Article Figures & Data
Figures
- Figure 1.
Isotopologue-specific EAG responses. Average EAG traces elicited by three concentrations of the normal (dark blue) or deuterated (magenta) isotopologues of four representative odorants are shown in A, F, K, and P. The shaded area indicates the timing and duration of odorant stimulation, while the scale is shown in the bottom right of each trace. The actual amplitude values elicited by the normal or deuterated isotopologues of HEL (B, C, D), BNZ (G, H, I), HEN (L, M, N), and BNL (Q, R, S) are represented by pairs of blue dots and magenta squares, respectively, shown for each individual, with the number of animals tested indicated in the abscissa as “fly number.” The ordinate scales have been adjusted to allow maximal resolution. The significance of isotopologue-specific amplitude differences were evaluated per fly using paired sample t tests and is indicated on the top left of each panel. Quantification of the collective differential response per odorant dilution was achieved by subtracting the absolute values of the h-odorant amplitude from that of the d-odorant for each fly tested, and the mean (Δ amplitude) ±SEM are shown in (E, J, O, and T). Therefore, the probabilities that Δ amplitude per odorant dilution is different than 0 are the same as shown on the respective panels. The actual Δ amplitude values and their SEMs are summarized as follows:
Dilution Mean h- SEM Mean d- SEM HEL 10−2 13.7872 0.5066 11.8815 0.4079 HEL 10−3 7.4245 0.2400 6.3346 0.2194 HEL 10−4 4.5079 0.1636 4.3553 0.1808 BNZ 10−2 8.5526 0.3489 9.3040 0.4044 BNZ 10−3 4.2038 0.1121 4.4785 0.1531 BNZ 10−4 1.8785 0.1916 1.9165 0.1940 HEN 10−2 11.4822 0.4242 9.6779 0.1681 HEN 10−3 4.2479 0.2185 3.4943 0.1723 HEN 10−4 2.7123 0.2251 2.6894 0.2495 BNL 10−2 4.4556 0.4427 4.5258 0.4009 BNL 10−3 2.7186 0.1895 2.7848 0.1660 BNL 10−4 2.1769 0.1641 2.1159 0.1596 - Figure 2.
The degree of deuteration does not affect the isotopologue-specific differential response. Average EAG traces of normal (dark blue) vs deuterated (magenta) odorants are shown in A and H. When two deuterated isotopologues are compared (E, L), the blue trace corresponds to the least deuterated species. The gray area on the left side of the traces indicates the timing and duration of odorant stimulation, while the scale is shown in the bottom right of each trace. A–C, Raw amplitudes elicited in response to normal and d2-hexanol (B), d5-hexanol (C), and d13-hexanol (D). The ordinate scales have been adjusted to allow maximal resolution. The number of flies tested with each isotopologue pair is shown in the abscissas, with each pair of blue dots and magenta squares representing the responses from single flies. Similarly, raw amplitudes in response to the di-deuterated hexanol (dark blue diamonds) vs the perdeuterated odorant (magenta squares) are shown in F. The significance of isotopologue-specific amplitude differences was evaluated per fly using paired sample t tests and is indicated on the top right of each panel. These differential responses are quantitatively represented in G, and ANOVA indicated significant differences (F(3,32) = 17.739, p < 0.0001), which were revealed by least square means (LSM) contrast analysis to be due to the difference of the d2 vs d13 Δ amplitude (open bar) compared with the other three (p = 0.0006, p < 0.0001, and p < 0.0001, respectively, in order of increasing deuteration). In contrast, comparing the Δ amplitudes of each partially deuterated odorant over the normal isotopologue with each other did not reveal significant differences (h/d2 vs h/d13, p = 0.087; h/d2 vs h/d5, p = 0.110; h/d5 vs h/d13, p = 0.940). I–K, Similarly, raw amplitudes elicited in response to normal and d3-acetophenone (I), d5-acetophenone (J), and d8-acetophenone (K). The ordinate scales have been adjusted to allow maximal resolution, while the number of flies tested with each isotopologue pair is shown in the abscissas. Each pair of blue dots and magenta squares represents the responses from single flies. Raw amplitudes in response to the d3-acetophenone (dark blue diamonds) vs the perdeuterated odorant (magenta squares) are shown in M. The significance of isotopologue-specific amplitude differences were evaluated per fly using paired sample t tests and is indicated on each panel. These differential responses are quantitatively represented in N, and ANOVA indicated significant differences (F(3,32) = 6.331, p = 0.002), which were revealed by LSM contrast analysis to be due to the difference of the d3 vs d8 Δ amplitude (open bar) from the other three (p = 0.0075, p = 0.0017, and p = 0.0005, respectively, in order of increasing deuteration). In contrast, comparing the Δ amplitudes of each partially deuterated odorant over the normal isotopologue with each other did not reveal significant differences (h/d3 vs h/d5, p = 0.438; h/d3 vs h/d8, p = 0.245; and h/d5 vs h/d8, p = 0.7287).
- Figure 3.
Isotopologue-specific EAG amplitudes of normal and deuterated alcohols. A–D, The maximal amplitudes for each fly tested with the normal (dark blue dots) or deuterated (magenta squares) isotopologue of the 2-carbon ethanol and d6-ethanol (ETL), the 5-carbon pentanol and d11-pentanol (PNL), the 6-carbon hexanol and d13-HEL, and the 8-carbon octanol and d17-octanol (OCT) at the 10−2 dilution. The number of flies tested with each isotopologue is shown in the abscissas, with each pair of blue and magenta dots representing the responses per single fly, while the ordinate scales have been adjusted for maximal resolution. The significance of the uncovered differences after paired t tests is shown on the graphs. The data in C are the same as in Figure 1D. E, Quantification of the collective differential response per odorant isotopologue shown as Δ amplitude ± SEM. The data for the hexanol isotopologues are the same as in Figure 1. The actual Δ amplitude values and their SEMs are summarized as follows:
Odorant Mean h- SEM Mean d- SEM ETL 3.0746 0.1671 4.1313 0.1733 PNL 10.6673 0.3975 11.1639 0.4391 HEL 13.7872 0.5066 11.8815 0.4079 OCT 6.4527 0.2811 7.0699 0.2752 - Figure 4.
Isotopologues elicit similar EAG activation and decay rates. A–P, The times required to achieve two-thirds of the maximal amplitude (time to rise, A–H) and times required to recover to one-third of the maximal amplitude (time to decay, I–P) are shown for isotopologues of all odorants used in this study. Dark blue and magenta triangles are used for the rise times due to normal and deuterated isotopologues, respectively, and conversely dark blue and magenta diamonds are used for decay times. The number of flies tested with each isotopologue is shown in the abscissas, and the ordinate scales have been adjusted for maximal resolution. The probability that paired t tests uncovered isotopologue-specific differences in rise and decay times is shown, with significant differences in bold and boxed. ETL, Ethanol; PNL, 1-pentanol; OCT, 1-octanol.
- Figure 5.
P450 inhibition does not alter the isotopologue-specific differential responses. A, Flies were exposed to PBO dissolved in acetone. The mean lethality ± SEM due to exposure to 0.25% (v/v) PBO in acetone alone (PBO), 5% and 7% methanol (MTL) alone (v/v in minimal food), and combinations thereof is shown (n = 8 for each). PBO significantly (t test-derived probabilities shown above the bars compared as indicated) augmented the lethality precipitated by either 5% or 7% methanol, demonstrating that under the conditions of the experiment it actually inhibits Drosophila P450s. B, Flies were exposed to PBO or the acetone vehicle (−PBO) for 18 h at 25°C. Another group of animals was exposed to PBO for 18 h and then allowed to recover on minimal food for another 18 h (REC). Average EAG traces from treated (+PBO), control (−PBO), and REC flies exposed to normal (h-HEL) and d13-hexanol (d-HEL) isotopologues. The gray area indicates the timing and duration of stimulation. C, The mean EAG amplitudes ± SEM of treated, untreated, and recovered animals demonstrated that the differential responses to HEL isotopologues remain significant (probabilities from paired t tests are shown above the relevant bars) despite the PBO treatment. D, Δ amplitudes calculated from the data in C. ANOVA (F(2,23) = 0.819) did not indicate significant differences (p = 0.453) among groups, indicating that the isotopologue-specific differences remain despite the PBO treatment. E, Mean EAG amplitudes ± SEM of PBO and vehicle-treated (−PBO) animals exposed to normal and d8-ACP show that the isotopologue-specific responses remain significant (paired t test probabilities above the respective bars) despite PBO treatment. F, Δ Amplitudes calculated from the data in E. The isotopologue-specific differences remain despite PBO treatment as ANOVA (F(1,14) = 0.087) did not indicate significant differences (p = 0.773) among groups. G, Mean EAG amplitudes ± SEM of PBO and −PBO animals exposed to normal and d17-octanol (OCT) indicate a marginal (p = 0.0152, paired t tests) isotopologue-specific response after PBO treatment. H, Δ Amplitudes calculated from the data in G. As indicated in G, the isotopologue-specific differences are decreased upon PBO treatment as ANOVA (F(1,16) = 7.727) indicated a significant difference (p = 0.013) among groups.
- Figure 6.
The differential response to isotopologues is conserved within the genus Drosophila. A–C, E–G, I–K, M–O, Raw EAG amplitudes from the antennae of D. simulans (D.sim), D. pseudoobscura (D.pse), and D. virilis (D.vir) in response to hexanol (A–C), benzaldehyde (E–G), hexanone (I–K), and benzonitrile (M–O) isotopologues (dark blue dots for the normal and magenta squares for the deuterated odorant). The ordinate scales have been adjusted to allow maximal resolution. The probability (paired t tests) that isotopologue-specific differences are uncovered is indicated on the panels. Δ Amplitudes calculated from the data in the previous panels are shown in D for hexanol, in H for benzaldehyde, in L for hexanone, and in P for benzonitrile. The relevant Δ amplitudes for D. melanogaster from Figure 1 are added for comparison. ANOVA did not indicate (F(3,34) = 0.992, p = 0.409) significant differences in Δ amplitudes for HEL (D) or BNL (F(3,28) = 1.518, p = 0.232; P). However, ANOVA indicated significant differences in Δ amplitudes for BNZ isopotologues (F(3,31) = 9.672, p < 3.25 × 10−5), which subsequent Tukey’s HSD test indicated were due to differences in the Δ amplitude values for D. simulans and D. pseudoobscura compared with that from D. virilis (α = 0.05). Similarly, ANOVA indicated differences (F(3,31) =10.802, p = 0.232) in Δ amplitudes elicited by HEN exposure (L). Tukey’s HSD test revealed that the Δ amplitude values of D. melanogaster and D. simulans were significantly different from those of D. virilis (α = 0.05).
- Figure 7.
Behavioral discrimination of 1-hexanol isotopologues within the genus Drosophila. The mean 1-hexanol isotopologue avoidance ± SEM of complementary experiments is shown. The Drosophila strains and species tested are indicated on the right of each pair of experiments. The graphs are shown horizontally to reflect the actual distribution of the flies in the left and the right arms of the T-maze. Room air is shown delivered on the right arm, whereas the odorant on the left, although in actuality the side of air and odorant delivery were alternated semi-randomly. Open bars indicate the naive response to the indicated isotopologue vs room air. A, C, E, G, I, Flies were exposed to 12–90 V electric footshocks (thunderbolts) in the presence of either normal (h-HEL, gray bars) or perdeuterated (d13-HEL, black bars) 1-hexanol and then tested for avoidance of the normal isotopologue vs air. The complementary experiments are shown in B, D, F, H, and J, with flies exposed to electric footshocks (thunderbolts) in the presence of either normal (h-HEL, gray bars) or perdeuterated (d-HEL, black bars) 1-hexanol and then tested for avoidance of the perdeuterated odorant vs air. Differences in the performance of each group were investigated by an initial ANOVA followed by least square means contrast analysis. The group trained to avoid the same isotopologue as used for testing was denoted as the control group, and the probabilities that it performed differently than naive or animals trained to the other isotopologue are shown above each relevant bar. n ≥ 8 for all groups.
Tables
h-hexanol+IPM d13-hexanol+IPM D. melanogaster (w1118 ) 5 μl/ml 1 μl/ml D. simulans 2 μl/ml 5 μl/ml D. pseudoobscura 2 μl/ml 2 μl/ml D. virilis 20 μl/ml 20 μl/ml D. melanogaster (Canton-S) 2 μl/ml 2 μl/ml h-1-Hexanol (Fluka Analytical) d2-1-Hexanol Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 4.314 BB 0.0273 2.69500 1.63299 0.04475 1 4.622 BB 0.0301 4.95090e-1 2.61661e-1 0.00676 2 5.653 BB 0.0369 7.43677e-1 3.22039e-1 0.01235 2 4.847 BB 0.0326 2.15994 1.02467 0.02950 3 6.214 BB 0.0578 6012.02588 1353.56238 99.82841 3 5.638 BV 0.0310 1.22029 6.18148e-1 0.01667 4 14.426 BB 0.1929 6.89491 4.49524e-1 0.11449 4 6.277 BB 0.0710 7312.0483 1446.4130 99.88262 5 6.953 BB 0.0404 1.71584 5.82760e-1 0.02344 6 9.090 BB 0.0251 8.65162e-1 5.27801e-1 0.01182 7 11.268 BB 0.0280 1.23281 7.18195e-1 0.01684 8 11.420 BB 0.0258 3.41531e-1 2.24189e-1 0.00467 9 13.871 BB 0.0324 5.62412e-1 2.92345e-1 0.00768 d5-1-Hexanol d13-1-Hexanol (lot X241P13) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.904 BB 0.0257 1.00243 6.49066e-1 0.01407 1 3.645 BB 0.0328 4.77799e-1 2.67615e-1 0.00709 2 4.796 BB 0.0339 6.67262 3.27978 0.09365 2 4.734 VB 0.0309 12.45923 6.33606 0.18490 3 5.215 BB 0.0326 2.84580 1.35165 0.03994 3 5.429 BB 0.0496 3.09593 8.68688e-1 0.04594 4 6.232 BV 0.0739 7097.00391 1337.32166 99.60153 4 5.695 BB 0.0364 8.49879e-1 3.48745e-1 0.01261 5 6.688 VB 0.0422 5.86693 2.26736 0.08234 5 6.127 BB 0.0810 6720.69971 1285.26160 99.73692 6 7.574 BB 0.0327 8.28919 3.91570 0.11633 6 7.432 BB 0.0368 8.44513e-1 3.97840e-1 0.01253 7 8.113 BB 0.0335 9.32212e-1 5.06874e-1 0.01308 8 10.646 BB 0.0317 2.78344 1.65248 0.03906 h-Benzaldehyde (Sigma) d6-Benzaldehyde (lot H468P23) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.083 BB 0.0214 2.73390e-1 2.07438e-1 0.00248 1 2.702 BB 0.0403 4.31283 1.78300 0.05008 2 2.445 BB 0.0212 8.67375e-1 6.65289e-1 0.00788 2 8.869 BB 0.0718 8558.87891 1929.26196 99.39384 3 5.213 BB 0.0273 3.35924e-1 2.02958e-1 0.00305 3 10.612 BB 0.0438 1.06592 4.46972e-1 0.01238 4 8.686 BB 0.0668 1.08576e4 2400.08643 98.68858 4 12.554 BB 0.0410 9.16474 5.10110 0.10643 5 9.365 BB 0.0290 3.97409 2.20585 0.3612 5 13.386 BB 0.0584 5.53546 1.52484 0.06428 6 10.762 BB 0.0309 2.21929 1.13301 0.02017 6 15.041 BB 0.0461 12.58218 4.87559 0.14612 7 19.732 BBA 0.0395 136.61024 50.65681 1.24170 7 19.918 BB 0.0484 19.53590 7.04030 0.22687 h-Benzoic acid d5-Benzoic acid Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 19.730 BBA 0.0439 85.00277 29.25834 1.00e2 1 19.658 BBA 0.0395 284.06458 105.29233 1.00e2 h-2-hexanone d5-2-hexanone Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.122 VV 0.0172 1.22825 1.17401 0.01777 1 2.567 BB 0.0252 3.98876 3.51804 0.05061 2 3.393 BV 0.0410 6898.30908 2597.11719 99.81266 2 2.792 BV 0.0310 4.36951e-1 2.68910e-1 0.00554 3 7.690 BB 0.0329 3.76977 1.76718 0.05455 3 3.342 VB 0.0505 7864.65332 2381.03467 99.79115 4 10.191 BB 0.0277 7.94950 4.47342 0.11502 4 3.766 BB 0.0427 11.39421 4.32301 0.14458 5 4.857 BB 0.0323 6.40037e-1 3.06994e-1 0.00812 h-Benzonitrile d5-Benzonitrile Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 10.234 BB 0.0557 9508.75098 2276.43311 1.000e2 1 4.644 BB 0.0344 2.18002 1.13441 0.01571 2 5.058 BB 0.0351 4.69176e-1 2.36558e-1 0.00338 3 6.316 BB 0.0311 18.41002 9.86647 0.13265 4 7.883 BB 0.0400 2.80853 1.02508 0.02024 5 10.188 BB 0.0808 1.38512e4 2657.15698 99.80494 6 15.243 BB 0.0389 6.83049e-1 2.95831e-1 0.00492 7 19.561 BB 0.0578 2.51985 7.02758e-1 0.01816 Results from gas chromatograms of normal, partially deuterated, and perdeuterated odorants used in the standard odorant set and relevant related odorants. The percentage (Area %) of the total that constitutes the main species within each preparation is shown in bold, as are those of all other species. For the hexanol isotopologues, the purity of all preparations was >99.6%. Contaminants varied with minimal overlap among the isotopologues and with the most abundant contaminant at 0.1–0.2%, but most others are at least 10-fold lower. Normal benzaldehyde purity was 98.7%, but the most abundant contaminant was benzoic acid (peak 7), while the contribution of others was negligible. Similarly, for d6-BNZ with 99.4% purity the most abundant contaminant was d5-benzoic acid (peak 7), as indicated by mass spectroscopy (data not shown). Solid normal and deuterated benzoic acid were dissolved in ethanol to generate a 1% solution, which did not show additional contaminants (benzoic acid isotopologues at 19.730 and 19.658 Ret times). Both hexanone isotopologues exhibited over 99.8% purity, while the contribution of contaminants was negligible. Normal benzonitrile was totally pure, while the deuterated isotopologue was 99.8% pure with negligible contribution from contaminants. Ret time, retention time; BB, Baseline to baseline; BV, Baseline to valley; VB, Valley to baseline; VV, Valley to valley.
h-1-Hexanol (Lluch Essence) d13-1-Hexanol (X421P8) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.196 BB 0.0369 5.72620 2.58805 0.10832 1 3.327 BB 0.0345 9.75216 6.15056 0.15369 2 2.569 BV 0.0214 5.22487 4.06881 0.09884 2 4.297 BB 0.0433 15.99059 8.01434 0.25201 3 2.726 VV 0.0331 4.29227 2.37241 0.08120 3 5.572 BB 0.0964 6313.49365 1076.56616 99.49862 4 2.856 VV 0.0308 2.58679 1.32312 0.04893 4 6.808 BB 0.0544 3.50448 1.18921 0.05523 5 3.148 VV 0.0475 3.80892 1.12580 0.07205 5 12.926 BB 0.0764 2.56706 5.30815e-1 0.04046 6 3.408 VB 0.0350 3.19802 1.62208 0.06050 7 4.004 BB 0.0818 2.04721 3.23609e-1 0.03873 8 5.228 BB 0.0435 3.19114 1.18053 0.06037 9 5.738 BB 0.0815 5256.20166 1146.01575 99.43107 h-Benzaldehyde (Fluka Analytical) d6-Benzaldehyde (X261P20) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.375 BB 0.0224 6.39770e-1 4.53907e-1 0.00680 1 2.377 BB 0.0267 5.28992e-1 3.69073e-1 0.00567 2 8.249 BB 0.0506 8977.63086 2349.54297 95.44216 2 8.105 BV 0.0544 1.22637 3.22554e-1 0.01314 3 8.921 BB 0.0426 9.60250 3.23823 0.10209 3 8.307 VB 0.0731 8838.80566 2099.49609 94.72651 4 10.373 BB 0.0419 8.93681e-1 3.07697e-1 0.00950 4 8.937 BB 0.0342 14.52237 7.00919 0.15564 5 12.829 BB 0.0340 6.93815e-1 3.11654e-1 0.00738 5 10.126 BB 0.0512 1.07901 3.21227e-1 0.01156 6 19.153 BB 0.0427 416.89743 139.98267 4.43208 6 11.935 BB 0.0376 1.19985 5.46938e-1 0.01286 7 12.754 BB 0.0581 2.19288 6.07426e-1 0.02350 8 14.382 BB 0.0350 1.63216 7.62619e-1 0.01749 9 19.128 BB 0.0530 469.68039 133.57043 5.03362 h-Acetophenone (Puriss grade, Fluka Analytical) d8-Acetophenone (G466P32) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 9.530 BB 0.0452 1.14808 3.80043e-1 0.01397 1 7.637 BB 0.0381 1.46471 6.08103e-1 0.01922 2 9.819 BB 0.0802 1.13668 2.06503e-1 0.01383 2 8.188 BB 0.0365 1.16085 4.74491e-1 0.01523 3 10.160 BB 0.0545 8210.81641 1976.72363 99.89956 3 10.084 BB 0.0605 7616.92383 1759.73987 99.93954 4 11.114 BV 0.0329 7.13131e-1 3.34774e-1 0.00868 4 11.941 BB 0.0893 1.98253 3.24316e-1 0.02601 5 11.176 VB 0.0833 3.74978 6.13817e-1 0.04562 6 13.617 BB 0.0512 1.50747 4.48821e-1 0.01834 Results from gas chromatograms of normal and perdeuterated odorants from distinct lots and sources, as indicated from those on Table 2 and Figure 1. The percentage (Area %) of the total that constitutes the main species within each preparation is shown in bold. The batch numbers of isotopologues distinct from those on Table 1 are indicated. Distinct trace impurities were present in the 1-hexanol preparation from Lluch Essence compared to that from Fluka Analytical (Table 2). The deuterated 1-hexanol contained fewer contaminants, which, however, constituted a slightly larger fraction of the sample, since pure d13-1-HEL exhibited 99.5% purity in this sample compared to 99.7% in the one in Table 2. Similarly, h-BNZ contained fewer contaminants than that in Table 2, but it constituted a lower percentage of the sample (94.4% vs 98.7% in Table 2), because a larger proportion was benzoic acid (Ret time, 19.153). d6-BNZ also contained additional contaminants from that in Table 2 (Ret times, 8.105, 8.937) and contained a larger percentage (5.03%) of deuterated benzoic acid than the batch in Table 2. Nevertheless, the EAG responses of the two sets of batches were similar. In contrast, the normal ACP sample was purer than the original one (Table 4), and the same was true for the d8-ACP sample. Based on retention times, there are no common contaminants in the two isotopologue preparations.
Odorant (from Table 3) Mean amplitude n Δ Amplitude p (t test) h-1-HEL (Lluch Essence) 11.9367 ± 0.6336 7 0.5841 ± 0.094 <8 × 10−4 d13-1-HEL (X421P8) 11.3525 ± 0.6546 h-BNZ (Fluka Analytical) 5.6187 ± 0.1380 8 −0.6275 ± 0.08 <1 × 10−4 d6-BNZ (X261P20) 6.2462 ± 0.1642 h-ACP (Puriss grade, Fluka Analytical) 6.0601 ± 0.4386 10 0.3103 ± 0.062 <7.1 × 10−4 d8-ACP (G466P32) 5.7498 ± 0.4073 Mean amplitudes ± SEMs of HEL, BNZ, and ACP isotopologue pairs distinct from those in Figures 1 and 2 derived from the indicated (n) animals are shown. Isotopologue pairs were used at the 10−2 dilution. The probability that the mean amplitudes evoked by the two isotopologues are significantly different is shown per odorant.
h-Acetophenone (Fluka analytical) d3-Acetophenone Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 8.634 VV 0.0532 1.75445 5.22483e-1 0.02044 1 9.212 BB 0.0459 6.59789e-1 2.27296e-1 0.00659 2 9.091 VB 0.0379 1.11003 4.33768e-1 0.01293 2 10.060 BB 0.0432 9.67617 3.62314 0.09659 3 9.499 BB 0.0409 1.46442 5.19445e-1 0.01706 3 10.384 BB 0.0443 9.54238 3.44315 0.09526 4 10.206 VB 0.0451 9.35422e-1 3.11201e-1 0.01090 4 10.844 BV 0.0763 9988.40723 1933.71594 99.71190 5 10.909 VV 0.0605 8552.97363 1899.87683 99.66466 5 11.217 VB 0.0400 7.89751e-1 3.28895e-1 0.00788 6 11.145 VV 0.0351 2.05978 8.85598e-1 0.02400 6 11.785 BB 0.0682 1.58633 5.11016e-1 0.01584 7 11.272 VV 0.0302 2.00964 1.05744 0.02342 7 12.807 BB 0.0334 5.71362 3.11383 0.05704 8 11.337 VV 0.0255 1.28859 7.71482e-1 0.01502 8 13.684 BB 0.0369 8.92039e-1 4.17685e-1 0.00891 9 11.391 VB 0.0300 1.06645 5.65312e-1 0.01243 10 11.826 BB 0.0296 7.87661 4.26206 0.09178 11 12.824 BB 0.0267 3.46171 1.94807 0.04034 12 12.967 BV 0.0318 7.98368e-1 3.91961e-1 0.00930 13 13.008 VB 0.0279 6.91700e-1 3.68989e-1 0.00806 14 14.378 BB 0.0265 4.26080 2.42068 0.04965 d5-Acetophenone d8-Acetophenone (lot G466P29) Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 10.866 BB 0.0933 9092.35840 1730.31287 99.98919 1 8.259 BB 0.0367 1.77587 8.40201e-1 0.01898 2 11.796 BB 0.0357 9.82952e-1 4.83287e-1 0.01081 2 8.850 BB 0.0328 1.34282 6.81408e-1 0.01435 3 10.820 BB 0.0933 9347.32031 1778.28320 99.90145 4 11.766 BB 0.0379 1.43155 6.45780e-1 0.01530 5 12.484 BB 0.0379 2.74861 1.23874 0.02938 6 13.712 BB 0.0391 1.48917 6.42145e-1 0.01592 7 16.677 BB 0.0313 4.32479e-1 2.30103e-1 0.00462 h-Ethanol d6-Ethanol Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.418 BB S 0.0205 4076.57593 3284.19873 1.000e2 1 2.387 BB 0.0360 4830.66064 2824.42505 1.000e2 h-1-Pentanol d11-1-Pentanol Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 2.711 BB 0.0231 6.06239e-1 4.13028e-1 0.00853 1 3.443 BB 0.0421 11.47077 6.04865 0.15594 2 4.421 BB 0.0363 1.78695 7.93818e-1 0.02515 2 3.984 BB 0.0381 24.50063 12.94281 0.33307 3 5.023 BB 0.0618 7100.83350 1662.52856 99.94445 3 4.528 BB 0.0792 7319.98291 1443.77832 99.51099 4 5.750 BB 0.0231 1.55354 1.05958 0.02187 h-1-Octanol d17-1-octanol Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%Peak
#Ret time
(min)Type Width
(min)Area
(pA*s)Height
(pA)Area
%1 5.607 BB 0.0376 8.33811 4.49661 0.10979 1 5.692 BB 0.0379 23.70400 12.64659 0.25730 2 6.696 BB 0.0325 2.99909 1.70881 0.03949 2 7.063 BB 0.0385 11.61009 6.02909 0.12603 3 8.018 BV 0.0456 1.06406 4.85292e-1 0.01401 3 8.644 BB 0.1126 9162.00488 1206.64636 99.45199 4 8.165 VB 0.0400 2.65737 1.29295 0.03499 4 9.924 BB 0.0483 1.24629 5.13031e-1 0.01353 5 8.821 BB 0.0560 27.26840 7.94571 0.35904 5 10.233 BB 0.0438 4.07517 1.99705 0.04424 6 9.302 BB 0.1036 7552.50098 1054.55908 99.44269 6 11.328 BB 0.0524 4.05706 1.45943 0.04404 7 13.140 BB 0.0522 2.07261 7.51371e-1 0.02250 8 13.614 BB 0.0537 1.37058 4.24300e-2 0.01488 9 14.565 BB 0.0490 2.34988 9.43809e-1 0.02551 Results from gas chromatograms of normal, partially deuterated, and perdeuterated additional odorants used herein. The percentage (Area %) of the total that constitutes the main species within each preparation is shown in bold. For acetophenone isotopologues, the purity of all preparations was >99.7%. The contribution of all contaminants was negligible as it ranged below 0.1%. Both ethanol isotopologues are 100% pure. For pentanol, the normal odorant was nearly pure (99.9%), while the deuterated isotopologue was highly pure at 99.5%, with two main, albeit low-level, contaminants: peak 1 at 0.16% and peak 2 at 0.33%. For octanol, the normal isotopologue was highly pure (99.4%), while the deuterated isotopologue is equally pure (99.5%). Although greater in number, the contribution of contaminants in the deuterated isotopologue is minor, except for the peak at 5.692 contributing 0.26% to the total. This peak is shared with the normal isotopologue at 5.607 and contributes 11% of the total while the major contaminating peak at 8.821 contributes 0.36%.
- Table 6:
Lack of differences in the amplitudes of partially and perdeuterated odorant pairs over a range of dilutions
Odorant Mean amplitude N Mean Δ amplitude p (t test) d2-1-HEL 1 × 10−4 4.0675 ± 0.1789 7 −0.0392 ± 0.009 0.3213 d13-1-HEL 1 × 10−4 4.1068 ± 0.1697 d2-1-HEL 1 × 10−3 6.4915 ± 0.1603 7 0.1438 ± 0.129 0.3085 d13-1-HEL 1 × 10−3 6.3477 ± 0.1641 d2-1-HEL 1 × 10−2 12.2325 ± 0.4389 7 −0.092 ± 0.195 0.653 d13-1-HEL 1 × 10−2 12.3246 ± 0.3804 d3-ACP 1 × 10−4 3.4181 ± 0.2490 7 0.0437 ± 0.054 0.4490 d8-ACP 1 × 10−4 3.3743 ± 0.2554 d3-ACP 1 × 10−3 4.6298 ± 0.2093 10 −0.1004 ± 0.064 0.1542 d8-ACP 1 × 10−3 4.7302 ± 0.2235 d3-ACP 1 × 10−2 6.2129 ± 0.6041 7 0.0447 ± 0.133 0.7478 d8-ACP 1 × 10−2 6.1682 ± 0.5080 The dilutions and the resultant mean amplitudes ± SEMs of perdeuterated and minimally deuterated HEL and ACP isotopologue pairs collected from the indicated (n) number of animals are shown. The resultant mean Δ amplitudes ± SEMs are also shown, as well as the probabilities from paired t tests (p value, t test) that the mean responses to each pair of isotopologues at each dilution are significantly different. Significant differences were not uncovered.
In this issue
