Chem. Percept. (2009) 2:118–132 DOI 10.1007/s12078-009-9049-1
Three TAS2R Bitter Taste Receptors Mediate the Psychophysical Responses to Bitter Compounds of Hops (Humulus lupulus L.) and Beer
Daniel Intelmann & Claudia Batram & Christina Kuhn & Gesa Haseleu & Wolfgang Meyerhof & Thomas Hofmann
Received: 15 January 2009 /Accepted: 18 May 2009 /Published online: 3 June 2009 # 2009 Springer Science + Business Media, LLC
Abstract In order to study the role of different haplotypes order of potency for the various compounds was similar in of taste receptor genes in food choice, it is necessary to first both experiments. Thus, the subjects perceived the bitter- identify the cognate hTAS2R bitter taste receptors for the ness of the investigated compounds at higher concentra- key bitter compounds in food products of our daily diet. In tions than those predicted by the results of the in vitro order to identify the candidate receptors mediating the bitter experiments. These differences were shown to be due, at taste of hop-containing beverages such as beer, we least in part, to interactions of the bitter substances with the transiently transfected plasmids encoding the 25 human oral mucosa. TAS2Rs into human embryonic kidney 293T cells, stably expressing the chimeric G-protein G16gust44. Thereby, we Keywords Beer . Hop . Iso-alpha Acids . hTAS2R . coupled the activation of hTAS2R receptors to the release hTAS2R1 . hTAS2R14 . hTAS2R40 . Bitter Taste . of Ca2+ from intracellular stores. The transfected cells were Oral Mucosa loaded with a calcium-sensitive fluorescence dye and challenged by 15 hop-derived compounds, including α-acids, β-acids, trans/cis-iso-α-acids, isoxanthohumol, Introduction xanthohumol, and 8-prenylnaringenin. Depending on their chemical structure, all these compounds activated various Bitter taste is considered a warning system for poisoning. combinations of the three bitter taste receptors hTAS2R1, Many toxic compounds appear to taste bitter; yet, toxicity hTAS2R14, and hTAS2R40 with distinct threshold concen- seems not to be directly correlated with the taste threshold trations and EC50 values. Notably, this is the first time that concentrations of bitter compounds (Glendinning 1994). an agonist for hTAS2R40 is reported. The threshold The instinctive rejection of intense bitter compounds may concentrations and EC50 values obtained from the taste be immutable because detecting poisonous substances has receptor assays were much lower than those determined by long been crucial for survival, and it appears that it still human psychophysical experiments, even though the rank affects our food choice to date. The oral sensing of the molecules evoking the bitter taste in foods is mediated in humans by a family of ~25 recently discovered G-protein- D. Intelmann and C. Batram contributed equally. coupled receptors encoded by the TAS2R gene family : : D. Intelmann G. Haseleu T. Hofmann (*) (Adler et al. 2000; Behrens et al. 2007; Chandrashekar et al. Chair of Food Chemistry and Molecular Sensory Science, 2000; Matsunami et al. 2000; Meyerhof 2005). Heterolo- Technische Universität München, gous expression experiments have successfully enabled the Lise-Meitner Str. 34, 85354 Freising, Germany identification of cognate bitter compounds for 13 out of the e-mail: [email protected] known 25 hTAS2Rs (Behrens et al. 2004; Brockhoff et al. : : 2007; Bufe et al. 2005; Bufe et al. 2002; Chandrashekar et C. Batram C. Kuhn W. Meyerhof al. 2000; Dotson et al. 2008; Kuhn et al. 2004; Maehashi et German Institute of Human Nutrition (DIFE) Potsdam-Rehbrücke, Arthur-Scheunert-Allee 114-116, al. 2008; Pronin et al. 2004; Sainz et al. 2007). The vast 14558 Nuthetal, Germany majority of these bitter compounds are synthetic molecules Chem. Percept. (2009) 2:118–132 119 or highly toxic alkaloids, which are not part of our daily bitter compound in beer, is generated from the hop-derived diet. However, to identify mechanisms affecting food prenylated chalcon 14, while the so-called xanthohumol is choice and determine if food products will be accepted or produced during wort boiling (Stevens et al. 1999). In rejected by the consumer, it is first necessary to find addition, the bitter tasting 8-prenylnaringenin (15) was hTAS2R/ligand combinations using the key bitter com- identified in hops and beer (Stevens et al. 1999). Although pounds in foods as stimuli. To date, only very few bitter multiple research groups tried to investigate the sensory receptors have been deorphanized using the key bitter contribution of these hop-derived bitter compounds in beer molecules that have been unequivocally confirmed to (Fritsch and Shellhammer 2007; Verzele 1970; Verzele et induce the typical bitterness of distinct foods or beverages al. 1970), the data reported in the literature are somehow (Brockhoff et al. 2007; Sandell and Breslin 2006). contradictory and, with the exception of the iso-α-acids 1-6 Examples for food-derived bitter compounds include (Fig. 1) (Kowaka and Kokubo 1976); (Guinard et al. 1994; absinthin from Artemisia absinthium L. and cascarillin Hughes and Simpson 1994, 1996), reliable data on human from the bark of Croton eluteria B. Both are used as bitter taste threshold concentrations in aqueous solution as well as flavorings for liquors and were reported to activate on psychometric functions of the individual bitter com- hTAS2R46 (Brockhoff et al. 2007). pounds are lacking. Driven by the need to discover the key players imparting The objectives of the present investigation were, the bitter taste of foods, the research area “sensomics” made therefore, to identify the hTAS2Rs bitter receptors respond- tremendous efforts to identify the most intense bitter ing to the hop-derived prenylated polyketides and flavo- molecules in fresh and processed foods such as, e.g. in noids 1-15 by functional expression studies in human carrots (Czepa and Hofmann 2003; Czepa and Hofmann embryonic kidney (HEK) cells, to determine the bitter 2004), roasted cocoa (Stark et al. 2005; Stark et al. 2006), threshold concentrations as well as psychometric functions stored cold-pressed linseed oil (Brühl et al. 2007), roasted of these compounds and to compare the human psycho- coffee (Frank et al. 2008; Frank et al. 2007; Frank et al. physical data with those obtained from the cell-based taste 2006), red wine (Hufnagel and Hofmann 2008a; Hufnagel receptor assay. and Hofmann 2008b), Gouda cheese (Toelstede and Hofmann 2008a; Toelstede and Hofmann 2008b), and orange juice (Glabasnia and Hofmann 2008), respectively. Materials and Methods Although a pronounced bitter taste is rejected in many foods including carrots (Czepa and Hofmann 2003; Czepa Chemicals and Materials and Hofmann 2004), linseed oil (Brühl et al. 2007), and orange juice (Glabasnia and Hofmann 2008), in some cases, The following compounds were obtained commercially: depending on the intensity, quality, and time/intensity formic acid, ethanol (Merck, Darmstadt, Germany). Deion- profile, bitterness contributes to the palatability of food ized water used for chromatographic compound purification and beverages and is accepted or even desired by consum- was prepared by means of a Milli-Q Gradient A10 system ers. Typical examples include coffee, cocoa, and beer. (Millipore, Billerica, USA). Bottled water (Evian; low Besides its sedative activity, beer has been attracting mineralization, 405 mg/L) was used for the sensory consumers over centuries due to its typical aroma and experiments. Formic acid, which is listed as generally bitter taste. The chemical structure of bitter compounds in recognized as safe flavoring agent for food and feed beer have been thoroughly investigated in the last decades, applications, was used to adjust the pH value of test and it is agreed that the typical beer bitterness is caused by solutions to pH 4.5 prior to sensory analysis. Trace amounts of adding hop (Humulus lupulus L.) during the wort boiling this acid do not influence the sensory profile of the test process. During wort boiling, a number of isomerization solution. An iso-α-acid extract (30%) prepared by pre- processes are of major importance for bitter taste develop- isomerization of a hop extract, a crude xanthohumol extract, ment in the final beer product. The trans- and cis-iso-α- and an ethanolic extract of hop was provided by the acids 1–6 (Fig. 1) have been identified as the major bitter Hallertauer Hopfenveredelungsgesellschaft mbH (Mainburg, contributors in beer (Kowaka and Kokubo 1976)and Germany). demonstrated to be generated upon a re-arrangement reaction of their hop-derived precursors, the α-acids 7–9 Bitter Compounds (Fig. 1) (De Keukeleire and Verzele 1971). Besides the α- acids, the second major constituents of hop are the β-acids Following the protocols reported recently (Intelmann et al. 10–12 (Fig. 1), but there are almost no data available on the 2009), the iso-α-acids trans-isocohumulone (1), trans- direct contribution of these compounds to beer bitterness. isohumulone (2), trans-isoadhumulone (3), cis-isocohumu- The so-called isoxanthohumol (13, Fig. 1), identified as a lone (4), cis-isohumulone (5), and cis-isoadhumulone (6) 120 Chem. Percept. (2009) 2:118–132
Fig. 1 Chemical structures of OO OO OH O OH O the iso-α-acids trans-isocohu- mulone (1), trans-isohumulone H H (2), trans-isoadhumulone (3), OOH OOH O OHOH O OHOH cis-isocohumulone (4), cis-iso- HO humulone (5), and cis-isoadhu- mulone (6), the α-acids cohumulone (7), humulone (8), and adhumulone (9), the β-acids 14 710 colupulone (10), lupulone (11), and adlupulone (12), as well as OO OO OH O OH O the prenylated flavonoids iso- xanthohumol (13), xanthohumol H H (14), and 8-prenylnaringenin OOH OOH O OHOH O OHOH HO (15)
25 811
OO OO OH O OH O
H H OOH OOH O OHOH O OHOH HO
36 912
OH OH OH
HOO HOHO HOO
O O O O OH O
13 14 15
were isolated from an iso-α-acid extract (30%), the α-acids constructs included the sequence for the first 45 amino cohumulone (7), humulone (8), and adhumulone (9), as well acids of the rat somatostatin receptor type 3 (SST) coupled as the β-acids colupulone (10), lupulone (11), and adlupu- to the N-terminal ends of the taste receptors in order to lone (12) were isolated from an ethanol extract of hop, improve membrane targeting. At the C termini of the xanthohumol (14) was purified from the commercial TAS2Rs, a herpes simplex virus (HSV) glycoprotein D xanthohumol extract, and isoxanthohumol (13) was prepared epitope was added for immunocytochemical detection. The from 14 by alkaline cyclization. Purified 8-prenylnaringenin SST-hTAS2R-HSV pcDNA5/FRT or pEAK-10 plasmid (15, Fig. 1) was provided by the Hallertauer Hopfenverede- constructs were transiently transfected in HEK-293T cells lungsgesellschaft mbH (Mainburg, Germany). Prior to the stably expressing the chimeric G-protein α subunit HEK- cell-culture assays and psychophysical tests, the purity 293T Gα16gust44 (Ueda et al. 2003). An “empty” (>98%) of the individual bitter compounds was checked pcDNA5/FRT plasmid was used as negative control. by means of high-performance liquid chromatography The HEK-293T Gα16gust44 cells were seeded into 96- (HPLC)/UV, HPLC/mass spectrometry (MS), as well as 1H well plates (Greiner Bio-One, Frickenhausen, Germany) NMR spectroscopy. under regular cell culture conditions [Dulbecco's modified Eagle medium (DMEM), 10% FCS, 1% penicillin/strepto-
Expression of TAS2Rs in Heterologous Cells mycin; 37°C, 5% CO2, 95% humidity] and, 24–26 h later, transfected with 150 ng plasmid DNA using 300 ng Lipofect- The bitter taste receptors were cloned into constructs amine 2000 (Invitrogen, San Diego, CA). About 24–26 h after derived from pcDNA5/FRT (Invitrogen, San Diego, CA) transfection, the cells were loaded with 2 µmol/L of the or pEAK-10 (Edge BioSystems, Gaithersburg, MD). The calcium-sensitive dye Fluo4-acetoxymethylester (Molecular Chem. Percept. (2009) 2:118–132 121
Probes, Karlsruhe, Germany) in serum-free DMEM medium. six wells per iso-α-acid. All experiments were averaged, The organic anion-transport inhibitor probenecid (Sigma and the obtained ratios were compared. Aldrich, Schnelldorf, Germany) was added to the dye/medium In order to examine whether or not signal amplitudes mixture to reduce leakage of the hydrolyzed indicator dye. correlate with EC50 values, the responses of hTAS2R1 to One hour after loading, the wells were washed three times stimulation with the iso-α-acids 1 to 6 at concentrations with 130 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES corresponding to their EC50 values were compared. All with pH 7.4, 2 mmol/L CaCl2, and 10 mmol/L glucose using compounds were measured on the same plate under the a Denley Cell Washer (Thermo Fisher Scientific Inc., same conditions. Waltham, MA). Cells were incubated in washing buffer in the dark for 30 min between the two last washing steps. The Sensory Experiments prenylated polyketides, flavonoids, and the chalcone were dissolved in dimethyl sulfoxide (DMSO) and diluted in General Conditions and Panel Training washing solution to avoid exposure of the transfected cells to toxic DMSO concentrations above 0.1% (v/v). The sensory panel consisted of 12 assessors (five women Calcium traces of the transfected cells were recorded and seven men, age 25–40 years), who have given the before and after application of the test substances by means informed consent to participate in the sensory tests of the of a fluorometric imaging plate reader (FLIPR, Molecular present investigation and had no history of known taste Devices, Munich, Germany). The vitality of the cells during disorders. The individuals participated for at least 2 years in the experiment was verified by a second application of weekly training sessions with reference solutions in order to 100 nmol/L somatostatin-14 (Bachem, Bubendorf, Switzer- familiarize them with the taste language and to get them land), an agonist for the endogenous somatostatin receptor trained in recognizing and distinguishing different qualities type 2. To exclude unspecific responses and false-positive of oral sensations in analytical sensory experiments. For signals, mock-transfected cells were always measured on training and classification of bitter taste, solutions of the same plate with the same compound concentrations as MgSO4 (166 mmol/L) representing a short-lasting, metallic the cells expressing hTAS2Rs. All experiments were bitter taste quality perceived mainly at the anterior part of performed in duplicates at least at three independent days the tongue, salicin (1.4 mmol/L) imparting a long-lasting in order to account for differences in cell passages and bitter taste sensation perceived mainly in the back of the transfection efficiency. tongue as well as the throat, and caffeine (8.0 mmol/L) For the calculation of concentration–response functions, providing a long-lasting bitterness perceived all over in the the amplitudes from mock-transfected cells were sub- oral cavity, were used as references. The assessors had tracted from the signal amplitudes of receptor expressing participated at regular intervals in sensory experiments and cells by means of the FLIPR384® software (Molecular were, therefore, familiar with the techniques applied. Devices, Munich, Germany). Next, the amplitudes were Sensory analyses were performed at 22–25°C in three corrected for dye loading efficiency and cell numbers. The independent sessions for each test. In order to minimize data were plotted half-logarithmically versus agonist cross-modal interactions with olfactory responses, nose concentrations. EC50 values were calculated using the clips were used as recommended in the literature (Lawless software SigmaPlot 9.01 (Systat Software GmbH, Erkrath, et al. 2004). Germany) using nonlinear regression to a sigmoid function
(Eq. 1). Within this equation, b represents the EC50 value, Precautions Taken for Sensory Analysis of Purified Bitter x the actual agonist concentration, a represents the Compounds maximal value (top) and d the minimal value (bottom), and c the Hill coefficient. Prior to sensory analysis, buffer compounds and solvent traces were removed from the purified bitter compounds 1– ðÞ ð Þ¼ a d þ ð Þ 15 isolated from the hop materials. To achieve this, the f x c d 1 ðÞb=x individual fractions were suspended in water and the remaining volatiles and solvent traces were removed in A subset of iso-α-acids (1, 2, 3, and 6) was chosen to high vacuum (<5 mPa, 35°C), then again taken up in water investigate potential additive effects on activation of and freeze-dried twice. High-resolution gas chromatogra- hTAS2R1. The responses of the receptor expressing cells phy–MS and ion chromatographic analysis revealed that the to iso-α-acids 1, 2, 3,or6 were measured individually at a bitter compounds treated by that procedure are essentially concentration of 10 µmol/L or in combinations of com- free of solvent traces and buffer compounds. In order to pounds 1+2 or 3+6 using concentrations of 5 µmol/L each. minimize the uptake of any toxic compound, the sensory The functional assay was repeated at least three times with analyses were performed by using the sip-and-spit method 122 Chem. Percept. (2009) 2:118–132 by which the test materials were not swallowed but In Mouth Oral Release Experiment expectorated. In order to investigate a potential binding of the bitter Recognition Threshold Concentrations compounds to the epithelium of the oral cavity, an aqueous solution (10 mL; 10 µmol/L each) of trans-isocohumulone The bitter recognition threshold values were determined by (1), cis-isohumulone (5), cohumulone (7), and colupulone 12 panelists by means of a triangle test according to the (10) was taken into the oral cavity, swirled around for 15 s, protocol detailed in ISO 4120 (Anonymus 2007). Using and then expectorated into a glass beaker (sample 0). bottled water (pH 4.5) as the solvent and an inter-stimulus Subsequently, the mouth was intensely rinsed with bottled interval length of 5 min, linear dilutions of the samples water (10 mL) for 30 s, expectorated, and collected in were presented in the order of increasing concentrations to another beaker (sample 1). Finally, an aliquot (10 mL) of the trained sensory panel in three different sessions. The 50% aqueous ethanol was taken in the mouth and swirled panelists were asked to swirl around the solution in the oral around to release bitter compounds adsorbed to the oral cavity for 10 s prior to expectoration. The individual threshold epithelium and was expectorated after 15 s (sample 2). In concentration of each panelist is calculated as the geometric order to counteract any adsorption of the analytes to glass mean between the last incorrect and the first correctly and filter materials used during the work-up, the individual identified sample solution. The bitter recognition threshold samples 0 and 1 were diluted 1:2 with ethanol and/or water of the panel was calculated from the geometric means of all to reach a final ethanol concentration of 50% (v/v) in each individual threshold concentrations. For statistical comparison sample solution. After ultrafiltration (Vivaspin 5 kDa, of the bitter threshold levels of the individual bitter substances, Sartorius Stedim Biotech GmbH, Goettingen, Germany) a one-way analysis of variance (ANOVA) was carried out of an aliquot (500 µL) of each solution, the filtrates using the individual threshold concentrations of each panelist. obtained were analyzed quantitatively for the bitter com- The relative standard deviation among subjects was less than pounds 1, 5, 7, and 10 by means of HPLC-MS/MS. As 0.6 for a given taste compound. control, another solution (10 mL) of these test compounds (10 µmol/L each) was separated directly by means of Psychophysical Concentration–Response Functions ultrafiltration, and the filtrate was analyzed by means of HPLC-MS/MS. For the recording of human concentration–response curves, the concentration range of the test samples was dependent Liquid Chromatography/Mass Spectrometry on the determined bitter threshold value and the maximum solubility of each individual bitter substance and was An Agilent 1100 Series HPLC system, consisting of a 5–512 µmol/L for the iso-α-acids 1–6,2–1,024 µmol/L pump, a degasser, and an autosampler (Agilent, Waldbronn, for cohumulone (7) and colupulone (10), and 1–480 µmol/L Germany), was connected to an API 4000 Q-TRAP mass for isoxanthohumol (13) and xanthohumol (14), respectively. spectrometer (AB Sciex Instruments, Darmstadt, Germany) All substances were dissolved in bottled water (pH 4.5) and used for quantitative analysis of the hop compounds. containing 5% of ethanol. In addition, the bitter intensities of Chromatography was performed using a 250×4.6 mm, these compounds at six equal concentration steps, namely 5 µm, Pursuit C18 column (Varian, Middelburg, The 5.5, 11.0, 22.0, 44.0, 88.0, and 176.0 µmol/L, were Netherlands) with a solvent gradient of acetonitrile, determined in direct comparison of the samples. For both containing 0.5% formic acid and aqueous formic acid assessments, six panelists were asked to evaluate the bitter (0.5% in water). LC/MS analysis was performed by means intensity of the individual sample solutions in the order of of the multiple reaction monitoring mode and negative increasing concentrations by rating the taste intensity of a electrospray ionization using the MS/MS parameters dilution of an individual compound against the intensity of reported recently (Intelmann et al. 2009). another dilution of the same compound and by cross- checking the bitter intensity between the test compound Statistical Analyses and a series of dilutions of salicin (0.07–140 mmol/L), which was presented in every session as a reference for direct Statistical variation was determined by one- or two-way comparison. In order to calculate EC50 values and the analysis of variance (ANOVA). When concentration repre- maximum intensities of the psychometric curves, the data sented a factor of the ANOVA, a mixed effect model was obtained for each individual were averaged and fitted to applied setting the concentration as a random factor. Results sigmoid functions (Eq. 1). For the statistical comparison of with P values <0.05 were considered as statistically the bitter intensities at distinct concentration steps, a two- significant. For multiple comparisons, a Fisher's least sig- factor ANOVA was carried out. nificant difference (LSD) test was applied with Bonferroni– Chem. Percept. (2009) 2:118–132 123
Holm correction of the α value to reduce the risk of type I errors. For these comparisons the global α level was set to 0.05.
Results and Discussion
Although various well-designed studies have been per- formed in recent years to understand the human bitter taste perception, a rather limited number of studies (e.g. Bufe et al. 2005) correlated in vitro data obtained in cell lines expressing bitter taste receptors with in vivo data recorded in human psychophysical experiments. In the present article, we investigated the recognition threshold concen- trations and the concentration–response curves of the beer bitter compounds 1–15 (Fig. 1) using a cell-based receptor assay and human psychophysical experiments. The indi- vidual test compounds were isolated from commercially available hop extracts, and a purity of more than 98% was confirmed by means of HPLC-UV, LC-MS, and 1HNMR spectroscopy, respectively. Fig. 2 Responses of cells expressing hTAS2R1 to four different hop bitter compounds (a) and of hTAS2R1, hTAS2R14, and hTAS2R40 Identification of the hTAS2Rs Responding to Bitter expressing cells to a single compound, cohumulone (b). Calcium Compounds from Hops and Beer responses of Fluo4-AM loaded HEK-293T-Gα16gust44 cells express- ing hTAS2R1 or mock-transfected cells elicited by bath application of 10 µmol/L trans-isocohumulone (1), 10 µmol/L cis-isohumulone (4), In order to identify the candidate taste receptors mediating 0.3 µmol/L cohumulone (7), or 0.3 µmol/L colupulone (10) are shown the bitter taste of the hop-derived prenylated polyketides, in a.Inb, calcium traces are shown of cells expressing hTAS2R1, flavonoids, and chalcones 1–15, we transiently expressed hTAS2R14, or hTAS2R40 or of mock-transfected cells following administration of 1.0 µmol/L cohumulone (7). Note that cohumulone the hTAS2Rs in HEK-293T cells stably transfected with a activates only two of the receptors but not hTAS2R14. All responses construct encoding the chimeric G-protein Gα16gust44. were recorded using a FLIPR system This construct was designed to couple the activation of hTAS2R receptors to the release of Ca2+ from intracellular stores, which can be measured using calcium-sensitive fluorescence dyes (Bufe et al. 2002; Chandrashekar et al. challenged by bath application of trans-isocohumulone (1), 2000; Ueda et al. 2003). In order to determine the highest cis-isohumulone (4), cohumulone (7), and colupulone (10) concentration of compounds 1–15 that can be employed in in comparison to a calcium trace of mock-transfected cells. the functional expression experiments, we first applied each Figure 2b reveals that cohumulone (7) was able to activate substance at increasing concentrations to non-transfected cells expressing hTAS2R1 or hTAS2R40 but not cells cells to monitor unspecific cellular responses (artifact test). expressing hTAS2R14 or mock-transfected cells. Next, we administered the 15 substances to HEK293T With the exception of 8-prenylnaringenin (15), all test Gα16gust44 cells transiently expressing the 25 hTAS2Rs at compounds gave robust increases in intracellular calcium the highest concentration that did not cause calcium signals levels in cells transfected with hTAS2R1 complementary in the previous artifact test. Most compounds could be DNA (cDNA) with threshold values of activation varying employed at 100 µmol/L, but in some cases, it was over two orders of magnitude between 0.03 µmol/L for necessary to reduce the concentration to 10 µmol/L (10, cohumulone (7; n=4) and 3.0 µmol/L for isoxanthohumol 11, and 12) or 3 µmol/L (7, 8, and 9). (13; n=2) (Table 1). The threshold concentration of the Using this strategy, we observed that cells expressing structurally related flavonoid xanthohumol (14) was com- hTAS2R1, hTAS2R14, or hTAS2R40 responded to several parable to that of isoxanthohumol (13), which showed a of the test compounds (Fig. 2, Table 1) and vice versa that threshold value of 1 µmol/L. In marked contrast, cells the test compounds stimulated various combinations of expressing hTAS2R14 were sensitive to 8-prenylnaringenin these receptors. Cells expressing any of the other TAS2Rs (15) with a threshold value of activation of 0.3 µmol/L, did not respond. As an example, Fig. 2a displays the whereas they were insensitive to the group of α-acids (7–9) calcium traces of cells expressing hTAS2R1 that have been up to the highest test concentration of 3 µmol/L and to the 124 Chem. Percept. (2009) 2:118–132
Table 1 Threshold concentrations of bitter compounds 1–15 in the taste receptor cell assay
Substance (no.)a Threshold concentrationb (µmol/L) in cells expressing
hTAS2R1 hTAS2R14 hTAS2R40 trans-isocohumulone (1) 1 1 n.r.c trans-isohumulone (2) 0.3 1 n.r.c trans-isoadhumulone (3) 0.3 1 n.r.c cis-isocohumulone (4) 1 1 n.r.c cis-isohumulone (5) 0.3 0.3 n.r.c cis-isoadhumulone (6) 0.3 1 n.r.c Cohumulone (7) 0.03 n.r.d 0.003 Humulone (8) 0.1 n.r.d 0.1 Adhumulone (9) 0.1 n.r.d 0.03 Colupulone (10) 0.1 n.r.e 0.03 Lupulone (11) 0.1 3 n.r.e Adlupulone (12) 1 3 n.r.e Isoxanthohumol (13)3 3 10 Xanthohumol (14)1 3 3 8-Prenylnaringenin (15) n.r.d 0.3 n.r.d a Substance number referring to the structure given in Fig. 1 b Threshold concentration is defined as the lowest concentration of the test substance that was used and led to a cellular response and is given as the mean of the data obtained by duplicate measurements performed at least at three independent days in order to account for differences in cell passages and transfection efficiency. Specificity of receptor activation was controlled by application of test substance on mock-transfected cells c No response to the stimulus up to a concentration of 30 µmol/L d No response to the stimulus up to a concentration of 3 µmol/L e No response to the stimulus up to a concentration of 10 µmol/L
β-acid colupulone (10) up to 10 µmol/L (Table 1). In α-andβ-acids the co-congener displays a distinctly lower addition, cells expressing hTAS2R40 were identified to EC50 value than the corresponding n- and ad-isomers. respond to the α-acids 7–9, the β-acid colupulone (10), as When compared to the α-andβ-acids, the group of iso- well as to isoxanthohumol (13) and xanthohumol (14). α-acids (1–6) showed somewhat higher EC50 values Interestingly, the colupulone (10) congener lupulone (11) between 2.5 and 10.6 µmol/L (Table 2). Notably, the and adlupulone (12) were unable to activate hTAS2R40. trans-isomers 1–3 showed higher EC50 values as the In order to investigate the activation of hTAS2R1, corresponding cis-isomers 4–6 with the EC50 value of hTAS2R14, and hTAS2R40 in more detail, cells expressing 9.0 µmol/L for trans-isohumulone (2) being about threefold these receptors individually were challenged with increas- above the value (3.3 µmol/L) of cis-isohumulone (5). ing concentrations of the bitter compounds 1–15, respec- Recording of concentration–response functions using tively. The concentration-dependent cellular responses cells transfected with hTAS2R14 or hTAS2R40 cDNA, followed the expected sigmoid functions when plotted respectively, gave robust responses of hTAS2R14 chal- semilogarithmically (Fig. 3), allowing EC50 values to be lenged with the iso-α-acids (1-6), the β-acids 11 and 12, determined as reliable measures for the potencies of and 8-prenylnaringin (15), as well as concentration- agonists at their receptors (Table 2). dependent activation of hTAS2R40 by the α-acids (7–9) With the exception of compounds 13 and 14, which did and colupulone (10) (Table 2). As we have already found not allow establishment of a concentration–response function for hTAS2R1, the trans-isomers 1–3 showed higher EC50 due to unspecific responses of the cells above 3.0 µmol/L, values also for activation of hTAS2R14 than for the
EC50 values between 0.2 (7) and 10.6 µmol/L (1)couldbe corresponding cis-isomers 4–6. Interestingly, we observed determined for all the test compounds in cells expressing a linear correlation between the EC50 values of the six iso- hTAS2R1. The α-andβ-acids exhibit comparatively low α-acids (1–6) for hTAS2R1 and hTAS2R14 (Fig. 4a). This
EC50 values, such as 0.2 µmol/L for the α-acid cohumulone suggests that the binding sites of the two receptors interact (7) and 0.7 µmol/L for the corresponding β-acid colupulone with similar structural motifs of the compounds 1–6. The (10), respectively (Table 2). Interestingly, within the group of same may also apply to hTAS2R40 as its ligand spectrum Chem. Percept. (2009) 2:118–132 125
100 trans-isocohumulone (1) mixture of both agonists (5 µmol/L each). The binary 75 mixture contained both agonists only in a concentration of 50 5 µmol/L, since the dose-dependent receptor activation 25 does not follow a linear relationship. Hence, an intermedi- 0 ate activation is expected for the case of additive behavior. 100 cis-isocohumulone (4) 75 In addition, a second set of experiments was performed 50 under the same conditions using trans-(3)andcis- 25 isoadhumulone (6) as agonists. The averaged fluorescence 0 intensities of the individual experiments are depicted in 100 cohumulone (7) Fig. 5. The data show that the relative signal amplitudes 75 evoked by separate application of compounds 1, 2, 3,or6 rel. intensity [%] 50 agree well with those that can be predicted from the data of 25 Tables 1 and 2. The signal amplitudes evoked by the binary 0 mixtures of compounds 1+2 or 3+6 reflect the arithmetic 100 colupulone (10) mean of the values generated by separate application of the 75 agonists. Thus, these results clearly confirm the additive 50 activation of hTAS2R1 by different polyketides when 25 present in mixtures. It is interesting to note that this 0 additive effect was observed for compounds with different 0.003 0.01 0.03 0.1 0.3 1 3 10 30 100 300 1000 concentration [µmol/L] alkanoyl side chains, such as trans-isocohumulone (1) and trans-isohumulone (2)orcis- and trans-epimers (3 and 6). Fig. 3 Concentration–response relation of hTAS2R1-transfected HEK cells (circle) and human concentration–response function (diamond) recorded for trans-isocohumulone (1), cis-isocohumulone (4), cohu- Human Bitter Threshold Concentrations mulone (7), and colupulone (10). Changes in fluorescence were plotted vs. log agonist concentration. Human psychometric functions Determination of bitter recognition thresholds of com- are normalized to equal the maximal activation of the receptor; error pounds 1–15 by means of a triangle test revealed low bars represent the confidential interval (P=0.05) threshold concentrations for all the test substances within the narrow range of 7–39 µmol/L (Table 3). Therefore, a overlaps with those of hTAS2R1 and hTAS2R14. However, one-factorial ANOVA was used to differentiate between the we are reluctant to conclude that the binding sites of the individual molecules. With an error probability level of P< hTAS2Rs for the compounds 1–15 are similar. It is more 0.001, significant differences were observed between the likely that the binding sites of the three receptors are bitter substances [F(14,165)=28.2, P<0.001]. Multiple composed of different structural motifs. This assumption is comparisons based on a Fisher's LSD test with Bonfer- supported by a phylogenetic consideration. The three genes roni–Holm correction revealed significantly lower threshold are located on different chromosomes, which is remarkable, concentrations of 7–10 µmol/L for the cis-iso-α-acids 4–6 given the fact that all 25 hTAS2R genes and their known when compared to the corresponding trans-iso-α-acids 1–3 pseudogenes cluster in only four loci on chromosomes 5, 7, (13–20 µmol/L) or the α-acids 7–9 (17–21 µmol/L). The and 12 (Bufe et al. 2002). Thus, these three genes are direct comparison of trans-isocohumulone (1), evaluated to derived from phylogenetically “old” ancestral genes and show a bitter threshold of 19 µmol/L, with its diastereo- thus are among those hTAS2R paralogs that display the meric antipode cis-isocohumulone (4) exhibiting a bitter highest degree of sequence divergence. Therefore, it would threshold of 7 µmol/L, clearly demonstrates the influence of be a challenging task to elucidate the binding motifs of the stereochemistry of the iso-α-acids on their bitter taste. these receptors in future work. Notably, the threshold concentrations of 13–20 µmol/L determined in this report for the individual trans-iso-α- Receptor Activation by Iso-α-Acid Combinations acids (1–3) are well in line with the threshold concentration of 4.54 mg/L (~13 µmol/L) published earlier for a trans- As iso-α-acids in mixtures were hypothesized to contribute iso-α-acid mixture (Weiss et al. 2002). No human threshold additively to bitter taste earlier in the literature (Verzele concentrations are available in the literature for purified 1970), the following experiments were performed to single compounds 1–15. investigate this phenomenon at the molecular level. Cells Although the complete group of trans-iso-α-acids (1–3) expressing hTAS2R1 were challenged separately either and α-acids (7–9) could not be significantly differentiated with trans-isocohumulone (1)ortrans-isohumulone (2), by means of multiple comparisons, the higher threshold each at a concentration of 10 µmol/L, or with a binary concentrations of 35–39µmol/L determined for the β-acid 126 Chem. Percept. (2009) 2:118–132
Table 2 EC50 values of bitter compounds 1–15 in the taste receptor cell assay
a b Substance (no.) EC50 values (µmol/L) in cells expressing
hTAS2R1 hTAS2R14 hTAS2R40 trans-isocohumulone (1) 10.6 14.5 n.r.c trans-isohumulone (2) 9.0 11.2 n.r.c trans-isoadhumulone (3) 6.7 9.0 n.r.c cis-isocohumulone (4) 7.4 9.4 n.r.c cis-isohumulone (5) 3.3 2.6 n.r.c cis-isoadhumulone (6) 2.5 2.8 n.r.c Cohumulone (7) 0.2 n.r.d 0.04 Humulone (8) 1.4 n.r.d 0.4 Adhumulone (9) 0.7 n.r.d 0.2 Colupulone (10) 0.7 n.r.e 0.2 Lupulone (11) 3.0 1.3 n.r.e Adlupulone (12) 2.2 4.1 n.r.e Isoxanthohumol (13) n.d.f n.d.f n.d.f Xanthohumol (14) n.d.f n.d.f n.d.f 8-Prenylnaringenin (15) n.r.d 1.5 n.r.d a Substance number referring to the structure given in Fig. 1 b The EC50 value is defined as the concentration required for a half-maximum activation of the receptor and is given as the mean of the data obtained by duplicate measurements performed at least at three independent days in order to account for differences in cell passages and transfection efficiency. Specificity of receptor activation was controlled by application of test substance on mock-transfected cells c No response to the stimulus up to a concentration of 30 µmol/L d No response to the stimulus up to a concentration of 3 µmol/L e No response to the stimulus up to a concentration of 10 µmol/L f Not determined because agonist could not been used in sufficiently high concentrations congeners 10–12 were significantly different from all the hydrophobic β-acid colupulone (10), exhibiting one addi- other bitter compounds evaluated (Table 3). In contrast to tional isoprenyl moiety when compared to the α-acid, the previous suggestion that bitter taste is correlated to exhibited a lower bitter threshold than the less hydrophobic hydrophobicity of a compound (Gardner 1979), the α-acid cohumulone (7).
15 350 A (1) B 300 (1)
(2) 250 10 (3) (4) 200 (2) 150 (4) human [µmol/L] hTAS2R14 [µmol/L] 5 50 (5) (3) 50 (6)
EC 100 (6) EC
(5) 50
0 0 0 5 10 15 0 5 10 15
EC50 hTAS2R1 [µmol/L] EC50 hTAS2R1 [µmol/L]
Fig. 4 Correlation of the EC50 value of the iso-α-acids 1–6 measured system and EC50 values were determined by a sigmoid fit to the for hTAS2R1 with those determined for hTAS2R14 (a) and with the function of fluorescence (ΔF/F) plotted vs. log agonist concentration. human psychophysical EC50 values (b). Receptor activation was Psychophysical EC50 values were determined by a sigmoid fit to the determined using HEK-293T-GR16gust44 cells transfected with averaged bitter intensities evaluated by six subjects against a salicin hTAS2R1 or hTAS2R14 cDNA, respectively, and loaded with reference curve (0.07–140 mmol/L) FLUO4-AM. Calcium traces of cells were recorded using the FLIPR Chem. Percept. (2009) 2:118–132 127
14, and 15, respectively, were rather similar to those of the 1.0 iso-α-acids and did not differ significantly among each 0.9 other (Table 3).
0.8 Human Concentration–Response Functions
rel. intensity 0.7
0.6 As supra-threshold measures have been suggested to be more suitable than threshold values to distinguish taste compounds 0.5 – 10 µmol/L 10 µmol/L 5 µmol/L (1) 10 µmol/L 10 µmol/L 5 µmol/L (3) (Bartoshuk 2000), concentration response functions were (1) (2) + 5 µmol/L (2) (3) (6) + 5 µmol/L (6) recorded to provide concentration-dependent information on – Fig. 5 hTAS2R1 receptor response to the bitter compounds trans- the bitter intensities of the prenylated polyketides 1 7 and 10 isocohumulone (1), trans-isohumulone (2), trans-isoadhumulone (3), as well as the flavonoids 13 and 14.Asanexample,the or cis-isoadhumulone (6) and binary mixtures of 1+2 and 3+6, averaged concentration–response curves of trans-isocohu- respectively. HEK-293T-GR16gust44 cells were transfected with mulone (1), cis-isocohumulone (4), cohumulone (7), and hTAS2R1 cDNA and were loaded with FLUO4-AM. Calcium traces were recorded in the FLIPR after stimulation of the cells with colupulone (10) are depicted in Fig. 3.Theintensityratings 10.0 μmol/L of single compounds or binary mixtures of each for the test solutions of increasing concentration followed 5.0 μmol/L sigmoid functions and are described by Eq. 1. The calculated
maximal intensities and EC50 values for each compound are In addition to the prenylated polyketides 1–12, the bitter listed in Table 3. Among the group of iso-α-acids, the EC50 tasting flavonoids isoxanthohumol (13), xanthohumol (14), values vary from 100 µmol/L for cis-isoadhumulone (6)to and 8-prenylnaringenin (15) were sensorially evaluated. 300 µmol/L for trans-isocohumulone (1), which is, on The threshold values of 16, 10, and 8µmol/L found for 13, average, a factor of 15 above the recognition threshold
Table 3 Human bitter threshold concentrations and characteristic values taken from the psychometric concentration–response functions of compounds 1–15
a b c d Substance (no.) TC (µmol/L) EC50 (µmol/L) Maximum intensity (−) Intensity at 88µmol/L (−) trans-isocohumulone (1) 19 (IV) 300 5.2 1.0 (I–III) trans-isohumulone (2) 20 (IV) 200 6.1 1.3 (III) trans-isoadhumulone (3) 13 (I-IV) 130 6.4 2.3 (IV) cis-isocohumulone (4) 7 (I) 180 8.1 3.0 (IV, V) cis-isohumulone (5) 10 (I-III) 110 7.4 3.8 (V,VI) cis-isoadhumulone (6) 8 (I, II) 100 7.3 3.9 (V) Cohumulone (7) 17 (III, IV) >500 7.8e 1.3 (III) Humulone (8) 21 (IV) n.d. n.d. n.d. Adhumulone (9) 21(IV) n.d. n.d. n.d. Colupulone (10) 39 (V) >500 5.4e 0.4 (I, II) Lupulone (11) 35 (V) n.d. n.d. n.d. Adlupulone (12) 37(V) n.d. n.d. n.d. Isoxanthohumol (13) 16 (II-IV) >500 8.8e 0.3 (I) Xanthohumol (14) 10 (I-III) 140 2.9 1.1 (III) 8-Prenylnaringenin (15) 8 (I, II) n.d. n.d. n.d.
Within a column, mean values marked with the same number (I–VI) given in parenthesis are not significantly different (Fisher's LSD test with Bonferroni–Holm correction, P=0.05) n.d. not determined a Substance number referring to the structure given in Fig. 1 b Threshold concentration is defined as the geometric mean of the individual thresholds of each panelist c The EC50 value is defined as the concentration required for a half-maximum bitter intensity taken from the human concentration–response function d Maximum intensity is the extrapolated value obtained by a sigmoid regression (term a from Eq. 1) e These data are regarded as estimations due to an insufficient number of values. 128 Chem. Percept. (2009) 2:118–132 concentrations of these bitter compounds. Like the threshold bitterness of 2.9 is only ~50% of that of the iso-α-acids. concentrations, also the EC50 values (100-180 µmol/L) of the This observation suggests that the compounds differ in their cis-iso-α-acids 4–6 were lower than those of the mode to activate the bitter taste receptors. corresponding trans-isomers 1–3 (130–300 µmol/L). Again, Unfortunately, the poor solubility of the substances 7, trans-isocohumulone (1) exhibited a distinctly lower bitter 10, and 13 prevented us from establishing reliable concen- potencywithanEC50 value of 300 µmol/L than its tration–response curves. In order to obtain some informa- corresponding diastereomeric antipode 4 (180 µmol/L). tion on the post-threshold bitterness of these compounds,
Comparing the EC50 values also indicated remarkable we directly compared the perceived bitter intensities of differences between the individual congeners within each selected compounds at concentrations of 5.5, 11, 22, 44, 88, group of tastants, e.g., the lowest EC50 values were found or 176 µmol/L. An ANOVA with two-factorial design for the ad-congeners 3 and 6, followed by the n-congeners (substance×concentration) revealed a significant effect for 2 and 5, and the co-congeners 1 and 4 (Table 3). With the the factor “substances” [F(9,45)=5.9, P<0.001] and, as exception of small shifts along the x-axis, the sigmoid already expected from the data above, significant differ- curves are rather similar in shape. Therefore, we expected a ences between the concentration steps [F(5,300)=314.0, similar influence of the congeners on their bitter threshold P<0.001]. This finding demonstrates that both factors concentrations. This assumption was confirmed by the impact on the perceived bitterness. Furthermore, also an linear correlation between threshold concentrations and interaction between the two factors substance and concen-
EC50 values (Fig. 6a). Thus, our data confirm a correlation tration was significant [F(45,300)=14.4, P<0.001]. This between the length of the alkanoyl side chain in the “substance×concentration” interaction is certainly due to congeners of hop bitter acids and their ability to elicit bitter the shape of the sigmoid functions, with small differences at taste (Gienapp and Schroeder 1975). lower concentration levels and bigger differences at higher Extrapolation of the sigmoid functions determined for concentrations. To differentiate the individual compounds, the six iso-α-acids 1–6 suggested that these bitter com- it was therefore necessary to have a look on the simple pounds differ in their maximal bitter intensities (Table 3). main effects by separately considering the factor “sub- Interestingly, the maximal intensities of the iso-α-acids stance” in all steps of the other factor “concentration.” were well correlated with their recognition threshold Application of this procedure revealed significant differ- concentrations (Fig. 6b). This observation is well in ences between the samples in concentrations above agreement with our previous observations that the isohu- 11 µmol/L. As an example, the bitterness intensity for mulone isomers 2 and 5 were more potent to elicit the factor “substance” at the concentration 88 µmol/L bitterness than the isocohumulone isomers 1 and 4 (Hughes [F(9,300)=50.8, P<0.001] is given in Table 3, as this is the and Simpson 1996). typical iso-α-acid concentration found in a Pilsner-type
We determined EC50 values also for xanthohumol (14) beers (Intelmann et al. 2009). Being well in line with and isoxanthohumol (13) (Table 3). Although the EC50 threshold concentrations and EC50 values, the multiple value (140 µmol/L) of xanthohumol (14) is in the range of comparisons showed a significant higher bitterness rating those of the cis-iso-α-acids, the calculated maximal for the cis-iso-α-acids (4–6) when compared to their
350 9 AB 300 (1) 8 (4) 250 (5) (6) 200 7 (2) 150 (4) (3) 6 (2) human [µmol/L]
50 (3)
100 maximum intensity
EC (6) (5) 5 (1) 50
0 4 0 5 10 15 20 25 0 5 10 15 20 25 taste threshold [µmol/lL] taste threshold [µmol/l]
Fig. 6 Correlation of the human bitter taste thresholds of the iso-α- means and were determined by 12 subjects by means of triangle tests acids 1–6 with their corresponding psychophysical EC50 values (a) in three independent sessions. Psychophysical EC50 values and and their maximum taste intensities extrapolated by using a sigmoid maximum taste intensities were determined using a sigmoid fit to function (b). Bitter recognition thresholds are presented as geometric the psychometric curves Chem. Percept. (2009) 2:118–132 129 corresponding trans-isomers (1–3). For example, the bitter 0.4 intensity of the 88 µmol/L solution of trans-isohumulone (2) showed an average value of 1.3, whereas an equimolar 0.3 solution of its cis-isomer (5) was evaluated with an intensity of 3.8 (Table 3). Compared to the cis-iso-α-acids, 0.2 the aqueous 88 µmol/L solutions of cohumulone (7) and rel. intensity xanthohumol (14) were judged only with mean bitter 0.1 intensity ratings of 1.3 and 1.1, respectively. As published work assumed only a weak impact of these substances on 0.0 12 3456 bitter taste (Fritsch et al. 2005; Verzele 1970), it is substance no. interesting to note that the bitterness intensities of 7 and 14 is similar to those of trans-isocohumulone and trans- Fig. 7 Amplitude of hTAS2R1 responses induced by the iso-α-acids 1–6 applied at their corresponding EC50 levels, that is, 10.6 µmol/L for isohumulone (1 and 2). From all compounds analyzed, the 1, 9.0 µmol/L for 2, 6.7 µmol/L for 3, 7.4 µmol/L for 4, 3.3 µmol/L for 88 µmol/L solutions of colupulone (10) and isoxanthohu- 5,and2.5µmol/Lfor6. Receptor activation was determined using mol (13) showed the lowest bitter intensities of 0.4 and 0.3, HEK-293T-GR16gust44 cells transfected with hTAS2R1 cDNA and respectively (Table 3). loaded with FLUO4-AM. Calcium traces of cells were recorded using the FLIPR system Moreover, increasing bitter intensities are observed for the individual congeners in the order co-, n-, and ad- congener, being well in agreement with the alkanoyl side diverse agonists is not significantly different. These data chains impacting on the EC50 values of the iso-α-acids. imply that the different sensory intensities perceived for the This trend is further strengthened at a concentration of six iso-α-acids at a given concentration are not only due to 176 µmol/L [F(9,300)=84.6, P<0.001], where these differ- differences in the taste receptor activation. ences became statistically significant (data not shown). This observation opens up the possibility to rank the individual Comparison of hTAS2R and Human Psychophysical Data iso-α-acids in terms of their bitter intensities. At the concentration of 176 µmol/L, the bitter intensities of the Taking all our data into account, it is obvious that the isomers increased in the following order: trans-isocohumu- threshold concentrations and EC50 values determined by the lone, 1 (1.8 units)
recovery [%] ments combined with the psychophysical characterization of the food-related bitter compounds in the present report 5 offer new insights in parameters influencing the perception 0 of dietary bitter compounds. Whereas receptor responses 012 step [-] clearly showed lower bitter thresholds for the unpolar α- and β-acids (7-12), the human sensory approach lead to Fig. 8 Recovery of bitter compounds (10 µmol/L each) after taking a contrary results and identified iso-α-acids (1-6) as most solution of trans-isocohumulone (1), cis-isocohumulone (4), cohumu- potent bitter compounds. lone (7), and colupulone (10) into the oral cavity, expectoration (sample 0), followed by rinsing with water (sample 1) and 50% Based on the findings of the present study, we conclude aqueous ethanol (sample 2) that, besides taste receptor activation, mechanisms that Chem. Percept. 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