Influence of chirality of amino acids on the growth of perceived taste intensity with concentration

Physiology & Behavior, Vol. 28, pp. 457-465. Pergamon Press and Brain Research Publ., 1982. Printed in the U.S.A.

Influence of Chirality of Amino Acids on the Growth of Perceived Taste Intensity with Concentration S U S A N S. S C H I F F M A N

Department of Psychiatry, Duke Medical Center, Durham, NC 27710 THOMAS

B. C L A R K I I I

School of Medicine, Medical University of South Carolina AND JEAN GAGNON

Department of Biochemistry, University of Oxford, Oxford, England R e c e i v e d 25 N o v e m b e r 1981 SCHIFFMAN, S. S., T. B. CLARK III AND J. GAGNON. Influence of chirality of amino acids on the growth of perceived taste intensity with concentration. PHYSIOL. BEHAV. 28(3) 457-465, 1982.--Amino acids have markedly

different taste properties depending upon their chirality and the structure of their side chains. They can modify the taste quality of foods in which they are found naturally or to which they are added depending on their concentrations. In this study, the influence of c hirality of amino acids on the growth in perceived taste intensity with concentration was examined. Serial dilutions of 19 D-amino acids were presented to young subjects who indicated the relative intensities of the dilutions using the magnitude estimation procedure. The slopes of the psychophysicai functions for 13 of the 19 D-amino acids which related log concentration and log perceived intensity were greater than the slopes for L-amino acids that have been reported in a previous study. The ratio (average slope D-amino acids)/(average slope L-amino acids) was found to be 1.21. The ratio of the slopes for individual enantiomers bears some relationship to taste quality. No conclusive relationships between the slopes of the psychophysical functions were found with chemical structure or thresholds, however. Implications for receptor mechanisms and nutrition are discussed. Amino acids

Taste

Magnitude estimation

Chirality

A M I N O acids have markedly different taste properties depending upon their chirality and the structure of their side chains [8, 24, 34--39, 41, 42, 46]. The degree to which their presence in food can modify taste quality is dependent upon their concentrations. Amino acids, peptides, and their derivatives have been added to foods to modify their taste properties as well as supplement nutrition [1-3, 12, 14, 25, 26]. Alterations in amino acid composition or modification of the amino acids themselves have been implicated in taste and olfactory changes during ripening of cheese [20,30], fermentation of wines [11, 17, 22], irradiation of foods [45], and other forms of food processing [7, 23, 28, 29, 31, 32, 43]. Amino acid diets have also been utilized in treatment of illnesses ranging from allergies [10] to cystic fibrosis [13] and uremia [21]. The purpose of this study was to determine the influence of chirality on the growth in perceived intensity with concentration for suprathreshold tastes of amino acids. The slopes of the psychophysical functions relating concentration and

perceived intensity for L-amino acids have been determined previously by Schiffman and Clark [34]. The slopes for D-forms were found here using Stevens' method of magnitude estimation [44]. Relationship between the slopes, taste detection thresholds, chemical structures, and suprathreshold taste qualities were examined for D- and L-enantiomers. METHOD

Subjects The subjects were 118 Duke University students, 63 males and 55 females, ranging in age from 18 to 29 years. A minimum of 12 and a maximum of 15 individuals tasted each amino acid. All the subjects were nonsmokers; none wore dentures. Subjects were requested to abstain from eating for 2 hours prior to the experiment. Magniture extimates for L-amino acids had been determined previously for a similar group of young subjects [34].

C o p y r i g h t © 1982 B r a i n R e s e a r c h P u b l i c a t i o n s I n c . a 0 0 3 1 - 9 3 8 4 / 8 2 / 0 3 0 4 5 7 - 0 9 5 0 3 . 0 0 / 0

458

S C H I F F M A N , C L A R K AND G A G N O N

Stimuli The stimuli shown in Table 1 were 19 D-amino acids obtained from Sigma Chemical Company, St. Louis, MO. The amino acids were dissolved in deionized water and presented to the subjects in 30 ml plastic medicine cups. The concentration ranges and number of dilutions for each of the D-amino acids were the same as those used by Schiffman and Clark [34] to determine the psychophysical functions for L-amino acids. These concentration ranges varied from just above average taste threshold for L-amino acids for elderly subjects (see [37]) to those concentrations which constituted maximum solubility with mechanical agitation at room temperature (72°F). The concentrations used for a given amino acid were serial dilutions which differed from one another by a factor of two. Thus, the stimulus range was greater (i.e., more dilutions) for highly soluble amino acids and those with low taste thresholds. The range in taste intensity varied greatly depending upon the amino acid.

Procedure The direct scaling procedure called "magnitude estimation" [5, 6, 40, 44] was used to determine the relationship between perceived intensity of D-amino acids and concentration in a manner analogous to that employed by Schiffman and Clark [34] for L-amino acids. First, subjects were given practice in assigning numbers to line lengths as described by Gent and McBurney [18]; they also participated in practice sessions using taste stimuli as well. In an experimental session, subjects, wearing noseplugs to reduce olfactory input, sampled 10 ml of each serial dilution for an amino acid, swirling it throughout the oral cavity. After tasting each dilution, the subjects assigned a number such that the ratios o f the applied numbers reflected the ratios of perceived taste intensities. The first stimulus presented for a given amino acid was a modulus (standard), shown in Table 1, which was in the middle of the range of the serial dilutions and was assigned the arbitrary value of 10 intensity units. The remaining serial dilutions were presented in randomized order with 3-minute interstimulus intervals; the modulus was repeated once again during the trials. Deionized water was used for an inter-stimulus rinse between presentation of the stimuli.

Analysis o f Data Each magnitude estimate was standardized by multiplying it by a scaling factor. This was done to normalize the data because individual subjects used different ranges of numbers. The scaling factor was computed as follows. First, the geometric mean of the magnitude estimates for each amino acid for a given subject was computed. The scaling factor was developed by dividing 100 by the geometric mean. RESULTS The logs of the concentrations of the amino acids are plotted against the logs o f the standardized magnitude estimates in Fig. 1. F o r all amino acids, a regression line could be fit to the points, indicating that a simple power function s = k c n (or log(s)=log(k) + nlog(c)) was appropriate to relate perceived magnitude and concentration. The values for the slopes for both D- and L-enantiomers as well as their ratios are given in Table 2. Thirteen of the slopes for the 19 enantiomer pairs given in Table 2 were found to be greater

TABLE 1 CONCENTRATION RANGES, MODULUS, AND NUMBER OF DILUTIONS EMPLOYED Range ( x 10 ~ M) Amino acid D-Alanine D-Arginine HCI D-Asparagine D-Aspartic acid D-Cysteine D-Cysteine He1 D-Glutamic acid D-Glutamine D-Histidine D-Histidine HCI D-Isoleucine D-Leucine D-Lysine HCI D-Methionine D-Phenylalanine D-Serine D-Threonine D-Tryptophan D-Valine

Modulus

Minimum Maximum (xl0 -~ M) 2.00 0.24 0.93 0.05 0.039 0.002 0.01 2.69 1.29 0.039 1.20 1.29 0.21 0.26 1.91 2.63 2.00 0.29 1.15

128.00 61.44 14.88 3.20 19.97 16.40 5.12 10.76 20.64 19.93 19.20 10.32 107.52 33.66 15.28 168.32 64.00 4.61 36.80

16.00 3.84 3.72 0.40 2.50 0.128 0.32 5.38 2.58 1.24 4.80 2.58 6.72 4.21 3.82 21.44 8.00 1.15 4.60

Number of dilutions 7 9 5 7 10 14 I0 3 6 10 5 4 10 8 4 7 6 5 6

for the D-form when compared with the L-form. The ratio: average slope D-amino acids/average slope L-amino acids= 1.21. Threshold values and brief taste descriptions are given as well for reference. Thresholds of D-amino acids were determined in an experiment which took place during the same time frame as determination of the slopes [38]. Because the concentration ranges for the D-enantiomers were based on the thresholds for L-enantiomers, it was found that the lowest concentration chosen for some D-amino acids could not be tasted by all the subjects, i.e., the lowest concentration of a range was below the geometric mean thresholds found by Schiffman et al. [38] for some of the D-amino acids. If a subject indicated that he/she was unable to taste the lowest concentration, that dilution was not included in determination of the slope because on a loglog plot, zeros would go to minus infinity probably with a zero statistical weight. DISCUSSION

Relationship of Slopes to Taste Quality The ratio of the slopes for L- and D-amino acids bears some relationship to taste quality as shown in Table 3. When the slope for the D-enantiomer is greater than that for the L-enantiomer, the taste quality characteristics can be divided into three groups. The first group is comprised of amino acids that have a definite bitter or meaty component in the L-form but not in the D-form. This group includes asparagine, glutamic acid, histidine, leucine, phenylalanine, and tryptophan. With the exception of glutamic acid, all of

TASTE INTENSITY OF AMINO ACIDS

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FIG. 1. Logarithms of the concentrations of the amino acids are plotted on the abscissa. Logarithms of the normalized magnitude estimates arc plotted on the ordinate. N o meaning should be attached to the absolute location of the psychophysical functions on the ordinate due to the normalization process described in Analysis of Data for the magnitude estimates.

TASTE INTENSITY OF AMINO ACIDS the D-forms in this group are sweet. The second group consists of alanine, aspartic acid, glutamine, and threonine, which are more flavorous or simple in the D-form than the L-form. The third group is comprised of sulfur-containing amino acids, which are unpleasant tasting in both forms. The amino acids crystallized from acid solution, arginine HCI, histidine HCI, and lysine HCI, have approximately equal slopes in the D- and L-forms. Three amino acids, isoleucine, serine, and valine, have steeper slopes in the L-form than in the D-form. The enantiomers of each of these amino acids have similar tastes according to Schiffman et al. [38]. It should be noted that the two D-amino acids for which the taste description found by Schiffman et al. [38] differs markedly from that given by Meister [24] are found in this last group. Meister classified D-valine as very sweet and D-isoleucine as sweet. These discrepancies in taste quality reported for D-amino acids may be due to individual variability in taste quality which have been reported previously for L-amino acids and dipeptides. The variability in reported taste quality for amino acids and dipeptides may result in part from individual differences in the detection of bitterness, as has been shown for phenylthiourea or 6-n-propylthiouracil [5,16].

Relationship of Slopes to Chemical Group, Threshold, and Number of Dilutions The slopes for the D-amino acids are given in descending order in Table 4 along with chemical groups of the side chains, rank order of the thresholds for D-amino acids from highest (ranked) to lowest (ranked 19), and number of dilutions used to determine the slope. Like the L-forms, no strong relationship between chemical structure and slope of the psychophysical functions was found. The rank order correlation between the slopes for L- and D-amino acids is Spearman's rho=0.258. The main consistencies in slope were for phenylalanine and tryptophan, which had relatively steep slopes in both forms, and cysteine and threonine, which had relatively fiat slopes for both enantiomers. No relationship between the slope for the D-amino acids and their detection thresholds was found here. The Spearman's rank order correlation was 0.033. Like L-amino acids, a small relationship was found between the slope and the number of dilutions, i.e., amino acids with flattened slopes

461 tended to have more dilutions. The rank order correlation between the slope and number of dilutions for D-amino acids was -0.39. A rank order correlation for L-amino acids of -0.36 was found previously by Schiffman and Clark [34]. L-amino acids with basic side chains were found to have steeper slopes when complexed with HCI (e.g., L-histidine HCI) than their free amino acid counterparts (e.g., L-histidine). In this study, D-histidine HC1 was found to have flatter slopes than D-histidine.

Implication of Slopes for Receptor Sites Receptors for amino acids with steep slopes presumably do not saturate as quickly as receptors for amino acids with flatter slopes. There may be several possible explanations for rapid saturation and thus relatively flatter slopes. First, there may be few receptor sites for a given amino acid, and these saturate quickly due to a much greater number of stimulus molecules relative to the number of receptors. Alternatively, there may be a large number of receptors to which amino acid molecules bind tightly (i.e., have high affinity) because the energetics favor the binding process. Whatever the ultimate reason for differences in the slopes for D- and L-amino acids, there is no reason to assume that enantiomer pairs bind to identical sites on the taste cell membrane.

Nutritive Effectiveness of D-Amino Acids D-amino acids can promote growth in animals and may contribute to nutrition either by transformation to the L-antipode by some form of steric conversion probably involving D-amino acid oxidase (DOX) or by providing nitrogen for the synthesis of nonessential amino acids [19]. The nutritive effectiveness of D-isomers is dependent on numerous factors, especially other components in the diet. For example, racemic tryptophan has been found to promote growth more effectively in rats on relatively crude diets when compared with more highly purified diets [9, 15, 27, 33]. The adequacy of diets consisting of racemic amino acids must be determined experimentally since the interactions cannot be predicted a priori. The availability of a D-isomer may be enhanced by the dietary presence of some L-isomer. In addition, the utilization of the D-forms of certain amino acids can be inhibited by other D-isomers.

462

SCHIFFMAN,

TABLE

Amino acid

With neutral side chains (aliphatic)

Glycine

(aromatic)

(amides)

With side chains containing basic (amine) groups

With side chains containing hydroxylic groups

With side chains containing acidic groups

With side chains containing sulfur atoms

L

D

Direction

AND

(iAGN()N

2

Slopes Chemical grouping

(?LARK

Thresholds D/L

0.561

L

D

Direction

D/L

3.09 × 10-2 M

Alanine

0.603

0.713

D>L

1.182

1.62 × 10-2 M

1.12 x 10 2 M

L>D

(I.69

Valine

0,726

0.625

L>D

0.861

0.416 × 10--2 M

0,295 × 10-2 M

L>D

0.71

Leucine

0.475

0.734

D>>L

1.546

0.645 × 10-2 M

0.501 × 10_2 M

L>D

0.78

Isoleucine

0.675

0.538

L>D

0.797

0.741 × 10 2 M

1.25 × 10-z M

D>L

1.69

Phenylalanine

0.796

1.02

D>L

1.282

0.661 × 1 0 - M

0.155 x 10-z M

L>>D

0.23

Tryptophan

0.667

0.759

D>L

1.138

0,229 × 10-2 M

0.048 × 10_2 M

L>>D

0.21

Asparagine

0.323

0.761

D>>L

2.358

0.162 × 10-2 M

0.977 × 10-2 M

D>>L

6.03

Glutamine

0,495

0.553

D>L

1,I 17

0.977 x 10 2 M

0.347 x 10 2 M

L>>D

0.36

Arginine HCI

0.645

0.623

L~D

0.966

0.123 × 10-2 M

0.162 × 10 ~ M

D>L

1.32

Lysine HCI

0.476

0.475

L~D

0.998

0.0447 × 10-2 M

0.133 × 10-2 M

D>>L

2.98

Histidine

0.415

0.758

D>>L

1.828

0.123 x 10-z M

0.186 × 10 2 M

D>L

1.51

Histidine HCI

0.574

0,563

L~D

0.981

0.00794 x 10-2 M

0.025 x 10-2 M

D>>L

3,16

Serine

0.788 0.671

L>D

0.852

2.09 × 10-2 M

6.48 x 10-2 M

D>>L

3.10

Threonine

0,396

0.448

D>L

1.131

2.57 × 10-2 M

3.37 x 10-2 M

D> L

t.31

Aspartic acid

0,498

0.652

D>L

1.309

0,0182 x 10-2 M

0.074 × 10-2 M

D>>L

4,07

Glutamic acid

0.305

0.600

D>>L

1.969

0.0063 × 10-2 M

0.0076 x 10-2 M

D>L

1.21

Cysteine

0.277

0.373

D>L

1.346

0,0063 x 10-z M

0.0085 × 10-2 M

D>L

1.35

Cysteine HCI

0.513

0.594

D>L

1.158

0.0016 × 10-2 M

Methionine

0.414

0.679

D>>L

1.639

0.372 × 10-2 M

0.501 × 10-2 M

D>L

1.35

T h e s l o p e s f o r D - a m i n o a c i d s d e t e r m i n e d in this s t u d y a r e c o m p a r e d w i t h t h o s e f o r L - f o r m s f o u n d p r e v i o u s l y b y S c h i f f m a n a n d C l a r k [34]. " D i r e c t i o n " i n d i c a t e s w h e t h e r t h e v a l u e f o u n d f o r o n e e n a n t i o m e r is " g r e a t e r t h a n " ( > ) , a p p r o x i m a t e l y e q u a l to ( ~ ) , o r less t h a n ( < ) its m i r r o r i m a g e . T h e a c t u a l r a t i o s f o r t h e v a l u e s f o r t h e s l o p e s ( D / L ) a r e g i v e n as well. T h e s y m b o l ( > > ) f o r " d i r e c t i o n " signifies " v e r y m u c h g r e a t e r t h a n " a n d is u s e d to i n d i c a t e r a t i o s g r e a t e r t h a n 1.5. T h r e s h o l d s a n d b r i e f t a s t e d e s c r i p t i o n s ( f r o m S c h i f f m a n et al. [38]) a r e g i v e n f o r comparison.

TASTE INTENSITY OF AMINO ACIDS

463

TABLE 2 (Continued) Brief Taste Description L

D sweet, pleasant, smooth, refreshing

sweet; possibly complex with bitter aftertaste flat to bitter; slightly sweet fiat to bitter (virtually indistinguishable from L-isoleucine) flat to bitter

bitter; possibly complex and strangling flat to bitter flat to bitter flat, sweet, meaty, somewhat unpleasant

bitter, complex, salty, sweet flat to bitter, minerally

flat to sweet; possibly sour, complex flat to sweet, possibly bitter, sour, or fatty fiat, sour, slightly bitter unique, possibly meaty, salty, bitter, sour, complex sulphurous, obnoxious sulphurous, obnoxious, concentrated, complex, poisonous flat to bitter, possibly sulfurous, meaty or sweet

sweet; simple with no bitter components somewhat tasteless; weak, alkaline, minerally, possibly sweet smooth, soft, moderately sweet

flat, bitter, minerally/ metallic; possibly salty and smokey indistinct; possibly sweet, minerally, possibly bitter and metallic smooth, sweet, possibly bitter, minerally sweet, simple, refreshing good, sweet, flavorous, smooth bitter, alkaline, complex with salty and sour elements, minerally bitter, minerally, posionpus, alkaline; metallic, soapy components sweet, fiavorous,

refreshing, fruity complex; sweet, sour, possibly salty; pungent sweet, smooth, fresh, dilute, possibly tingling somewhat tasteless; simple, weak, possibly sweet strong, sour, salty, slightly bitter yet flavorous sour, constant, pungent, possibly salty obnoxious, repulsive, slightly bitter with persistent aftertaste obnoxious, concentrated, sour, sharp, poisonous, possibly bitter alkaline, stale, bitter, minerally, meaty, sour, sweet components

464

SCHIFFMAN. TABLE 3 RELATIONSHIP OF SLOPES TO TASTE QUALITY Slope D>slope L (1)

(3)

C2)

L-form but not D-form has definite bitter or meaty component

D-form more fiavorous and simple than L-form

Asparagine Glutamic acid Histidine Leucine Phenylalanine Tryptophan

Both forms unpleasant tasting, sulfurcontaining amino acids

Alanine Aspartic acid Glutamine Threonine

Cysteine Cysteine HCI Methionine

Slope L~slope D Arginine HCI Histidine HCI Lysine HCI Slope L>siope D L- and D-forms have similar tastes lsoleucine Serine Valine

TABLE 4 SLOPES OF D-AMINO ACIDS GIVEN IN DESCENDING ORDER ALONG WITH CHEMICAL GROUPS OF SIDE CHAINS, RANK ORDER OF THRESHOLDS, AND NUMBER OF DILUTIONS USED TO DETERMINE THE SLOPE

Amino acid

Slope

Chemical group

Rank order of threshold

Number of dilutions

D-Phenylalanine D-Asparagine D-Tryptophan D-Histidine D-Leucine D-Alanine D-Methionine D-Serine D-Aspartic acid D- Valine D-Arginine HCI D-Glutamic acid D-Cysteine HCI D-Histidine HCI Glycine D-Glutamine D-Isoleucine D-Lysine HCI D-Threonine D-Cysteine

1.02 0.761 0.759 0.758 0.734 0.713 0.679 0.671 0.652 0.625 0.623 0.600 0.594 0.563 0.561 0.553 0.538 0.475 0.448 0.373

VI IV VI V I I III II IV I V IV III V I IV I V II III

13 6 16 11 7 5 8 1 15 I0 12 19

4 5 5 6 4 6 8 7 7 6 9 10 14 10 6 3 5 10 6 10

17 3 9 4 14 2 18

Chemical group: I, with aliphatic side chains; II, with side chains containing hydroxylic groups; III, with side chains containing sulfur atoms; IV, with side chains containing acidic groups of their amides; V, with side chains containing basic groups; VI, containing aromatic rings; VII, imino acid.

CLARK AN D GAGN()N

TASTE INTENSITY OF AMINO ACIDS

465 REFERENCES

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