U.S. patent application number 09/757782 was filed with the patent office on 2001-06-28 for method and compositions for increasing the anaerobic working capacity in tissues.
Invention is credited to Dunnett, Mark, Harris, Roger.
Application Number | 20010005579 09/757782 |
Document ID | / |
Family ID | 25427356 |
Filed Date | 2001-06-28 |
United States Patent
Application |
20010005579 |
Kind Code |
A1 |
Harris, Roger ; et
al. |
June 28, 2001 |
Method and compositions for increasing the anaerobic working
capacity in tissues
Abstract
A method for increasing the synthesis and accumulation of
beta-alanylhistidine dipeptides, with a simultaneous increase in
the accumulation of creatine, in bodily tissues of humans and
animals is described. This is accomplished by causing an increase
in the blood plasma concentrations of beta-alanine and creatine, or
the blood plasma concentrations of beta-alanine, L-histidine and
creatine, by the ingestion or infusion of a composition including
beta-alanine, beta-alanine and creatine, or beta-alanine,
L-histidine and creatine, or active derivatives thereof.
Inventors: |
Harris, Roger; (Newmarket,
GB) ; Dunnett, Mark; (Tuddingharu, GB) |
Correspondence
Address: |
GREGORY R. EINHORN
Fish & Richardson P.C.
Suite 500
4350 La Jolla Village Drive
San Diego
CA
92122
US
|
Family ID: |
25427356 |
Appl. No.: |
09/757782 |
Filed: |
January 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09757782 |
Jan 9, 2001 |
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09318530 |
May 25, 1999 |
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6172098 |
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09318530 |
May 25, 1999 |
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08909513 |
Aug 12, 1997 |
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5965596 |
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Current U.S.
Class: |
435/4 ; 514/400;
514/5.5; 514/6.7 |
Current CPC
Class: |
A61K 31/415 20130101;
A61K 35/16 20130101; A61K 35/16 20130101; A23K 20/142 20160501;
A61K 31/198 20130101; A61K 38/01 20130101; A61K 31/415 20130101;
A23L 33/175 20160801; A61K 31/197 20130101; A61K 31/195 20130101;
A61K 31/415 20130101; A23L 33/18 20160801; A61K 38/05 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/195 20130101;
A61K 31/195 20130101; A61K 2300/00 20130101; A61K 31/195
20130101 |
Class at
Publication: |
435/4 ; 514/400;
514/2 |
International
Class: |
A61K 031/415; A01N
043/50 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 1996 |
GB |
9621914.2 |
Aug 12, 1996 |
GB |
9616910.7 |
Claims
What is claimed is:
1. A method of increasing the anaerobic working capacity of a
tissue comprising: providing an amount of beta-alanine to blood or
blood plasma effective to increase beta-alanylhistidine dipeptide
synthesis in a tissue; providing an amount of L-histidine to the
blood or blood plasma effective to increase beta-alanylhistidine
dipeptide synthesis; and exposing the tissue to the blood or blood
plasma, whereby the concentration of beta-alanylhistidine is
increased in the tissue.
2. The method of claim 1, further comprising increasing a
concentration of creatine in the tissue.
3. The method of claim 1, wherein the providing steps include
ingestion of a composition including the amount of beta-alanine and
the amount of L-histidine.
4. The method of claim 1, wherein the providing step includes
infusion of a composition including the amount of beta-alanine and
the amount of L-histidine.
5. The method of claim 1, further comprising increasing a
concentration of insulin in the blood or blood plasma.
6. The method of claim 1, wherein the tissue is a skeletal
muscle.
7. The method of claim 1, wherein the tissue is a human tissue.
8. The method of claim 1, wherein the tissue is an animal
tissue.
9. A composition consisting essentially of: a peptide source
including beta-alanine; between about 39 and about 99 percent by
weight of a carbohydrate; and up to about 60 percent by weight of
water, wherein the composition includes between about 1 and about
20 percent by weight of the beta-alanine.
10. The composition of claim 9, wherein the peptide source includes
L-histidine and the composition includes between about 1 and about
20 percent by weight of L-histidine.
11. A composition consisting essentially of: a peptide source
including beta-alanine; between about 1 and about 98 percent by
weight of a creatine source; and up to about 97 percent by weight
of water, wherein the composition includes between about 1 and
about 98 percent by weight of the beta-alanine.
12. The composition of claim 11, wherein the peptide source
includes L-histidine and the composition includes between about 1
and about 98 percent by weight of L-histidine.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates to methods and compositions for
increasing the anaerobic working capacity of muscle and other
tissues.
[0002] Natural food supplements are typically designed to
compensate for reduced levels of nutrients in the modern human and
animal diet. In particular, useful supplements increase the
function of tissues when consumed. It can be particularly important
to supplement the diets of particular classes of animals whose the
normal diet may be deficient in nutrients available only from meat
and animal produce (e.g., human vegetarians and other animals
consume an herbivorous diet).
[0003] For example, in the sporting and athletic community, natural
food supplements which specifically improve athletic ability are
increasingly important, such as supplements that promote or enhance
physical prowess for leisure or employment purposes. In another
example, anaerobic (e.g., lactate-producing) stress can cause the
onset of fatigue and discomfort that can be experienced with aging.
Anaerobic stress can also result from prolonged submaximal
isometric exercise when the local circulation is partially or
totally occluded by the increase in intra-muscular pressure (e.g.,
during rock climbing, free diving, or synchronized swimming).
Excessive lactate production can result in the acidification of the
intracellular environment.
[0004] Creatine (i.e., N-(aminoiminomethyl)-N-glycine,
N-amidinosarcosine, N-methyl-N-guanylglycine, or
methylglycocyamine) is found in large amounts in skeletal muscle
and other "excitable" tissues (e.g., smooth muscle, cardiac muscle,
or spermatozoa) characterized by a capacity for a high and variable
energy demand. Creatine is converted into phosphorylcreatine in
energy-generating biochemical pathways within cells. In mammalian
skeletal muscle, the typical combined content of creatine (i.e.,
creatine and phosphorylcreatine) may vary from less than 25 to
about 50 mmol per kilogram fresh muscle (i.e., 3.2 to 6.5 grams per
kilogram fresh muscle).
[0005] Creatine formed is formed in the liver and taken up into
tissues, such as muscle, by means of an active transport system.
Creatine synthesis in the body may also be augmented by the
ingestion of creatine present in meat (e.g., 5-10 milligrams per
kilogram body weight per day in the average meat-eating human and
approximately zero in a vegetarian diet).
[0006] During sustained intensive exercise, or exercise sustained
under conditions of local hypoxia, the accumulation of hydronium
ions formed during glycolysis and the accumulation of lactate
(anaerobic metabolism) can severely reduce the intracellular pH.
The reduced pH can compromise the function of the
creatine-phosphorylcreatine system. The decline in intracellular pH
can affect other functions within the cells, such as the function
of the contractile proteins in muscle fibers.
[0007] Dipeptides of beta-alanine and histidine, and their
methylated analogues, include carnosine (beta-alanyl-L-histidine),
anserine (beta-alanyl-L-1-methylhistidine), or balenine
(beta-alanyl-L-3-methylhis- tidine). The dipeptides are present in
the muscles of humans and other vertebrates. Carnosine is found in
appreciable amounts in muscle of, for example, humans and equines.
Anserine and carnosine are found in muscle of, for example,
canines, camelids and numerous avian species. Anserine is the
predominant beta-alanylhistidine dipeptide in many fish. Balenine
is the predominant beta-alanylhistidine dipeptide in some species
of aquatic mammals and reptiles. In humans, equines, and camelids,
the highest concentrations of the beta-alanylhistidine dipeptides
are found in fast-contracting glycolytic muscle fibers (type IIA
and IIB) which are used extensively during intense exercise. Lower
concentrations are found in oxidative slow-contracting muscle
fibers (type I). See, e.g., Dunnett, M. & Harris, R. C. Equine
Vet. J., Suppl. 18, 214-217 (1995). It has been estimated that
carnosine contributes to hydronium ion buffering capacity in
different muscle fiber types; up to 50% of the total in equine type
II fibers.
SUMMARY OF THE INVENTION
[0008] In general, the invention features methods and compositions
for increasing the anaerobic working capacity of muscle and other
tissues. The method includes simultaneous accumulation of creatine
and beta-alanylhistidine dipeptides, or beta-alanine and
L-histidine analogues, within a tissue in the body. The methods
include ingesting or infusing compositions into the body. The
compositions are mixtures of compounds capable of increasing the
availability and uptake of creatine and of precursors for the
synthesis and accumulation of beta-alanylhistidine dipeptides, in
human and animal muscle. The composition induces the synthesis and
accumulation of beta-alanylhistidine dipeptides in a human or
animal body when introduced into the body.
[0009] The compositions include mixtures of creatine and
beta-alanine, creatine, beta-alanine and L-histidine, or creatine
and active derivatives of beta-alanine or L-histidine. Each of the
beta-alanine or L-histidine can be the individual amino acids, or
components of dipeptides, oligopeptides, or polypeptides. The
beta-alanine or L-histidine can be active derivatives. An active
derivative is a compound derived from, or a precursor of, the
substance that performs in the same or similar way in the body as
the substance, or which is processed into the substance and placed
into the body. Examples include, for example, esters and
amides.
[0010] In one aspect, the invention features a method of regulating
hydronium ion concentrations in a tissue. The method includes the
steps of providing an amount of beta-alanine to blood or blood
plasma effective to increase beta-alanylhistidine dipeptide
synthesis in a tissue, and exposing the tissue to the blood or
blood plasma, whereby the concentration of beta-alanylhistidine is
increased in the tissue. The method can include the step of
providing an amount of L-histidine to the blood or blood plasma
effective to increase beta-alanylhistidine dipeptide synthesis.
[0011] In another aspect, the invention features a method of
increasing the anaerobic working capacity of a tissue. The method
includes the steps of providing an amount of beta-alanine to blood
or blood plasma effective to increase beta-alanylhistidine
dipeptide synthesis in a tissue, providing an amount of L-histidine
to the blood or blood plasma effective to increase
beta-alanylhistidine dipeptide synthesis in a tissue, and exposing
the tissue to the blood or blood plasma. The concentration of
beta-alanylhistidine is increased in the tissue.
[0012] In embodiments, the methods can include the step of
increasing a concentration of creatine in the tissue. The
increasing step can include providing an amount of creatine to the
blood or blood plasma effective to increase the concentration of
creatine in the tissue (e.g., by providing the amount of creatine
to the blood or blood plasma).
[0013] The providing steps of the methods can include ingestion or
infusion (e.g., injection) of a composition including the amount of
beta-alanine, or the amounts of beta-alanine and L-histidine, or a
combination of ingestion and infusion.
[0014] The methods can include increasing a concentration of
insulin in the blood or blood plasma. The concentration of insulin
can be increased, for example, by injection of insulin.
[0015] The tissue can be a skeletal muscle.
[0016] In another aspect, the invention features a composition
consisting essentially of a peptide source including beta-alanine,
between about 39 and about 99 percent by weight of a carbohydrate,
and up to about 60 percent by weight of water. The composition
includes between about 1 and about 20 percent by weight of the
beta-alanine. The peptide source can include L-histidine. The
composition can include between about 1 and about 20 percent by
weight of L-histidine.
[0017] The carbohydrate can be a simple carbohydrate (e.g.,
glucose). In another aspect, the invention features a composition
consisting essentially of a peptide source including beta-alanine,
between about 1 and about 98 percent by weight of a creatine
source, and up to about 97 percent by weight of water. The
composition includes between about 1 and about 98 percent by weight
of the beta-alanine. The peptide source can include L-histidine and
the composition includes between about 1 and about 98 percent by
weight of L-histidine.
[0018] The peptide source can be a mixture of amino acids,
dipeptides, oligopeptides, polypeptides, or active derivatives
thereof.
[0019] The composition can be a dietary supplement. The creatine
source can be creatine monohydrate.
[0020] The concentrations of components in blood or blood plasma
can be increased by infusion (i.e., injection) or ingestion of an
agent operable to cause an increase in the blood plasma
concentration. The composition can be ingested in doses of between
about 10 grams and about 800 grams per day. The doses can be
administered in one part or multiple parts each day.
[0021] An increase in the muscle content of creatine and
beta-alanylhistidine dipeptides can increase the tolerance of the
cells to the increase in hydronium ion production with anaerobic
work, and to lead to an increase in the duration of the exercise
before the onset of fatigue. The compositions and methods can
contribute to correcting the loss of beta-alanine, L-histidine, or
creatine due to degradation or leaching of these constituents
during cooking or processing. The compositions and methods can also
contribute to correcting the absence of these components from a
vegetarian diet.
[0022] The methods and compositions can be used to increase
beta-alanylhistidine dipeptide by, for example, sportsmen,
athletes, body-builders, synchronized swimmers, soldiers, elderly
people, horses in competition, working and racing dogs, and game
birds, to avoid or delay the onset of muscular fatigue.
[0023] Other advantages and features of the invention will be
apparent from the detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIG. 1 is a graph depicting changes in the concentrations of
beta-alanine in blood plasma of five horses, before and at 2 hour
intervals following the feeding of beta-alanine and L-histidine
(100 milligrams per kilogram body weight and 12.5 milligrams per
kilogram body weight, respectively, three times per day) over a
period of 30 days.
[0025] FIG. 2 is a graph depicting changes in the concentrations of
L-histidine in blood plasma of five horses, before and at 2 hour
intervals following the feeding of beta-alanine and L-histidine
(100 milligrams per kilogram body weight and 12.5 milligrams per
kilogram body weight, respectively, three times per day) over a
period of 30 days.
[0026] FIGS. 3a and 3b are graphs depicting the contrast in the
changes in the concentrations of beta-alanine in blood plasma of
six horses, before and at hourly intervals following the feeding of
beta-alanine and L-histidine (100 milligrams per kilogram body
weight and 12.5 milligrams per kilogram body weight, respectively,
three times per day) on the first and last day of a 30 day period
of dietary supplementation.
[0027] FIGS. 4a and 4b are graphs depicting the contrast in the
changes in the concentrations of L-histidine in blood plasma of six
horses, before and at hourly intervals following the feeding of
beta-alanine and L-histidine (100 milligrams per kilogram body
weight and 12.5 milligrams per kilogram body weight, respectively,
three times per day) on the first and last day of a 30 day period
of dietary supplementation.
[0028] FIG. 5 is a graph depicting the contrast in the changes in
the mean concentrations of beta-alanine in equine blood plasma
(n=6), before and at hourly intervals following the feeding of
beta-alanine and L-histidine (100 milligrams per kilogram body
weight and 12.5 milligrams per kilogram body weight, respectively,
three times per day) on the first and last day of a 30 day period
of dietary supplementation.
[0029] FIG. 6 is a graph depicting the contrast in the changes in
the mean concentrations of L-histidine in equine blood plasma
(n=6), before and at hourly intervals following the feeding of
beta-alanine and L-histidine (100 milligrams per kilogram body
weight and 12.5 milligrams per kilogram body weight, respectively,
three times per day) on the first and last day of a 30 day period
of dietary supplementation.
[0030] FIG. 7 is a graph depicting the correlation between the
increase in 6 thoroughbred horses in the carnosine concentration in
type II skeletal muscle fibers (the average of the sum of type IIA
and IIB fibres) and the increase, between the 1st and 30th day of
supplementation, in the area under the blood plasma beta-alanine
concentration-time curve over the first 12 hours of the day
(AUC.sub.(0-12 hr)).
[0031] FIG. 8 is graph depicting the mean results of the
administration of beta-alanine, broth, or carnosine to test
subjects.
[0032] FIG. 9 is a graph depicting mean changes in plasma
beta-alanine over nine hours of treatment.
[0033] FIG. 10 is a graph depicting the mean changes in plasma
beta-alanine over 9 hours following the oral ingestion of 10
milligrams per kilogram body weight of beta-alanine.
[0034] FIG. 11 is a graph depicting the mean (n=6) plasma
beta-alanine concentration over the 24 hour of Day 1 and Day 30 of
the treatment period.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] Beta-alanylhistidine dipeptides such as carnosine, anserine,
and balenine have pKa values between approximately 6.8 and 7.1 and
are involved in the regulation of intracellular pH homeostasis
during muscle contraction and the development of fatigue. The
content of other substances involved in hydronium ion buffering,
such as amino acid residues in proteins, inorganic and organic
phosphates and bicarbonate, is constrained by their involvement in
other cell functions. The beta-alanylhistidine dipeptides can
provide an effective way of accumulating pH-sensitive histidine
residues into a cell. Variations in the muscle beta-alanylhistidine
dipeptide concentrations affect the anaerobic work capacity of
individual athletes.
[0036] The beta-alanylhistidine dipeptides are synthesized within
the body from beta-alanine and L-histidine. These precursors can be
generated within the body or are made available via the diet,
including from the breakdown of an ingested beta-alanylhistidine
dipeptide. Beta-alanine within the body is transported to tissues
such as muscle. In a typical fed state, the concentration of
beta-alanine is low in comparison with the concentration of
L-histidine in human and equine blood plasma. These concentrations
should be viewed in relation to the affinity of the carnosine
synthesizing enzyme, carnosine synthetase, for its substrates as
determined by the Michaelis-Menton constant (Km). The Km for
histidine is about 16.8 .mu.M. The Km for beta-alanine is between
about 1000 and 2300 .mu.M. The low affinity of carnosine synthetase
for beta-alanine, and the low concentration of beta-alanine in
muscle, demonstrate that the concentration of beta-alanine in
muscle which is limiting to the synthesis of the
beta-alanylhistidine dipeptides.
[0037] Increasing the amount of beta-alanylhistidine dipeptides
within a muscle favorably affects muscular performance and the
amount of work that can be performed by the muscle. Accordingly,
the synthesis and accumulation of beta-alanylhistidine dipeptides
is increased in a tissue in a human or animal body.
[0038] The synthesis and accumulation of beta-alanylhistidine
dipeptides in a human or animal body can be increased with an
increase in the content within the body of creatine, by increasing
the blood or blood plasma concentrations of beta-alanine,
increasing the blood or blood plasma concentrations beta-alanine
and creatine, or increasing the blood or blood plasma
concentrations beta-alanine, L-histidine, and creatine. The
increase in dipeptide can be simultaneous with the increase in
beta-alanine concentration.
[0039] The blood plasma concentrations of beta-alanine, L-histidine
and creatine can be increased by ingestion or infusion of
beta-alanine, L-histidine, and creatine, or active derivatives
thereof. The composition can be administered orally, enterally, or
parenterally. The beta-alanine and creatine, or beta-alanine,
L-histidine and creatine, are preferably orally ingested.
[0040] The composition can include carbohydrates (e.g., simple
carbohydrates), insulin, or agents that stimulate the production of
insulin.
[0041] The composition can be ingested as a dietary supplement.
Preferably, the composition can be administered in one or more
doses per day. The beta-alanine dosage can be between about 5
milligrams and about 200 milligrams per kilogram body weight. The
creatine (e.g., creatine monohydrate) dosage can be between about 5
milligrams to 200 milligrams per kilogram body weight. The
L-histidine dosage can be between about 1 milligrams to 100
milligrams per kilogram body weight. The simple carbohydrate (e.g.,
glucose) dosage can be between about 0.5 and 2.0 grams per kilogram
body weight.
[0042] In an 80 kilogram person, suitable dosages per day can be
between 0.4 grams to 16.0 grams of beta-alanine, 0.4 grams to 16.0
grams of creatine monohydrate, 0.08 grams to 8.0 grams of
L-histidine, or 40 grams to 160 grams of glucose or other simple
carbohydrate. The composition can be in solid form or liquid form
or the form of a suspension which is ingested, or in liquid form or
suspension for infusion into the body. The composition is ingested
in humans in an amount of between 2 grams and 1000 grams per day
(e.g., between 10 grams and 800 grams), which may be taken in one
or more parts throughout the day. In animals the daily intake will
be adjusted for body weight.
[0043] For humans and animals, the compositions can be:
[0044] (a)
[0045] 1% to 99% by weight of beta-alanine;
[0046] 1% to 99% by weight of creatine monohydrate; and
[0047] 0% to 98% by weight of water;
[0048] (b)
[0049] 1% to 98% by weight of beta-alanine;
[0050] 1% to 98% by weight of L-histidine;
[0051] 1% to 98% by weight of creatine monohydrate; and
[0052] 0% to 97% by weight of water;
[0053] (c)
[0054] 1% to 20% by weight of beta-alanine;
[0055] 39% to 99% by weight of glucose or other simple
carbohydrate; and
[0056] 0% to 60% by weight of water; or
[0057] (d)
[0058] 1% to 20% by weight of beta-alanine;
[0059] 1% to 20% by weight of L-histidine
[0060] 39% to 99% by weight of glucose or other simple
carbohydrate; and
[0061] 0% to 60% by weight of water.
[0062] The following are specific examples of the methods of
methods and compositions for increasing the anaerobic working
capacity of muscle and other tissues.
EXAMPLE 1
[0063] The effect of supplementation of a normal diet with multiple
daily doses of beta-alanine and L-histidine on the carnosine
concentration in type I, IIA, and IIB skeletal muscle fibers of
thoroughbred horses was assessed. Six experimental thoroughbred
horses of normal health (three fillies and three geldings), aged 4
to 9 years, underwent one month (30 days) of dietary conditioning
(pre-supplementation period) prior to the commencement of the
supplementation period. During the dietary conditioning period each
horse was fed a diet comprising 1 kilogram of pelleted feed
(Spillers racehorse cubes) and 1 kilogram of soaked sugar beet pulp
as a source of complex and simple carbohydrates, three times per
day (at 08:30, 12:30, and 16:30, respectively). Soaked hay (3
kilograms dry weight) was also provided twice daily (at 09:00 and
17:00). Water was provided ad libitum.
[0064] During the supplementation period an identical feeding
regime was implemented. However, each hard feed meal was
supplemented with beta-alanine and L-histidine (free base).
Beta-alanine and L-histidine were mixed directly into the normal
feed. Individual doses of beta-alanine and L-histidine were
calculated according to body weight. Beta-alanine was administered
at 100 milligrams per kilogram body weight and L-histidine at 12.5
milligrams per kilogram body weight. Dietary supplementation was
begun on day 1 of the protocol and discontinued at the end of day
30. Heparinized blood samples (5 milliliters) were collected on
days 1, 6, 18, 24, and 30. On day 1 and day 30, blood samples were
collected prior to the first feed and at hourly intervals for a
total of 12 hours each day. On the three intervening sampling days,
blood was collected prior to the first feed and 2 hours after each
subsequent feed. On the day before the start of supplementation
(day 0) a muscle biopsy was taken, following application of local
anaesthesia of the skin, from the right middle gluteal muscle (m.
gluteus medius) of each horse using a Bergstrom-Stille percutaneous
biopsy needle. Subsequent muscle biopsies were collected
immediately after the end of the supplementation period (day 31) as
close as possible to the original sampling site. Clinical
monitoring of the horses was performed daily. This comprised a
visual examination and measurement of body weight, twice-daily
measurement of rectal temperature, and weekly blood sampling for
clinical biochemistry and hematology. During the course of the
study the horses received no formal training or exercise, although
they were allowed one hour of free exercise each day.
[0065] Fragments of individual muscle fibers dissected from
freeze-dried muscle biopsies were characterized as either type I,
IIA or IIB by histochemical staining for myosin ATPase activity at
pH 9.6 following pre-incubation at pH 4.50 by a modification of the
method described in, Kaiser and Brook, Arch. Neurol., 23:369-379
(1970).
[0066] Heparinized blood plasma samples were extracted and analyzed
for beta-alanine and L-histidine concentrations by high-performance
liquid chromatography (HPLC). Individual weighed muscle fibers were
extracted and analyzed for carnosine by HPLC according to the
method described in, Dunnett and Harris, "High-performance liquid
chromatographic determination of imidazole dipeptides, histidine,
1-methylhistidine and 3-methylhistidine in muscle and individual
muscle fibers," J. Chromatogr. B. Biomed. Appl., 688:47-55
(1997).
[0067] Differences in carnosine concentrations within fiber types
before and after supplementation were established within horses
using one-way analysis of variance (ANOVA). In instances where
differences were detected, significance was determined using a
multiple comparison test (Fisher's PLSD).
[0068] No palatability problems were encountered with the addition
of beta-alanine and L-histidine to the feed. No adverse
physiological or behavioral effects of the supplemented diet were
observed in any of the horses during the thirty days of
supplementation. No significant changes in body weight were
recorded, and rectal temperatures remained within the normal range.
No acute or chronic changes in clinical biochemistry or hematology
were observed. Beta-alanine was not detected in the plasma of any
of the horses prior to the start of supplementation. The lower
limit of quantitation for beta-alanine in plasma by the assay used
was 3 micromolar (.mu.M). Plasma L-histidine concentrations in the
six horses prior to the start of supplementation were between 36.6
and 54.4 .mu.M.
[0069] Individual changes in blood plasma beta-alanine and
L-histidine concentrations for five of the six horses over on all
the sampling days are shown in FIGS. 1 and 2, respectively. There
was a trend towards an increase in the pre-feeding concentrations
of blood plasma beta-alanine and L-histidine with increasing time
of supplementation. Furthermore, over the thirty day
supplementation period, the blood plasma concentration response to
supplementation was also increased. The response was greater for
beta-alanine.
[0070] Comparisons of the changes in blood plasma beta-alanine and
L-histidine concentrations prior to the first feed of the day, and
hourly thereafter between the first and last days of the
supplementation period, for the six individual horses, are shown in
FIGS. 3a and 3b, and FIGS. 4a and 4b, respectively. The mean
(.+-.SD) changes (n=6) in blood plasma beta-alanine concentration
over time during the 24 hours of the first (day 1) and last (day
30) days of the supplementation period are contrasted in FIG. 5.
The area under the mean blood plasma beta-alanine concentration
versus time curve over 24 hours (AUC.sub.(0-24 hr)) was much
greater on day 30 of the supplementation.
[0071] The mean (.+-.SD) changes (n=6) in blood plasma L-histidine
concentration over time during the 24 hours of the first (day 1)
and last (day 30) days of the supplementation period are contrasted
in FIG. 6. The area under the mean blood plasma beta-alanine
concentration vs. time curve over 24 hours (AUC.sub.(0-24 hr)) was
greater on day 30 of the supplementation. The greater AUC for blood
plasma beta-alanine on the last day of supplementation (day 30) in
contrast to the first day of supplementation (day 1) suggests the
increased uptake of beta-alanine from the equine gastro-intestinal
tract with progressive supplementation. A similar effect was
observed for changes in blood plasma L-histidine concentration
during the supplementation period. Peak blood plasma concentrations
of beta-alanine and L-histidine occurred approximately one to two
hours post-feeding in each case.
[0072] A total of 397 individual skeletal muscle fibers (192
pre-supplementation; 205 post-supplementation) from the six horses
were dissected and analyzed for carnosine. Mean (.+-.SD) carnosine
concentration, expressed as millimoles per kilogram dry weight
(mmol kg.sup.-1 dw), in pre- and post-supplementation type I, IIA,
and IIB skeletal muscle fibers from the six individual horses are
given in Table 1 where n is the number of individual muscle fibers
analyzed. Following thirty days of beta-alanine and L-histidine
supplementation the mean carnosine concentration was increased in
type IIA and IIB fibers in all six horses. These increases were
statistically significant in seven instances. The increase in mean
carnosine concentration in type IIB skeletal muscle fibers was
statistically significant in five out of six horses. The increase
in mean carnosine concentration in type IIA skeletal muscle fibers
was statistically significant in two out of six horses.
1TABLE 1 Horse Day Type 1 n Type IIA n Type IIB n 6 0 32.3 3 72.1
11 111.8 14 31 (14.5) (47.7) 17 (22.8) 12 -- 16.2 117.7 (20.9)
(38.7) 5 0 59.5 2 102.6 12 131.2 26 31 (3.9) 1 (12.7) 18 (26.6) 22
55.5 112.2 153.3 (17.1) (28.0)** 4 0 44.8 4 59.9 13 108.6 19 31
(6.6) 2 (19.5) 17 (41.5) 19 37.0 88.0 152.4 (9.3) (34.2)* (65.0)* 1
0 56.7 2 88.5 15 101.3 13 31 (5.3) 1 (20.9) 19 (15.2) 11 57.8 96.1
14.3 (17.3) (13.3)* 2 0 -- 89.6 13 104.2 14 31 65.9 4 (16.2) 18
(22.2) 12 (13.2) 102.2 142.0 (22.1) (35.4)*** 3 0 30.9 2 85.1 6
113.5 23 31 (4.0) (20.3) 23 (20.4) 9 105.0 135.4 (17.6)* (24.9)*
Mean 0 44.8 13 83.0 70 111.8 109 31 54.1 8 96.6* 112 135.9** 85
*significantly different to pre-supplementation, p < 0.05
**significantly different to pre-supplementation, p < 0.01
***significantly different to pre-supplementation, p < 0.005
[0073] The absolute (e.g. mmol kg.sup.-1 dw) and percentage
increases in the mean carnosine concentrations in type IIA and IIB
skeletal muscle fibers from the six horses are listed in Table
2.
2TABLE 2 Type IIIA Type IIA % Type IIB Type IIB Horse Absolute
increase increase Absolute increase % increase 6 4.1 5.7 5.6 5.3 5
9.6 9.4 22.1 16.8 4 28.1 46.9 43.8 40.3 1 7.6 8.6 13.0 12.8 2 12.6
14.1 37.8 36.3 3 19.9 23.4 21.9 19.3 Mean 13.6 18.0 24.1 21.8
[0074] It was observed that the individual horses which showed the
greater increase in muscle carnosine concentration following thirty
days of supplementation also demonstrated the greater increase in
blood plasma beta-alanine AUC between day 1 and day 30 of the
supplementation period. Referring to FIG. 7, a significant
correlation (r=0.986, p<0.005) for five of the six horses was
observed between the increase in mean carnosine concentration,
averaged between type IIA and IIB skeletal muscle fibers and the
increase, between the 1st and 30th day of supplementation, in blood
plasma beta-alanine AUC, over the first 12 hours (AUC.sub.(0-12
hr)). Only five horses were used to calculate the regression line.
Horse 6 (filled circle) showed no appreciable increase in blood
plasma beta-alanine concentration greater than that observed on day
1 until the last day of supplementation. This was unlike the other
five horses which showed a progressive increase with each sampling
day. For this reason horse 6 was excluded from the calculation of
the regression equation.
[0075] Increases in muscle carnosine concentration following thirty
days of supplementation with beta-alanine and L-histidine will
cause a direct increase in total muscle buffering capacity. This
increase can be calculated by using the Henderson-Hasselbach
Equation. Calculated values for the increases in muscle buffering
capacity in type IIA and IIB skeletal muscle fibers in the six
thoroughbred horses are shown in Table 3.
3TABLE 3 Type IIA Type Type .DELTA..beta.m- Type Type Type IIB IIA
IIA total IIB IIB .DELTA..beta.m-total Horse Day .beta.mcar
.beta.mtotal (%) .beta.mcar .beta.mtotal (%) 6 0 23.9 93.9 37.1
107.1 31 25.3 95.3 +1.5 39.0 109.0 +1.8 5 0 34.0 104.0 43.5 113.5
31 37.2 107.2 +3.1 50.8 120.8 +6.4 4 0 19.9 89.9 36.0 106.0 31 29.2
99.2 +10.3 50.5 120.5 +13.7 1 0 29.3 99.3 33.6 103.6 31 31.9 101.9
+2.6 37.9 107.9 +4.2 2 0 29.7 99.7 34.5 104.5 +12.1 31 33.9 103.9
+4.2 47.1 117.1 3 0 28.2 98.2 37.6 107.6 31 34.8 104.8 +6.7 44.9
114.9 +6.8 Mean 0 27.5 97.5 37.1 107.1 31 32.1 102.1 +4.7 45.0
115.0 +7.5
EXAMPLE 2
[0076] The effect of supplementation of a normal diet with multiple
daily doses of beta-alanine and L-histidine on the carnosine
content of type I, IIA, and IIB skeletal muscle fibers of humans
was assessed. The plasma concentration of beta-alanine in six
normal subjects following the consumption of a broth delivering
approximately 40 milligrams per kilogram body weight of
beta-alanine was monitored. Doses of 10 and 20 milligrams per
kilogram body weight of beta-alanine were also given.
[0077] The broth was prepared as follows. Fresh chicken breast
(skinned and boned) was finely chopped and boiled for fifteen
minutes with water (1 liter for every 1.5 kg of chicken). Residual
chicken meat was removed by course filtration. The filtrate was
flavored by the addition of carrot, onion, celery, salt, pepper,
basil, parsley and tomato puree, and reboiled for a further fifteen
minutes and then cooled before final filtration though fine muslin
at 4.degree. C. The yield from 1.5 kilograms of chicken and one
liter of water was 870 mL of broth. A portion of the stock was
assayed for the total beta-alanyl-dipeptide content (e.g.,
carnosine and anserine) and beta-alanine. Typical analyses
were:
4 total beta-alanyl-dipeptides 74.5 mM free beta-alanine 5.7 mM
[0078] The six male test subjects were of normal health and between
25-53 years of age, as shown in Table 4. The study commenced after
an overnight fast (e.g., a minimum of 12 hours after the ingestion
of the last meat containing meal). Subjects were given the option
to consume a small quantity of warm water prior to the start of the
study. Catheterization was begun at 08:30 and the study started at
09:00.
[0079] As a control, 8 milliliters per kilogram body weight of
water was ingested (e.g., 600 mL in a subject weighing 75
kilograms).
[0080] In one session, 8 milliliters per kilogram body weight of
broth containing approximately 40 milligrams per kilogram body
weight of beta-alanine (e.g., in the form of anserine and
carnosine) was ingested. For a subject weighing 75 kilograms this
amounted to the ingestion of 600 milliliters of broth containing 3
grams of beta-alanine. In another session, 3 milliliters per
kilogram body weight of a liquid containing the test amount of
beta-alanine with an additional 5 milliliters per kilogram body
weight of water was ingested. In all sessions, subjects
additionally consumed a further 8 milliliters per kilogram body
weight of water (in 50 mL portions) during the period of 1 to 2 h
after ingestion. A vegetarian pizza was provided after 6 hours. An
ordinary diet was followed after 8 hours.
[0081] 2.5 milliliter venous blood samples were drawn through an
indwelling catheter at 10 minute intervals for the first 90 minutes
and then after 120, 180, 240 and 360 minutes. The blood samples
were dispensed into tubes containing lithium-heparin as
anti-coagulant. The catheter was maintained by flushing with
saline. Plasma samples were analyzed by HPLC according to the
method described in Jones & Gilligan (1983) J. Chromatogr.
266:471-482 (1983).
[0082] Table 4 summarizes the allocation of treatments during the
beta-alanine absorption study. The estimated equivalent doses of
beta-alanine are presented in Table 3.
5TABLE 4 Broth .beta.-ala .beta.-ala .beta.-ala .beta.-ala 40 0 10
20 40 Carnosine Age Weight mg/kg mg/kg mg/kg mg/kg mg/kg 20 mg/kg
Subject yrs kg bwt bwt bwt bwt bwt bwt 1 53 76 .check mark. .check
mark. .check mark. .check mark. 2 33 60 .check mark. .check mark.
.check mark. 3 29 105 .check mark. .check mark. .check mark. .check
mark. 4 31 81 .check mark. .check mark. .check mark. .check mark. 5
30 94 .check mark. .check mark. .check mark. .check mark. 6 25 65
.check mark. .check mark. .check mark. .check mark.
[0083] Plasma concentration curves following each treatment are
depicted graphically in FIG. 8. Mean results of the administration
of beta-alanine, broth, or carnosine according to the treatments
schedule in Table 4. Plasma beta-alanine was below the limit of
detection in all subjects on the control treatment. Neither
carnosine or anserine were detected in plasma following ingestion
of the chicken broth or any of the other treatments. Ingestion of
the broth resulted in a peak concentration in plasma of 427.9 (SD
161.8) .mu.M. Administration of carnosine equivalent to 20
milligrams per kilogram body weight of beta-alanine in one test
subject resulted in an equivalent increase in the plasma
beta-alanine concentration.
[0084] Administration of all treatments except control resulted in
an increase in the plasma taurine concentration. The changes in
taurine concentration mirrored closely those of beta-alanine.
Administration of broth, a natural food, caused the an equivalent
increase in plasma taurine, indicating that such a response is
occurring normally following the ingestion of most meals.
EXAMPLE 3
[0085] The effect of administration of three doses of 10 milligrams
per kilogram body weight of beta-alanine per day (i.e.,
administered in the morning, noon, and at night) for seven days on
the plasma concentration profiles of beta-alanine and taurine were
investigated. The plasma concentration profiles following
administration of 10 milligrams per kilogram body weight of
beta-alanine were studied in three subjects at the start and end of
a seven day period during which they were given three doses of the
beta-alanine per day.
[0086] Three male subjects of normal health, aged between 33-53
years were studied. Test subjects received three doses per day of
10 milligrams per kilogram body weight of beta-alanine for eight
days. In two subjects, this was followed by a further 7 days (days
9-15) when three doses of 20 milligrams per kilogram body weight
per day were given. Subjects reported at 8 am to the blood
collection laboratory on days 1 (prior to any treatment given), 8
and 15 following an overnight fast. Subjects were asked not to
consume any meat containing meal during the 12 hours preceeding the
study. On each of these three test days subjects were catheterized
and an initial blood sample taken when the beta-alanine was
administered at or close to 9 am, 12 noon, and 3 pm. Blood samples
were drawn after 30, 60, 120 and 180 minutes, and analyzed for
changes in the plasma concentration of beta-alanine and taurine. 24
hour urine samples were collected over each day of the study and
analyzed by HPLC to determine the excretion of beta-alanine and
taurine. The treatments are summarized in Table 5.
6TABLE 5 Treatment Day Day 1 Day 8 Day 15 beta-alanine 10 mg/kg bwt
10 mg/kg bwt 20 mg/kg bwt 1 .check mark. .check mark. .check mark.
2 .check mark. .check mark. .check mark. 3 .check mark. .check
mark.
[0087] The plasma beta-alanine concentrations are summarized in
FIG. 9. Each dose resulted in a peak beta-alanine concentration at
one-half hour or one hour after ingestion followed by a decline to
a 0-10 micromolar basal level at three hours, just prior to
administration of the next dose. The response on day 8 of the
treatment tended to be less than on day 1 as indicated by the area
under the plasma concentration curve.
EXAMPLE 4
[0088] The effect of administration of three doses of 40 milligrams
per kilogram body weight of beta-alanine per day (i.e.,
administered in the morning, noon, and at night) for 2 weeks on the
carnosine content of muscle, and isometric endurance at 66% of
maximal voluntary contraction force.
[0089] Six normal male subjects, aged 25 to 32 years, that did not
have evidence of metabolic or muscle disease were recruited into
the study. The subjects were questioned regarding their recent
dietary and supplementary habits. None of subjects was currently
taking supplements containing creatine, or had done so in recent
testing supplementation procedures. The physical characteristics of
the test subjects are summarized in Table 6.
7TABLE 6 Subject Age (years) Weight (kg) 1 29 78 2 31 94 3 29 105 4
25 65 5 31 81 6 25 75 7 53 76
[0090] Two days before treatment, a preliminary determination of
maximal voluntary (isometric) contraction force (MVC) of knee
extensors with the subject in the sitting position was carried out.
MVC was determined using a Macflex system with subjects motivated
by an instantaneous visual display of the force output. For each
subject, two trials were carried out to determine endurance at 66%
MVC sustained until the target force could no longer be maintained
despite vocal encouragement. This first contraction was
subsequently followed by a rest period of 60 seconds, with the
subject remaining in the isometric chair. After the rest period, a
second contraction was sustained to fatigue. Following a second
rest of 60 seconds, a third contraction to fatigue was
undertaken.
[0091] One day before treatment, the subjects reported to the
isometric test laboratory between 8 and 10 am. MVC was determined
and endurance at 66% MVC over three contractions with 60 second
rest intervals, as described above, was determined. Measurements
were determined using the subject's dominant leg. A biopsy of the
lateral portion of the vastus lateralis was taken again from the
dominant leg.
[0092] On day 1 of the treatment study, subjects reported to the
blood sampling laboratory at 8 am following an overnight fast and a
minimum of 12 hours since the last meat containing meal. Following
catheterization and a basal blood sample, each subject followed the
supplementation and blood sampling protocol described in Example 3.
A dose of 10 milligrams per kilogram body weight of beta-alanine
was administered at time 0 (9 am), 3 hours, and 6 hours.
[0093] On days 2-15, subjects continued to take three doses of 10
milligrams per kilogram body weight of beta-alanine.
[0094] In the morning of day 14, post-treatment isometric exercise
tests were conducted on the dominant leg to determine MVC and
endurance at 66% MVC relative to the 66% MVC measured on the day
prior to treatment. In the afternoon, a muscle biopsy was taken of
the vastus lateralis from close to the site of the biopsy taken on
the day before treatment.
[0095] On day 15, the procedures followed on day 1 were repeated to
determine any overall shift in the plasma concentration profile of
beta-alanine and taurine over the 15 days of supplementation. Mean
changes in plasma beta-alanine over 9 hours following the oral
ingestion of 10 milligrams per kilogram body weight of beta-alanine
at 0, 3 and 6 hours on days 1 and 15 when dosing at 3.times.10
milligrams per kilogram body weight per day are shown in FIG.
10.
[0096] One additional test subject (number 7) followed the study,
taking three doses 10 milligrams per kilogram body weight for 7
days followed by three doses of 20 milligrams per kilogram body
weight for 7 days. No muscle biopsies were taken from this test
subject.
[0097] There was no apparent change in the muscle carnosine content
in the muscle of the six subjects biopsied. Changes in plasma
taurine concentrations in the six subjects mirrored those of
beta-alanine, as noted in Example 2.
[0098] Values from the MVC and endurance at 66% MVC measurements
one day before treatment and after 14 days after treatment with
three doses of 10 milligrams per kilogram body weight of
beta-alanine are listed in Table 7. The mean endurance time at 66%
MVC increased in 5 of the 6 subjects. An increase was also seen in
subject 7 taking the higher dose.
8TABLE 7 time @ time @ time @ Total MVC MVC 66% MVC 66% MVC 66% MVC
Contraction 1st try 2nd try 1st 2nd 3rd Time Subject N N seconds
seconds seconds seconds Pre 1 784.5 821.9 48.53 29.03 23.78 100.83
2 814.4 886.2 48.40 26.03 16.90 91.33 3 984.9 970.4 38.15 26.03
16.78 80.95 4 714.6 740.4 89.03 56.15 45.65 190.83 5 1204.8 1217.2
37.65 27.64 21.53 86.83 6 722.4 716.8 46.78 29.40 21.90 98.08 Pre
mean 870.9 892.1 51.4 32.4 24.3 108.1 Pre SD 190.6 184.6 19.1 11.7
10.8 41.2 Post 1 895.6 908.0 47.08 30.38 24.03 101.48 2 832.2 908.0
46.65 31.28 18.40 96.33 3 973.7 952.2 42.65 25.03 16.03 83.70 4
814.1 863.9 114.40 64.28 48.53 227.20 5 1246.6 1233.0 42.03 22.78
19.40 84.20 6 760.8 773.3 52.28 31.53 25.95 109.73 Post mean 920.5
939.7 57.5 34.2 25.4 117.1 Post SD 175.7 156.0 28.1 15.2 11.9 54.9
Subject 7 Pre 858.18 861.54 54.0 Post 792.54 851.41 62.0
[0099] Other embodiments are within the claims.
* * * * *