0.157 .+-.
0.012 35.853 .+-. 4.215 22.697 .+-. 1.231 46.900 .+-. 4.117 120 mM
0.243 .+-. 0.163 0.420 .+-. 0.036 0.140 .+-. 0.017 33.363 .+-.
2.542 23.333 .+-. 3.312 48.827 .+-. 4.095 150 mM 0.220 .+-. 0.132
0.403 .+-. 0.012 0.163 .+-. 0.015 36.037 .+-. 4.510 21.967 .+-.
1.260 45.653 .+-. 2.449 180 mM 0.227 .+-. 0.134 0.420 .+-. 0.040
0.160 .+-. 0.020 38.117 .+-. 3.356 24.210 .+-. 0.698 46.070 .+-.
3.290 200 mM 0.207 .+-. 0.074 0.427 .+-. 0.080 0.163 .+-. 0.006
43.203 .+-. 4.646 29.410 .+-. 0.115 81.437 .+-. 4.319
TABLE-US-00012 TABLE 12 The Effect of Cl.sup.- Concentration On The
Solubility Of Zinc (g/L) In Different Formulations At pH 1 Cl.sup.-
Conc. Caltrate .TM. Ca ACE A1 A4 A5 A6 0 mM 0.007 .+-. 0.006 0.030
.+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637 .+-. 0.143 1.440
.+-. 0.140 50 mM 0.030 .+-. 0.000 0.027 .+-. 0.006 0.917 .+-. 0.156
1.500 .+-. 0.216 2.073 .+-. 0.598 1.237 .+-. 0.110 100 mM 0.130
.+-. 0.026 0.067 .+-. 0.025 1.120 .+-. 0.010 1.683 .+-. 0.100 2.353
.+-. 0.057 1.293 .+-. 0.025 120 mM 0.217 .+-. 0.047 0.103 .+-.
0.031 1.113 .+-. 0.112 1.687 .+-. 0.196 2.487 .+-. 0.273 1.363 .+-.
0.095 150 mM 0.277 .+-. 0.091 0.073 .+-. 0.015 1.360 .+-. 0.144
1.803 .+-. 0.121 2.320 .+-. 0.106 1.280 .+-. 0.046 180 mM 0.180
.+-. 0.060 0.117 .+-. 0.031 1.193 .+-. 0.211 1.927 .+-. 0.015 2.590
.+-. 0.061 1.313 .+-. 0.032 200 mM 0.190 .+-. 0.056 0.123 .+-.
0.040 1.413 .+-. 0.187 2.230 .+-. 0.265 3.173 .+-. 0.248 2.083 .+-.
0.112
B. Chloride Effects at pH 7
[0075] At pH 7, the solubility of calcium in Caltrate.TM. is
dramatically lower than that at pH 1 in the presence of chloride
(Compare values in Tables 10 and 13). As chloride concentration
increased, the solubility of calcium in Caltrate.TM. increased. The
pH and chloride effects are not pronounced for the acetate
formulations. In general, maximum calcium solubility is reached at
chloride concentrations between 50 to 100 mM.
[0076] In the presence of chloride, pH has less of an effect on
magnesium solubility (compare values between Tables 11 and 14). In
general, the solubility of magnesium at pH 7 is slightly lower for
all formulas and the chloride effect is not pronounced.
[0077] In the presence of chloride, the solubility of zinc in
Caltrate.TM. at pH 7 is less than half of that at pH 1 (compare
values between 11 and 14). However, this difference is not as
pronounced in the acetate formulas. There is a tendency for zinc
solubility to increase with the increase of chloride concentration.
Maximum zinc solubility is reached at 120 mM chloride when
Caltrate.TM. was evaluated. For the acetate formulas, maximum zinc
solubility occurred when chloride concentration reached 200 mM.
TABLE-US-00013 TABLE 13 The Effect of Cl.sup.- Concentration On The
Solubility Of Calcium In Different Formulations At pH 7 Cl.sup.-
Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4 [g/L] A5 [g/L]
A6 [g/L] 0 mM 0.133 .+-. 0.051 80.017 .+-. 3.505 84.170 .+-. 16.834
34.640 .+-. 3.268 49.497 .+-. 8.097 20.627 .+-. 3.821 50 mM 0.340
.+-. 0.082 99.373 .+-. 6.182 80.703 .+-. 13.103 47.473 .+-. 2.381
61.537 .+-. 6.436 31.490 .+-. 2.399 100 mM 0.557 .+-. 0.040 87.263
.+-. 13.984 77.660 .+-. 19.779 47.867 .+-. 7.511 66.743 .+-. 13.191
29.053 .+-. 6.684 120 mM 0.370 .+-. 0.165 71.440 .+-. 5.851 61.437
.+-. 8.616 35.400 .+-. 0.864 45.060 .+-. 6.166 22.353 .+-. 2.351
150 mM 0.567 .+-. 0.075 70.923 .+-. 3.240 73.773 .+-. 12.437 33.017
.+-. 2.455 42.980 .+-. 2.603 20.313 .+-. 2.005 180 mM 0.560 .+-.
0.165 77.823 .+-. 12.314 59.720 .+-. 7.467 34.003 .+-. 0.846 42.890
.+-. 5.516 17.490 .+-. 0.916 200 mM 0.600 .+-. 0.132 73.930 .+-.
7.785 84.707 .+-. 15.685 33.223 .+-. 2.093 46.403 .+-. 4.643 18.627
.+-. 2.238
TABLE-US-00014 TABLE 14 The Effect of Cl.sup.- Concentration On The
Solubility Of Magnesium In Different Formulations At pH 7 Cl.sup.-
Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4 [g/L] A5 [g/L]
A6 [g/L] 0 mM 0.093 .+-. 0.006 0.460 .+-. 0.035 0.153 .+-. 0.015
30.197 .+-. 2.818 21.677 .+-. 3.127 36.983 .+-. 7.234 50 mM 0.280
.+-. 0.202 0.503 .+-. 0.031 0.140 .+-. 0.020 43.190 .+-. 2.792
29.203 .+-. 1.107 56.003 .+-. 3.989 100 mM 0.280 .+-. 0.149 0.480
.+-. 0.017 0.143 .+-. 0.006 45.253 .+-. 6.350 30.917 .+-. 6.111
52.953 .+-. 14.721 120 mM 0.110 .+-. 0.026 0.390 .+-. 0.030 0.147
.+-. 0.012 31.983 .+-. 3.302 19.333 .+-. 2.217 42.463 .+-. 1.448
150 mM 0.227 .+-. 0.096 1.750 .+-. 2.382 0.167 .+-. 0.015 29.087
.+-. 0.957 19.383 .+-. 1.482 42.643 .+-. 0.446 180 mM 0.253 .+-.
0.129 0.430 .+-. 0.046 0.167 .+-. 0.006 32.633 .+-. 2.372 19.733
.+-. 2.149 36.160 .+-. 10.009 200 mM 0.283 .+-. 0.107 0.427 .+-.
0.065 0.203 .+-. 0.025 32.923 .+-. 0.802 23.067 .+-. 2.175 47.133
.+-. 1.598
TABLE-US-00015 TABLE 15 The Effect of Cl.sup.- Concentration On The
Solubility Of Zinc In Different Formulations At pH 7 Cl.sup.- Conc.
Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4 [g/L] A5 [g/L] A6
[g/L] 0 mM 0.007 .+-. 0.012 0.023 .+-. 0.006 1.237 .+-. 0.223 1.770
.+-. 0.132 2.493 .+-. 0.372 0.990 .+-. 0.157 50 mM 0.113 .+-. 0.006
0.180 .+-. 0.089 0.997 .+-. 0.195 2.057 .+-. 0.189 3.177 .+-. 0.289
1.457 .+-. 0.244 100 mM 0.140 .+-. 0.026 0.213 .+-. 0.102 0.903
.+-. 0.280 2.413 .+-. 0.144 3.063 .+-. 0.287 1.540 .+-. 0.380 120
mM 0.050 .+-. 0.017 0.167 .+-. 0.202 0.760 .+-. 0.118 1.573 .+-.
0.146 1.997 .+-. 0.254 1.110 .+-. 0.036 150 mM 0.087 .+-. 0.025
0.177 .+-. 0.085 0.987 .+-. 0.110 2.030 .+-. 0.615 2.010 .+-. 0.165
1.177 .+-. 0.072 180 mM 0.093 .+-. 0.015 0.143 .+-. 0.071 0.780
.+-. 0.151 1.637 .+-. 0.127 2.090 .+-. 0.167 1.077 .+-. 0.163 200
mM 0.093 .+-. 0.012 0.160 .+-. 0.079 1.117 .+-. 0.202 1.663 .+-.
0.078 1.643 .+-. 1.217 1.303 .+-. 0.060
C. Bicarbonate Effects at pH 7.
[0078] The solubility of calcium in Caltrate.TM. increased with the
increase of bicarbonate concentration (Table 16). However, the
opposite is true for calcium acetate. The solubility was reduced at
least 40%. The reduction for all the pearl extract formulas was
less, approximately 20 to 25%.
[0079] The solubility of magnesium in Caltrate.TM. increased with
bicarbonate concentration (Table 17). Bicarbonate effect was
minimal for the acetate formulas.
[0080] The solubility of zinc in Caltrate.TM. increased in the
presence of bicarbonate (Table 18). Maximum zinc solubility was
reached at 70 mM. For calcium acetate, the trend is similar to that
of Caltrate.TM.. Bicarbonate has very little effect on the pearl
extract formulas.
TABLE-US-00016 TABLE 16 The Effect of HCO.sub.3.sup.- Concentration
On The Solubility Of Calcium In Different Formulations At pH 7
HCO.sub.3.sup.- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0 mM 0.133 .+-. 0.051 80.017 .+-. 3.505
84.170 .+-. 16.834 34.640 .+-. 3.268 49.497 .+-. 8.097 20.627 .+-.
3.821 50 mM 0.217 .+-. 0.214 50.243 .+-. 3.312 72.030 .+-. 7.103
36.007 .+-. 3.807 42.577 .+-. 0.779 21.737 .+-. 1.255 70 mM 0.213
.+-. 0.098 62.090 .+-. 8.524 70.933 .+-. 4.812 33.420 .+-. 5.263
42.130 .+-. 4.734 22.343 .+-. 0.847 100 mM 0.380 .+-. 0.075 56.367
.+-. 9.062 83.640 .+-. 10.870 34.997 .+-. 6.049 46.167 .+-. 4.546
25.260 .+-. 10.191 120 mM 0.440 .+-. 0.167 46.023 .+-. 2.463 67.010
.+-. 3.767 31.060 .+-. 2.23 46.973 .+-. 2.919 20.817 .+-. 1.664 150
mM 0.433 .+-. 0.120 70.637 .+-. 3.622 65.617 .+-. 1.475 30.410 .+-.
2.888 41.567 .+-. 4.620 19.163 .+-. 1.568 180 mM 0.930 .+-. 1.290
46.847 .+-. 2.741 65.270 .+-. 1.781 28.680 .+-. 1.362 38.073 .+-.
3.465 18.870 .+-. 1.679
TABLE-US-00017 TABLE 17 The Effect of HCO.sub.3.sup.- Concentration
On The Solubility Of Magnesium In Different Formulations At pH 7
HCO.sub.3.sup.- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0 mM 0.093 .+-. 0.006 0.460 .+-. 0.035
0.153 .+-. 0.015 30.197 .+-. 2.818 21.677 .+-. 3.127 36.983 .+-.
7.234 50 mM 0.090 .+-. 0.035 0.297 .+-. 0.055 0.190 .+-. 0.056
34.600 .+-. 4.638 20.427 .+-. 1.272 48.140 .+-. 1.653 70 mM 0.093
.+-. 0.012 0.347 .+-. 0.031 0.160 .+-. 0.000 32.057 .+-. 4.407
22.000 .+-. 0.141 42.767 .+-. 0.460 100 mM 0.223 .+-. 0.111 0.343
.+-. 0.101 0.167 .+-. 0.065 41.580 .+-. 12.984 26.393 .+-. 4.720
43.883 .+-. 1.288 120 mM 0.220 .+-. 0.069 0.303 .+-. 0.015 0.483
.+-. 0.551 30.960 .+-. 2.164 22.877 .+-. 1.082 46.990 .+-. 5.278
150 mM 0.227 .+-. 0.072 0.410 .+-. 0.061 0.150 .+-. 0.017 28.950
.+-. 2.262 18.850 .+-. 2.169 42.877 .+-. 7.608 180 mM 0.240 .+-.
0.095 0.293 .+-. 0.049 0.163 .+-. 0.015 30.787 .+-. 1.021 19.607
.+-. 1.529 36.957 .+-. 0.839
TABLE-US-00018 TABLE 18 The Effect of HCO.sub.3.sup.- Concentration
On The Solubility Of Zinc In Different Formulations At pH 7
HCO.sub.3.sup.- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0 mM 0.007 .+-. 0.012 0.023 .+-. 0.006
1.237 .+-. 0.223 1.770 .+-. 0.132 2.493 .+-. 0.372 0.990 .+-. 0.157
50 mM 0.057 .+-. 0.015 0.050 .+-. 0.010 0.953 .+-. 0.101 1.663 .+-.
0.205 2.010 .+-. 0.142 1.260 .+-. 0.017 70 mM 0.070 .+-. 0.020
0.100 .+-. 0.061 0.990 .+-. 0.082 1.560 .+-. 0.236 2.237 .+-. 0.099
1.147 .+-. 0.081 100 mM 0.070 .+-. 0.017 0.157 .+-. 0.055 1.190
.+-. 0.101 2.067 .+-. 0.654 2.660 .+-. 0.442 1.193 .+-. 0.023 120
mM 0.093 .+-. 0.025 0.210 .+-. 0.096 0.907 .+-. 0.042 1.513 .+-.
0.127 2.290 .+-. 0.115 1.317 .+-. 0.182 150 mM 0.087 .+-. 0.021
0.137 .+-. 0.083 0.863 .+-. 0.081 1.427 .+-. 0.059 1.887 .+-. 0.144
1.237 .+-. 0.235 180 mM 0.070 .+-. 0.017 0.160 .+-. 0.078 0.933
.+-. 0.072 1.517 .+-. 0.119 1.997 .+-. 0.157 1.023 .+-. 0.042
D. Effects of Phosphates at pH 7
[0081] Phosphates have insignificant effects on the solubility of
calcium in Caltrate.TM. (Table 19). As phosphate concentrations
increased the solubility of calcium decreased in all acetate
formulations. Maximum reduction (up to 40%) of the solubility of
calcium was observed in formulas containing higher percentage of
magnesium (A4, A5 and A6). Considering the range of phosphate
concentration tested, 10,000-fold, the change of calcium solubility
is not significant.
[0082] Magnesium solubility decreased as phosphate concentration
increased (Table 20). The reduction (80%) is most significant for
the magnesium in Caltrate.TM.. For the other formulas, the maximum
reduction was approximately 50%. Again, the effect of phosphates
was not that significant considering the range of concentration
tested.
[0083] Among the three elements, phosphates have the most intense
effect on the solubility of zinc (Table 21). All formulas were
affected to the same extent and the maximum reduction was
approximately 70%. Considering the range of phosphate concentration
tested, again, the effects of phosphates were not that
significant.
TABLE-US-00019 TABLE 19 The Effect of PO.sub.4.sup.3- Concentration
On The Solubility Of Calcium In Different Formulations At pH 7
PO.sub.4.sup.3- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0.01 mM 0.587 .+-. 0.200 77.517 .+-. 6.084
84.270 .+-. 9.511 34.950 .+-. 6.725 47.823 .+-. 3.080 22.287 .+-.
2.539 1 mM 0.510 .+-. 0.252 68.220 .+-. 19.638 56.450 .+-. 9.879
39.923 .+-. 10.060 42.363 .+-. 3.572 23.530 .+-. 0.159 10 mM 0.430
.+-. 0.046 78.417 .+-. 7.046 64.697 .+-. 9.058 25.703 .+-. 7.033
41.287 .+-. 3.584 21.687 .+-. 1.156 100 mM 0.453 .+-. 0.158 64.770
.+-. 1.548 58.607 .+-. 9.415 25.090 .+-. 3.181 34.650 .+-. 6.972
15.437 .+-. 2.428
TABLE-US-00020 TABLE 20 The Effect of PO.sub.4.sup.3- Concentration
On The Solubility Of Magnesium In Different Formulations At pH 7
PO.sub.4.sup.3- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0.01 mM 0.280 .+-. 0.070 0.493 .+-. 0.025
0.203 .+-. 0.006 38.017 .+-. 2.532 24.733 .+-. 0.886 52.000 .+-.
5647 1 mM 0.317 .+-. 0.087 0.450 .+-. 0.095 0.217 .+-. 0.031 35.647
.+-. 10.790 18.583 .+-. 1.676 48.967 .+-. 1.486 10 mM 0.240 .+-.
0.050 0.477 .+-. 0.035 0.173 .+-. 0.012 20.837 .+-. 5.545 18.163
.+-. 1.368 37.140 .+-. 2.681 100 mM 0.073 .+-. 0.006 0.350 .+-.
0.017 0.127 .+-. 0.012 21.490 .+-. 1.830 16.720 .+-. 4.514 31.163
.+-. 4.838
TABLE-US-00021 TABLE 21 The Effect of PO.sub.4.sup.3- Concentration
On The Solubility Of Zinc In Different Formulations At pH 7
PO.sub.4.sup.3- Conc. Caltrate .TM. [g/L] Ca ACE [g/L] A1 [g/L] A4
[g/L] A5 [g/L] A6 [g/L] 0.01 mM 0.117 .+-. 0.042 0.190 .+-. 0.070
1.193 .+-. 0.097 1.950 .+-. 0.040 2.750 .+-. 0.135 1.470 .+-. 0.154
1 mM 0.100 .+-. 0.044 0.197 .+-. 0.110 0.780 .+-. 0.151 1.800 .+-.
0.394 1.993 .+-. 0.093 1.380 .+-. 0.079 10 mM 0.070 .+-. 0.010
0.180 .+-. 0.089 0.767 .+-. 0.137 0.937 .+-. 0.253 1.740 .+-. 0.173
1.023 .+-. 0.060 100 mM 0.033 .+-. 0.015 0.053 .+-. 0.023 0.527
.+-. 0.119 0.623 .+-. 0.087 1.013 .+-. 0.345 0.510 .+-. 0.131
Example 5
Effects of Cations on the Solubility of Calcium, Magnesium and Zinc
in the Test Preparations
[0084] The following analyses using cations which are present in
abundance in gastro-intestinal tract fluids were performed on the
four test formulas (A1, A4, A5 and A6), Caltrate.TM. and calcium
acetate in order to assess the solubility and subsequently, their
rate of absorption and bioavailability.
A. Effects of Na.sup.+ at pH 1
[0085] The effects of Na.sup.+ concentration on the solubility of
the three elements in the four formulations (A1, A4, A5, and A6),
Caltrate.TM. and CaACE were investigated at gastric pH (pH=1) and
intestinal pH (pH=7), respectively. Tables 22 and 23 show the
results tested at pH 1. No significant effects of Na.sup.+
concentration on calcium and magnesium solubility of all
formulations were observed. Solubility of zinc in Caltrate.TM. and
calcium acetate, which contained trace amounts of Zn, increased
significantly with an increase in sodium concentrations; however,
no significant differences were obtained for all the acetate
formulations (Table 24).
TABLE-US-00022 TABLE 22 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Calcium Of Each Formula At pH 1 Na+ Solubility of
calcium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 4.597
.+-. 0.276 98.950 .+-. 19.224 101.353 .+-. 12.947 37.637 .+-. 2.509
48.670 .+-. 2.102 23.337 .+-. 3.162 5 5.447 .+-. 2.061 84.800 .+-.
13.912 72.233 .+-. 1.501 36.467 .+-. 5.173 46.100 .+-. 0.721 22.000
.+-. 1.323 10 4.340 .+-. 0.035 66.967 .+-. 17.377 80.000 .+-. 1.852
40.033 .+-. 4.623 49.833 .+-. 2.503 27.900 .+-. 3.736 50 4.640 .+-.
0.707 90.167 .+-. 9.343 83.467 .+-. 3.313 36.633 .+-. 1.877 49.033
.+-. 4.452 25.467 .+-. 0.231 80 5.530 .+-. 0.946 87.167 .+-. 3.630
83.067 .+-. 6.813 37.033 .+-. 1.069 55.733 .+-. 5.372 30.600 .+-.
1.709 100 5.360 .+-. 0.742 79.233 .+-. 15.964 84.900 .+-. 11.609
39.100 .+-. 5.696 48.733 .+-. 3.968 25.067 .+-. 0.153 Data are
expressed as mean .+-. S.D. No statistical differences in all
Na.sup.+ concentrations tested for all formulations tested.
TABLE-US-00023 TABLE 23 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Magnesium Of Each Formula At pH 1 Na+ Solubility of
magnesium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.197
.+-. 0.015 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-. 4.836
23.927 .+-. 1.747 41.797 .+-. 5.622 5 0.223 .+-. 0.006 0.700 .+-.
0.183 0.283 .+-. 0.040 36.400 .+-. 4.854 24.467 .+-. 1.361 39.933
.+-. 1.343 10 1.037 .+-. 1.109 0.483 .+-. 0.115 0.317 .+-. 0.050
38.967 .+-. 5.745 23.900 .+-. 1.800 49.100 .+-. 3.305 50 0.807 .+-.
0.889 0.620 .+-. 0.115 0.237 .+-. 0.031 35.733 .+-. 1.909 22.667
.+-. 2.055 45.500 .+-. 2.211 80 1.087 .+-. 1.264 0.580 .+-. 0.061
0.960 .+-. 1.031 35.033 .+-. 3.625 27.767 .+-. 3.700 50.900 .+-.
7.375 100 0.577 .+-. 0.525 0.497 .+-. 0.025 0.223 .+-. 0.032 36.000
.+-. 5.629 21.267 .+-. 2.120 46.233 .+-. 1.401 Data are expressed
as mean .+-. S.D. No statistical differences in all Na.sup.+
concentrations tested for all formulations tested.
TABLE-US-00024 TABLE 24 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Zinc Of Each Formula At pH 1 Na+ Solubility of zinc
(g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.007 .+-. 0.006
0.030 .+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637 .+-. 0.143
1.440 .+-. 0.140 5 0.087 .+-. 0.006 0.123 .+-. 0.006 0.660 .+-.
0.128 1.393 .+-. 0.316 2.180 .+-. 0.413 1.183 .+-. 0.121 10 0.173
.+-. 0.015 0.317 .+-. 0.106 0.883 .+-. 0.080 1.767 .+-. 0.280 2.080
.+-. 0.160 1.760 .+-. 0.617 50 0.240 .+-. 0.053 0.400 .+-. 0.139
1.023 .+-. 0.075 1.727 .+-. 0.060 2.250 .+-. 0.114 1.410 .+-. 0.125
80 0.210 .+-. 0.026 0.397 .+-. 0.163 0.907 .+-. 0.211 1.730 .+-.
0.479 2.613 .+-. 0.270 1.747 .+-. 0.015 100 0.223 .+-. 0.031 0.363
.+-. 0.095 0.947 .+-. 0.188 1.490 .+-. 0.105 2.207 .+-. 0.506 1.493
.+-. 0.630 Data are expressed as mean .+-. S.D
B. Effects of Na.sup.+ at pH 7
[0086] Tables 25-27 show the effects of sodium ion at pH 7.
Na.sup.+ has no significant effects on calcium, magnesium and zinc
solubility in general. It is interesting to note that all three
elements in Caltrate.TM. could be not detected in the presence of
Na.sup.+ at pH 7.
TABLE-US-00025 TABLE 25 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Calcium Of Each Formula At pH 7 Na+ Solubility of
calcium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.133
.+-. 0.051 98.950 .+-. 19.224 101.353 .+-. 12.947 37.637 .+-. 2.509
48.670 .+-. 2.102 23.337 .+-. 3.162 10 -- 83.300 .+-. 26.469 67.433
.+-. 4.460 37.433 .+-. 4.822 43.800 .+-. 4.703 39.367 .+-. 16.110
50 -- 69.000 .+-. 1.015 99.333 .+-. 21.548 35.533 .+-. 0.814 48.367
.+-. 4.359 23.833 .+-. 2.219 100 -- 71.467 .+-. 10.891 71.433 .+-.
1.193 36.867 .+-. 3.139 46.267 .+-. 1.380 24.567 .+-. 4.104 140 --
83.067 .+-. 6.596 68.900 .+-. 7.400 32.300 .+-. 1.153 47.200 .+-.
6.023 25.633 .+-. 3.754 170 -- 72.333 .+-. 15.467 71.433 .+-. 0.551
37.567 .+-. 10.473 43.133 .+-. 4.876 25.867 .+-. 3.175 Data are
expressed as mean .+-. S.D. No statistical differences in all
Na.sup.+ concentrations tested for all formulations tested.
TABLE-US-00026 TABLE 26 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Magnesium Of Each Formula At pH 7 Na+ Solubility of
magnesium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.093
.+-. 0.006 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-. 4.836
23.927 .+-. 1.747 41.797 .+-. 5.622 10 -- 0.740 .+-. 0.165 0.110
.+-. 0.010 35.300 .+-. 3.579 19.500 .+-. 1.769 75.167 .+-. 34.360
50 -- 0.427 .+-. 0.081 0.193 .+-. 0.015 35.933 .+-. 5.139 23.000
.+-. 4.327 52.167 .+-. 4.852 100 -- 0.510 .+-. 0.066 0.157 .+-.
0.006 33.267 .+-. 3.889 20.667 .+-. 0.493 45.867 .+-. 3.329 140 --
0.497 .+-. 0.099 0.157 .+-. 0.021 28.867 .+-. 2.255 20.567 .+-.
2.610 51.000 .+-. 6.963 170 -- 0.530 .+-. 0.036 0.167 .+-. 0.021
45.633 .+-. 11.097 21.600 .+-. 2.476 53.500 .+-. 3.650 Data are
expressed as mean .+-. S.D.
TABLE-US-00027 TABLE 27 Effect Of Concentration Of Na.sup.+ On The
Solubility Of Zinc Of Each Formula At pH 7 Na+ solubility of zinc
(g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.007 .+-. 0.012
0.030 .+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637 .+-. 0.143
1.440 .+-. 0.140 10 -- 0.213 .+-. 0.102 0.600 .+-. 0.040 1.453 .+-.
0.185 1.543 .+-. 0.215 2.337 .+-. 1.351 50 -- 0.280 .+-. 0.118
0.963 .+-. 0.280 1.700 .+-. 0.779 2.317 .+-. 0.798 1.687 .+-. 0.466
100 -- 0.293 .+-. 0.129 0.707 .+-. 0.107 1.243 .+-. 0.211 1.790
.+-. 0.087 1.667 .+-. 0.275 140 -- 0.320 .+-. 0.165 0.690 .+-.
0.137 1.113 .+-. 0.144 1.770 .+-. 0.056 1.643 .+-. 0.402 170 --
0.223 .+-. 0.102 0.730 .+-. 0.079 2.230 .+-. 0.397 1.933 .+-. 0.838
1.577 .+-. 0.529 Data are expressed as mean .+-. S.D.
C. Effects of K.sup.+ at pH 1
[0087] There is a tendency for the solubility of calcium to
increase with an increase in potassium ion concentration (Table
28). However, most of the differences are not statistically
different (p<0.05). In A5, the calcium solubility increased by
more than 50%; this difference is significant (p<0.05).
[0088] Magnesium solubility profiles for the acetate formulas show
a similar trend (Table 29) to that of calcium. There is a
three-fold increase in the magnesium solubility in Caltrate.TM.,
(p<0.05). However, the magnitude of increase in inconsequential
when compared to that of A4, A5 and A6.
[0089] An increase in potassium is associated with an increase in
zinc solubility for Caltrate.TM. and CaACE (Table 30). Potassium
has insignificant effect on the solubility of zinc in the four
formulas (p>0.05). Again, the magnitude of increase in zinc
solubility is inconsequential when compared to A4, A5 and A6.
TABLE-US-00028 TABLE 28 Effect Of Concentration Of K.sup.+ On The
Solubility Of Calcium Of Each Formula At pH 1 K.sup.+ Solubility of
calcium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 4.597
.+-. 0.276 98.950 .+-. 19.224 101.353 .+-. 12.947 37.637 .+-. 2.509
48.670 .+-. 2.102 23.337 .+-. 3.162 2 4.300 .+-. 0.403 78.933 .+-.
1.320 71.833 .+-. 9.338 34.033 .+-. 1.739 35.833 .+-. 5.314 24.067
.+-. 1.474 5 3.607 .+-. 0.540 71.033 .+-. 13.079 73.733 .+-. 3.412
36.967 .+-. 1.159 47.500 .+-. 5.272 23.500 .+-. 1.778 10 6.497 .+-.
3.381 158.333 .+-. 40.624 83.733 .+-. 14.093 40.467 .+-. 7.823
66.567 .+-. 21.033 30.867 .+-. 10.262 15 6.877 .+-. 0.956 161.667
.+-. 46.918 92.167 .+-. 14.793 41.867 .+-. 7.019 63.333 .+-. 7.651
26.667 .+-. 0.473 20 3.567 .+-. 0.501 100.800 .+-. 3.811 103.333
.+-. 15.822 42.633 .+-. 4.674 103.567 .+-. 64.463 29.300 .+-. 3.751
Data are expressed as mean .+-. S.D.
TABLE-US-00029 TABLE 29 Effect Of Concentration Of K.sup.+ On The
Solubility Of Magnesium Of Each Formula At pH 1 K.sup.+ Solubility
of magnesium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0
0.197 .+-. 0.015 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-.
4.836 23.927 .+-. 1.747 41.797 .+-. 5.622 2 0.223 .+-. 0.087 0.693
.+-. 0.283 0.203 .+-. 0.029 34.933 .+-. 1.716 21.633 .+-. 4.300
49.700 .+-. 1.249 5 0.490 .+-. 0.419 0.453 .+-. 0.112 0.170 .+-.
0.030 32.667 .+-. 2.542 23.433 .+-. 3.408 43.000 .+-. 2.406 10
0.703 .+-. 0.846 0.820 .+-. 0.193 0.270 .+-. 0.130 38.733 .+-.
5.552 30.067 .+-. 8.429 55.400 .+-. 18.187 15 0.730 .+-. 0.912
0.687 .+-. 0.215 0.327 .+-. 0.185 41.467 .+-. 8.617 31.067 .+-.
4.050 54.800 .+-. 3.897 20 0.660 .+-. 0.764 0.650 .+-. 0.020 0.883
.+-. 1.140 52.067 .+-. 2.859 55.733 .+-. 34.208 54.233 .+-. 14.632
Data are expressed as mean .+-. S.D.
TABLE-US-00030 TABLE 30 Effect Of Concentration Of K.sup.+ On The
Solubility Of Zinc Of Each Formula At pH 1 K+ Solubility of zinc
(g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.007 .+-. 0.006
0.030 .+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637 .+-. 0.143
1.440 .+-. 0.140 2 0.053 .+-. 0.015 0.077 .+-. 0.006 0.607 .+-.
0.108 1.377 .+-. 0.221 1.937 .+-. 0.591 1.360 .+-. 0.122 5 0.173
.+-. 0.035 0.240 .+-. 0.075 0.790 .+-. 0.147 1.297 .+-. 0.169 2.593
.+-. 0.821 1.143 .+-. 0.278 10 0.203 .+-. 0.058 0.357 .+-. 0.111
1.127 .+-. 0.142 1.630 .+-. 0.185 2.373 .+-. 0.658 1.627 .+-. 0.225
15 0.193 .+-. 0.023 1.307 .+-. 1.199 1.060 .+-. 0.600 1.953 .+-.
0.590 2.963 .+-. 0.309 1.630 .+-. 0.161 20 0.167 .+-. 0.015 0.293
.+-. 0.093 1.100 .+-. 0.140 2.500 .+-. 0.236 5.450 .+-. 3.159 2.540
.+-. 1.424 Data are expressed as mean .+-. S.D.
C. K.sup.+ Effects at pH 7
[0090] There was a tendency for the solubility of calcium to
increase with an increase in potassium concentration, however, the
difference is not significant, p>0.05 (Table 31). No calcium
could be detected in preparations using Caltrate.TM..
[0091] Similar observations to that of calcium were obtained for
the solubility of magnesium and zinc (p>0.05) in all formulas
containing acetate salts (Tables 32-33). No measurable magnesium
and zinc was reported for preparations using Caltrate.TM..
TABLE-US-00031 TABLE 31 Effect Of Concentration Of K.sup.+ On The
Solubility Of Calcium Of Each Formula At pH 7 K+ The solubility of
calcium (g/L) Conc.(mM) Caltrate .TM. Ca ACE A1 A4 A5 A6 0 0.133
.+-. 0.051 98.950 .+-. 19.224 101.353 .+-. 12.947 37.637 .+-. 2.509
48.670 .+-. 2.102 23.337 .+-. 3.162 10 -- 144.000 .+-. 14.731
66.800 .+-. 1.539 32.100 .+-. 0.361 64.033 .+-. 8.892 17.100 .+-.
0.173 50 -- 174.467 .+-. 79.146 68.533 .+-. 3.259 33.933 .+-. 2.515
64.867 .+-. 17.244 19.033 .+-. 3.630 100 -- 156.333 .+-. 64.361
68.600 .+-. 5.356 30.500 .+-. 3.672 82.000 .+-. 35.508 20.667 .+-.
2.363 140 -- 130.033 .+-. 32.461 60.400 .+-. 25.999 56.767 .+-.
32.771 68.400 .+-. 7.100 42.000 .+-. 18.340 170 -- 134.567 .+-.
55.048 126.133 .+-. 72.997 68.433 .+-. 29.905 64.800 .+-. 26.352
30.900 .+-. 14.912 Data are expressed as mean .+-. S.D. No
statistical differences in all K.sup.+ concentrations tested for
all formulations tested.
TABLE-US-00032 TABLE 32 Effect Of Concentration Of K.sup.+ On The
Solubility Of Magnesium Of Each Formula At pH 7 K+ The solubility
of magnesium (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0
0.093 .+-. 0.006 0.527 .+-. 0.121 0.173 .+-. 0.015 34.697 .+-.
4.836 23.927 .+-. 1.747 41.797 .+-. 5.622 10 -- 0.767 .+-. 0.189
0.140 .+-. 0.010 32.033 .+-. 2.829 30.967 .+-. 2.136 46.800 .+-.
3.158 50 -- 1.027 .+-. 0.587 0.347 .+-. 0.316 33.533 .+-. 2.084
31.867 .+-. 8.151 48.200 .+-. 1.253 100 -- 0.807 .+-. 0.278 0.183
.+-. 0.047 34.067 .+-. 3.465 39.233 .+-. 16.350 54.000 .+-. 2.955
140 -- 0.817 .+-. 0.303 0.160 .+-. 0.035 57.833 .+-. 34.279 32.833
.+-. 5.541 90.467 .+-. 42.518 170 -- 0.760 .+-. 0.310 0.230 .+-.
0.062 64.200 .+-. 26.513 31.333 .+-. 12.507 61.900 .+-. 30.685 Data
are expressed as mean .+-. S.D. No statistical differences in all
K.sup.+ concentrations tested for all formulations tested.
TABLE-US-00033 TABLE 33 Effect Of Concentration Of K.sup.+ On The
Solubility Of Zinc Of Each Formula At pH 7 K.sup.+ The solubility
of zinc (g/L) Conc.(mM) Caltrate .TM. CaACE A1 A4 A5 A6 0 0.007
.+-. 0.012 0.030 .+-. 0.010 1.283 .+-. 0.220 1.980 .+-. 0.256 2.637
.+-. 0.143 1.440 .+-. 0.140 10 -- 0.293 .+-. 0.110 0.727 .+-. 0.064
1.173 .+-. 0.163 3.243 .+-. 0.725 1.090 .+-. 0.070 50 -- 0.627 .+-.
0.437 1.140 .+-. 0.036 1.447 .+-. 0.135 3.127 .+-. 0.720 1.247 .+-.
0.045 100 -- 0.257 .+-. 0.110 1.197 .+-. 0.068 1.587 .+-. 0.106
3.417 .+-. 1.252 1.460 .+-. 0.122 140 -- 0.387 .+-. 0.186 0.827
.+-. 0.506 2.583 .+-. 0.755 2.747 .+-. 1.432 2.607 .+-. 1.301 170
-- 0.287 .+-. 0.142 1.223 .+-. 0.541 2.437 .+-. 0.618 2.873 .+-.
0.771 1.720 .+-. 0.624 Data are expressed as mean .+-. S.D.
Example 6
In Vivo Evaluation of Calcium, Magnesium and Zinc Balance
[0092] The objectives of the balance studies were to evaluate the
effects of dietary conditions and formulations on calcium,
magnesium and zinc balance.
A. Dietary Conditions
[0093] Two diets, one with normal calcium and the other is calcium
free, were used for the studies. The nutrient composition of the
diets is listed on Table 34:
TABLE-US-00034 TABLE 34 Composition Of Normal And Calcium Free Diet
Normal Calcium Free Protein, % 24.0 19.0 Fat, % 4.5 (ether extract)
10.0 6.0 (acid hydrolysis) Cholesterol, ppm 101 48 Fiber, % 5.3 5.4
Carbohydrates, % 21.5 (starch) 60.6 0.2 (Glucose) 0.2 (Fructose)
3.4 (Sucrose) 0.6 (Lactose) Potassium, % 1.20 0.62 Sodium, % 0.40
0.27 Chlorine, % 0.70 0.27 Calcium, % 0.95 0.0 Magnesium, % 0.25
0.07 Zinc, % 0.011 0.0031 Iron, ppm 290 60 Manganese, ppm 110 65
Copper, ppm 17 23.9 Vitamin K, ppm 3.2 10.4 Riboflavin, ppm 12 20.0
Pyridoxine, ppm 8.0 16.5
B. Materials and Methods
[0094] Male Sprague-Dawley rats (about 6-7 weeks), with an initial
weight between 220 g to 250 g, were randomly divided into different
treatment groups. All the rats were housed in individual metabolic
cages in a temperature-controlled room. Each rat received free
access to the normal diet (Table 34) before the experiment. Both
normal and calcium free diets (Table 34) were used in this set of
studies. De-ionized water was provided ad libitum. All the rats
were weighed before treatment.
C. Treatments
[0095] Two sets of studies were performed: a normal diet and
calcium free diet. In each study, there were seven treatment
groups. Thirty five animals were randomly assigned to one of the
treatment groups in which one of the following were administered:
Caltrate.TM., Calcium Acetate (Ca ACE), A1, A4, A5, A4 plus vitamin
D.sub.3 and A5 plus vitamin D.sub.3 (n=5 per group). Rats
participating in the normal diet study received normal diet ad
libitum throughout. Rats participating in the group of calcium free
diet received the calcium free food ad libitum starting five days
before and throughout treatment. In both study groups, animals
received one dose a day for five days. Amounts of calcium,
magnesium and zinc in individual formulation and in each diet were
determined using ICP-OES. Values of dosage and dietary intake were
measured for the calculation of elemental balance. For rats that
were fed the normal diet, average daily elemental intake of
calcium, magnesium and zinc was 625, 155 and 10 mg/kg/day,
respectively. Daily elemental dosages, similar to that of human's,
are 53.14 mg/kg for calcium, 0.38 to 55 mg/kg/day for magnesium and
0.017 to 2.5 mg/kg/day for zinc. Vitamin D.sub.3, 1.06 .mu.g/kg/day
(42.512 IU/kg/day; 1 IU=0.025 .mu.g), was added to each dosage
preparation prior to administration. The vehicle for preparing each
dose was de-ionized water. The concentration of calcium in all
dosage preparations was 15.94 mg/mL. One mL of each preparation was
administered by gavage. Body weight, elemental dosage and diet
consumption were recorded daily.
D. Sample Collection, Handling and Analysis
[0096] Animals were housed individually in a metabolic cage five
days before the study. Food consumption was evaluated daily. Urine
and feces were collected daily for four days and the content of
calcium, magnesium and zinc was determined. On Day 5, each animal
received its treatment. These treatments were administered once a
day for four days. After the last treatment, each animal was
anesthetized shortly before peak elemental blood concentration was
achieved. Blood was collected using a heparinized syringe via
cardiac puncture. Immediately after blood collection, the animal
was then sacrificed with an overdose of isoflourane. Each blood
sample was centrifuged at 1900 rpm at room temperature; plasma was
harvested and stored at -20.degree. C. until analysis. Urine was
measured daily; it was diluted with de-ionized water, filtered and
an aliquot was stored at -20.degree. C. until analysis. Daily fecal
output was collected and lyophilized. Each sample was weighed and
digested using a mixture of three volume of nitric acid and one
volume of perchloric acid. For every gram of dried feces, 10 mL of
acid mixture was added. Each sample was digested for three days.
The volume of the digested sample was measured and an aliquot of
the digest was stored at -20.degree. C. until analysis. The content
of calcium, magnesium and zinc in plasma, feces and urine were
determined using ICP-OES.
[0097] Daily calcium balance was calculated using equation 1:
Ca Balance=total Ca intake(dose and dietary intake)-Ca excreted in
urine-Ca excreted in feces (1)
[0098] While, percentage of Ca balance was determined using
equation 2:
% Ca balance=Ca balance/(total Ca intake).times.100% (2)
[0099] Cumulated calcium balance and % cumulated net calcium
balance were calculated using equations (1) and (2), except, the
sum of daily intake and excretion was used for calculation. The
balance for magnesium and zinc was also calculated using the
concept of equations (1) and (2). Cumulated elemental balance and %
cumulated net elemental balance were calculated in a similar
fashion as described above.
[0100] In general, urinary excretion accounted for less than 5% of
fecal excretion. Therefore, fecal excretion practically determines
the quantity of elemental balance.
E. Statistical Analysis
[0101] All results were analyzed using two-way ANOVA. P<0.05 was
considered to be significantly different. The data are presented as
mean.+-.S.D. and mean.+-.S.E.M. in tables and figures,
respectively.
F. Results: Calcium Free Diet
[0102] Table 35 shows the body weight of rats during the study.
Stools from study animals were soft and this observation could be
related to low elemental intake. Insufficient elements from the
diet and dosage may have also caused the lack of weight gain for
this set of animals. There is a statistical difference (p<0.05)
among the starting body weights of the study animals (Table 35).
There is also a slight in decline in body weight during the
treatment period; is not the difference significantly
different.
TABLE-US-00035 TABLE 35 Body Weight Of Rats In Each Treatment Group
With Calcium Free Diet (n = 5) Treatment Body weight of rats (g)
group Day 1 Day 2 Day 3 Day 4 Day 5 Caltrate .TM. 184.6 .+-. 7.7
178.6 .+-. 9.9 177.2 .+-. 8.8 179.2 .+-. 13.7 175.4 .+-. 14.2 Ca
ACE 202.4 .+-. 9.3 194.8 .+-. 9.3.sup.$ 196.8 .+-. 10.9 193.4 .+-.
11.9.sup.$ 188.0 .+-. 12.2.sup.$ A1 190.4 .+-. 11.9 185.6 .+-.
14.0* 187.6 .+-. 10.9 186.6 .+-. 11.3.sup.$ 182.8 .+-. 15.4* A4
188.4 .+-. 12.9.sup.$*.sup.+ 184.2 .+-. 13.2.sup.$*.sup.+ 184.0
.+-. 12.7 182.4 .+-. 13.5.sup.$*.sup.+ 183.2 .+-. 14.0* A5 187.8
.+-. 8.8.sup. 184.0 .+-. 6.0.sup.$ 184.2 .+-. 5.6.sup.$*.sup.#
185.4 .+-. 6.0.sup.$*.sup.+ 182.4 .+-. 9.2*.sup.# A4 + Vit D 207.6
.+-. 11.9.sup.$*.sup.+& 200.0 .+-. 5.2.sup.$+ 198.6 .+-.
4.5.sup.$*.sup.+& 204.2 .+-. 4.4.sup.$&@ 199.8 .+-.
6.4.sup.$+& A5 + Vit D 204.8 .+-. 14.4.sup.$*.sup.+&% 195.6
.+-. 8.3.sup.$+% 196.4 .+-. 7.7.sup.+&% 201.0 .+-.
5.0.sup.$&% 196.8 .+-. 8.2.sup.$+ .sup.$P < 0.05, compared
with Caltrate .TM.; *P < 0.05, compared with Ca ACE; .sup.+P
< 0.01, compared with A1; .sup.&P < 0.05, compared with
A4; .sup.%P < 0.001, compared with A5; .sup.#P < 0.001,
compared with A4 + Vit D; .sup.@P < 0.05, compared with A5 + Vit
D.
[0103] The addition of magnesium and zinc to a formula promotes the
retention of calcium. A1, a composition with miniscule amounts of
magnesium and zinc, has a lower calcium retention (17%, Table 36);
whereas the retention of calcium is significantly higher when the
ratio of Ca/Mg was increased to 2/1 (A5), the calcium retention is
49% (Table 36). A higher proportion of magnesium, such as that
present in A4, does not produce more changes in calcium retention
(49%, Table 36). With respect to the minimum amount of magnesium
required to provide the highest calcium retention, it appears a 2/1
Ca/Mg ratio is optimal.
[0104] The addition of vitamin D.sub.3 increases calcium retention
significantly (FIG. 2 and Table 36). Calcium retention increased to
62% when vitamin D.sub.3 was added to A5 (Table 36). This value is
more than five times higher than that of the Caltrate.TM. and CaACE
groups.
TABLE-US-00036 TABLE 36 Cumulative Net Percentage Of Calcium In
Rats Treated With Elemental Supplements While Receiving Calcium
Free Diet (n = 5 per group) Treatment Cumulative net percentage of
calcium (%) group Day 1 Day 2 Day 3 Day 4 Caltrate .TM. 23.8 .+-.
15.9* 22.3 .+-. 16.8 -2.27 .+-. 40.0 0.734 .+-. 35.7 Ca ACE -30.6
.+-. 51.3 -9.88 .+-. 26.5 4.88 .+-. 24.0 11.1 .+-. 20.0 A1 37.5
.+-. 18.7* 20.9 .+-. 15.5 20.8 .+-. 15.0 17.2 .+-. 12.1 A4 40.9
.+-. 19.1* 48.6 .+-. 13.7* 49.1 .+-. 10.2.sup.$* 49.1 .+-.
7.7.sup.$* A5 36.4 .+-. 24.1* 46.8 .+-. 19.5* 48.7 .+-. 18.4.sup.$*
48.6 .+-. 19.1.sup.$* A4 + Vit D 46.6 .+-. 22.3* 50.3 .+-. 10.9*
47.9 .+-. 14.8.sup.$* 50.8 .+-. 11.2.sup.$* A5 + Vit D 43.7 .+-.
19.2* 52.7 .+-. 11.8* 59.2 .+-. 7.6.sup.$*.sup.+ 62.0 .+-.
5.2.sup.$*.sup.+ .sup.$P < 0.05, compared with Caltrate .TM.; *P
< 0.05, compared with Ca ACE; .sup.+P < 0.05, compared with
A1; .sup.#P < 0.05, compared with A4 + Vit D.
[0105] Magnesium appears to be required in order to maintain
magnesium balance (i.e. to avoid magnesium depletion) (Table 37).
Formulas (Caltrate.TM., CaACE and A1) that have miniscule amounts
of magnesium caused a net loss of magnesium (FIG. 3 and Table
37).
[0106] The addition of vitamin D.sub.3 has no significant effect on
the retention of magnesium. The cumulative net percentage of
magnesium did not change significantly after vitamin D.sub.3 was
added to A4 and A5 (FIG. 3 and Table 37).
TABLE-US-00037 TABLE 37 Cumulative Net Percentage Of Magnesium In
Rats Treated With Elemental Supplements While Receiving Calcium
Free Diet (n = 5 per group) Cumulative net percentage of magnesium
(%) Treatment group Day 1 Day 2 Day 3 Day 4 Caltrate .TM. -191.9
.+-. 139.1 -125.6 .+-. 51.0 -111.8 .+-. 39.1 -116.5 .+-. 37.7 Ca
ACE -197.2 .+-. 105.2 -150.4 .+-. 88.9 -115.3 .+-. 62.7 -93.6 .+-.
37.2 A1 -47.3 .+-. 22.4.sup.$* -67.9 .+-. 33.3* -55.2 .+-. 13.4
-64.4 .+-. 24.6 A4 66.5 .+-. 8.7.sup.$*.sup.+ 68.1 .+-.
6.4.sup.$*.sup.+ 65.8 .+-. 5.9.sup.$*.sup.+ 60.9 .+-.
4.7.sup.$*.sup.+ A5 23.7 .+-. 46.3.sup.$* 37.6 .+-.
37.1.sup.$*.sup.+ 41.1 .+-. 34.2.sup.$*.sup.+ 39.6 .+-.
33.3.sup.$*.sup.+ A4 + Vit D 46.3 .+-. 27.5.sup.$*.sup.+ 49.3 .+-.
18.8.sup.$*.sup.+ 49.3 .+-. 15.3.sup.$*.sup.++ 48.9 .+-.
15.5.sup.$*.sup.+ A5 + Vit D 16.0 .+-. 20.0.sup.$* 23.9 .+-.
21.5.sup.$*.sup.+ 28.9 .+-. 17.9.sup.$*.sup.+ 27.2 .+-.
23.0.sup.$*.sup.+ .sup.$P < 0.05, compared with Caltrate .TM.;
*P < 0.05, compared with Ca ACE; .sup.+P < 0.05, compared
with A1.
[0107] The retention of zinc is highly variable; it is particularly
true with formulas such as Caltrate.TM., calcium acetate and A1
that contain minute amounts of zinc (Table 38). The results also
show that zinc balance became negative when the amount of zinc is
low.
[0108] The addition of zinc to formulas such as A4 and A5 did not
significantly improve zinc balance (Table 38). The addition of
magnesium to the formulas may have caused zinc balance to stay
negative (FIG. 4).
[0109] However, the addition of vitamin D.sub.3 to A4 and A5 made
zinc balance positive (FIG. 4 and Table 38). The importance of
vitamin D.sub.3 on zinc is clearly demonstrated in this set of
studies.
[0110] FIG. 5 shows plasma elemental profiles after each treatment.
There were no significant differences observed after elemental
treatments.
TABLE-US-00038 TABLE 38 Cumulative Net Percentage Of Zinc In Rats
Treated With Elemental Supplements While Receiving Calcium Free
Diet (n = 5 per group) Treatment Cumulative net percentage of zinc
group Day 1 Day 2 Day 3 Day 4 Caltrate .TM. -50.6 .+-. 50.0 -38.7
.+-. 23.8 -36.9 .+-. 26.4 -39.5 .+-. 23.7 Ca ACE -107.1 .+-. 85.5
-77.7 .+-. 59.0 -65.7 .+-. 66.7 -50.5 .+-. 46.4 A1 10.1 .+-.
8.7.sup.& -0.348 .+-. 22.2.sup.& 4.22 .+-. 7.3.sup.&
-2.79 .+-. 6.4 A4 -61.0 .+-. 38.8.sup.& -55.3 .+-.
29.3.sup.& -58.3 .+-. 24.5.sup.$ -33.8 .+-. 23.9.sup.$ A5 -8.05
.+-. 45.3.sup.$& 9.737 .+-. 39.5.sup.$*.sup.& 9.96 .+-.
40.3.sup.$*.sup.& 8.76 .+-. 40.1.sup.$* A4 + Vit D 27.2 .+-.
40.7.sup.$*.sup.& 43.7 .+-. 18.8.sup.$*.sup.& 51.2 .+-.
15.1.sup.$*.sup.& 54.2 .+-. 11.2*.sup.& A5 + Vit D 22.8
.+-. 17.9*.sup.& 35.8 .+-. 17.8*.sup.& 42.9 .+-.
12.9*.sup.& 44.6 .+-. 10.1*.sup.& .sup.$P < 0.05,
compared with Caltrate .TM.; *P < 0.05, compared with Ca ACE;
.sup.&P < 0.05, compared with A4
G. Results: Normal Diet
[0111] Rats that received normal diet gained weight (Table 39).
Elemental treatments have no significant effect on weight gain
(p>0.05).
TABLE-US-00039 TABLE 39 Body Weight Of Rats Receiving Normal
Calcium Diet (n = 5) Treatment Body weight of rats (g) group Day 1
Day 2 Day 3 Day 4 Day 5 Caltrate .TM. 228.8 .+-. 4.6 232.8 .+-. 2.6
233.8 .+-. 3.5 243.6 .+-. 8.9 243.8 .+-. 5.1 Ca ACE 242.0 .+-. 7.4
237.0 .+-. 12.5 239.2 .+-. 13.9 238.6 .+-. 13.9 244.0 .+-. 12.8 A1
230.0 .+-. 4.5 233.8 .+-. 8.0 238.2 .+-. 6.1 244.6 .+-. 7.2 244.6
.+-. 3.5 A4 234.8 .+-. 7.7 238.6 .+-. 5.1 238.2 .+-. 5.9 239.0 .+-.
5.1 245.8 .+-. 4.9 A5 239.6 .+-. 10.3 243.0 .+-. 13.9 245.4 .+-.
13.6 245.4 .+-. 13.4 248.6 .+-. 14.4 Data are expressed as mean
.+-. S.D.
[0112] The pattern of calcium retention appears to be similar to
that obtained from rats that received calcium free diet (compare
Tables 36 and 40); suggesting calcium balance is dependent upon
elemental treatments, despite the fact that the amount of calcium
administered was approximately 10% of the animal's daily dietary
intake (.about.130 to 140 mg of calcium per day). This observation
strongly suggests that dietary calcium, present in the least
absorbable carbonate form, was enhanced by elemental treatments.
The treatment with Caltrate.TM. has minimal effect. It is not
surprising because Caltrate.TM. contains only calcium carbonate.
The treatment with A5 has the most pronounced effect (FIG. 6 and
Table 40).
TABLE-US-00040 TABLE 40 Cumulative Net Percentage Of Calcium In
Rats Treated With Elemental Supplements While Receiving Normal Diet
(n = 5 per group) Treatment Cumulative net percentage of calcium
(%) group Day 1 Day 2 Day 3 Day 4 Caltrate .TM. -6.9 .+-. 24.6 17.3
.+-. 7.5 21.3 .+-. 10.0 17.5 .+-. 10.2 Ca ACE 14.4 .+-. 24.0 26.9
.+-. 9.0 30.3 .+-. 4.9 31.9 .+-. 3.0 A1 .sup. 31.4 .+-. 33.5.sup.$
.sup. 49.2 .+-. 38.8.sup.$ 39.2 .+-. 27.3 31.3 .+-. 21.9 A4 19.3
.+-. 12.6 23.7 .+-. 9.4 26.2 .+-. 9.6 22.7 .+-. 7.3 A5 .sup. 52.1
.+-. 21.7.sup.$*.sup.& .sup. 49.0 .+-. 19.8.sup.$ 48.9 .+-.
20.4 45.3 .+-. 22.7 .sup.$P < 0.05, compared with Caltrate .TM.;
*P < 0.05, compared with Ca ACE; .sup.&P < 0.05, compared
with A4
[0113] Average dietary intake of magnesium by the study animals was
approximately 35 mg. Magnesium balance for all study groups was
positive (FIG. 7 and Table 41). This observation is consistent with
the observation obtained from animals receiving the calcium free
diet, in that magnesium intake is required to maintain a positive
balance (Tables 37 and 41). Interestingly, the day to day trend
showed that animals treated with acetate formulas (CaACE, A1, A4
and A5 vs. Caltrate.TM.) had consistently higher percentages of
magnesium balance.
TABLE-US-00041 TABLE 41 Cumulative Net Percentage Of Magnesium In
Rats Treated With Elemental Supplements While Receiving Normal Diet
(n = 5 per group) Treatment Net accumulative percentage of
magnesium (%) group Day 1 Day 2 Day 3 Day 4 Caltrate .TM. -2.82
.+-. 19.6.sup.% .sup. 23.3 .+-. 8.1.sup.% 27.3 .+-. 10.0 24.6 .+-.
6.9 Ca ACE 16.7 .+-. 17.2.sup.% 29.9 .+-. 3.8 34.3 .+-. 2.5 37.7
.+-. 2.7 A1 11.7 .+-. 11.7.sup.% 44.1 .+-. 30.7 38.9 .+-. 22.9 31.5
.+-. 17.0 A4 28.2 .+-. 9.1.sup.$ 34.0 .+-. 7.8 36.8 .+-. 7.2 35.0
.+-. 4.4 A5 48.9 .+-. 25.3 .sup. 48.9 .+-. 20.9 50.6 .+-. 20.1 48.6
.+-. 21.0 .sup.$P < 0.05, compared with Caltrate .TM.; .sup.%P:
<0.05, compared with A5
[0114] There were no statistical differences among elemental
treatments in terms of zinc balance (FIG. 8 and Table 42). The
quantity of zinc administered via elemental formulas was no more
than 30% of the daily dietary intake. It was noted that the
addition of a high quantity of magnesium tended to lower zinc
balance, a trend observed with A4 treatment (FIG. 8 and Table 42).
This observation is similar to that observed in the calcium free
diet study (Table 38).
[0115] Contrary to the calcium free diet study (Table 38), zinc
balance was positive in this study (Table 42). This was achieved
without vitamin D.sub.3 (FIGS. 4 and 8, Tables 38 and 42). This
apparent discrepancy may be due to the quantity of total zinc
intake and/or the rate at which zinc was consumed. Elemental
consumption, along with other nutrients, occurred throughout the
feeding period which may last up to 12 hours; whereas elemental
treatments were given as a bolus. Concentration and ratio of
nutrients presented to the intestinal wall may have a huge
difference between bolus administration and dietary consumption.
These differences could account for the difference in zinc
balance.
[0116] The results from the calcium free and normal diet studies
clearly suggest that adequate dietary intake of elements is key to
elemental balance. Elemental and vitamin D.sub.3 supplementation
are necessary if the diet in deficient in these nutrients.
[0117] FIG. 9 shows plasma concentration of calcium, magnesium and
zinc after individual elemental treatments. There were no
statistical differences in the concentration of these elements in
plasma after elemental treatments (P>0.05).
TABLE-US-00042 TABLE 42 Cumulative Net Percentage Of Zinc In Rats
Treated With Elemental Supplements While Receiving Normal Diet (n =
5 per group) Treatment Cumulative net percentage of zinc (%) group
Day 1 Day 2 Day 3 Day 4 Caltrate .TM. 0.67 .+-. 34.7.sup.% 29.5
.+-. 7.5 33.8 .+-. 10.2 32.0 .+-. 7.8 Ca ACE 27.5 .+-. 16.0.sup.%
40.9 .+-. 7.3 45.6 .+-. 5.9 48.4 .+-. 4.4 A1 26.6 .+-. 11.2.sup.%
50.8 .+-. 26.8 46.3 .+-. 20.4 38.7 .+-. 18.8 A4 17.7 .+-.
10.3.sup.% .sup. 24.9 .+-. 6.3.sup.% 27.6 .+-. 7.2 27.6 .+-. 5.0 A5
54.7 .+-. 21.9 .sup. 52.6 .+-. 21.7 53.8 .+-. 21.0 51.2 .+-. 23.0
.sup.%P < 0.05, compared with A5
H. Results: Calcium Free Diet with Daily Consumed Doses of
Calcium
[0118] The objective of this study was to evaluate elemental
balance when the daily intake of calcium, magnesium and zinc was
replaced with elemental treatments. Animals, received de-ionized
water ad libitum (DI Water group), were fed normal calcium diet.
Animals, substituting their daily calcium intake by A1 or A5, were
fed calcium free diet. It is apparent that the gavage procedure did
not have an effect on the body weight of the animals (Table 43).
Elemental treatments, however, induced a significant reduction in
body weight.
TABLE-US-00043 TABLE 43 Body Weight Of Rats Receiving Calcium Free
Diet And Daily Consumed Doses Of Calcium (n = 4) Treatment Body
weight of rats (g) group Day 1 Day 2 Day 3 Day 4 Day 5 DI Water
200.8 .+-. 2.50 207.0 .+-. 3.9 209.0 .+-. 8.7 209.5 .+-. 9.9 215.5
.+-. 11.7 A1 198.0 .+-. 9.1 183.5 .+-. 7.7 178.3 .+-. 8.1 180.8
.+-. 10.2 186.0 .+-. 8.0 A5 194.0 .+-. 8.2 182.3 .+-. 7.1 179.8
.+-. 7.2 179.0 .+-. 7.7 181.5 .+-. 6.8 Note: There is no
statistical significant difference between A1 and A5. There is
statistical difference between A1 and DI (p < 0.001), and
between A5 and DI (p < 0.001).
[0119] Contrary to the results obtained from the normal and calcium
free diet studies, magnesium has a minor effect in enhancing
calcium retention (FIG. 10 and Table 44). The administration of a
soluble form of calcium, calcium acetate, significantly enhanced
calcium balance (FIG. 10 and Table 44).
TABLE-US-00044 TABLE 44 Cumulative Net Percentage Of Calcium In
Rats Treated With A Daily Consumed Dose Of Calcium While Receiving
Calcium Free Diet (n = 4 per group) Treatment Net accumulative
percentage of Ca (%) group Day 1 Day 2 Day 3 Day 4 DI Water 2.87
.+-. 5.4 3.89 .+-. 7.6 .sup. 5.72 .+-. 4.3 5.41 .+-. 5.2 A1 46.3
.+-. 14.7* 37.7 .+-. 8.9* .sup. 37.4 .+-. 1.3* 42.7 .+-. 3.1* A5
54.9 .+-. 12.7* 56.7 .+-. 10.3*.sup.@ 50.4 .+-. 7.5* 47.4 .+-. 8.0*
*P < 0.05, when compared with DI; .sup.@P < 0.05 m when
compared to A1
[0120] Consistent with the calcium free diet study described above,
magnesium was required to maintain a positive magnesium balance
(FIG. 11 and Table 45).
TABLE-US-00045 TABLE 45 Cumulative Net Percentage Of Magnesium In
Rats Treated With A Daily Consumed Dose Of Calcium While Receiving
Calcium Free Diet (n = 4 per group) Treatment Net accumulative
percentage of Mg (%) group Day 1 Day 2 Day 3 Day 4 DI Water -32.2
.+-. 12.4 -17.0 .+-. 10.4 -7.9 .+-. 10.0 -2.59 .+-. 10.4 A1 -75.9
.+-. 50.0* -27.6 .+-. 27.7 -6.54 .+-. 19.4 3.6 .+-. 18.0 A5 18.3
.+-. 12.7*.sup.@ .sup. 14.4 .+-. 8.6.sup.@ 7.0 .+-. 5.2 4.4 .+-.
8.4 *P < 0.05, when compared with DI; .sup.@P < 0.05 m when
compared to A1
[0121] Despite a higher amount of zinc administered with A5, zinc
balance was significantly lower than that of the DI Water group,
providing further support that high calcium and magnesium
concentration in the intestine could have diminished zinc
absorption. (FIG. 12 and Table 46). The amounts of zinc
administered between the DI Water and A1 groups were similar.
However, similar to that of A5, zinc balance was significantly
lower than that of DI Water (FIG. 12 and Table 46); suggesting high
solution concentration of calcium in the intestine may interfere
with zinc absorption.
[0122] This set of results suggest that elemental dietary intake of
elements does not produce the same effects when compared to that of
an equivalent bolus dose.
[0123] Taking all the study results into consideration, A5 produces
the most consistent calcium balance under different
experimental/dietary conditions (compare results on Tables 36, 40
and 44). The addition of vitamin D.sub.3 enhances calcium retention
of A5 when the subject is deficient in dietary elements (Table
36).
[0124] FIG. 13 shows plasma concentrations of calcium, magnesium
and zinc after each elemental treatment. No statistical differences
were found in these profiles (P>0.05).
TABLE-US-00046 TABLE 46 Cumulative Net Percentage Of Zinc In Rats
Treated With A Daily Consumed Dose Of Calcium While Receiving
Calcium Free Diet (n = 4 per group) Treatment Net accumulative
percentage of Zn (%) group Day 1 Day 2 Day 3 Day 4 DI Water -26.5
.+-. 37.7 -10.8 .+-. 22.9.sup. -4.45 .+-. 17.3 -1.80 .+-. 12.2 A1
-42.9 .+-. 25.9 -67.3 .+-. 16.3* -69.5 .+-. 7.3* -58.5 .+-. 6.2* A5
23.7 .+-. 16.2*.sup.@ -9.09 .+-. 19.3.sup.@ -45.1 .+-. 11.8* -63.2
.+-. 16.2* *P < 0.05, when compared with DI; .sup.@P < 0.05,
when compared to A1
Example 7
[0125] The objectives of this study were to evaluate the effects of
salt, mineral composition and vitamins on the rate of bone loss in
an ovariectomized rat model.
[0126] One hundred 4.5-month-old female Sprague-Dawley rats were
used and housed at the Laboratory Animal Services Center at the
Chinese University of Hong Kong with 12-h light-night cycle. Free
cage movement was allowed with access to the normal calcium pellets
and tap water. Daily consumption of calcium was approximately 140
mg, similar to that recorded in animals who participated in the
balance studies. Ovariectomy (OVX), the removal of ovaries from the
female rats, was performed on all rats at 6-month of age with the
exception of the sham control.
[0127] Three weeks after OVX, all the rats recovered from the
trauma of the surgery. The rats were randomly divided into
different treatment groups or control groups and each group
contained six rats. Four calcium formulas (A1, A4, A5 and A6) and
Caltrate.TM. were investigated in the present study. The
Caltrate.TM. group served as an elemental treatment control. All
formulas were dissolved in distilled water, while Caltrate.TM. was
in suspension in distilled water. The solution or suspension was
given to the rats daily for 8 weeks by gavages. The dose of all
formulas was calculated based on a calcium dose of 53.14 mg/kg/day.
Dose of vitamin D.sub.3 and vitamin K.sub.2 was 12.75 IU/kg/day
(equivalent to 800 IU/70 kg man/day) and 1.71 .mu.g/kg/day
(equivalent to 120 .mu.g/70 kg man/day), respectively. All the
treated rats were weighed daily and the mass data were recorded.
The rats in two control groups (sham control and normal control)
were given the equivalent volume of distilled water in parallel.
For the groups with the treatment of bisphosphonate, alendronate
(14 .mu.g/kg/2-week) was injected subcutaneously on the back of the
rats once every two weeks.
[0128] At the end of 8 weeks, the rats were anesthetized using
isoflourane. Blood sample was then taken via heart puncture. The
rats were then euthanized under anesthesia by neck dislocation, and
right hip, right femur and right tibia of each rat were collected
for analysis. Plasma was collected from blood samples centrifuged
at 1500 g for 15 min. Plasma concentrations of calcium, magnesium,
and zinc were measured using ICP-OES.
[0129] Results show that plasma calcium levels were not
statistically different from that of the sham control (p>0.05)
and the values are all within normal levels (90-110 mg/L). All
plasma concentrations of Mg were within the normal range (18-36
mg/L). No significant difference in magnesium plasma concentrations
was observed except normal control (without surgery) has a mean
value higher than that of A4+Vit D+Vit K (p<0.05). Similarly,
plasma concentrations of Zn in all rats reached the rat normal
concentration at about 1.26 mg/L. Zn plasma concentrations of rats
in the normal control was significantly higher than that of sham
control rats and also the rats treated with A5+vitamin D and
A4+vitamin D+vitamin K (p<0.05).
[0130] Body weight changes for different treatment groups are shown
in FIG. 14. As expected, weight gains in the OVX rats were
significantly greater than the normal rats (p<0.05).
[0131] The effects of test substances on bone mineral density (BMD)
are shown on FIGS. 15 and 16. Trabecular BMD of Distal Femur BMD
values of groups A1, A5+Vit D, Bis+A1+Vit D, Bis+A4+Vit D,
Bis+A5+Vit D and Bis+Caltrate+Vit D are significantly higher than
that of the OVX control (FIG. 15), suggesting these treatments
significantly slow down the rate of loss of bone mass. The addition
of vitamin K did not have any significant effect on reducing the
rate of bone loss. Similar observations were obtained for the
average values of trabecular BMD of Proximal Tibia, except the
value of Caltrate.TM. was high enough to become statistically
different (p<0.05, FIG. 16). Again, vitamin K did not have any
significant contribution. The treatment with A5+Vit D provided
consistently higher BMD at distal femur and proximal tibia,
suggesting this formula may have an advantage over the other
elemental formulas. Although, the addition of bisphosphonate
provides consistently better results, the difference, when compared
to A5+Vit D and other elemental formula, such as A1, was not
significant (FIGS. 15 and 16).
[0132] The BMD results of A1 are similar to that of A5+vit D. This
is not surprising because A1 animals were fed normal calcium diet
which contains a significant amount of magnesium.
[0133] The OVX rat model used in this study did not permit
evaluation of maximum bending force and failure energy after each
treatment because the values obtained from the OVX control and that
of the Sham were insignificantly different from each other
(P>0.05).
Example 8
Fortification of Juices with A5
[0134] Fruit juices contain a number of acids such as malic acid,
citric acid, etc. which may alter the solubility and hence the
recovery of the three key elements in the formulae, hence changing
the absorbability of these elements when administered in juice
format.
[0135] The objectives of this study were to evaluate the effects of
temperature and storage on the recovery of calcium, magnesium and
zinc in A5 after mixing with filtered and unfiltered orange, grape
and carrot juice.
[0136] A 2.6 g or 500 mg amount of A5 was weighed accurately and
mixed with 330 ml of water or either filtered or unfiltered grape,
orange or carrot juice. The specimens were prepared at either 4 or
21.degree. C. The elemental content was measured using ICP-OES.
[0137] Small quantities of calcium, magnesium and zinc were found
in orange, grape and carrot juice (Tables 47, 50 and 53).
Temperature and filtration had no effects on the recovery of
calcium, magnesium and zinc of A5 when 2.6 g of A5 was used for the
study (Tables 48, 51 and 54).
TABLE-US-00047 TABLE 47 Content of the three key elements in fresh
orange juice Content (mg/L) Sample Ca Mg Zn Fresh orange 87.7 .+-.
0.87 115 .+-. 0.9 0.47 .+-. 0.03 juice Data are expressed as Mean
.+-. S.D. (n = 3)
TABLE-US-00048 TABLE 48 Elemental recovery of the 3 key elements of
A5 (2.6 g) in orange juice at 4.degree. C. and 21.degree. C.
Solubility (g/L) Ca Mg Zn Sample 4.degree. C. 21.degree. C.
4.degree. C. 21.degree. C. 4.degree. C. 21.degree. C. Unfiltered
0.968 .+-. 0.006 0.997 .+-. 0.002 0.528 .+-. 0.007 0.542 .+-. 0.012
0.047 .+-. 0.004 0.045 .+-. 0.000 Filtered 0.978 .+-. 0.008 0.994
.+-. 0.008 0.521 .+-. 0.007 0.527 .+-. 0.002 0.045 .+-. 0.001 0.045
.+-. 0.003 Data are expressed as mean .+-. S.D. (n = 3)
TABLE-US-00049 TABLE 49 Elemental recovery from 500 mg of A5 in 330
ml orange juice stored at 4.degree. C. Solubility (g/L) Ca Mg Zn
Filtered Unfiltered Filtered Unfiltered Filtered Unfiltered Fresh
0.273 .+-. 0.005 0.274 .+-. 0.009 0.170 .+-. 0.001 0.167 .+-. 0.003
0.0105 .+-. 0.0012 0.0089 .+-. 0.0003 One 0.272 .+-. 0.005 0.172
.+-. 0.017*** 0.171 .+-. 0.001 0.104 .+-. 0.010*** 0.0104 .+-.
0.0015 0.0139 .+-. 0.0088 Week Data are expressed as Mean .+-. S.D.
(n = 3) ***P < 0.001 comparing with fresh group
TABLE-US-00050 TABLE 50 Content of the three key elements in fresh
grapefruit juice Content (mg/L) Sample Ca Mg Zn Fresh grapefruit
48.2 .+-. 0.79 104 .+-. 1.6 0.536 .+-. 0.008 juice Data are
expressed as mean + S.D. (n = 3)
TABLE-US-00051 TABLE 51 Comparison of elemental recovery of A5 (2.6
g) in grapefruit juice at 4.degree. C. and 21.degree. C. Solubility
(g/L) Ca Mg Zn Sample 4.degree. C. 21.degree. C. 4.degree. C.
21.degree. C. 4.degree. C. 21.degree. C. Unfiltered 0.958 .+-.
0.010 0.968 .+-. 0.016 0.55 .+-. 0.005 0.518 .+-. 0.010 0.046 .+-.
0.001 0.046 .+-. 0.001 Filtered 0.981 .+-. 0.018 0.975 .+-. 0.004
0.516 .+-. 0.027 0.520 .+-. 0.005 0.045 .+-. 0.002 0.048 .+-. 0.002
Data are expressed as mean .+-. S.D. (n = 3)
TABLE-US-00052 TABLE 52 Elemental recovery from A5 (2.6 g) in
distilled water at 4 and 21.degree. C. Solubility (g/L) Temperature
Ca Mg Zn 4.degree. C. 0.875 .+-. 0.018 0.407 .+-. 0.000 0.024 .+-.
0.002 21.degree. C. 0.897 .+-. 0.016 0.404 .+-. 0.009 0.028 .+-.
0.001 Data are expressed as Mean .+-. S.D. (n = 3)
[0138] Similarly, temperature has no effect on the recovery of A5
elements in distilled water (Table 52).
[0139] Storage at 4.degree. C. for a week did not change the
recovery of calcium, magnesium and zinc when 2.6 g of A5 was
dissolved in 330 ml of filtered and unfiltered orange and grape
juice (Tables 48 and 51). However, when 500 mg of A5 was used
instead, the recovery of calcium and magnesium was significantly
lowered from the unfiltered orange juice (Table 49). The lower
recovery of calcium from unfiltered orange juice suggests that the
pulp in orange juice may bind Ca and Mg in A5. Carrot juice did not
have this problem (Table 54).
TABLE-US-00053 TABLE 53 Content of the three elements in fresh
carrot juice Content (mg/L) Sample Ca Mg Zn Fresh carrot 37.499
.+-. 4.613 75.279 .+-. 6.183 0.7045 .+-. 0.0195 juice Data are
expressed as Mean .+-. S.D. (n = 3)
TABLE-US-00054 TABLE 54 Elemental recovery from A5 in 330 ml carrot
juice stored at 4.degree. C. Solubility (g/L) Ca Mg Zn 2.6 grams
500 mg 2.6 grams 500 mg 2.6 grams 500 mg Fresh 0.907 .+-. 0.018
0.196 .+-. 0.009 0.482 .+-. 0.013 0.151 .+-. 0.001 0.0235 .+-.
0.0022 0.0031 .+-. 0.0001 One Week 0.935 .+-. 0.033 0.188 .+-.
0.006 0.481 .+-. 0.016 0.150 .+-. 0.008 0.0491 .+-. 0.0094** 0.0031
.+-. 0.0000 Data are expressed as Mean .+-. S.D. (n = 3) ***P <
0.01 comparing with fresh group
[0140] This set of studies suggests that A5 can be used to fortify
a number of juices and water. The 2.6 g of A5 provides a daily
requirement of the three key elements for the prevention of
osteoporosis: 300 mg of calcium, 150 mg of magnesium and 5.6 mg of
zinc. 500 mg of A5 is intended to provide a serving of these
elements in the functional food format.
Example 9
Tablet Formulation Compositions
[0141] The relatively low calcium content in A5 has posed a
challenge in creating a solid form with a size that is acceptable
to end-users. The following formulation was created in tablet form
(Table 55):
TABLE-US-00055 TABLE 55 Wt. for 2000 Wt./Tablet tablets Ingredients
(mg) % Comp. batch (g) Calcium 550 93.43 1100 Acetate blend Dry
Vitamin D.sub.3 3.25 0.55 6.5 100 GFP/HP* Kollidon VA 64 32.5 5.52
65 Magnesium 2.93 0.50 5.86 Stearate Total 588.68 100.00 1177.36
*Equivalent to 250 IU of Vitamin D.sub.3
[0142] The calcium acetate blend in the above table comprises 14%
calcium acetate, 7% magnesium acetate and 0.7% zinc acetate.
Magnesium stearate was used as a lubricant.
[0143] The Dry Vitamin D.sub.3 100 GFP/HP composition (as mentioned
in the certificate of analysis provided by BASF) is as follows:
TABLE-US-00056 Ingredients (CAS No.) Concentration (w/w) Sucrose
(57-50-1) 30.0-40.0% Starch (9005-25-8) 20.0-30.0% Gum Arabic
(9000-01-5) 15.0-25.0% Glycerides (73398-61-5) 5.0-15.0% Water
(7732-18-5) 1.0-5.0% Tricalcium phosphate <1.0% (7758-87-4)
Vitamin D.sub.3 (67-97-0) >=0.25% D,L-alpha-Tocopherol <0.2%
(10191-41-0)
[0144] Assay value: 100,000 IU Vitamin D.sub.3/g (=2500 microgram
cholecalciferol/g). The target weight of Vitamin D.sub.3 per tablet
is 2.5 mg. 30% extra Vitamin D.sub.3 has been added per tablet as
overage. The manufacturer assay value is 100000 IU/g i.e. 100
IU/mg. Since 2.5 mg (3.25 mg with 30% overage) has been used each
tablet has .about.250 IU of Vitamin D.sub.3.
[0145] The tablets were created according to the following
steps:
[0146] Step 1: Calcium Acetate blend provided was sieved through 40
mesh screen and 100/120 mesh screen. The fraction that passed
through the 40 mesh screen and was retained on 100/120 mesh screen
was used for formulation. The fraction of calcium acetate above 40
mesh and below 100 mesh was not used for formulation. This fraction
was chosen to keep the particle size similar to other
ingredients--Vitamin D.sub.3 and Kollidon Va 64.
[0147] Step 2: Blending 01: 6.5 g of dispensed Dry Vitamin D.sub.3
100 GFP/HP and 65 g OF Kollidon VA 64 were blended for 5 minutes at
a speed of 25 rpm using a small tumble blender to produce Blend
01.
[0148] Step 3: Blending 02: 250 g of dispensed Calcium Acetate
blend (Blend 01*3.49) prepared in Step 1 was mixed with Blend 01
prepared in Step 2 for 5 minutes to produce Blend 02 (using tumble
blender at 25-30 rpm).
[0149] Step 4: Blending 03: 250 g of dispensed Calcium Acetate
blend prepared in Step 1 was mixed with Blend 02 prepared in Step 3
for 5 minutes to produce Blend 03 (using double cone blender at
25-30 rpm).
[0150] Step 5: Blending 04: 600 g of dispensed Calcium Acetate
blend prepared in Step 1 was mixed with Blend 03 prepared in Step 4
for 9 minutes to produce Blend 04 (using double cone blender at
25-30 rpm).
[0151] Step 6: Blending 05: 5.86 g of dispensed Magnesium Stearate
was mixed with Blend 04 prepared in Step 5, for 2 minutes.
[0152] Step 7: The final blend prepared above was dispensed using a
Rotary table press with target tablet weight of 588.7 g.
[0153] The following formulation was also created in tablet
form:
TABLE-US-00057 Wt. for 2000 Wt./Tablet tablets Ingredients (mg) %
Comp. batch (g) Calcium 1100 93.43 2200 Acetate blend Dry Vitamin
D.sub.3 6.5 0.55 13 100 GFP/HP* Kollidon VA 64 65 5.52 130
Magnesium 5.86 0.50 11.72 Stearate Total 1177.36 100.00 2354.72
*Equivalent to 500 IU of Vitamin D.sub.3
[0154] The calcium acetate blend in the above table comprises 14%
calcium acetate, 7% magnesium acetate and 0.7% zinc acetate.
Magnesium stearate was used as a lubricant.
[0155] The Dry Vitamin D.sub.3 100 GFP/HP composition (as mentioned
in the certificate of analysis provided by BASF) is as presented
above.
[0156] Assay value: 100,000 IU Vitamin D.sub.3/g (=2500 microgram
cholecalciferol/g). The target weight of Vitamin D.sub.3 per table
is 5 mg. 30% extra Vitamin D3 has been added per tablet to account
for loss due to degradation. The manufacturer assay value is 100000
IU/g i.e. 100 IU/mg. Since 5 mg (6.5 mg with 30% overage) has been
used each tablet has .about.500 IU of Vitamin D.sub.3.
[0157] The tablets were created according to the following
steps:
[0158] Step 1: Calcium Acetate blend provided was sieved through 40
mesh screen and 100/120 mesh screen. The fraction that passed
through the 40 mesh screen and was retained on 100/120 mesh screen
was used for formulation. The fraction of calcium acetate above 40
mesh and below 100 mesh was not used for formulation. This fraction
was chosen to keep the particle size similar to other
ingredients--Vitamin D.sub.3 and Kollidon Va 64.
[0159] Step 2: Blending 01: 13 g of dispensed Dry Vitamin D.sub.3
100 GFP/HP and 130 g OF Kollidon VA 64 were blended for 5 minutes
at a speed of 25 rpm using a small tumble blender to produce Blend
01.
[0160] Step 3: Blending 02: 500 g of dispensed Calcium Acetate
blend (Blend 01*3.49) prepared in Step 1 was mixed with Blend 01
prepared in Step 2 for 5 minutes to produce Blend 02 (using double
cone blender at 25-30 rpm).
[0161] Step 4: Blending 03: 500 g of dispensed Calcium Acetate
blend prepared in Step 1 was mixed with Blend 02 prepared in Step 3
for 5 minutes to produce Blend 03 (using double cone blender at
25-30 rpm).
[0162] Step 5: Blending 04: 1200 g of dispensed Calcium Acetate
blend prepared in Step 1 was mixed with Blend 03 prepared in Step 4
for 9 minutes to produce Blend 04 (using double cone blender at
25-30 rpm).
[0163] Step 6: Blending 05: 11.72 g of dispensed Magnesium Stearate
was mixed with Blend 04 prepared in Step 5, for 2 minutes.
[0164] Step 7: The final blend prepared above was dispensed using a
Rotary table press with target tablet weight of 1.17 g.
[0165] The size of these two formulations has proven to be
acceptable to a test population.
Example 10
Gel Cap Formula Consisting of Fish Oil
[0166] A gel cap formula for the Calcium Acetate blend was created
to enhance end user acceptance, increased solubility of vitamin
D.sub.3 and increased efficacy on bone mineral density.
[0167] Vitamin D.sub.3 is an oil soluble vitamin. It can be
dissolved using lipophilic substances.
[0168] Fish oil containing omega 3-6-9 fatty acids is known to have
beneficial effects on bone health (32). This oil also has the
advantage of dissolving vitamin D.sub.3, obviating the granulation
process of Calcium Acetate blend as described in Example 9.
[0169] Fish oil has been found to have the ability to increase the
bulk density of Calcium Acetate blend by displacing air from the
powder.
[0170] Examples of oil to Calcium Acetate blend ratios include, but
are not limited to, about 1:1, 1.5:1 and 2:1.
[0171] Examples of oil to calcium ratios include, but are not
limited to, about 1:0.14, 1.5:1 and 2:1.
[0172] Examples of oil to magnesium ratios include, but are not
limited to, about 1:0.07, 1.5:0.07 and 2: 0.07.
[0173] Examples of oil to zinc ratios include, but are not limited
to, about 1:0.007, 1.5:0.007 and 2:0.007.
[0174] The dosage of vitamin D.sub.3 ranges from 30 to 300 IU.
[0175] Soft gel capsules can be manufactured using conventional
methods (33).
[0176] Gel capsules made with this blend in dose sizes amounting to
two to four capsules a day will be acceptable. The size of a gel
capsules will be equivalent to or smaller than that described in
Example 9.
Example 11
Optimization of Elemental Formula
[0177] The objective of this example is to design an elemental
formula which would provide an optimal mix of vitamin D.sub.3 and
acetate salts of calcium, magnesium and zinc for supporting bone
health.
[0178] It is a general belief that the bioavailability of calcium
is independent of the solubility of calcium salts (Heaney, 1999).
Low levels of magnesium and zinc are associated osteoporosis (Mutlu
et al., (11). Vitamin D.sub.3 enhances calcium absorption
(Christakos et. al., 2011) and therefore, is an important component
of an ideal elemental formula.
[0179] Results presented in this invention clearly show that the
bioavailability calcium is dependent on the solubility of a calcium
salt in the gastrointestinal fluids. An optimal ratio of calcium to
magnesium is required to enhance calcium absorption. Vitamin
D.sub.3 is responsible for increasing calcium absorption and
preventing zinc depletion.
[0180] A formula containing calcium, magnesium, zinc and vitamin
D.sub.3 may not work because the form of the elements and the
amount of vitamin D.sub.3, are not necessarily formulated in the
right ratios in terms of absorbable fractions. The lack of clinical
effect of a blend of calcium, magnesium, zinc and vitamin D.sub.3
is a good example (Braam et. al., 2003). The confusion in the
literature relating to calcium absorption and the equivocal
clinical trial results on bone mineral density by calcium
supplementation has created problems for experts skilled in the art
in designing an optimal formula of a calcium blend.
[0181] Using the acetate salts of calcium, magnesium and zinc with
the appropriate addition of vitamin D.sub.3, an optimum calcium
supplement is designed. The ratio of calcium to magnesium is
generally 2:1, the ratio of magnesium to zinc is 10:1 and the daily
dosage of vitamin D.sub.3 ranges from 500 to 1000 IU.
[0182] The bioavailability of calcium described in this invention
is appropriately 2 to 3 times higher than that of Caltrate. The
dosage of calcium should be half to one third of that of
Caltrate.TM..
[0183] The recommended intake of calcium from all sources is 1000
mg. The average intake of calcium from dietary sources is 400 mg.
It is recommended that 600 mg of calcium should be provided as a
supplement; usually this implies that the source of calcium is from
calcium carbonate. The recommended dose of calcium from this
invention is 200 to 300 mg. This will provide 100 to 150 mg of
magnesium and 5 to 7.5 mg of zinc. In addition to dietary intake,
the supplementation of magnesium and zinc will also provide an
adequate daily requirement of the elements.
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