A paper screening test to assess genetic taste sensitivity to 6-n-propylthiouracil

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Abstract

The purpose of this study was to develop a quantifiable method to fix 6-n-propylthiouracil (PROP) onto filter paper disks and to test the validity of the method relative to the three-solution test, previously developed in this laboratory. Filter paper disks were impregnated with 50 mmol/l PROP or 1.0 mol/l NaCl then dried. The concentration of PROP per disk was determined to be 0.280 mg±2.2% (CV) by ethanol extraction. Subjects were studied in two groups (n=62 per group). All subjects were initially classified by the three-solution test and then were independently classified by the paper disk test. In the paper disk test, group means for PROP (±95% confidence interval) were calculated and used to establish numerical cutoff scores for taster group classification. Cutoff scores for Group 1 were used to classify subjects in Group 2, verifying that the cutoffs were applicable to another subject group. The percentage of nontasters, medium tasters, and supertasters identified by paper disk was 27%, 42%, and 31%, respectively, which is consistent with expected frequencies in the population. For Group 1, the agreement between the two classification methods ranged from 86% to 94% across taster groups. For Group 2, the agreement ranged from 83% to 100%. The contingency coefficient (P) of the degree of association between the two classification methods was high (P=0.77 and P=0.74 for Groups 1 and 2, respectively; P≤.001). These results demonstrate that the paper disk method is a reliable screening tool for assessing sensitivity to PROP that has numerous applications in basic and applied research.

Introduction

6-n-Propylthiouracil (PROP) and phenylthiocarbamide (PTC) are members of a class of bitter-tasting compounds known as thioureas. All thioureas contain the chemical moiety NCS, which is responsible for their bitter taste. In 1932, Fox [1] accidentally discovered that ∼30% of the population is genetically taste-blind to PTC. Subsequent research [2] suggested that taste blindness to PTC and PROP was inherited as a Mendelian recessive trait, with nontasters having two recessive alleles (tt) and tasters having at least one dominant allele (Tt or TT). However, further work indicated that heterozygous tasters (Tt) might constitute a separate group from homozygous tasters (TT). Bartoshuk [3] identified these groups as “medium tasters” and “supertasters” and showed that supertasters perceived the most extreme bitterness from PTC/PROP. The precise mode of inheritance for this polymorphism remains elusive. Yet, evidence suggests that more than one genetic locus or multiple alleles [4] might be involved. A recent linkage study revealed that the primary PROP locus maps to chromosome 5p15 [5] near a cluster of genes expressing bitter taste receptors [6], [7], with a second locus on chromosome 7. These findings are intriguing because they suggest that individual differences in bitterness perception might have a genetic component, and that PROP screening of individuals may provide a convenient tool for studying this phenotypic variation.

A growing body of research has shown that PROP tasters (medium and supertasters) perceive a greater intensity than nontasters from a wide range of oral stimuli, including other basic taste sensations such as sweetness, oral irritation (chili and ethanol), and the mouthfeel of fats [8], [9], [10]. Some studies have sought to define a role for PROP status in guiding food preferences and dietary habits that could ultimately influence nutritional status and health. One line of inquiry has examined the association between PROP taste responsiveness and selection of fat-containing foods. Studies have shown that PROP nontasters give higher acceptance ratings to high-fat salad dressings [10] and full-fat milk [11] and reportedly consume more discretionary fats per day than tasters [11]. In another work, women who were less sensitive to the taste of PROP preferred a wide range of high-fat foods, including meats, dairy products, and sweets [12]. Emerging evidence also suggests that nontasters maintain higher body mass indices than tasters [10], [13]. Preliminary studies reported by Duffy et al. [14] have revealed a modest negative relation between PROP taste responsiveness and plasma cholesterol in men, suggesting that PROP status associates with other indices of health status. Other investigators have examined associations between PROP status and rejection of bitter-tasting fruits and vegetables as a risk factor for breast cancer [15], [16]. Additional associations between PROP status and disease states such as alcoholism have been investigated [17], [18], and still others remain to be identified.

Despite these positive findings, several studies have not observed meaningful associations between PROP status and a variety of outcome measures, including taste responses [19], [20], [21], food selection, or body weight [22], [23]. There could be many underlying reasons for this lack of consensus, including the dependence on small sample sizes, which could distort PROP effects (e.g., [19], [20]), or the use of scaling techniques that lack sensitivity or produce ceiling effects (e.g., [21], [23]). Scaling issues have been discussed previously [24], [25]. One important source of variation could be the use of different methods for PROP screening and group assignment that might not be equivalent. A variety of screening methods are currently in use, including threshold, suprathreshold, and paper tests. Except for the work of Lawless [26], methodological studies comparing different methods are rare.

The use of filter papers impregnated with PTC crystals has a long history of use in the field (e.g., [26], [27], [28], [29], [30]). However, the method has been criticized for producing a high rate of false-positive/false-negative responses and poor agreement with more traditional threshold methods [26]. Bartoshuk et al. [31] recently introduced a screening procedure in which filter papers are dipped in a supersaturated PROP solution to capture the precipitate onto the paper then dried. This method is reminiscent of the early procedures used by Blakeslee and Fox [32] in which PTC crystals were placed directly on the tongue. According to Bartoshuk et al. [33], the PROP filter paper method was intended as a rough screening procedure. But the simplicity of this method has made it an appealing choice for investigators (e.g., [5], [15], [23], [34], [35]). Correlations between PROP paper and suprathreshold scaling were reported in two of the abovementioned studies (r=.60–.75) [33], [35]. However, neither study classified individuals into discrete groups then compared the reliability of those assignments across the two methods. Since a supersaturated solution is not uniform, this method cannot assure a consistent concentration of PROP across papers, which could be a significant source of variation in the classification of subjects.

A recent study used PROP filter papers [31] to classify more than 400 subjects responding to a mail-in health surveillance survey [23]. Approximately 40% of the male respondents and ∼55% of the female respondents were identified as high tasters (i.e., supertasters). Among young women, the percentage of supertasters exceeded 70%. These findings are not consistent with estimated frequencies in the US population [33], suggesting that this method overestimates the proportion of supertasters.

Tepper et al. [36] recently described brief screening methods based on a well-known suprathreshold procedure [33]. The original procedure involves the presentation of five aqueous solutions each of PROP and NaCl. The procedure was simplified to three concentrations of each stimulus (three-solution method) and then to one concentration of each stimulus (one-solution method). Both methods were shown to be reproducible and valid as compared to the parent procedure [36]. In the three-solution test, three concentrations of PROP (0.032, 0.32, and 3.2 mmol/l) and three concentrations of NaCl (0.01, 0.1 and 1.0 mol/l) are evaluated, and subjects are classified by visually comparing the taste function for PROP to that of NaCl [36]. Subjects who give higher ratings to NaCl than to PROP are classified as nontasters. Those who rate PROP higher in intensity than NaCl are classified as supertasters. Medium tasters give similar ratings to NaCl and PROP. Classification by the three-solution test can be subjective because group assignment depends on visual examination of the data. However, reclassification of subjects using the PROP ratio method, which compares the slope of the line for PROP to that of NaCl, did not alter the results [36]. In the one-solution test, subjects evaluate one concentration each of PROP (0.32 mmol/l) and NaCl (0.1 mol/l). A key feature of the one-solution test is the use of numerical cutoff scores for subject classification. The cutoff scores are obtained by calculating the 95% confidence interval around the group means for PROP intensity for nontasters, medium tasters, and supertasters, respectively. The paper disk method described herein utilizes the same procedures for subject classification as the one-solution method.

The first objective of the present study was to develop a quantifiable method to impregnate filter paper disks with PROP. Ethanol extraction was used to verify the concentration of PROP on the disks and to determine the disk-to-disk variation. The second objective was to test the validity of the paper disk method relative to the three-solution test and its reproducibility. Subjects were studied in two groups. All subjects completed the three-solution test and paper disk test. Numerical cutoff scores for the paper disk test were calculated for Group 1 and used to classify subjects in Group 2. This was done to verify that the cutoff scores established for one subject population were applicable to another comparable subject population.

Section snippets

Subjects

Subjects were healthy adults who were recruited and tested at two different sites. All subjects were screened prior to testing and were excluded from the study if they reported any oral or nasal disease, or if they were taking any medications that interfered with taste or olfactory perception. Informed consent was obtained from all subjects. The protocol was approved by the Institutional Review Board at Rutgers University and a safety committee at International Flavor and Fragrances.

Group 1

Three-solution test

The three-solution test was identical to that described previously [36]. The test consisted of three solutions (0.032, 0.32, and 3.2 mmol/l) of PROP (Aldrich Chemical, Milwaukee, WI) and three solutions (0.01, 0.1, and 1.0 mol/l) of sodium chloride (NaCl; Fischer Scientific, Fairlawn, NJ). The PROP solutions were prepared by dissolving the powder in bottled spring water on a stirring hotplate under mild heat. The solutions were prepared prior to the test day and stored at 4° C in a

Three-solution test

ANOVA revealed a significant three-way interaction of Taster Group×Stimulus Type×Concentration on the intensity ratings for subjects in Group 1 [F(4,114)=33.11, P≤.001]. As shown in Fig. 1, post-hoc comparisons showed that for Group 1 (panel A), nontasters gave lower intensity ratings to the two highest concentrations of PROP (0.32 and 3.2 mmol/l) than that which they gave to the two corresponding concentrations NaCl (0.1 and 1.0 mol/l). Supertasters gave higher intensity ratings to all

Discussion

The results of this study show that the paper disk method is a valid and reliable screening tool for classifying individuals by PROP taster status. The classifications obtained by the PROP paper disks were comparable to those obtained using the three-solution method, which was previously validated against the traditional Bartoshuk procedure [33] (five concentrations each of PROP and NaCl). In addition, taster-group cutoffs established for Group 1 clearly identified three independent taster

Acknowledgments

This work was supported by International Flavors and Fragrances (Union Beach, NJ) and a Rutgers University Busch Biomedical Research Grant to B.J.T. The authors thank Richard Ludescher for assistance in conducting the ethanol extractions.

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