Taste effectiveness of anomers of sugars and glycosides as revealed from hamster taste responses

Camp. Biochem. Phytioi., 1974, Vo’of.48A, pp. 249 to 262. Pmgmmm Press. Printed in Great Bdaii

TASTE

EFFECTIVENESS OF ANOMERS OF SUGARS GLYCOSIDES AS REVEALED FROM HAMSTER TASTE RESPONSES

AKINORI

NOMA,

MASAYASU

SATO,l

and YOJIRO

AND

TSUZUKIa

Medical School, Kumamoto; ‘Department of Physiology, Kumam oto Giver&y aDepartment of Chemistry, Tokyo University of Science, Tokyo, Japan

and

(Receiered20 June 1973)

Abstract-l.

The effectiveness of anomers of sugars and glycosides for hamster taste receptors was examined by recording responses in the whole nerve or single fibers of the chorda tympani. 2. or-Anomers of D-ghCOSe and D-galactose were less effective than their @namers while the reverse was true for the anomers of n-methylglucoside and ~-me~ylg~ac~side. 3. Little difference was observed between the D- and L-forms of glucose and xylose.

INTRODUCTION

the sweetness of sugars varies with the experimental method and the physical state of the compounds, common sugars can be listed in order of decreasing sweetness as fructose, sucrose, glucose, galactose, mannose, lactose and rafiose (Biester et al., 1925 ; Cameron, 1947; Schutz & Pilgrim, 1957). On the other hand, to determine the relative taste effectiveness of various sugars, a number of electrophysiological studies has been made by recording the chorda tympani responses to lingual stim~ations in the dog (Andersen et af., 1963) and in the rat (Hagstrom & Pfa~~, 1959; Noma et al., 1971). Both sets of experiments, psychophysical and ele~rophysiologi~, no~ithstanding the difference in species and method of experiments, yielded results indicating an approximate agreement in the order of effectiveness of sugars. Reports indicate further that the sweetness of sugars depends on their stereochemical structures, i.e. pairs of enantiomorphic sugars present different degrees of sweetness from each other. According to Boyd & Matsubara (1962) D-glucose is about two-thirds as sweet as sucrose, but L-glucose has no taste, and D-mannose is sweeter than L-mannose. This, however, was later denied by Shallenberger et aZ. (1969), who stated that pairs of enantiomorphs did not differ in their ability to elicit a sweet taste. Differences in sweetness between a- and j%anomers of sugars have also been noted in the past. The sweetness of cY-D-fructose is about one-third of that of @-fructose (Tsuzuki & Yamazaki, 1953), and the sweetness of a-r..-rhamnose is ALTHOUGH

249

250

AKINORI NOMA, MASAYASU SATO AND YOJIRO TSUZUKI

less than

two-fifths of that of @-L-rhamnose (Tsuzuki & Mori, 1954). Tsuzuki (1947) pointed out that the sweeter form of the reducing sugars always has cishydroxyl groups on the carbonyl and adjacent carbon atoms while in the less sweet isomer the hydroxyls on the two carbon atoms are trans. It has also been reported that the a-anomers of D-ghCOSe and n-galactose are sweeter than /Lanomers (Schutz & Pilgrim, 1957; Pangborn & Gee, 1961) while ar-n-lactose is not as sweet as p-n-lactose (Pangborn & Gee, 1961; Shallenberger, 1963). However, Shallenberger (1963) reported that, when crystals of the two D-glucose anomers were compared, the /3-anomer was slightly sweeter, although cy-n-galactose is sweeter than the p-form (Shallenberger et ai., 1965 ; Shallenberger & Acree, 1971). The difference in taste between the w and p-forms of glycosides is also well known. The or-forms are, in general, sweeter than the p-forms and the latter possesses a bitter after-taste (Tsuzuki, 1947; Shallenberger & Acree, 1971). von Frisch (1935) reported in his classical studies on the taste response of bees that a-methyl-glucoside was one-fourth as effective as sucrose while /?-methylglucoside was ineffective. Detheri (1955) also found that ol-methylglucoside was more effective than /I-methylglucoside in stimulating the labellar and tarsal hairs of the blow fly. The present study was started first to determine the difference between the 01-and /5-anomers of D-glucose and n-galactose as well as of n-methylglucoside and D-methylg~actoside in the effect on the gustatory receptors by recording the response from the whole chorda tympani of hamsters. As single chorda tympani fibers of hamsters respond to more than two kinds of the stimuli representing the four primary tastes (Ogawa et aZ., 1968; Frank, 1972) and consequently the whole chorda tympani response reveals the collective response of the population of receptors in the tongue, in later experiments impulse discharges in response to the above anomers from single chorda tympani fibers were examined. The differences in effectiveness between D- and L-glucose, D- and L-xylose and among various common sugars were also examined in the present study. MATERIALS

AND METHODS

Golden hamsters (~esoc~ice~us awatus) were chosen for the mater&I because of their high sensitivity to sugars (Beidler et al., 1955). The hamsters, weighing 100-150 g, were anesthetized by intraperitoneal injection of sodium amobarbital (70 mg/kg body wt.). The chorda tympani was surgically exposed at the submandibular region and centraily cut. Responses of the chorda tympani were monitored by an oscilloscope and recorded with a pen-writing recorder, using a pair of Ag-AgCl electrodes, an amplifier and an integrator circuit of a time-constant of 0.5 set (Beidler, 1953). For recording impulses in a single chorda tympsni unit, a few fibers were dissected alive with a pair of needles from the distal portion of the cut chorda tympani, and impulse discharges resulting from chemical stimulation of the tongue were photographed by a kymographic camera. However, for carrying out experiments on single units, response characteristics of each unit were examined first by applying O-1 M NaCI, 0.02 M quinine hydrochloride, 0.01 N HCI and 0.5 M sucrose as basic stimuli, and the units highly responsive to sucrose or quinine were selected for the experiments.

TASTEEFFECTIVENESS OF SUGARANOMERS

251

For stimulation of gustatory receptors the tongue was enclosed in a flow chamber made of glass (Yamashita & Sato, 1965), through which 100 ml of a test solution were passed at a constant rate. The tongue was rinsed with 500 ml of tap water after each stimulation. In order to avoid the effect of the preceding stimulus, the interval between successive stimulations was kept at about 3 min. The temperature of both test solutions and water was maintained at 30°C to avoid the thermal response in the nerve (Yamashita 8z Sato, 1965). As stimuli for examining the responses of the chorda tympani to sugars, 0.5 M solutions of sucrose, maltose, D-fructose, D-mannose, D-glucose, D-@lactose, D-xyloSe, L-sorbose and L-rhamnose were used. For the experiments on single-fiber responses sucrose, D-fiWCtOSe, D-glucose and D-mannose of varying concentrations were used and, in addition, the effects of sorbitol and mannitol were examined in a few experiments. All of these sugars were of reagent grade and commercially available. For examining the difference in neural response between the a- and fl-anomers of sugars, the a- and p-forms of D-glucose, D-galactose, D-methylglucoside and D-methylgalactoside were employed at a concentration of O-5 M. The purity of these anomers was confirmed both by melting point and specific rotation determinations (Table 1). Since a-glucose and a-galactose become gradually less sweet after dissolution because of the conversion in part into p-anomers (Tsuxuki & Mori, 1954), the above anomers were applied to the tongue at 30°C about 3 min after the solutions had been made up. In a few experiments, in which differences in effect between pairs of enantiomorphic sugars were investigated, 0.5 M solutions of the D- and L-forms of glucose and xylose (guaranteed reagent grade, Sigma Chemical Company) were used as stimuli. In these experiments the effect of 3-0-methylglucoside was also examined to compare ita effect with those by a- and p-methylglucosides. TABLE l-ClWWJTERISTIC

CONSTANTS OF ANOMERS Melting point (“C)

Anomers

IaID” (“)

a-D-Glucose B-D-Glucose

143-5 145-8

+116 + 18 (OOC)

a-n-&lactose p-D-&lactose

165-6 164-5

+152 + 59

Methyl-a-D-glucoside Methyl-j?-D-glucoside

162-3 109-10

+ 158.4 - 37

Methyl-a-D-galactoside Methyl-p-D-galactoside

101-3 175-7

+174 + 4

RESULTS

Relative effectiveness of various sugars and their anomers, determined by the chorda tympani nerve response Integrated responses of the chorda tympani nerve to O-5 M solutions of sugars attained a peak within a few seconds after stimulation and declined gradually to a steady-response magnitude after the peak response. The relative magnitudes of

252

AKINORI NOMA, MASAYASU SATO AND YOJIRO TSLZUKI

responses to nine kinds of common sugars, obtained from five experimental results, are shown in Table 2. The order of effectiveness of these sugars, determined from the magnitudes of responses at 5 and 10 set after stimulation, is sucrose > fructose > mannose > glucose > sorbose > maltose + rhamnose > galactose > xylose. This order is in approximate agreement with that obtained from the chorda tympani of rats by Noma et al. (1971) except that in the hamster sorbose and xylose are relatively less effective than in the rat.

TABLE 2-RELATIW

Sugars (0.5 M) Sucrose D-Fructose D-Mannose D-Glucose L-Sorbose Maltose L-Rhamnose n-Galactose D-Xylose

MAGNITUDE OF RESPONSES OF THE CHORDA TYMPANI NERVE TO NINE SUGARS

Initial peak magnitude

100

82 + 4.4 69 + 13.0 6.5 + 8.2 64+ 6.2 66 + 12.1 61 + IO.5 59+ 8.0 52 + 8.9

Magnitude at 5 set after stimulation

(1) (2) (3) (5) (4) (6) (7) (8) (9)

100

76+ 5.1 66 rt: 13.3 57+ 6.9 50 + 12.9 48 f 13.3 47f 4.1 43 f 7.0 42 It 5.3

(1) (2) (3) (4) (5) (6) (7) (8) (9)

Magnitude at IO set after stimulation 100 61 + 7.4 51 + 9.3 44+ 7.7 38 f 5.0 37f II.8 38& 7.4 33 f 8.6 30f 4.6

(I) (2) (3) (4) (5) (7) (6) (8) (9)

Order of effectiveness in rat*

(1) (2) (4) (5) (3) (6) (8) (9) (6)

* Results reported by Noma et al. (1971). All values indicate means + SD. of five experimental results, expressed relative to the magnitude of response to 0.5 M sucrose, except for the initial peak magnitudes, which were obtained from four experiments. Numerals inside parentheses represent the order of effectiveness of sugars, determined by using the Kendall coefficient of concordance (Siegel, 1956).

Integrated responses of the chorda tympani to the w and /3-anomers of Dglucose, D-galactose, D-methylglucoside and D-methylgalactoside are presented in Fig. 1. In this figure the ol-anomers of n-glucose and n-galactose produced a response smaller than that by the /3-anomers, while the responses to the ol-anomers of n-methylglucoside and D-methylgalactoside are greater in magnitude than those produced by the /Lanomers. Similar results can also be seen in Table 3, in which the average magnitudes of responses to these sugars, obtained from three experimental results and expressed relative to that for 0.5 M sucrose, are presented. Although, because of a small number of experiments, it was impossible to test the significance of difference in magnitude between the responses to LY-and /3-anomers, in all the three experiments carried out /3-anomers of glucose and galactose yielded a greater response magnitude than ol-anomers and the reverse was true for the anomers of methylglucosides and methylgalactosides.

TASTE

EFFECTIVENRSS

OF SUGAR

253

ANOhBRS

20 s?c

O.~t~~~actoside

RDgalactosea.~~~~~oside

a~taciose

FIG. 1. Integrated responses of the chorda tympani nerve of a hamster to 0.5 M solutions of the ar-and p-anomers of D-ghCOSe, n-galactose, n-methylglucoside and n-methylgalactoside.

TABLE

3---RELATrW

B -ANOMERS

OF

MAGNITUDES

D-GLUCOSE,

OF

RESPONSES

D-GALACTOSE,

sugars (0.5 M) a-D-Gkxose

~-~*~a~~e /kkilactose

or-n-Methylglucoside p-n-Methylglucoside a+n-Methylgalactoside p-n-Methylgalactoside

OF

TI-IE

CHORDA

D-METHYLGLUCOSIDE

Initial Peak magnitude

56 75 63 110 81 48 59 51

TYMPIU’I

AND

TO

THE

a-

AND

D-MRTHYLGALACTOSIDE

Magnitude at 5 set after stimulation

Magnitude at 10 set after stimulation

37 52 61 112 75 42 56 47

30 52 47 81 54 38 47 41

All values were the means of three experimental results, in which each response magnitude was expressed relative to that for 0.5 M sucrose.

Fiber cJaa~act~~t~cs and responses to sugars Responses of eight chorda tympani fibers to lingual stimulations by sugars were successfully recorded in the experiments. These units were selected for the experiments because of their high sensitivity to sucrose or quinine. Response profiles of these units for O-5 M sucrose (S), O-1 M NaCl (Na), 0.01 N HCl (H) and 0.02 M quinine (Q) are presented in Fig. 2. Fibers A, B, C and H were predominantly responsive to sucrose. Fibers D and E responded most strongly to NaCl and to HCl, respectively, and next to sucrose. However, fibers F and G yielded their greatest response to quinine, but responded little to sucrose. Responses of the above fibers to various sugars of varying concentrations were examined in the experiments. A typical example of the relationship between the stimulus concentration and the response magnitude for four kinds of sugars as well as sorbitol and mannitol, obtained from a fiber highly sensitive to sucrose (fiber B), is demonstrated in Fig. 3. As shown in this figure, sucrose is most effective for producing impulses in this unit, its threshold was below 0.01 M, while the

254

AKINORI NOMA, MASAYASU SATO AND YOJIRO TSUZUKI

250 -

n

,I7 200 -

x 150I% N 2 J!?looz. ,E

1

A

i

B D H C

50 -

E

O-

E-

s

NaH

Q

FIG. 2. Response profile of eight hamster chorda tympani fibers for 0.5 M sucrose (S), O-1 M NaCl (Na),O*Ol N HCl(H) and 0*02M quinine hydrochloride (Q). Each fiber was represented by different symbols. 0 sucrose 0 maltose A fructose A glucose n sorbitol l mannitol

g200In Lo .

O0.01

0.02

0.05

a1

02

I

Concentration (Tl) FIG. 3. Relationship between the concentration of sugars and the numbers of impulses elicited during the initial 5 set after lingual stimulation in a chorda tympani fiber (fiber B in Fig. 2).

threshold for other stimuli was between 0.1 and 0.2 M. The effectiveness of the four sugars demonstrated in Fig. 3 is in the order of sucrose > fructose > glucose > maltose, and sorbitol and mannitol are less effective than the sugars. Such facts were commonly observed also in other fibers examined. The relative response magnitudes for 0.5 M sucrose, fructose, glucose and maltose, calculated from the

TASTE EFFECTIVEXIBS OF SUGAR ANOMEFIS

255

number of impulses discharged during the initial 5 set after stimulation, are 100 : 67 : 42 : 38 in Fig. 3 and the average values from three units (fibers B, C and D) are 100 : 51 : 36 : 15. Two fibers highly sensitive to quinine also responded to sugars at high concentrations such as 1 or 2 M, but their responses were different from those highly sensitive to sucrose in that the former showed a higher threshold for sugars than the latter and responded to fructose and glucose at high concentrations better than sucrose. The response of quinine-sensitive fibers to sugars was also characterized by their temporal pattern of impulse discharges. Impulse discharges produced by various sugars in fibers highly sensitive to sucrose usually consisted of the initial discharge at a high rate and a subsequent steady discharge (Fig. 4a). However, in fibers sensitive to quinine impulses in response to fructose and glucose appeared with a very long latency, such as about 10 set after stimulation, and thereafter the number of impulses increased gradually, although the response to quinine attained a maximum about 5 set after stimulation (Fig. 4b).

OL

I I I I I II 1

5

9

11 a

l7

0 ““I 1 5

9

“1

13

17

Time (set)

FIG. 4. Time course of impulse discharges produced by 0.5 M sucrose, 0.02 M quinine hydrochloride, 2 M fructose and 2 M glucose in two chorda tympani fibers. A. Unit sensitive to sucrose (fiber E in Fig. 2). B. Unit predominantly sensitive to quinine (fiber F in Fig. 2).

Dajkence in chorda tympaniJiber response between pairs of enuntiomorphs Responses to O-5 M solutions of D- and L-glucose, and of D- and L-xylose were examined in four chorda tympani fibers predominantly responsive to sucrose and in two fibers highly sensitive to quinine. No detectable responses were recorded from the latter. Impulse discharges elicited by the four basic taste stimuli and D- and L-glucose and xylose in a sucrose-sensitive fiber (fiber A in Fig. 2) are demonstrated in Fig. 5. As shown in this figure the L-forms of sugars elicited impulse discharges to nearly the same extent as those produced by the n-forms. The numbers of impulses elicited by two pairs of enantiomorphic sugars in four

256

AKXNORI

~~M~,~~~A~~su

SATO AND YOJXRO TSWWKI

sucrose-sensitive fibers are presented in Table 4. No substantial difference in effect can be found between n- and t-xylose. The effect of n-glucose in fiber E is neariy the same as that by L-glucose, but in fiber A the latter is far smaller than the former. However, because of the small number of experiments it cannot be concluded here whether the effect by n-glucose is really greater than that by t-glucose. It is most probable that little difference in effect would exist between n- and Lglucose as in the case of xylose. TABLE

~--MAGNITUDES

OF RESPONSES TO D- ANO L-RNANT~~MoRPH~ CHORDA TYMPANI FIBERS

of s_rGiuxsIN FOUR

Fibers A

E SUCtOW n-Glucose L-Glucose D-X&W2 L-Xylase

44 41 42 25 32

(40) (12) (6) (19) (11)

193 109 62 114 118

(124) (21) (21) (33) (42)

B

c

150 (100) 5Q (13)

90 (49) 27 (1) 16 (0) 23 (0)

52;) 50 (13)

Mean 119 57 52 52 56

(78) (12) (14)* (15) (19)

* Mean values of two experimental results. Numerals outside and inside parentheses indicate numbers of impulses elicited during the initial 5 set and the next 5 set after stimulation in an individual fiber respectively.

ofa-

E~~c~iv~~~~s

and ~-fa~~

of.w..ws

Responses of seven chorda tympani fibers to 0.5 M solutions of IY-and P-D@-n-galactose, 01-and /3-n-methylglucoside and 01-and p-n-methylgalactoside were examined. Five out of these fibers were highly sensitive to sucrose (fibers A, B, C, D and E in Fig. 2), while the other two were sensitive to quinine but scarcely to sucrose (fibers F and G). The five sucrose-sensitive fibers responded well to the above anomers, but the latter two quinine-sensitive fibers yielded little response. Responses to the anomers in one of the sucrose-sensitive fibers (fiber A) are demonstrated in Fig. 5, As shown in this Figure, all these anomers yielded impulse discharges, characterized by rhythmic bursts of impulses, which are especially marked in the response to ~-m~thyl~lu~oside= The numbers of impulses produced by the cy- and p-anomers during the initial 5 set and the next 5 set after stimulation in five sucrose-sensitive fibers are presented in Fig. 7 together with those produced by sucrose. The average magnitudes of responses to the anamers, obtained from responses in five units, are also summarized in Table 5. As shown in Fig. 7 and Table 5, little difference is found in effect between the a+ and /3-forms of glucose and galactose, though the magnitude of response to ,%glucose during 5-10 set after stimulation is greater in four units than that for c+glucose. Therefore, it was not possibIe from the present experiments on single chorda tympani fibers to reveal significant differences in effect between the OL-and /I-anomers of glucose, cr-and

TASTEEFFECTIVBNESS

OFSUCAR

257

ANOMRRS

4

quinine

,

sucrose

...... ................................................................................

FIG. 5. Impulse discharges elicited in a chorda tympani fiber (fiber B in Fig. 2) by 0.1 M NaCl, 0.02 M quinine hydrochloride, 0.01 N HCI, 0.5 M sucrose and 0.5 M solutions of the D- and L-forms of glucose and xylose. Arrows indicate the first spike after stimulation.

TABLE

~-MEAN

MAGNITUDES OF RESPONSES TO THE C+ AND p-ANOMERS GLYCOSIDES IN CHORDA TYMPANI FIBERS OF HAMSTERS

Sugars (0.5 M) Sucrose o-n-Glucose @-Glucose o-n-Calactose /I-n-Calactose or-n-Methylglucoside /?-n-Methylglucoside ar-n-Methylgalactoside /?-n-Methyigalactoside

No. of impulses during the initial 5 set after stimulation 121+ 51 55 f 24 58 + 29 35 + 21 32 + 12 103 f 55 35+15 48zb25 34 f 19

(100) (45) (48) (29) (27) (85) (29) (40) (28)

OF SUGARS AND

No. of impulses during the next 5 set after stimulation 85 + 34 21+ 25 35 + 43 13 + 7 12f 6 46 + 28 13+11 24 + 23 17 + 14

(100) (25) (41) (16) (15) (54) (16) (28) (21)

Numerals outside parentheses represent means + S.D. of number of impulses during the initial 5 set after stimulation, obtained from five experimental results, but those for n-galactose are the means of four experiments. Numerals inside parentheses indicate mean values expressed relative to the magnitude of the response to sucrose.

AKINORI NOMA,

258

MASAYASU SATO AND YOJIRO TSUZUKI

4

a-Dgl3: i3-D-qhxcse I

1

,,

811 1 I D &_&#&

1. III

I.

1 1.

II

. I

I

30.methylglucosidc

I FIG. 6. Impulse discharges elicited in a chorda tympani fiber predominantly sensitive to sucrose (fiber B in Fig. 2) by 0.5 M solutions of the (Y- and @namers of D-&COSe, D-g&CtOSe, n-methylglucoside, D-methylgalactoside and 3-O-methyiglucoside. The arrow indicates the first spike after stimulation.

FIG. 7. Response profiles of five chorda tympani fibers (A, B, C, D and E) for O-5 M solutions of sucrose and four pairs of the anomers of sugars and glycosides. The upper figure indicates the number of impulses elicited during the initial 5 set after stimulation, while the lower figure represents the number during the next 5 sec. In both figures empty blocks represent the magnitudes of responses to or-anomers while the filled ones represent those for the /&nomers. S, Gu, Ga, Me-Gu, Me-Ga represent sucrose, D-glucose, D-galactose, methylglucoside and methylgalactoside, respectively,

.,

TASTE EFFECT-

OF SUGAR ANOMERS

259

glucose and galactose, such as is demonstrated from the whole nerve response. The inability to obtain a difference in the response in single fibers between the OL-and /3-anomers may be due to the fact that the difference is so small that it may be masked by the fluctuation of the response magnitude in individual fibers. However, in all the five fibers the ol-anomers of u-methylglucoside and D-methylgalactoside elicited greater responses than those produced by the /&anomers. The magnitudes of response to these four anomers, expressed relative to that for 0.5 M sucrose and shown in Table 5, are in good agreement with those obtained from the whole nerve response (Table 3). The effect of 3-0-methylglucoside was examined in four fibers. An example of these is shown in Fig. 5. The effectiveness of 3-0-methylglucoside was intermediate between LY-and /3-methylglucosides. The average impulse numbers during 5 set after stimulation, obtained from four fibers, were 45 for 3-0-methylglucoside and 107 and 31 for the OL-and fi-anomers, respectively.

DISCUSSION In the present experiments the relative taste effectiveness of various sugars was determined by recording taste responses in the chorda tympani and chorda tympani fibers of hamsters. The order of effectiveness for nine kinds of sugars obtained from the whole nerve response was sucrose > fructose > mamrose > glucose > sorbose > maltose ‘Yrhamnose > galactose > xylose. This order is in approximate agreement with the order demonstrated on rats by Noma et at. (1971), but sorbose and xylose are relatively less effective in hamsters than in rats. Experiments on single-fiber responses to four kinds of sugars yielded similar results, in which the order of effectiveness, determined by both the threshold concentration and the relative response magnitude, was sucrose > fructose > glucose > maltose. Two fibers, which were most sensitive to quinine, were found to respond to sugars, but their responses to sugars were characterized by a high threshold, a very gradual increase in impulse discharges and a greater magnitude of response to monosaccharides such as fructose and glucose at high concentrations than that for sucrose. Ogawa et al. f1968) analyzed the response profiles of forty-eight chorda tympani fibers of rats and twenty-eight fibers of hamsters. Their results indicate that about 90 per cent of the fibers in the hamster are sensitive to sucrose while fibers responsive to sucrose occupied only 50 per cent of the population in the rat. On the other hand, fibers sensitive to quinine occupied 50 per cent of all the units examined in both animals. Such a difference in the relative number of fibers responsive to sugars and quinine between hamsters and rats would lead to the difference in the order of relative effectiveness of sugars between the two animals. Noma et al. (1971) showed that the responses in the rat chorda tympani to sorbose and sorbitol at 1 M increase very slowly in magnitude after stimulation and attained a maximum about 20 set after stimulation and that rats prefer sorbose and sorbitol at low concentrations to water but reject them above O-3 M. These facts may easily be explained if one assumes the presence in the rat chorda tympani of

260

AKINORI NOMA, MASAYASU SATO AND YOJIRO TSUZ~KI

quinine-sensitive fibers, which respond to sorbose and sorbitol at high concentrations and with gradually increasing impulse discharges with time. Tsuzuki & Mori (1954) pointed out that the sweeter sugar anomers have the cis configuration between the OH groups on the anomeric and the adjacent carbon atoms, although /I-D-mannose and /?-D-lactose are obvious exceptions. On this basis it might be expected that ol-D-glucose would be sweeter than the p-anomer. A few studies on the taste of sugars (Schutz & Pilgrim, 1957; Pangborn & Gee, 1961) indicated the greater sweetness of a-D-glucose. However, Shallenberger (1963) reported that crystalline /?-D-glucose appeared to taste sweeter than crystalline ol-D-glucose, although a later study by Shallenberger et al. (1965) indicated that the difference in sweetness was statistically nonsignificant. In the latter study they also reported the greater sweetness of ol-D-galactose and mannose anomers. The results of our experiments on the hamster indicate that the /3anomers of D-glucose and D-galactose are more effective in eliciting impulses in the chorda tympani than the ol-anomers. Therefore, our results are consistent with those of Shallenberger et al. (1965) in the relative taste effectiveness of CL-and fl-D-glucose anomers, but do not agree with the results by Pangborn & Gee (1961) and Shallenberger et al. (1965) in the effectiveness of the CX-and fi-D-galactose anomers. Regarding the difference in taste between the w and S-forms of D-methylglucoside it has been reported that the ol-anomer is sweeter than the /3-anomer (Tsuzuki, 1947; Shallenberger & Acree, 1971). Also a similar conclusion has been drawn from studies on the behaviour of insects (von Frisch, 1935 ; Dethier, 1955 ; Pflumm, 1972). Our results that the a-forms of D-methylglucoside and D-methylgalactoside are more effective in eliciting gustatory impulses than the p-forms are therefore consistent with the earlier reports. Dethier (1955) compared the effectiveness of D- and L-stereoisomers of some carbohydrates in stimulating single labellar hairs of the blowfly, and reported that D-arabinose was markedly more stimulating than L-arabinose while no difference between D- and L-xylose could be demonstrated. Shallenberger et al. (1969) compared the sweet taste characteristics of enantiomorphic pairs of several sugars including arabinose, xylose and glucose, and reported that no significant difference could be found in the sweet taste intensity of a given D-sugar and its enantiomorph. In the present experiments no significant difference was observed between D-Xyl0S.e and its enantiomorph in eliciting impulses in chorda tympani fibers. Also the results suggested an insignificant difference in effect between D-glucose and its enantiomorph, though the conclusive evidence for this was not obtained because of a small number of experiments. It has sometimes been stated that between 01- and /3-anomers of certain sugars or D- and L-enantiomorphs a difference exists not only in the sweet taste intensity but also in the quality of taste. For example, a-D-mannose is sweet while P-Dmannose is bitter (Stewart et al., 1971). In the present study in which the effects of 0.5 M solutions of iy- and /3-anomers of two hexoses and two glycosides were compared, both the oc- and P-anomers initiated impulses in the chorda tympani

TASTE EFFECTIWNESS

OF SUGAR ANOMERS

261

fibers highly sensitive to sucrose but not in those predominantly responsive to quinine. Also no essential difference was observed in effect between D-and Lenantiomorphs of glucose and xylose. Therefore, the above results indicate the absence of difference between either the a- and j3-anomers or D- and L-enantiomorphs in the taste quality produced in the hamster. However, the fact that glucose and fructose at a very high concentration elicit impulses increasing slowly with time in fibers highly responsive to quinine may account for the fact that j3-D-mannose tastes bitter (Stewart et al., 1971) and that p-methylglucoside yields a bitter taste (Tsuzuki, 1947; Shallenberger & Acree, 1971). SUMMARY

1. The taste effectiveness of various sugars and their anomers were investigated by recording the integrated responses of the chorda tympani of hamsters. The effectiveness of sugars examined is in the order of sucrose > fructose > mannose > glucose > sorbose > maltose % rhamnose > galactose > xylose. 2. Responses to the ar-anomers of n-glucose and n-galactose were smaller in magnitude than those to the j?-anomers while the reverse was true for the anomers of u-methylglucoside and D-methylgalactoside. 3. The difference in effectiveness between the LX-and &anomers of D-methylglucoside and n-methyl~lactoside was further confirmed by recording impulses in single chorda tympani fibers highly sensitive to sucrose. 4. A significant difference between cy- and ,!I-anomers of n-glucose and Dgalactose was not obtained from the experiments on single fibers. The reason for this was attributed to the fact that the difference in effectiveness between the two anomers is too small to be detected from a small number of experiments on singlefiber responses. 5. Experiments to record impulses in response to D- and L-forms of glucose and xylose in single chorda tympani fibers were carried out, and their results indicated little difference between pairs of the enantiomorphs in eliciting impulses, 6. The responses of quinine-sensitive fibers to various sugars were examined. These fibers yielded responses to fructose and glucose at high concentrations with a very long latency. However, no significant impulse discharges were elicited by O-5 M solutions of the LX.-and fi-anomers of sugars and glycosides. The results indicate that all the CX-and @anomers tested act as sweet stimuli for the hamster gustatory receptors. Ac~ow~edg~ts-ace

authors are indebted to Mr. K. Komatsu

and Miss Y. Doi for

preparation of the anomers. REFERENCES H. T., FUNAKOSHI M. & ZOTTERMAN Y. (1963) Electrophysiological responses to sugars and their depression by salt. In Olfaction and Taste (Edited by ZOTTERMAN Y.), Vol. I, p. 177. Pergamon Press, Oxford. BEIDLERL. M. (1953) Properties of chemoreceptors of tongue of rat. J. ~~~~~y~o~. 16, 595-607.

ANDERSEN

262

AKIN~RINOMA, MA~AYASU SATOANDYOJIROTsuzurrr

BEIDLERL. M., FIS~MAIQI. Y. & HARDIMANC. W. (1955) Species differences in taste responses. Am. r. Physiol. 181, 235-239. BIESTERA. M., WOOD W. & WAHLIN C. S. (1925) Carbohydrate studies-I. The relative sweetness of pure sugars. Am. J. Physiol. 73, 387-396. BOYD W. C. & MATSUBARAS. (1962) Different tastes of enantiomorphic hexoses. Science, Wush. 137, 669. CA~V~ERON A. T. (1947) The taste sense and the relative sweetness of the sugars and other sweet substances. Sugar Research Found. Inc. Scienti’c Report No. 9, pp. l-72. New York. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Quart. Rev. Biol. 30, 348-371. FRANK M. (1972) Taste responses of single hamster chorda tympani nerve fibers. In ~~~~tion and Taste (Edited by SCHNEIDERD.), Vol. IV, p. 287. Wissensch~t~che Verlagsgesellschaft MBH, Stuttgart. VONFRISCHK. (1935) uber den Geschmackssinn der Biene. 2. vergt. Physiot. 21, l-157. HAGSTROME. C. & PFAFFMANNC. (1959) The relative taste effectiveness of different sugars for the rat. r. camp. Physiol. Psychot, 52, 259-262. NOMA A., GOTO J. & SATO M. (1971) The relative taste effectiveness of sugars and sugar alcohols for the rat. Kumamoto MedJ. 24, 1-9. OGAWA H., SATO M. & YAMASHITAS. (1968) Multiple sensitivity of chorda tympani fibers of the rat and hamster to gustatory and thermal stimuli. J. Physiot., Lond. 199,223-240. PANCBORNR. M. & GEE S. C. (1961) Relative sweetness of (Y- and /?-forms of selected sugars. Nature, Lond. 191, 810-811. PFLUMMW. W. (1972) Molecular structure and stimulating effectiveness of oligosaccharides and glycosides. In Olfaction and Taste (Edited by SCHNEIDER,D.), Vol. IV, p. 364. Wissenschaftliche Verlagsgesellschaft MBH, Stuttgart. SCHUTZH. G. & PILGRIM F. J. (1957) Sweetness of various compounds and its measurement. Food Res. 22, 206-213. SHALLENBERGER, R S. (1963) Hydrogen bonding and the varying sweetness of the sugars. J. Food Sci. 28, 584-589. SHALLENBERCER R. S. & ACREET. E. (1971) Chemical structure of compounds and their sweet and bitter taste. In IIandbook of Sensory Physiology (Edited by BEIDLERL. M.) Vol. IV, p. 221. Springer, Berlin. SHALLENBERGER R. S., ACREET. E. & GUILD W. E. (1965) Con~guration, confo~ation, and sweetness of hexose anomers. J. Food Sci. 30, 560-563. SHALLENBERGER R. S., ACREET. E. & LEE C. Y. (1969) The sweet taste of D- and L-sugars and amino-acids and the steric nature of their receptor site. Nature, Lond. 221, 555-556. SIEGELS. (1956) Nonparametric Statisticsfor the Behavioral Sciences, pp. 229-239. McGrawHill, New York. STEWARTR. A., CARRICOC. K., WEBSTERR. L. & STEINHARDT R. G. (1971) Physicochemical stereospecificity in taste perception of cu-n-mannose and ~-n-mannose. Nature, Lond. 234, 220. ‘I’suzu~r Y. (1947) Relation between sweetness and configuration in sugars. Kuguku (Science) 17, 342-346. TSUZUKIY. & MORI N. (1954) Sweetness and configuration in rhamnose. Nature, Lond. 174, 458-459. TSUZUKIY. & YAMAZAKIJ. (1953) On the sweetness of fructose and some other sugars, especially its variation with temperature. Biochem. 2. 323, 525-531. YAMASHITAS. & SATO M. (1965) The effects of temperature on gustatory response of rats. J. cell. camp. Physiol. 66, 1-18. Key Word Index-Taste effectiveness ; sugars and glycosides ; anomers ; enantiomorphs ; chorda tympani response; single fiber analysis; hamster; Mesocricetus auratus.