Department of Biological Sciences, Louisiana State University,
Baton Rouge, Louisiana 70803
 |
INTRODUCTION |
Citric acid, a six-carbon tricarboxylic hydroxy
acid, is heavily used in the food and beverage industries as an
acidulant, a compound that renders food more palatable, and serves a
variety of other functions, such as pH buffer, preservative, synergist to antioxidants, melting and viscosity modifier, and a curing agent
(Gardner 1972
). Citric acid is also known to enhance the palatability of animal foods. For example, citric acid increased daily
food consumption as an additive in horse feed (Betz and Lantner
1980
) and in combination with phosphoric acid enhanced the
flavor of cat food (Kealy 1975
). In the herbivorous fish
Tilapia zillii citric acid increased the palatability of a
nonpreferred diet to a level equivalent to that of the most preferred
feed (Adams et al. 1988
). Although citric acid and its
salt (trisodium citrate) are used to modify the flavor of foods and
beverages, little is known concerning the specific food target(s) or
mechanism(s) of its action. A recent report indicated that citrate
enhanced the preference for sweet compounds and glycine in rats and
that citrate enhanced taste cell responses to both saccharin and
glycine (Gilbertson et al. 1997
). Our study indicates
that trisodium citrate specifically enhances the taste response of the
glossopharyngeal nerve in the carnivorous largemouth bass,
Micropterus salmoides, to particular amino acids. Although
citric acid is often used as a representative sour stimulus (i.e.,
proton donor) in the study of vertebrate taste, the current enhancing
effect is not due to an acidic effect on taste cells as gustatory
enhancement occurred only with stimuli at pH 7-9. A portion of these
results appeared in abstract form (Ogawa and Caprio
1995
).
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METHODS |
Largemouth bass, Micropterus salmoides (~17-25 cm
in length), were obtained from a fish farm (Ken's Hatchery and Fish
Farms; Alapaha, GA) and shipped overnight to Louisiana State
University, where they were maintained in a 250-l fiberglass aquarium
in aerated, charcoal-filtered tap water (artesian well water) at 25°C
and tested within 3 wk of shipment. Before experimentation, the bass were immobilized with an intramuscular injection of Flaxedil (gallamine triethiodide; 0.4 mg/100 g body weight), wrapped in wet tissue paper,
and positioned on a wax block in a Plexiglas container. Aerated
artesian tap water at room temperature (21°C) containing the
anesthetic MS-222 (ethyl-m-aminobenzoate methane sulfonic acid) at 0.05% continually bathed (~500 ml/min) the gills on one side of the animal throughout the experiments. Supplemental Flaxedil was administrated as required. The glossopharyngeal nerve (cranial nerve IX) was targeted for the electrophysiological taste recordings because scanning electron microscopy indicated a higher density of
taste buds occurring on the ventral oral epithelium innervated by IX
than on the lips or rostral palate innervated by the facial nerve
(cranial nerve VII) (Ogawa and Caprio, unpublished). The dorsal portion of the operculum was surgically removed contralateral to
the side receiving the respiratory water flow. An incision was made
along the margin of the first gill arch parallel to the supporting
cartilage to expose the glossopharyngeal (IX) nerve that innervates
taste buds on the floor of the oral cavity. Once freed from adjacent
tissue, the IX nerve was transected and desheathed, and its peripheral
cut end was separated with fine forceps into small nerve bundles. To
create a recording cavity, one or two stitches were made with a fine
surgical needle in the tissue ventral to the incision. Slight tension
was placed on the surgical thread to open the incision and to create a
cavity. Muscle tissue previously surgically removed and collected was
positioned along the periphery of the cavity to create a dam. The taste
activity of the different IX nerve bundles to amino acid stimulation of
the oral cavity was sampled to find one that was responsive to amino
acid stimuli. Preliminary studies indicated better signal-to-noise
taste responses when recording from a smaller nerve bundle than from
the entire nerve. The end of the selected IX bundle was placed over a
platinum hook electrode, and the recording chamber cavity was filled
with halocarbon oil to prevent the nerve bundle from drying during the
recording session. An insect pin placed into tissue adjacent to the
gill arch served as the reference electrode, and the fish was grounded
via a hypodermic needle embedded in the flank musculature. The neural
activity was AC amplified, integrated (0.5 s rise time), monitored
aurally, displayed on an oscilloscope and chart recorder, and recorded
on videotape. The height of peak magnitude of the integrated taste
response was quantified and reported as a percentage of the response to
a standard (either 3 × 10
3 M or 10
1 M
L-arginine for most analyses or 10
1 M
D-arginine). Responses were recorded from 29 dissected IX
nerve branches obtained from 26 fish. Gustatory "enhancement"
occurred if the integrated taste response to the mixture of citrate and an amino acid was significantly greater than the sum of the responses to the components tested separately at the same concentrations as
presented in the binary mixture. Gustatory "suppression" (i.e., response decrement) is therefore a significantly smaller taste response
under the same conditions as stated previously. The effects of
concentration and treatment on the taste response were analyzed as a
two-factor factorial randomized block design (Proc Mixed, SAS 6.12;
= 0.05). Fish were treated as random effects. The interaction between
concentration and treatment was also tested, and when significant the
effect of treatment was tested for significance at each concentration.
Trisodium citrate and individual amino acids (Table
1) were purchased from Sigma Chemical
(St. Louis, MO) and were of the highest quality available. Stimulus
solutions were prepared weekly at 10
1 M in
charcoal-filtered tap water, stored in glass bottles at 4°C, diluted
to the desired experimental concentrations daily, and tested at room
temperature (21°C). Stimulus solutions were added to a 0.5-ml Teflon
loop of a manual sample injection valve (model No. 1106, Omnifit USA;
Atlantic Beach, NY) and injected into the water flow (12 ml/min)
directed to the floor of the oral cavity. The maximum stimulus
concentration delivered was 75% of the concentration injected as
determined by photodensitometry of dye solutions. The undiluted
concentrations of the stimuli are reported in the text. Interstimulus
intervals were
3 min. For experiments reported in Fig. 3, pH of the
test solutions was adjusted with either NaOH or HCl.
 |
RESULTS |
The glossopharyngeal (IXth) taste system in the largemouth bass,
which innervates taste buds on the floor of the oral cavity, responds
moderately but is highly selective to the basic amino acids
L-arginine and L-lysine (Table 1). However,
when presented with 10
3 M trisodium citrate, itself
nonstimulatory, as a component in a binary mixture with L-
or D-arginine, the taste response was significantly
enhanced in comparison with the response to arginine alone (Fig.
1A). Other tested citrate
concentrations in a binary mixture with L-arginine resulted
in either no significant effect (10
5 M and 10
4
M citrate) or in a significant response suppression (10
2
M and 10
1 M citrate) (Fig. 1B). Citrate
(10
3 M) enhanced the taste activity to 10
2
M L-lysine but not to other tested concentrations and had
no significant effect on the response to 10
3 M,
10
2 M, or 10
1 M L-proline (Fig.
2).

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Fig. 1.
A: paired integrated glossopharyngeal taste responses to
10 3 M trisodium citrate (Aa1) to 3 × 10 4 M L-arginine (Aa2) and to
the binary mixture of 10 3 M trisodium citrate and 3 × 10 4 M L-arginine (Aa3);
paired integrated glossopharyngeal taste responses to 10 3
M trisodium citrate (Ab1) to 3 × 10 4
M D-arginine (Ab2) and to the binary
mixture of 10 3 M trisodium citrate and 3 × 10 4 M D-arginine (Ab3); paired
responses are to indicate the repeatability of the responses.
B: dose-response relation of the response to trisodium
citrate ( ) and to the binary mixture of trisodium
citrate and 3 × 10 4 M L-arginine
( ); *: significantly greater taste response to the
binary mixture than to the sum of the responses to 3 × 10 4
M L-arginine (=100) and citrate tested
independently; **: significantly smaller taste response to the binary
mixture than to the sum of the responses to 3 × 10 4
M L-arginine and citrate tested independently; bars,
±SD; mean control response to water + 1 SD is indicated by the line
parallel to the abscissa; pH 9.0; n = 5 fish.
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Fig. 2.
Dose-response relations of the integrated glossopharyngeal taste
response to L-lysine alone ( ), the
binary mixture of L-lysine and 10 3 M Na3
citrate (+ citrate; ), to
L-proline alone ( ), and to the binary
mixture of L-proline and 10 3 M Na3
citrate (+ citrate; ); *: significantly
greater response to the binary mixture than to the sum of the responses
of the components tested individually; : response
to 10 3 M Na3 citrate; pH 9.0;
n = 3 fish.
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The enhancement effect of trisodium citrate on the gustatory response
to L-arginine was due to the citrate ion and not the sodium
cation. A binary mixture of nonstimulatory 10
3 M
trisodium citrate [2.4 ± 1.6% (SD) of the response to
10
1 M L-arginine; n = 7; pH
9.0] and 3 × 10
4 M L-arginine
(26.3 ± 13.4%) dissolved in charcoal-filtered artesian tap water
(cftw), which naturally contains 3 × 10
3 M NaCl,
resulted in gustatory enhancement (53.0 ± 28.6%); however, no
enhancement but a significant response decrement occurred in response
to 3 × 10
4 M L-arginine dissolved in
cftw to which an additional 3 × 10
3 M NaCl was
added (16.9 ± 10.6%; repeated-measure analysis of variance,
P < 0.05; n = 7 fish).
The enhancement effect of citrate on the amino acid taste response was
pH dependent and was observed over the pH range of 7-9 (Fig.
3).

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Fig. 3.
Effect of pH on the integrated glossopharyngeal taste response to
charcoal-filtered tap water adjusted with either NaOH or HCl to pH
4-9, trisodium citrate (10 3 M), L-arginine
(3 × 10 3 M), and to the binary mixture of trisodium
citrate and arginine. *: significant enhancement of the taste response
to the binary mixture of citrate and arginine compared with the
response to 3 × 10 3 M L-arginine and
citrate; the SD of the responses to citrate and the adjusted tap-water
over the range of pH 9-4 was too small to indicate by bars;
n = 3 fish.
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The enhancing effect of 10
3 M citrate was dependent on
the L-arginine concentration. Trisodium citrate
(10
3 M) enhanced significantly the taste responses to
3 × 10
4 M and 10
3 M
L-arginine, and this enhancement occurred whether the
citrate was presented with the arginine as a brief bolus of binary
mixture (Fig. 4; Table
2) or whether the taste buds
innervated by IX were continuously adapted to citrate to which arginine
was then presented (Fig. 5). Although
10
2 M and 10
1 M L-arginine in a
binary mixture with 10
3 M citrate had no significant
effect on the magnitude of the integrated taste response (Fig. 4),
during continuous adaptation of the taste-receptive field to
10
3 M citrate, the responses to 10
2 M and
10
1 M L-arginine were significantly
suppressed compared with the taste response to L-arginine
alone (Fig. 5). Response enhancement also occurred when the
taste-receptive field was continuously adapted to
L-arginine, to which citrate was presented (Fig.
6). In this paradigm gustatory
enhancement occurred at all tested concentrations of citrate
10
2 M.

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Fig. 4.
Dose-response relation of the integrated glossopharyngeal taste
response to L-arginine alone (circle) and to the binary
mixture of L-arginine and 10 3 M
Na3citrate (+ citrate, star). *: significant enhancement of
the taste response to the binary mixture compared with the sum of the
responses to the components tested individually; square, response to
10 3 M citrate; numbers adjacent to each response indicate
the number of fish tested from a total of 10; pH 9.0
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Fig. 5.
Dose-response relation of the integrated glossopharyngeal taste
response to L-arginine alone (unadapted, circle) and to
L-arginine during continuous presentation of 10 3
M trisodium citrate (adapted, star) to the taste-receptive
field. *: significant enhancement of the taste response in comparison
to the sum of the responses to arginine and trisodium citrate tested
independently. **: significant inhibition of the taste response in
comparison to the sum of the responses to the components tested
independently; square, response to 10 3 M
trisodium citrate; numbers adjacent to each response indicates the
number of fish tested from a total of 7; pH 9.0.
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Fig. 6.
Dose-response relation of the integrated glossopharyngeal taste
response to trisodium citrate alone (preadaptation, star; post
adaptation, diamond) and to trisodium citrate during adaptation of the
taste-receptive field to 10 2 M L-arginine
(square). *: significant enhancement of the taste response; pH 8.0;
n = 5 fish.
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The enhancement of the taste response to arginine by citrate is not
stereospecific to the L-amino acid as a binary mixture of
D-arginine and citrate also resulted in a significantly
greater response magnitude than to D-arginine alone (Figs.
1A and 7). A significant
enhancing effect of citrate on the D-arginine taste response occurred only with 10
3 M D-arginine.
This same concentration of L-arginine with
10
3 M citrate resulted in taste enhancement in either the
unadapted (Fig. 4) or adapted (Fig. 5) states (Table 2).

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Fig. 7.
Dose-response relation of the integrated glossopharyngeal taste
response to D-arginine alone (circle) and to the binary
mixture of D-arginine and 10 3 M Na3
citrate (+ citrate, star). *: significant enhancement of the
taste response to the binary mixture in comparison to the response to
D-arginine plus control; square, response to
10 3 M Na3 citrate; pH 8.0;
n = 2 fish.
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DISCUSSION |
This paper shows that the citrate anion enhances the
glossopharyngeal taste response to specific amino acids in the
largemouth bass. A previous study indicated that citric acid caused
increased food consumption, bite size, and rate of feeding in the
herbivorous fish Tilapia zillii (Adams et al.
1988
). Citrate threshold for this enhancing effect in
Tilapia was estimated to be between 10
3 M and
10
2 M, approximately one log unit higher in concentration
than indicated here for the electrophysiological enhancement of citrate
on the taste response to amino acids in the largemouth bass. However, the citrate effect in Tilapia may have in large part been a
pH effect as 1) the citric acid solutions tested had the
lowest pH of the several potential enhancers studied, 2)
Tilapia showed a general feeding response to acidic
substances, and 3) feeding enhancement by the various
potential enhancer substances tested increased dramatically at pH
values of <4.0. In this study, enhancement of IX taste activity in the
largemouth bass was also pH sensitive, but the effect was not due to
acidity of the test solutions as the synergistic effect occurred only
between pH 7 and 9, pH values that are found naturally in pond water
where the fish live (unpublished observations).
The enhancing effect of citrate in the IX taste system of the
largemouth bass is selective for the more stimulatory amino acids.
Citrate enhanced the taste response in the largemouth bass to
L- and D-arginine and L-lysine but
was ineffective in modifying the taste response to the less stimulatory
amino acid L-proline. Although the percent enhancement of
the taste response to both D-arginine and
L-lysine was greater than that reported for
L-arginine (Table 2), the absolute value of the integrated
taste responses to all three amino acids was similar (compare Figs. 2,
4, and 7). In the channel catfish, citrate selectively enhanced the
glossopharyngeal taste response to L-proline but had no
effect on the taste response to either L-alanine or
L-arginine (Davis and Caprio 1996
). These results in both the largemouth bass and the channel catfish are consistent with the hypothesis that the target of the citrate action is
the taste cell and either a specific amino acid receptor site or its
immediate environment.
The enhancing effect of citrate on amino acid taste responses in
carnivorous teleosts observed in the laboratory likely occurs naturally
in the aquatic habitat. Amino acids are naturally occurring in the
environment emanating from all living and decaying organisms. Concentrations of amino acids can be rather high (up to a few hundred
millimoles) in living tissue (Carr 1988
; Carr et
al. 1996
). In addition, because of the citric acid cycle (i.e.,
Kreb's cycle) citrate concentrations in the hemolymph of a variety of
potential food organisms (e.g., insects) for both catfish and bass
exceed 10
3 M and can reach over 3 × 10
2
M (Wyatt 1961
). Although citrate, a cellular
metabolite during aerobic respiration, enhances gustatory neural
activity (Davis and Caprio 1996
; this report) and
ingestion (Adams et al. 1988
) in fish, lactic acid, a
cellular by-product of anaerobic respiration, also increases ingestion
in fish. Lactic acid enhanced the palatability of a synthetic white
muscle extract of the jack mackeral (Trachurus japonicus)
for young yellowtail (Seriola quinqueradiata)
(Kohbara et al. 1993
) and a synthetic mixture of squid
muscle for plaice (Pleuronectes platessa) (Mackie
1982
). Similar to citrate, lactic acid also occurs in tissues
of fish and crustaceans in concentrations as high as 2 × 10
2 M to 7 × 10
2 M (Carr et
al. 1996
). Although the mechanism for the lactic acid effect is
unknown, it is tempting to speculate that lactic acid like citrate may
enhance the taste of specific amino acids present in the extracts.
Citrate also enhanced the taste responses of amino acids in mammals
(Gilbertson et al. 1997
). In 2- and 4-day two-bottle
preference tests, citrate (10
3 M to 2.5 × 10
2 M; pH 7.0) significantly enhanced the preference of
Sprague-Dawley rats for glycine [and also saccharin (5 × 10
4 M), sucrose (10
1 M), and the
synthetic sweetener SC-4567 (5 × 10
5 M)] over
control (noncitrate containing) solutions. Perforated patch-clamp
recordings of isolated rat fungiform taste cells in current-clamp mode
indicated that citrate (5 × 10
3 M) enhanced the
frequency of action potentials in response to glycine (5 × 10
2 M) [and saccharin (2 × 10
2 M)],
which confirmed that citrate has a direct effect on taste cells
(Gilbertson et al. 1997
). Because citrate alone produced small depolarizations of rat taste receptor cells that were
insufficient to generate taste cell action potentials, it was proposed
that the additive depolarizing effects of the citrate and a
depolarizing taste stimulus summed to result in the enhanced action
potential output.
The report of citrate effects in the rat taste system
(Gilbertson et al. 1997
) is consistent with the current
results in the largemouth bass. These data collectively suggest that
citrate might act as a calcium chelator at the surface of the taste
cell microvilli and not as an effector at specific gustatory amino acid
receptor sites. The citrate ion is a tricarboxylic anion that can bind
calcium ions at the surface of taste cell microvilli reducing the
surface potential and shifting sodium channel activation to more
negative potential (Hille 1992
). This chelating effect of the citrate ion on extracellular calcium ions is likely to cause
taste cells of largemouth bass to be depolarized slightly as it did for
rat taste cells (Gilbertson et al. 1997
) and more ready
to activate voltage-gated ion channels (Roper 1983
) on
effective taste cell stimulation. This hypothesis for the mechanism of
action of citrate is consistent with the experimental observations in this report of pH dependence (Fig. 3) and the effects of citrate at
both high arginine (Figs. 4, 5, and 7) and citrate (Fig. 1) concentrations. The lack of a taste-enhancing effect of citrate at pH
values of
6.0 (Fig. 3) is consistent with the hydration of one of the
carboxyl groups of the citrate ion whose dissociation constant at
25°C is 4.0 × 10
7 M. Also, the lack of a
taste-enhancing effect at 10
2 M and 10
1M
L- and D-arginine concentrations (Figs. 4 and
7) is consistent with the large number of positively charged arginine
molecules neutralizing the negative charge of the carboxyl groups of
the citrate ions. It is currently unknown why a suppression of taste responses to 10
2 M and 10
1 M
L-arginine occurred during adaptation to 10
3
M citrate (Fig. 5) but did not occur during their testing in an unadapted preparation as a binary mixture (Fig. 4). It is possible, however, that the continuous presence of citrate as an adapting stimulus lowered the extracellular calcium sufficiently to have a
modulating effect on the gustatory transduction mechanism for arginine.
We thank Dr. T. Gilbertson and two anonymous reviewers for
critically reviewing this manuscript. We also thank R. Bouchard for
assistance with the figures and Dr. E. Obata for partial financial support for K. Ogawa.
Present address of K. Ogawa: Dept. of Otolaryngology, Kagoshima
University Medical School, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.
Address for reprint requests: J. Caprio, Dept. of Biological Sciences,
Louisiana State University, Baton Rouge, LA 70803.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.