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,**1,
*
Department of Clinical Science/Division of Clinical Nutrition,
Center for Nutritional Sciences, and
Multidisciplinary Ph.D. Program in Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40506 and
**
Department of Community Health/Division of Human Biology, Wright State University School of Medicine, Kettering, Ohio 45425
1To whom correspondence should be addressed at Division of Human Biology, Department of Community Health, Wright State University, School of Medicine, 3171 Research Blvd., Kettering, OH 45420. E-mail: leah.maynard{at}wright.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05). Muscle vitamin E concentrations were not significantly
affected by dietary carnosine. Thus, very high levels of dietary
carnosine are associated with increases in carnosine and histidine
concentrations in rat soleus muscle.
KEY WORDS: • fast-twitch glycolytic muscle • fast-twitch oxidative-glycolytic muscle • slow twitch oxidative muscle • histidine-containing dipeptides • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Crush 1970
). The amount of carnosine in skeletal muscle is fairly
constant for specific muscles within a species; however, there are
large variations in concentrations between different muscles within an
individual animal and between the same type of muscles of different
species (Davey 1960
). Carnosine levels are generally
higher in fast-twitch muscles and lower in slow-twitch muscles
(Bump et al. 1990
, Dunnett et al. 1997
).
Only a few studies have reported the effect of carnosine feeding on
muscle concentrations of carnosine (Boissonneault et al. 1998
, Chan et al. 1994
, Hama et al. 1976
, Tamaki et al. 1984
), and an
interrelationship between carnosine and the well-known antioxidant
vitamin E has been documented (Chan et al. 1994
,
McManus 1960
, Neifakh 1966
, Tallan 1955
). Vitamin E deficiency was associated with a concomitant
decrease in carnosine concentrations of skeletal muscle (McManus 1960
, Tallan 1955
), and others suggested an
enhanced antioxidative effect of vitamin E by carnosine (Neifakh 1966
). Chan et al. (1994
) reported elevated
liver concentrations of carnosine when vitamin E was supplemented
jointly with dietary carnosine.
Because the fiber composition of various skeletal muscles
determines the capacity of specific muscles to perform glycolytic and
oxidative processes, it is likely that the role of carnosine in
skeletal muscle may depend in part on the fiber composition of the
skeletal muscle in which carnosine is found. The purpose of this
investigation was to examine the effect of dietary supplementation of
carnosine and/or vitamin E on concentrations of carnosine, histidine
and vitamin E in four specific skeletal muscles: soleus
(SOL),2
lateral gastrocnemius (GAS), red vastus lateralis (RVL) and white vastus lateralis (WVL). These muscles were selected for their varying
percentages of fast-twitch glycolytic (FG), fast-twitch
oxidative glycolytic (FOG) and slow-twitch oxidative (SO) fibers.
The SOL contains 0% FG fibers, 13% FOG fibers and 87% SO fibers; the
GAS contains 8% FG fibers, 62% FOG fibers and 30% SO fibers; the RVL
contains 35% FG fibers, 56% FOG fibers and 9% SO fibers and the WVL
contains 97% FG fibers, 3% FOG fibers and 0% SO fibers
(Armstrong and Phelps 1984
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats were fed a modified AIN-76 diet (American Institute of Nutrition 1977
). Experimental diets were either not
supplemented with vitamin E or carnosine (0 E, 0 C) or were
supplemented as a percentage of diet as follows: 0% vitamin E plus
0.1% carnosine (0 E, 0.1 C), 0.001% vitamin E plus 0% carnosine
(0.001 E, 0 C), 0.001% vitamin E plus 0.1% carnosine (0.001 E, 0.1
C), 0.01% vitamin E plus 0% carnosine (0.01% E, 0 C), 0.01% vitamin
E plus 0.1% carnosine (0.01 E, 0.1 C) and 0.001% vitamin E plus 1.8%
carnosine (0.001 E, 1.8 C). These diets were selected to represent
deficient (0%), minimally adequate (0.001%) and sufficient (0.01%)
levels of vitamin E and no (0%), moderately high (0.1%) and
pharmacological (1.8%) levels of carnosine. Vitamin E was supplemented
as dl-
-tocopherol acetate (Roche Vitamins & Fine
Chemicals, Nutley, NJ). Carnosine was purchased from Sigma Chemical
Company (St. Louis, MO). Glycine (Dyets, Bethlehem, PA) was added to
the carnosine-deficient diets isonitrogenously relative to the
0.1% carnosine diet. Rats were fed the specified diets for 8 wk.
Of the 84 rats used in this investigation, 6 randomly selected
rats from each experimental diet were exercised on a motorized
treadmill according to a modified procedure of Alessio and Goldfarb (1988
). The exercise protocol was designed to elicit
in vivo oxidation within skeletal muscle and consisted of a single 70-s
run on a motorized treadmill at a grade of 0% and a speed sequence of
16 m/min for 5 s, 31 m/min for 5 s and 45 m/min for 60 s. Rats were familiarized with the treadmill by walking at 16 m/min for
4 min on the evening before their sprinting activity. Exercise had no
significant effect on muscle concentrations of carnosine and histidine;
therefore, all data for these variables are reported regardless of the
exercise treatment. Vitamin E concentrations in the GAS were
significantly lower in exercised rats, so only the data from
nonexercised rats were used for the vitamin E analyses.
Rats were anesthetized with sodium pentobarbital (35 mg/kg body weight) and were killed by cervical dislocation 6 h after exercise. The SOL, GAS, RVL and WVL from the right and left hindquarters were removed. Muscles were weighed and immediately frozen in liquid nitrogen. Frozen samples were stored at -70°C for later analyses. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.
Thawed muscle samples were divided for determination of the carnosine, histidine and vitamin E concentrations. The SOL was not large enough to perform all tests, so only carnosine and histidine concentrations were determined in this muscle.
Carnosine and histidine analyses.
An 8% muscle homogenate was made with 0.36 mol/L perchloric acid, and to this we added 50 µL of 2 mmol/L homocarnosine as the internal standard. The homogenate was heated in a boiling water bath for 5 min and centrifuged for 10 min. The supernatant was stored in the freezer at -20°C until later analysis.
Carnosine and histidine concentrations of skeletal muscle were analyzed via HPLC. The solvent delivery system (model 510), manual injector (model U6K), system interface module and column oven were from Waters (Millipore, Milford, MA). Fluorescence was measured with a Spectroflow 980 programmable fluorescence detector from ABI Analytical (Kratos Division, Ramsey, NJ). Millivolts were converted to analogue signals and sent to an IBM computer, where a Maxima software package (Waters, Millipore) was used to analyze data. A 5-µm Hypersil C18 ODS column (250 mm x 46 mm) from Alltech Associates (Deerfield, IL) was used for sample separation.
Procedural conditions for the measurement of muscle levels of carnosine
and histidine were modified from those of Teahon and Rideout (1992
). Thawed supernatant was filtered with a 0.45-µm Gelman
filter, and 50 µL of the filtered sample was derivatized with 150
µL of o-phthaldehyde reagent. The column was initially
equilibrated with 90% solvent A (10% methanol in 0.3 mol/L sodium
acetate, pH 5.5) and 10% solvent B (80% methanol in 0.3 mol/L sodium
acetate, pH 5.5). Glacial acetic acid was used to adjust pH in both
solvents. The gradient was varied during a 50-min run (90% A and 10%
B for 20 min, 75% A and 25% B for 15 min, 65% A and 35% B for 5
min, 40% A and 60% B for 2 min and 90% A and 10% B for 8 min). The
flow rate was constant at 1.5 mL/min, and the column temperature was
maintained at 30°C. Fluorescence was determined at an excitation
wavelength of 315 nm and an emission wavelength of 370 nm. Retention
times of unknown compounds were compared with retention times of
carnosine, histidine and homocarnosine standards. Data were expressed
as mol carnosine or histidine/mg protein. Total protein was determined
using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,
CA).
Carnosine, histidine and homocarnosine standards were >99% pure
and were obtained from Sigma Chemical Company. Sodium acetate was
purchased from Sigma Chemical Company. Methanol (HPLC grade),
perchloric acid and acetic acid were purchased from Fisher Scientific
(Pittsburgh, PA). o-Phthaldehyde reagent was obtained
from Pierce (Rockford, IL). Protein Assay Dye Reagent Concentrate and
bovine 
-globulin were purchased from Bio-Rad Laboratories.
Vitamin E analysis.
Thawed muscle samples were diluted in precooled phosphate buffer (1.15% KCl in 0.05 mol/L, pH 7.4) to obtain a 10% homogenate and then extracted with ethanol/hexane (1:4.5 vol/vol). A 4-mL portion of the hexane layer was removed and evaporated under nitrogen. Samples were stored at -20°C for later analysis of vitamin E.
Extracted samples were reconstituted with 100% methanol and were
analyzed for vitamin E concentration using HPLC according to the
modified procedure of Hatam and Kayden (1979
). Solvent
delivery system (model 501), sample processor (WISP model 712) and
system interface module were from Waters (Millipore). Fluorescence was
determined using a Spectroflow 980 programmable fluorescence detector
from ABI Analytical (Kratos Division) at an excitation wavelength of
205 nm and an emission wavelength of 345 nm. An RCM-100 radial
compression separation system (Waters, Millipore) with a 5-µm C18
Nova Pak (5 mm x 10 cm) radial-pack cartridge column (Waters)
was used to separate samples. The isocratic solvent system consisted of
100% methanol. Flow rate was variable (1 mL/min for 5 min and then 1.5
mL/min for 1 min).
Standard dl--tocopherol was obtained from Sigma
Chemical Company. Methanol (HPLC grade) was purchased from Fisher
Scientific.
Statistical analyses.
To determine the effect of a moderately high dose of carnosine on muscle concentrations of carnosine, histidine and vitamin E at deficient, minimally adequate and sufficient levels of dietary vitamin E, rats fed the two lower doses of carnosine (0 and 0.1%) at each of the three levels of vitamin E (0, 0.001 and 0.01%) were compared (analysis 1). In a separate analysis (analysis 2), rats fed the pharmacological dose of 1.8% carnosine at the minimally adequate level (0.001%) of vitamin E were compared with rats fed no (0%) and moderately high (0.1%) levels of carnosine at the same level of vitamin E.
Data were analyzed using a mixed-effects model with muscle
concentrations of carnosine, histidine and vitamin E as dependent
variables and diet, exercise treatment, muscle fiber type and all
possible interactions between diet, exercise and muscle type as the
independent variables (SAS Institute, Inc., Cary, NC). The diet and
exercise treatments were nested within each individual rat in the
random effects statement. Data are presented as least square means
(lsmeans) ± SEM. A Tukey post hoc test was used to
determine significant differences between means. Significant
differences were defined at P 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Diet had no effect on food consumption, body weight or muscle weight of
rats (data not shown). There were no significant dietary effects on
muscle carnosine (Table 1
). Higher levels of carnosine were observed in the WVL than in the other
three muscle groups (P 0.0001; Fig. 1
).
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, and were greater in SOL muscle than in the
other three muscles (P 0.0001; Fig. 1
).
Concentrations of vitamin E in GAS were higher in rats fed 0.01%
vitamin E than in rats fed the lower doses of vitamin E (P
0.0001) (Table 1)
. Concentrations of vitamin E were greater in
the GAS than in the RVL or WVL (P 0.0001). A
significant muscle x diet interaction on concentrations of
vitamin E (P 0.0001) was also observed.
Analysis 2.
A significant effect of diet was found on food consumption
(P 0.008; data not shown). Rats supplemented with
1.8% dietary carnosine consumed significantly more food than did rats
deficient in carnosine (P 0.008) and slightly more
than those supplemented with 0.1% carnosine (P 0.066). Despite the greater food consumption by rats supplemented with
1.8% dietary carnosine, there were no differences in body or muscle
weight among groups.
Rats fed 1.8% carnosine had significantly greater concentrations of
carnosine in the SOL than did rats fed either of the other two doses of
carnosine (P 0.0001; Fig. 2
). A significant muscle x diet interaction (P 0.007) was also associated with muscle carnosine levels.
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0.0001) (Fig. 2)
. The muscle x diet interaction was also significant (P 0.0003).
No significant diet effect due to carnosine supplementation was
observed at minimally adequate levels of vitamin E; however, high doses
of carnosine tended to be associated with increased vitamin E levels
within each muscle group (P 0.13; data not shown). A
significant effect due to muscle type was observed (P
0.0001); vitamin E levels were significantly different between
each muscle type (data not shown).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Boldyrev 1993
), intracellular buffering
(Mannion et al. 1995
, Sewell et al. 1992
), calcium influx (Batrukova and Rubtsov 1997
) and other functions (Boldyrev and Severin 1990
, Tan and Candlish 1998
). Because the fiber
composition of various skeletal muscles determines the capacity of
specific muscles to perform glycolytic and oxidative processes, it is
likely that the role of carnosine in skeletal muscle may depend in part
on the fiber composition of the skeletal muscle in which carnosine is
found. Therefore, the focus of this investigation was to describe the
effect of dietary carnosine and vitamin E on concentrations of
carnosine, histidine and vitamin E in specific skeletal muscles
selected for their varying percentages of FG, FOG and SO fibers.
Feeding of 0.1% carnosine did not significantly increase carnosine
concentrations in any of the muscles studied (SOL, GAS, RVL or WVL).
This finding is in agreement with Chan et al. (1994
),
who reported that feeding of 0.0875% dietary carnosine did not
increase carnosine levels in skeletal muscle. In the present study,
dietary carnosine at levels of 1.8% of the diet were associated with
elevated muscle concentrations of carnosine. The point between 0.1 and
1.8% at which dietary carnosine begins to be associated with increased
muscle concentrations is not known, although Tamaki et al. (1984
) reported that dietary intake as high as 0.9% did not
significantly increase muscle carnosine levels. Tamaki et al. (1984
) also reported that 1.8% dietary carnosine increased
muscle concentrations of carnosine in the GAS to approximately twice
that observed in control animals. In the current investigation, 1.8%
dietary carnosine tended to increase GAS levels nearly 100%,
whereas increases in SOL muscle were more pronounced, increasing
approximately fivefold. Dietary carnosine at 1.8% did not
significantly alter levels of carnosine in RVL or WVL.
High dietary carnosine (1.8%) appears to preferentially increase carnosine concentrations in red, SO muscle. When dietary intake of carnosine was 0.1%, carnosine levels were not affected and followed the classic trend reported in the literature of higher levels measured in the muscles with the highest glycolytic activity and lower levels in muscle with the highest oxidative ability. A continual utilization of carnosine in the more oxidative muscle may account for the lower levels in this muscle type.
The mechanism by which carnosine becomes preferentially incorporated
into the red muscle is not known. Dietary carnosine appears to be
readily absorbed intact, primarily in the jejunum by a
carrier-mediated transport system (Ferraris et al. 1988
). Although some hydrolysis of carnosine may occur within
the cells of the small intestine (Ferraris et al. 1988
),
most of the carnosine is thought to be hydrolyzed in the plasma, where
only very small amounts of carnosine are detected (Chan et al. 1994
, Tamaki et al. 1984
). Thus, these previous
studies suggest that the precursors of carnosine are likely
incorporated into the muscle, where carnosine is then resynthesized.
Although our investigation was not designed to elucidate a metabolic
mechanism, data obtained from the present investigation support the
suggestion that precursors of carnosine are preferentially incorporated
into the SO muscle. High levels of dietary carnosine (1.8%) increased
histidine concentrations by approximately twofold in SOL muscle.
To speculate why carnosine and histidine would be preferentially
incorporated in SO muscle, one might consider the role of carnosine as
an antioxidant. If the primary role of carnosine is to serve as an
antioxidant, then it is reasonable to expect that the formation of
carnosine would take place in the muscle that is primarily oxidative.
Previous investigations have measured higher carnosine concentrations
in white, FG muscle, suggesting that carnosine is preferentially
incorporated into these sites where it serves primarily in a buffering
capacity (Davey 1960
, Sewell et al. 1992
). However, if carnosine is continually utilized as an
antioxidant in SO muscle such that pools of carnosine are diminished,
then its presence (as well as its possible biological importance) may
have gone undetected. Recent evidence by Tanaka (1995
)
supports this possibility. Tanaka (1995
) reported a
reduction of carnosine levels in FG and SO muscle of rats after 5–20
wk of endurance training. Although Tanaka (1995
)
suggested an adaptive mechanism resulting from training, it is also
possible that the carnosine levels were lower in exercised muscle due
to the utilization of carnosine during this highly oxidative activity.
Until further investigations designed to discern the role of carnosine
in in vivo skeletal muscle are conducted, explanations for the
preferential incorporation of carnosine and histidine in SO muscle with
high doses of dietary carnosine are only speculative.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received July 10, 2000. Initial review completed August 15, 2000. Revision accepted November 21, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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