JP7301327B2 - Depression and/or anxiety biomarkers in mice - Google Patents

Description

The present invention relates to mouse depression and/or anxiety biomarkers and the like.

WHO news reports that depression and anxiety cost the economy $1 trillion each year, and investing in prevention and treatment development for these diseases could yield a fourfold economic return. It's about. In addition, WHO studies show that people with schizophrenia, which affects more than 21 million people worldwide, have a 2-2.5 times higher mortality rate from cardiovascular, metabolic, and infectious diseases. In addition, according to the 2014 patient survey by the Ministry of Health, Labor and Welfare, the ratio of patients with neuropsychiatric disorders to the total number of patients was as high as 8%, and hyperlipidemia, heart disease, malignant neoplasm, respiratory disease, and cerebrovascular disease were reported. It overtakes and ranks second after diabetes. From the above, it is useful to develop drugs for depression, anxiety, and schizophrenia (negative symptoms). Required. Examples of such research and development include those shown in Patent Documents 1 to 3 (prior art documents are summarized at the end).

However, the research and development described above was not sufficient, leaving room for further research.
The present invention has been made in view of the circumstances described above, and an object of the present invention is to provide depression and/or anxiety biomarkers and the like for mice.

The inventor used GC-MS to identify substances found in the urine of wild-type mice and α2,3-sialyltransferase (ST3Gal4) gene-deficient mice (ST3Gal4 KO mice; model mice for anxiety and depressive disorders). . Dozens of substances were found in mouse urine, and it was found that certain compounds among these could be used as biomarkers.
Thus, the depression and/or anxiety biomarkers in mice according to the invention for achieving the above objects are Methylamine, N,N-dimethyl-; RI955; RI1101; -2-one (3-penten-2-one); Pentanoic acid, 4-methyl-, ethyl ester; 3-Heptanone, 6-methyl (3-Heptanone, 6- RI1227; RI1237; Styrene; 3-Heptanone, 5-methylene-/5-Heptene 3-one, 5-methyl (5-Heptene 3-one, 5 β-Farnesene; α-Farnesene; 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2 ,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)); 2,2,4-trimethyl-1,3-pentanediol 3-monoisobutyrate (2,2,4-trimethyl ,1,3-pentandiol, 3-monoisobutylate (texanol isomer)) and 5,9-Undecadien-2-ol,6,10-dimethyl (5,9-Undecadien-2-ol, 6,10-dimethyl) It is characterized by containing at least one compound selected from the group.

In addition to the present invention, the present inventor obtained texanol and texanol isomers as substances capable of evaluating depression and/or anxiety in human urine/blood. These two substances have also been identified in mouse urine or blood. In other words, it was found that the same substances in the blood or urine of mammals, humans and mice, respond to stress. Therefore, it is very likely that the above texanol and texanol isomers are conserved in many mammals as substances associated with depression and/or anxiety.
Thus, another inventive mammalian depression and/or anxiety biomarker is 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)) and 2,2,4-trimethyl-1,3-pentanediol 3-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer)). At this time, mammals include humans, monkeys, gorillas, orangutans, chimpanzees, horses, rhinos, tapirs, hippos, camels, giraffes, cows, pigs, goats, sheep, monkeys, serows, wild boars, bears, dogs, cats, and rabbits. , guinea pig, rat, mouse, squirrel, capybara, sloth, anteater, armadillo, bat, wolf, bear, panda, otter, sea otter, mongoose, hyena, puma, lion, tiger, jaguar, leopard, cheetah, kangaroo, koala, sea lion , seals, elephants, whales, killer whales, dolphins and dugongs.
According to this configuration, by examining the increase or decrease of a predetermined substance in the specimen of a mammal, the state of the mammal's psychiatric disorder can be determined, so that it can also be applied to drug discovery screening.

In addition, a method for testing depression and/or anxiety in mice according to another invention comprises the steps of: (1) collecting mouse-derived urine or blood as a sample; RI955; RI1101; 3-penten-2-one; Pentanoic acid, 4-methyl, ethyl ester (Pentanoic acid, 4-methyl- RI1227; RI1237; Styrene; 3-Heptanone, 5-methylene (3-Heptanone, 5-methylene-) / 5-Heptene 3-one, 5-methyl; Benzaldehyde; beta-Farnesene; α-Farnesene; 2,2, 4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)); 2,2,4-trimethyl-1, 3-pentanediol 3-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer)) and 5,9-undecadien-2-ol,6,10-dimethyl A step of detecting at least one substance selected from the group consisting of (5,9-Undecadien-2-ol, 6,10-dimethyl) from the sample.

In addition, a method for testing mammalian depression and/or anxiety according to another invention comprises the steps of: (1) collecting urine or blood derived from a mammal as a sample; -trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)) and 2,2,4-trimethyl-1,3 Detecting at least one substance selected from the group consisting of -pentanediol/3-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer)) from the sample. the step of
At this time, mammals include humans, monkeys, gorillas, orangutans, chimpanzees, horses, rhinos, tapirs, hippos, camels, giraffes, cows, pigs, goats, sheep, monkeys, serows, wild boars, bears, dogs, cats, and rabbits. , guinea pig, rat, mouse, squirrel, capybara, sloth, anteater, armadillo, bat, wolf, bear, panda, otter, sea otter, mongoose, hyena, puma, lion, tiger, jaguar, leopard, cheetah, kangaroo, koala, sea lion , seals, elephants, whales, killer whales, dolphins and dugongs.
According to this configuration, by examining the increase or decrease of a predetermined substance in the specimen of a mammal, the state of the mammal's psychiatric disorder can be determined, so that it can also be applied to drug discovery screening.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide biomarkers for knowing the state of depression and anxiety in mice and mammals. Since the mouse model is associated with human diseases, the biomarkers of the present invention can also be used as human biomarkers. In addition, this biomarker can be used not only for psychiatric disorders in general, but also for examining the state of transition to frailty in the elderly. Furthermore, since the state of psychiatric disorders can be determined from the increase or decrease of this biomarker, it can be applied to drug discovery screening.
In addition, according to the guidelines of the Ministry of Health, Labor and Welfare in recent years, Texanol and TXIB were scheduled to be newly designated as substances that cause sick house syndrome in the "Guidelines for Indoor Air Pollution" (http://www.mhlw.go). jp/file/05-Shingikai-11121000-Iyakushokuhinkyoku-Soumuka/0000166137.pdf). It has also been reported that Texanol is degraded from TXIB by the action of esterase in rats (Astill et al., 1972; Nielsen et al., 1997). Furthermore, isobutylate in Texanol and TXIB, like butylate, is metabolized in colonocytes and is thought to be used as a carbon source for energy and anaplerotic reactions (Jaskiewicz et al., 1996; Bourassa et al., 1996; et al., 2016). Among these substances, Texanol was included in the biomarkers of depression and/or anxiety in mammals in the present invention. We can provide animals and test methods.

Representative TIC chromatograms of urinary VOCs from wild-type mice (A) and ST3Gal4 knockout (KO) mice (B). The method for acquiring the TIC chromatogram is as shown in <Test method>. Numbers in the figure refer to metabolites with an SI of 85% or greater. The compounds with respective numbers are as follows. 1) Methylamine, N,N-dimethyl-; 2) 4-Octen-3-one, 6-ethyl-7-hydroxy-; 3) 2-Pentanone; 4) 2-Hexenal, 2-ethyl-; 6) 2-Heptanone; 7) 3-Heptanone, 6-methyl-; 8) 2-Penten-1-ol, acetate, (Z)-; 9) 5-Hexen-2-one, 10) 5-Oxohexanenitrile; 11) 5-Hepten-2-one; 12) 1,3,5,7-Cyclooctatetraene; 13) 6-Hepten-3-one, 4-methyl-; 14) 15) Pentane, 2-nitro-; 16) 3-Heptanone, 5-methylene-; 17) 5-Hepten-2-one, 6-methyl-; 18) Dodecane, 2,6 20) (E) beta-Famesene; 21) alpha-Farnesene; 22) 2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol); 23) 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate; 24) 2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer); 25) 5,9-Undecadien-2-ol , 6,10-dimethyl-; 26) p-Cresol; 27) 2,5-cyclohexadien-1-one, 2,6-bis(1,1-dimethylethyl)-4-hydroxy-4-methyl-; 28) Methyl octadecyl ether; 29) 1-Docosanol, acetate (Eicosyl acetate). FIG. 10 is a three-dimensional graph (A) showing the results of grouping 17 VOCs by PCA score, and a diagram (B) showing the results of hierarchical cluster analysis of each VOC. The 17 are VOCs from Table 1 excluding 3-Hepten-2-one. VOCs used for hierarchical cluster analysis are Pentanoic acid, 4-methyl-, ethyl ester; 3-Heptanone, 6-methyl; RI1227; RI1237; Benzaldehyde; Beta-Farnesene; Alpha-Farnesene; 5,9-Undecadien-2-ol, 6,10-dimethyl. An analysis was performed. FIG. 10 is data showing the relationship between startle responses and principal component analysis score plots for 6 VOCs. FIG. RI1237; Styrene; 3-Heptanone 5-methylene / 5-Heptene 3 -one, 5-methyl; Benzaldehyde; 5,9-Undecadien-2-ol, 6,10-dimethyl. is a correlation diagram. (A) is a graph showing the difference in startle response of 120 dB between ST3Gal4 KO mice (KO) and wild-type mice (WT). (B) Graph showing results of second-order polynomial regression curve analysis of startle response and PC1 of 6 VOCs. As a result of analysis, F (2,16) = 29.336, ****p = 4.44E-006, r = 0.886 (multiple correlation coefficient), Y = 2.1942 - 0.7805*x+1.4666*x^2 . When ST3Gal4-KO mice excreted large amounts of 6 VOCs, the 6 principal components correlated with the startle response. 1 is a graph showing the results of comparison of urinary VOC levels before and after exposing preestrus/estrous female mice to male mice in a field test. In the graph, WT means wild-type mice, KO means ST3Gal4 knockout mice, P/E means pro-estrus/estrus, and D means late estrus. (A) is a graph showing the total number of accesses to females, (B) to (Q) are the urinary excretion amount of the compound shown on the vertical axis (and the structural formula shown in the upper right of the graph), and before and after contact with females. is a graph showing the relationship between Urine was collected for 1-3 weeks before and after exposure to females. Among the graphs, in F, G, I, K, L and Q, the ST3Gal4 KO mouse showed a higher excretion amount than the wild-type mouse excretion amount with less variation. D, M and N showed less excretion before exposure to females. O and P showed higher excretion after exposure to females. Kruskal-Wallis test and Dunn's multiple comparison test were performed. FIG. 4 is a chart summarizing the results regarding the amount of VOCs excreted. The numbers in parentheses after each component refer to the same component as the number attached to the column in Table 1. FIG. 10 is a chart showing the results of GC-MS analysis of Texanol and TXIB samples that mice were allowed to sniff. FIG. The left shows the results of Texanol and the right shows the results of TXIB. When the Texanol standard was confirmed by NMR, 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)) and 2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer)) , 60%:40% (molar ratio). FIG. 3 shows the results of confirming the effects of Texanol and TXIB on behavior during fear conditioning test using mice. (A) A diagram showing an experimental schedule. (B-1) 500 μL of water in which Texanol is dissolved is dropped into the cage, and the concentrations in the cage atmosphere are 0.24 μg/m ³ (0.03 ppb), 7.2 μg/m ³ (0.81 ppb) and 216 μg/m ³ (24 ppb), respectively. ), and on the 6th day, mice were given a conditioned stimulus that linked a sound (CS) and a foot shock (USw), and the mice underwent the first fear conditioning training. 3 is a graph showing the traveled distance (cm) of . As a control, the data obtained when only 500 μL of water containing no Texanol was dropped is shown. The underfloor paper chips and VOC were replaced four times a day (the same is true for (B-2), (B-3), (C-1) to (C-3)). (B-2) A graph showing the distance traveled when a contextual memory test the day after the conditioned stimulus was performed on the 7th day (the mice were placed in the same environment for 300 seconds without the conditioned stimulus on the first day). (B-3) Sound fear memory test after 2 days of conditioned stimulation on day 8 It is a graph showing the movement distance when listening to CS) for 60 seconds and then staying in the same environment for 120 seconds). (C-1) 500 μL of water in which TXIB was dissolved was dropped into the cage, and the concentrations in the cage atmosphere were 10 μg/m ³ (0.85 ppb), 50 μg/m ³ (4.3 ppb), and 100 μg/m ³ (8.5 ppb), respectively. And, it is a graph showing the traveled distance (cm) when maintaining 216 μg/m ³ (20 ppb) and performing the first fear conditioning training on the 6th day. As a control, data obtained when only 500 μL of water containing no TXIB was dropped is shown. (C-2) A graph showing the distance traveled when the contextual memory test was conducted on the 7th day. (C-3) A graph showing the distance traveled when a phonophobic memory test was conducted on the 8th day. The total distance traveled for each mouse was calculated, both maximum and minimum values were excluded, and the remaining values were used for analysis. Line graphs (mean ± SEM) show the total distance traveled during the 30-s period up to each period. Statistical analysis was performed by two-way repeated measures analysis of variance (ANOVA, VOC concentration × cm) for distance per time, and Tukey's multiple comparison test compared each VOC concentration group to the 0 μg/ ^m3 concentration group. (control). As a result, (B-1) 0 μg/m ³ : F (3, 12) = 2.5706, p = 0.1029; (B-2) F (3, 12) = 3.7556, ^* p = 0.0412, Turkey ^* p = 0.0338 [0.24 vs. 0 μg/m ³ ]; F (3, 12) = 5.3794, ^* p = 0.0140 in (B-3), ^* p = 0.0104 [216 vs. 0 μg/m ³ in Turkey ]; F (4, 21) = 1.5774, p = 0.2172 in (C-1); F (4, 21) = 1.5918, p = 0.2135 in (C-2); F (4, 21) = 4.3747, ^** p = 0.0099, Turkey obtained ^** p = 0.0089 [216 vs. 0 μg/m ³ ]. 1 is a graph showing the results of confirming the effects of Texanol and TXIB in a phonophobia test using mice. Results for Texanol (A)-(D) and TXIB (E)-(H) are shown. Behavioral freezing times were measured when freezing times longer than 2 seconds were observed. Total moving distance (cm) and behavior stop time (%) for 300 seconds of contextual fear memory are shown in (A), (B), (E) and (F), and total moving distance for 240 seconds of sound fear memory test. (cm) and behavior stop time (%) are shown in (C), (D), (G) and (H). Statistical analysis was performed by conventional one-way analysis of variance, and comparisons between groups were performed by Tukey's multiple comparison test. As a result, F (3, 12) = 3.7638, ^* p = 0.0409 in (A), ^* p = 0.0336 [0.24 vs. 0 μg/m ³ ] in Turkey; F (3, 12) = 1.0355 in (B) , p = 0.4118; in (C) F (3, 12) = 8.9663, ^** p = 0.0022, in Turkey ^* p = 0.0304 [0.24 vs. 0 μg/m ³ ], ^** p = 0.0016 [216 vs. 0 μg/m ³ ], ^* p = 0.0333 [216 vs. 7.2 μg/m ³ ]; in (D) F (3, 12) = 5.5875, ^* p = 0.0124, in Turkey ^* p = 0.0474 [0.24 vs. 0 μg/m ³ ], ^** p = 0.0099 [216 vs. 0 μg/m ³ ]; F (4, 21) = 0.8671, p = 0.4999 in (F); = 4.3772, ^** p = 0.0099 for Turkey ^** p = 0.0089 [216 vs. 0 μg/m ³ ]; for (H) F(4, 21) = 4.2410, ^* p = 0.0114 for Turkey ^* p = 0.0186 [10 vs. 0 μg/m ³ ], ^* p = 0.0467 [100 vs. 0 μg/m ³ ]. In (E), a significant difference ( ^* p = 0.0162) was observed in the ANOVA Bartlett's test, indicating that the variance was different between the groups with different TXIB concentrations. Therefore, the inventor decided that the parametric test could not be used, and used the Kruskal Wallis test as a non-parametric method. No significant difference was observed (p = 0.3881). Fig. 10 is a graph showing the results of examining the effect of TXIB on the head swing of mice on day 8 in a phonophobia test using mice. (A) Graph comparing results of 0, 10, 50, 100 and 216 μg/m ³ , (B) Graph comparing 0 and 216 μg/m ³ . Statistical analysis was performed by conventional one-way analysis of variance, and comparisons between groups were performed by Tukey's multiple comparison test. As a result, one-way ANOVA: F (4, 21) = 1.9680, p = 0.1365, Mann-Whitney U test (two-sided test): p = 0.1409 [10 vs. 0 μg/m ³ ], p = 0.6064 [50 vs. 0 μg/m ³ ], p = 0.3007 [100 vs. 0 μg/m ³ ], ^* p = 0.0240 [216 vs. 0 μg/m ³ ]. FIG. 10 shows the results of confirming the effect of TXIB on the immobility (%) of mice during the forced swimming test. (A) A diagram showing an experimental schedule. From day 3 to day 8, mice were administered water (500 μL) containing 100 μg/m ³ (8.5 ppb) or 216 μg/m ³ (20 ppb) of TXIB, and forced swimming test was performed on day 8. gone. The time during which the animal showed immobility for 1 second or longer was measured between 1 minute and 6 minutes during the forced swimming test. The maximum and minimum values were removed and the remaining counts were used for statistical analysis. (B) Graph showing the results of the immobility test of mice during the forced swim test (mean±standard error (SEM)). Statistical analysis was performed by two-way repeated measures analysis of variance (ANOVA, VOC concentration x %) yielding F (2, 9) = 0.31908, p = 0.7347. (C) Bar graph showing the results of the full 6 minute immobility test. A conventional one-way ANOVA was performed for statistical analysis, and F (2, 9) = 0.28327, p = 0.7598 was obtained.

Next, embodiments of the present invention will be described with reference to the drawings. can be implemented in

<Test method>
1. Experimental animals All animals were treated in accordance with the "Guidelines for Appropriate Animal Experimentation" (Science Council of Japan, 2006). The experimental protocol was approved by the Animal Experiment Ethics Committee of Kyoto Sangyo University (approval number 2017-08).
The ST3Gal IV gene-deficient mouse (C57BL/6-ST3Gal4<tm1.1Bsi>, RBRC02286) was prepared according to a previous report (Non-Patent Document 1: Srimontri et al., 2014). For the experiments, we used C57BL/6J mice subcultured from 13 to 20 generations. Mice were housed in a 12-hour day/night facility at a temperature of 22°C to 24°C. Feed (AIN93G and MF: manufactured by Oriental Yeast Co., Ltd.) and water were allowed ad libitum. Four mice were housed in open-top plastic cages (width 225 mm, length 338 mm, height 140 mm). The upper surface of the cage was covered with a stainless steel wire grid lid. The floor of the cage was covered with sawdust (Pure Chip, manufactured by Shimizu Jikken Seizai Co., Ltd.) and paper chips (Echo Chip, manufactured by Clea Japan, Inc.), which were replaced twice a week.

2. Reagents The following reagents were used as standards. Methylamine, N,N-dimethyl (25% pure ethanol solution, catalog number T2892 (Tokyo Chemical Industry Co., Ltd.)), 3-pentene, 2-one (70% purity, catalog number 145017 (Sigma)), pentanoic acid, 4-methyl, ethyl ester (purity of 97% or higher, catalog number 58730 (Sigma)), styrene (purity of 99% or higher, catalog number S0095 (Tokyo Chemical Industry Co., Ltd.)), 3-heptene-2-one (purity of 95%) Catalog number H0838 (Tokyo Chemical Industry Co., Ltd.)), benzaldehyde (purity 99.9%, catalog number B1334 (Sigma)), 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate ( Texanol: Catalog No. 40366 (Alpha Aesal)), 2,2,4-trimethyl-1,3-pentanediol isobutyrate (TXIB: >98.5% purity, Catalog No. 41601 (Sigma)), n-alkanes Mixed solutions (C9-C40: 50 µg/mL; C10, 20, 30 and 40: 100 µg/mL) were purchased from GL Sciences (catalog No. 1021-58321). β-farnesene was provided by Nippon Terpene Chemical Co., Ltd.

3. Urine Collection Urine was collected from ST3Gal4 knockout (KO) and wild-type (WT) male littermates. The collected urine was freshly frozen using liquid nitrogen and stored at -80°C until use. Urine collected from one mouse on the same day was collected in one tube, divided into 200 µL aliquots in 1.5 mL glass vials, and analyzed. The urinary creatinine concentration was measured using a lab assay creatinine colorimetric determination kit (Wako Pure Chemical Industries, Ltd.) based on the Jaffe method (Jaffe method: Bonsnes and Taussky, 1945).

4. Extraction of VOCs by Solid Phase Microextraction Urinary VOCs were extracted using headspace solid phase microextraction (SPME) fibers. 2 cm 50/30 μM DVB/CAR/PDMS (divinylbenzene/carboxene/polydimethylsiloxane, manufactured by Supelco, USA) was used as the SMPE fiber in order to secure a wide range of VOC molecular weights. SPME fibers were pretreated at 250°C for 30 minutes and equilibrated at 45°C for 1 minute to harvest VOCs. VOCs in the headspace were extracted at 45°C for 60 minutes with SPME fibers inserted into the headspace of vials containing urine.

5. Gas Chromatography and Mass Spectrometry (GC-MS)
VOCs were analyzed by gas chromatography/quadrupole mass spectrometry (TQ-8040 (manufactured by Shimadzu Corporation)). SPME fibers containing VOCs were inserted into the injection port of GC and desorbed at 240°C for 3 minutes. The injection was performed by a 3-minute pulse using the splitless injection method. InertCap Pure-WAX (60m + 10m pro-guard line and 2m transfer line, inner diameter 0.25mm, film thickness 0.5μm (GL Sciences)) equipped with ProGuard and TL column was used as the GC-MS instrument. . The chromatographic analysis conditions used prior to the mass spectrometer were as follows. It was held at 40°C for 10 minutes, heated to 240°C at a temperature increase of 5°C per minute, and then held at 240°C for 10 minutes. The injector temperature was 250°C. Helium was used as the carrier gas, and the column flow rate was constant at 2 mL/min from 0 to 3 minutes after injection and 1 mL/min from 3 to 60 minutes.

The processing parameters of the mass spectrometer were as follows in <Test 1>. The ion source temperature was 200°C, the ionization energy was 70 eV, the scanning frequency was 0.3 seconds from 35 m/z to 300 m/z, and the column length was 30 m. In <Test 2>, the parameters were the same as in <Test 1> except that the scan frequency was 0.2 seconds per scan and the column length was 65 m (Fig. 1). Analysis of VOC peaks in urine of 10- to 15-week-old mice was performed using QP-2010 Plus (Shimadzu Corporation) according to the method of <Test 2>, except that the helium gas flow rate was 20 cm/s. rice field. VOC peak identification was performed by comparison with retention times of VOC standards and by matchability using the NIST14 standard reference database (NIST/EPA/NIH Mass Spectral Library (Wiley10)).

In order to determine the retention time index (RI) of a polar column (InertCap Pure-WAX) and a non-polar column (DB-1), the retention time of VOCs was measured using a GC-MS system (Shimadzu Corporation) for n-alkane standards. compared. SPME fibers containing VOCs were inserted into the injection port of GC and desorbed at 240°C for 10 minutes. The injection was performed by a 3-minute pulse using the splitless injection method. In order to separate volatile components, a DB-1 column (30 m line, 0.25 mm inner diameter, 0.25 μm film thickness (manufactured by Agilent), InertCap Pure-WAX (60 m + 10 m pro-guard line and 60 m + 10 m pro-guard line) with ProGuard and TL columns was used. A 2 m transfer line, inner diameter 0.25 mm, film thickness 0.5 μm (manufactured by GL Sciences Inc.) was used.The heating program for the gas chromatogram was as follows: Hold at 40° C. for 10 minutes, then 1 minute. After heating to 240°C at a rate of 5°C per hour, the temperature was maintained for 10 minutes, the injector temperature was 240°C, helium was used as the carrier gas, and the column flow rate was 50 cm/s for DB-1. , InterCap Pure-WAX was set to 20 cm/sec.Analysis parameters of the mass spectrometer were as follows: ion source temperature 200 °C, interface temperature 240 °C, ionization energy 70 eV, scan frequency 35 m/z to 300 m /z is 0.2 seconds per time.

6. Data Analysis and Statistical Analysis Chromatographic peak areas were determined using GCMSsolution ver.4.45 software (manufactured by Shimadzu Corporation). Detection and calculation of ion peaks obtained by electron ionization were performed using R software ver3.2.3 (http://cran.r-project.org/) and XCMS software package ver1.3.2 (http://masspec. scripps.edu). The TIC (total ion current) of each peak was manually identified and the peaks detected were confirmed. Precise measurement of VOCs was performed by manual integration calculations based on retention times and peak shapes of various ion peaks (m/z).
First, we examined differences in urinary VOCs between ST3Gal4 KO mice and their littermate wild-type mice at 20 to 35 weeks of age. VOCs in mouse urine were measured three times by gas chromatography. To examine the differences in the ion peaks of urinary VOCs from ST3Gal4 KO mice (n = 3 in both Tests 1 and 2) and littermate wild-type mice (n = 3 in both Tests 1 and 2), It was examined by XCMS (Table 1).

Ion peak areas were then calculated using the GCMSsolution software. To evaluate each data set together, the absolute ion peak areas of knockout and littermate wild-type mice (n=6 for each pair; 20–35 weeks of age) were averaged and the relative mean peak areas were calculated. strength was calculated. In contrast, when determining VOCs using a GC-MS system (manufactured by Shimadzu Corporation) equipped with an autosampler, after determining all urinary VOCs, the urinary VOCs of knockout mice and littermate wild-type mice were The absolute areas of the ion peaks were compared (Table 2).

Multivariate analysis was performed using Excel and Kyence 5.0, and principal component analysis (PCA) was performed from the relative values of 15 VOCs obtained from 6 pairs of knockout mice and littermate wild type mice. A score plot was obtained. Then, a three-dimensional drawing was obtained using Graph-R ver.1.24.3 (Tohru Itoh). For the classification of VOCs belonging to the groups extracted by PCA, the absolute value (area) of VOCs was determined by hierarchical cluster analysis using Excel and KyPlot 5.0 (manufactured by Kaiens). Next, to assess whether VOCs and the startle reflex are correlated, polynomial regression curves were obtained using Excel and Kyens 5.0 (Srimontri et al., 2014).
Next, the ion peak area was determined by GCMS for VOCs in urine collected from male mice before and after pairing with female mice, and the absolute value of m/z was calculated (Fig. 3). Chromatography was used once per mouse to determine peak areas. Differences in peak areas were analyzed by Mann-Whitney U test (Table 2) and Kruskal-Wallis test using Excel and Prism6. The results are shown in FIG.
VOC identification was performed by comparison with mass spectral libraries (NIST14 and Wiley10) with manual interpretation and comparison with commercially available standards. VOCs not commercially available were identified by comparison with mass spectral libraries.

7. Startle reflex test Acoustic startle response (ASR) by mice was examined in order to observe sensory stimulus responses using a startle reflex measurement device for mice (manufactured by O'hara & Co) (Non-Patent Document 3: Buccafusco et al., 2009). A mouse was introduced into the acrylic chamber, the chamber was placed on the test unit, and both the chamber and the test unit were clamped. A continuous 70 dB background noise (white noise) was played from the loudspeaker during the 5 min acclimation and test periods. Mice were exposed to ten 20 msec noise pulses (120 dB) every 500 msec. The vibrations induced by the whole-body startle response of mice were displayed numerically by an accelerometer. The numerical signal was amplified, converted to voltage, and displayed as a wave shape. The peak-to-peak value (pp value) of the waves was taken as the strength of the startle response.

8. Field Trials Field trials to meet females were performed using 10-16 week old ST3Gal4 knockout mice and littermate wild-type male mice. Male mice were placed alone in a separate waiting room at least 30 minutes before the start of the test. Behavioral testing was performed between 08:00 and 13:00 by an experimenter blinded to the mouse genotype. Field trials were performed by placing mice in a square arena (490 mm wide, 490 mm long, and 195 mm high) with a ceiling height of 125 cm above the field floor. The intensity of the fluorescent lights illuminating the open field and waiting room was set at 17 lux.
Vaginal smear tests were performed on ddY female mice (8-12 weeks old) immediately prior to field testing. The smears were stained with Giemsa solution (Wako Pure Chemical Industries, Ltd.) and examined with a bright field microscope. The stages of the estrus cycle were determined according to previous reports (Allen, 1922).
First, a male mouse was placed in an open field, and after 1 minute, a proestrus female mouse was placed in the open field for 30 minutes. Both mice were then returned to their cages. Data were collected by TimeOFCR4 (manufactured by O'hara & Co) using a camera placed 470 mm above the field floor. Behavioral testing was performed by an experimenter blinded to the genotype of the mice. During the first 10 minutes of recording, the number of male accesses per second from the male's nose to the female's nose, from the male's nose to the female's vaginal circumference, and from the male's forelimbs to the female's body was counted.

9. A Study of Texanol and TXIB in C57Bl/6J Mice A search for urinary biomarkers of depression in the elderly identified five known VOCs as candidates. One of these five was Texanol. On the other hand, the present inventors reported that gene-deficient mice (St3gal4-KO mice) lacking alpha2,3-sialyltransferase (St3gal4), an enzyme that causes epilepsy, exhibit depression and anxiety (Srimontri et al. ., 2014). St3gal4-KO mice exhibiting depression and anxiety exhibited increased urinary excretion of texanol isomers with age (Fujita et al., 2018a). Along with TXIB, Texanol has been discussed as a possible cause of sick house syndrome (http://www.mhlw.go.jp/file/05-Shingikai-11121000-Iyakushokuhinkyoku-Soumuka/0000166137.pdf). TXIB has two isobutyl groups, while Texanol has one isobutyl group. It has been reported that Texanol is degraded from TXIB by the action of esterase in the rat body (Astill et al., 1972; Nielsen et al., 1997). In addition, isobutylate, like butylate, is metabolized in colon cells and is thought to be used as a carbon source for energy and anaplerotic reactions (Jaskiewicz et al., 1996; Bourassa et al., 2016). ). On the other hand, Texanol and TXIB that enter the body from the environment such as food packages (Kemf et al, 2009) are expected to be oxidized or conjugated and excreted in the urine (Nielsen et al, 1997). .

Texanol and TXIB have traditionally been considered the safest indoor solvents (Weschler, 2009). However, in recent years, there is a possibility that it may be designated as a causative agent of sick house syndrome in Japan (Ministry of Health, Labor and Welfare, 2017; Ghaffarianhoseini et al., 2018). Sick building syndrome is known to be accompanied by neuropsychiatric symptoms (Kinman et al., 2011; Runeson-Broberg et al., 2013; Miyajima et al., 2015). On the other hand, Texanol has been reported to affect mucous membranes (Ghaffarianhoseini et al., 2018). However, to date, there is no evidence of a relationship between Texanol or TXIB and neuropsychiatric symptoms.
Therefore, we investigated the possibility that Texanol and TXIB, which were detected as urinary biomarkers, actually induce symptoms similar to those in St3gal4-KO mice.

Four littermate mice (8-week-old, male, C57Bl/6J strain: CLEA Japan, Inc.) were placed in open-top plastic cages (width 225 mm, length 338 mm, height 140 mm) to prevent transportation stress. They were kept for more than a week for recovery. All animals were treated according to "Guidelines for Proper Animal Experimentation" (Science Council of Japan, 2006). The experimental protocol was approved by the Animal Experiment Ethics Committee of Kyoto Sangyo University (approval number 2017-08, 2018-07). Mice were housed in a 12-hour day/night facility (lights on at 8:00 am) at a temperature of 22°C to 24°C. Feed and water were given ad libitum.
A rat cage (capacity: 11.1 L, width: 234 mm, length: 360 mm, height: 148 mm (Clea Japan)) separated from the individual ventilation system and isolated from the environment by HEPA was used. Each cage housed two littermate male mice to allow them to inhale volatile organic compounds (VOCs). The cage was opened, and regardless of the presence or absence of VOCs, paper chips (φ5 mm, made by Clair Japan) under the floor were changed three times every 4 hours during the daytime for 12 hours, and changed 3 times during the night for 12 hours. was exchanged only once (at 20:00).

Significant effects of TXIB were determined by effects after repeated oral exposure in animals. Specifically, the NOAEL and NOEL for TXIB are 50 mg/kg/day (Nielsen et al., 1997) and 30 mg/kg/day (OECD, 2005, CAS , N°: 6846-50-0). This value is attributed to decreased kidney weight and changes in blood parameters in rats. Based on NOAEL and NOEL values, the provisional inhalation limit for TXIB is 1 mg/m ³ for a 70 kg male (Nielsen et al., 1997). A slightly higher value is 2.5 mg/m ³ (Eastman Kodak Company, 2007) and a tenfold lower value is 30 mg/kg/day for a 50 kg male (Azuma et al., 2016). Next, Texanol and TXIB, which contain mono- and di-isobutyrate, suggest that isobutyrate functions as an energy source in colonocytes and as a carbon source for anaerobes in low-butyrate environments (Jaskiewicz et al. ., 1996). Therefore, the inventor selected 100 μg/m ³ as the maximum concentration of TXIB. Next, the maximum concentration of Texanol was 260 μg/m ³ [≒100 (μg/m ³ )×2×286.4 (g/mol)/216.3 (g/mol)].

Two behavioral tests were performed on 10- to 13-week-old mice. All behavioral experiments (fear conditioning and forced swim test) were automatically performed from 08:00 to 12:00 using Time FZ1 (Ohara Medical Sangyo Co., Ltd., Japan) by the experimenter blinded to which mouse to use. went between Anxious behavior was evaluated by the auditory fear conditioning test (Fig. 7(A)), and depressive behavior was evaluated by the forced swimming test. Auditory fear conditioning was performed according to a previous report (Kato, 2015). Briefly, it was as follows. On the training day (day 6), a conditioned stimulus (CS) of 65 dB for 10 sec and an unconditioned stimulus (US) of foot shock of 0.3 mA for 1 sec were administered twice at 20 sec intervals (200 lux). bottom. On the following day (day 7), mice were left without CS or US (200 lux) for 5 minutes in the same clear square cages used on the training day. Furthermore, on the next day (day 8), the mouse was placed in a different box (50 lux) from the previous day, and the mouse was given a 1-minute search time, a 1-minute CS (tone), and then a 2-minute search time. A cue test to give was conducted. Immobility behavior was defined as when the mouse stopped for longer than 2 seconds as measured using Time FZ1.
A forced swimming test (1010-lux) was also performed according to a previous report (Kato, 2015). Statistical analysis was performed using mean ± standard error (SEM) using Excel and GraphPad Prism 7 (manufactured by GraphPad Software). Analysis was performed using values other than the maximum and minimum values (n=6 for each concentration of VOC).

<Test results and discussion>
The inventors have previously shown (Kato, 2015) that emotional behavior is affected by the quality and quantity of lipids (triglycerides) containing different fatty acids in the diet given to mice. In addition, changes in emotional behavior induced by eating a lipid-containing diet differed between ST3Gal4 knockout mice, which are anxiety and depression model mice, and littermate wild-type mice. This suggests that differences in lipid metabolism are involved in emotional and cognitive behavior. Therefore, in order to investigate whether different metabolism affects behavior, the present inventors examined the differences in lipid metabolism between ST3Gal4 knockout mice and wild-type mice by measuring the urinary VOCs of each mouse with GC- Investigated using MS.

1. Specific differences in urinary VOCs in ST3Gal4 KO mice
Urine samples (200 μl) from 6 pairs of ST3Gal4 knockout mice and wild type littermates were analyzed using HS-SPME GC-MS. As described in <Test method>, urinary VOCs were investigated by two types of tests (Test 1 and Test 2). Representative total ion current (TIC) peaks and similarity index (SI) are shown in Table 1. In test 2, increasing the number of scans increased the number of detected VOC types from 42 to 75. GC-MS showed that the urine of C57B1/6J mice contained at least 97 VOCs. Since ST3Gal4 knockout mice and littermate wild-type mice were obtained by gene editing from mice of the C57B1/6J strain (Non-Patent Document 1: Srimontri et al., 2014), they are thought to have similar metabolic pathways. Conceivable.

Triplicate urine samples per mouse (n=3 each for ST3Gal4 knockout and littermate wild-type mice in studies 1 and 2) were analyzed by GC-MS. Thirty-six VOCs were obtained by XCMS (Non-Patent Document 4: Smith, 2006; Non-Patent Document 5: Benton et al, 2008). XCMS software was used for peak identification, non-linear retention time alignments and quantification of mass spectra and ion intensities. The alignment yielded information for each ion peak including retention time, m/z value, intensity, fold change, p-value and retention time index (RI). As a result of test 1 and test 2, 558 and 729 ion peaks were obtained from urine of ST3Gal4 KO mice. Ion peaks obtained from the urine of ST3Gal4 KO mice were extracted with a risk factor of less than 0.05 (p<0.05). 93 and 244 ion peaks were obtained for Test 1 and Test 2, respectively. The ion peaks belonging to each retention time were collected, the TIC peak spectra were compared with the NIST14 library, and the similarity of the peaks was examined with each SI as a control. As a result, 18 VOCs were identified with a similarity of over 85%.

Next, in order to investigate whether the different areas of the ion peaks to which these 18 VOCs belong are correct, the base peak of the candidate VOCs (third column in Table 1) included in the ion peaks extracted by XCMS is selected, and the VOC ions were calculated using GCMSsolution ver. 4.45 software. Comparison of relative ratios of quantification areas of VOCs obtained from knockout and wild-type mice in six pairs of knockout and littermate wild-type mice, fold changes, standard errors (S.E.M.) and Mann-Whitney U The p-value for the test was calculated (columns 11-13 in Table 1). Thirteen of the 18 VOCs were identified with a high degree of similarity to the database. Furthermore, for 9 of the 13 VOCs, the mass spectra were in agreement with the commercially available standard products, and the concentrations in the VOCs could be measured. On the other hand, 4 of the 13 VOCs could not be identified due to lack of information. The RIs of VOC (No. 10) were 1291 (InertCap pure-WAX column) and 972 (DB-1 column). The RI pattern showed no difference between the two compounds (3-heptane, 5-methylene/5-heptene 3-one, 5-methyl). 3-pentene and 2-one were not found in the urine of 3 pairs of mice, so no statistical analysis was performed. In ST3Gal4 KO male mice, an anxiety and depression model, 2 VOCs decreased and 10 VOCs increased in urine at 20-35 weeks of age (see Table 1).

2. Metabolic characterization of urinary VOCs analyzed by GC-MS Excel and KyPlot 5.0 were used to evaluate the relationship of VOCs obtained by XCMS using total ion peaks of knockout and wild-type mice. A principal component analysis (PCA) was performed from the relative values of the base peaks of 17 VOCs obtained from mice and littermate wild-type mice (Fig. 2). 3-heptene,2-one was not found in the urine of 3 pairs of knockout mice and littermate wild-type mice and was therefore excluded. In PCA, the cumulative contribution ratio should be 0.8 or higher. In the present results, the total of principal component 1 (PC1) to principal component 3 (PC3) was 0.83. We then displayed a PCA score plot containing the areas of the X-, Y- and Z-axes and found two groups of 2 and 11 VOCs out of 17 VOCs (Fig. 2A). In FIG. 2A, the numbers inside the circle are the same as the numbers in the first column of Table 1. 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (texanol) (15); 2,2,4-trimethyl-1,3-pentanediol 3-monoisobutyrate rate (texanol isomers) (16) is a group of two VOCs, pentanoic acid, 4-methyl-, ethyl ester (5); 3-heptanone, 6-methyl (6); RI1227 (7); RI1237 (8); styrene (9); 3-heptanone, 5-methylene/5-hepten-3-one, 5-methyl (10); benzaldehyde (12); β-farnesene (13); 5,9-undecadien-2-ol, 6,10-dimethyl (17); RI2297 (18) were the group of 11 VOCs. In addition, methylamine, N,N-dimethyl (1); RI955 (2); RI1101 (3); showed the direction.

Next, in Test 2, urine samples obtained from 10 to 16-week-old ST3Gal4 knockout mice (n = 9) and wild-type mice (n = 11) were analyzed by HS-SPME GS-MS ( Table 2). The purpose of Study 2 was to directly compare absolute peak areas (rather than relative values) between samples from individual mice, and to assess the correlation between urinary VOC levels and emotional responses. Young male mice aged 10-16 weeks were used to observe male contact with pre-estrous/estrous female mice. Of the 18 VOCs extracted from the urine of 20- to 35-week-old male mice (Table 1), 15 VOCs were detected from the urine of 10- to 16-week-old male mice (Table 2). This indicates that 3-heptene, 2-one and RI2297 were hardly found in the urine of young male mice.
In order to classify the groups containing 11 VOCs shown in FIG. 2A, hierarchical cluster analysis was performed using the absolute values of mouse urine areas shown in Table 2. A PCA score was calculated using the absolute values of 10 VOCs excluding RI2297.

Next, using PCA principal component 1 (PC1) to principal component 6 (PC6) (cumulative contribution of 0.99), a dendrogram of 10 VOCs was created. As a result, three categories were identified: (1) styrene only, (2) a group containing RI1227, β-farnesene, α-farnesene, and (3) three VOCs (pentanoic acid, 4-methyl-, ethyl ester; 3-heptanone, 6-methyl; 3-heptanone, 5-methylene/5-heptene 3-one, 5-methyl), benzaldehyde, and RI1237 and 5,9-undecadien-2-ol, 6,10-dimethyl (Fig. 2B). Regarding the Y-axis, regardless of whether the ST3Gal4 knockout mouse group or the wild-type mouse group, the distance between VOCs was short when the changes in VOC values in individual mice were similar. VOCs produced on metabolic and degradative pathways close to each other were thought to reduce the distance.

3. Relationship between startle reflex and urinary VOC production A startle response test was performed to investigate the relationship between the amount of urinary VOCs and emotional reactions. As a result, the difference in startle reflex between ST3Gal4 knockout mice and wild-type mice was small (p=0.1882 in Mann-Whitney U test; FIG. 3A). This indicates that there is no difference between auditory activity and the startle response. Urine was collected following the startle response test. Next, in order to examine the relationship between the amount of urinary VOCs and the startle reflex, PCA scores were determined using the absolute values of the 6 VOCs classified as shown in FIG. 2B. Since the contribution ratio of principal component 1 (PC1) was 0.90 and the loading of PC1 in all 6 VOCs exceeded 0.90, PC1 and startle response were distributed on the XY axes, and the second-order polynomial regression curve was high. It showed compatibility (r=0.886, Fig. 3B). Twenty-five percent of ST3Gal4 KO mice produced more of the 6 VOCs in their urine, showing a strong correlation between the production of 6 VOCs and startle response. On the other hand, the remaining 75% of the KO mice had low levels of the 6 VOCs. No correlation was observed between the production of the 6 VOCs and the startle response in wild-type mice (Fig. 3B).

4. Relationship between contact level of male mice with female mice and production of urinary VOCs In order to investigate the relationship between urinary VOC levels and emotional reactions, a social behavioral test was conducted using male and female mice. Specifically, the number of accesses per second by male mice to preestrus to early estrous (P/E) female mice (male nose to female nose, male nose to female vaginal area, and male forelimbs of females) were counted for 10 minutes (Fig. 4A). As a result, wild-type male mice averaged 2.2 times more accesses to P/E female mice than ST3Gal4 KO male mice. In wild-type male mice, the average number of accesses to P/E female mice was 2.0 times higher than that to late estrus female mice. This trend was the same as that of ST3Gal4 KO male mice. These results indicate that ST3Gal4 KO male mice have less access to P/E female mice. Therefore, in order to investigate the relationship between urinary VOC levels and emotional responses, we investigated the number of accesses of male mice to P/E female mice.

Urine was collected from male mice before and after contact with P/E stage female mice, and VOCs were analyzed (Table 2). As a result, we found three types of VOC patterns with significant correlations between urinary VOC levels and the number of accesses in males. First, the amount of RI1101, β-farnesene and α-farnesene in the urine was low in wild-type male mice before exposure to female mice. The amount in urine increased after wild-type male mice were exposed to female mice (Fig. 4D, M, N). The increase in β-farnesene and α-farnesene was similar to that in ST3Gal4 KO male mice (Table 2). However, no p-values were significant (Kruskal-Wallis p-values were 0.2467 and 0.1398, respectively). On the other hand, RI1101 increased significantly (only 0.57-fold compared to ST3Gal4 KO male mice) after wild-type male mice were exposed to female mice (p = 0.0077 by Kruskal-Wallis test). Fig. 4D).

The following patterns are 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (texanol) and 2,2,4-trimethyl-1,3-pentanediol 3-monoisobutyrate Butyrate (Texanol isomer). These VOCs were significantly increased after contact with female mice in both ST3Gal4 knockout male mice and wild-type male mice (p=0.042, 0.0154; FIG. 4O, P). Specifically, texanol increased 2.0-fold (knockout male mice) and 2.6-fold (wild-type male mice) after contact with female mice, and texanol isomers increased 1.4-fold (knockout male mice) and 1.7-fold, respectively. (wild-type male mice). According to the inventor's studies, texanol and texanol isomers have increased urinary levels in humans exhibiting anxiety when compared to controls. When an immature male mouse first comes into contact with a P/E stage female mouse, it can have high emotional stress on the female mouse. Therefore, increased urinary texanol and texanol isomers may indicate symptoms similar to those in depressed humans.
The third pattern was the opposite response to Texanol and Texanol isomers, showing a decrease after contact with female mice in both ST3Gal4 knockout male mice and wild-type male mice (p=0.0280; Fig. 4J). It is styrene.

In contrast, urinary amounts of six VOCs in ST3Gal4 knockout mice were higher than those in wild-type mice, regardless of whether they were in contact with female mice (Figures 4 and 5, Table 2). ). Regarding these VOCs, no changes were observed in the urinary amounts of male mice before and after contact with female mice. Furthermore, the variability of the absolute ion peak values of urinary VOCs in wild-type mice was low (Fig. 4). Six VOCs were shown not to be involved in the correlation between urinary VOC levels and access numbers of young male mice to P/E female mice. Six VOCs were identical to substances that correlated with the startle response in 25% of ST3Gal4-deficient mice. This suggests that urinary VOCs, which correlate with emotional reactions, are not involved in all emotional reactions.

5. Test results using C57BL/6 mice Japan) and Texanol (Alfa Aesar, Lancashire, UK) standards were checked. The results of GC-MS analysis are shown in FIG. The TXIB standard was almost pure, while the Texanol standard contained about 40% (molar ratio) of Texanol isomers [Texanol:Texanol isomers = 12.98:8.652 (ng)].
On the first day of fear conditioning training (day 6), there was no effect of inhalation of Texanol (Fig. 7 (B-1)) and TXIB (Fig. 7 (C-1)) on the distance traveled by mice.
On the second day, the behavior of the mice was examined in the experimental area (170 mm wide x 100 mm long x 100 mm high square cage) without CS or US. Evaporation of Texanol slightly decreased the distance traveled. At the lowest dose (0.24 μg/m ³ ), there was a slight effect on the distance traveled for 30 seconds in each period (*p = 0.0338 in Figure 7 (B-2)), but the total distance for 300 seconds A 1/2 reduction in distance was observed (*p = 0.0336 (Figure 8 (A); Tukey's multiple comparison test). 2)) or the total distance of 300 seconds (Fig. 8(E)), there was almost no statistical effect.When TXIB was evaporatively aspirated to 10-216 μg/m ³ , 60-200 seconds As shown by the average distance (cm) for 140 seconds of 140 seconds, there was a tendency to decrease the moving distance (Fig. 7 (C-2)). (Fig. 8(B), Fig. 8(F)) At least, 4 days of Texanol evaporation had little effect on fear memory associated with spatial cognition.

On the third day, the behavior of the mice was examined in a separate laboratory (solid, gray, square area 170 mm wide x 100 mm long x 100 mm high). At this time, only the sound stimulus (CS) was given for 60 seconds from 60 seconds to 120 seconds. Evaporation of Texanol and TXIB decreased the total distance traveled for 240 seconds (Fig. 7 (B-3) and (C-3)) during each period (Fig. 7 (B-3) and (C-3)), and the degree of reduction was Scaled up gradually according to dose. Furthermore, evaporative administration of Texanol and TXIB increased total immobility time (%) per 4 minutes. Specifically, evaporative administration of Texanol increased total immobility time (%) by 2.05-2.35-fold, depending on the dose of 0.24-216 μg/m ³ . On the other hand, in mice, doses of 10 to 100 μg/m ³ of TXIB increased total immobility time (%), but doses of 216 μg/m ³ reduced the extent of the increase in total immobility time (%).
We also found that measuring head shake was more sensitive for assessing anxiety (Yang et al, 2015). There was almost no difference ⁱⁿ the number of head swings between 0 and 216 μg/m ³ (p=0.1365; Fig. 9(A)). The head swing number increased 1.7 times when evaporated to 216 μg/m ³ (*p=0.024). There was no difference in head swing numbers at 10, 50 and 100 μg/m ³ compared with 0 μg/m ³ . Thus, evaporating high volumes of TXIB induced a head shake in mice that remained motionless for longer than 2 seconds. Finally, the study found that Texanol and TXIB were administered evaporatively over 5 days, and auditory stimulation increased anxiety, based on baseline values that the Ministry of Health, Labor and Welfare attempted to standardize. The effect of inhalation of 0, 100 and 216 μg/m ³ of TXIB was evaluated over an 8-day period. On the other hand, as a result of measuring the forced water test of mice inhaled TXIB for 8 days, it was found that TXIB had no effect on immobility (%) (Fig. 10).

In the olfactory system, sensory receptors are localized in the peripheral olfactory epithelium within the nasal cavity. Following binding of odor molecules to receptors, odor stimuli propagate from the receptors to the olfactory bulb. In the olfactory bulb, mitral and tufted cells are arranged as output neurons. The tufted cells exchange information with the mitral cells, which form a single synapse from the periphery and link their axons to other sensory systems, the piriform cortex, which is the emotional center. To the amygdala, the entorhinal cortex sends axons to the hippocampus to function in learning and working memory. Texanol and TXIB bind to sensory receptors as odor molecules and can send odor information directly to the amygdala. The amygdala plays an important role in fear conditioning testing (Srimontri 2014). Neurons in the medial geniculate body of the thalamus and the auditory cortex of the cerebral cortex transmit auditory information to neurons in the lateral amygdala, especially during sound-associated conditioning. In the present study, stimulation of the amygdala via the olfactory bulb by inhalation of Texanol and TXIB induced acoustic fear in mice.
In contrast, the forced swim test has been used to examine antidepressant activity, and the duration of immobility is affected by the degree of disruption of hippocampal-frontal lobe interconnectivity, with disruption increasing duration. Become. This hippocampal-frontal lobe interconnection is relayed by thalamic-connected nucleus neurons (Kafetzopoulos et al., 2018). In the forced swim test, no effects of inhalation of Texanol or TXIB were observed. Since olfactory stimuli input from the olfactory nerve of the olfactory epithelium reach the hippocampus via two or more synapses, it is thought that olfactory information from inhalation of Texanol and TXIB could not reach the hippocampus via the entorhinal cortex. .

It was also suggested that Texanol and TXIB strongly induce anxiety in mice. If we understand the mechanism of anxiety mediation by Texanol and TXIB, we will be able to understand the effects on the nervous system related to sick house syndrome and chemical hypersensitivity. Both Texanol and TXIB are designated indoor air pollutants, and it has been suggested that sick house chemicals may cause symptoms such as anxiety and depression in addition to chemical hypersensitivity and allergic diseases such as asthma. (Choi et al., 2015; Ghaffarianhoseini et al., 2018). We have previously measured the amount of VOCs in vials containing urine in depressed elderly people and in anxiety model mice (St3gal4-ko), and found that the amounts of Texanol and Texanol isomers in depressed elderly people were similar to anxiety in middle age. We have found that the amount of Texanol isomers is enhanced in disease model mice (35 weeks old). So far, it has been confirmed that Texanol is elevated in both human and mouse urine, regardless of whether Texanol passes through the body and is excreted in the urine or is collected in the urine from the environment. In this study, Texanol and TXIB were used at concentrations near the environmental standard values (which were assumed in August 2017, but were later reconsidered and were not standard values as of October 2018), causing concern for healthy young mice. found to cause illness. The symptom was an enhancement of tone fear memory similar to that exhibited by the anxiety model mouse (St3gal4-ko). These results suggest that anxiety may also occur in humans, and that the frequency of onset increases with age. Thus, it was found that environmental TXIB and Texanol can be used as a screening system for anxiety and depression.

6. The following considerations were obtained from the results of this test and the findings of the present inventor.
(1) RI955, RI1227, and RI1237 were highly likely to be lipid metabolites. On the other hand, the present inventor conducted another test to examine the relationship between human depression and urinary substances. As a result of another study, Texanol was obtained as a substance capable of assessing depression in humans. In other words, it was found that the same urinary substance responds to stress in both humans and mice. Therefore, at least Texanol and Texanol isomers were highly likely to be the result of reactions conserved in many mammals as depression-related urinary substances.
Also, these substances can be extracted from urine, which can be collected non-invasively, so there is no need to harm the animal.

(2) In this study, urine was collected with care to avoid contamination from feces to urine. However, there were some VOCs whose urinary and blood levels were not proportional. This indicates that active reabsorption may be occurring. However, 3-penten-2-one (salivary, endogenous), RI1227 (from food), 5,9-undecadien-2-ol, and 6,10-dimethyl were not detected in blood. In this study, the substance was measured in the SPME column used to trap VOCs, so it is highly soluble in blood and may be difficult to volatilize.
(3) 1-Docosanol, acetate (Eicosyl acetate) was not observed in young mice, so it may increase with aging.
(4) When Texanol or TXIB was added to the atmosphere, phonophobic memory was enhanced, showing a reaction similar to anxiety symptoms. From this, Texanol or TXIB could be effectively used to evaluate depression and anxiety.

Thus, according to this embodiment, a biomarker for knowing the state of depression and anxiety in mice and mammals could be provided. Since the mouse model is associated with human diseases, the biomarkers of this embodiment can also be used as biomarkers for non-invasive depression and/or anxiety in humans. In addition, this biomarker can be used not only for psychiatric disorders in general, but also for examining the state of transition to frailty in the elderly. Furthermore, since the state of mental illness can be determined from the increase or decrease of this biomarker, it can be used for drug discovery screening.

JP 2014-224759 A JP-A-2014-13257 JP 2012-13415

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Claims (4)

Methylamine, N,N-dimethyl- ; 3 -penten-2- one ; Pentanoic acid, 4-methyl, ethyl ester (Pentanoic acid, 4 -methyl-, ethyl ester; 3-Heptanone, 6-methyl- ; Styrene ; 3-Heptanone, 5-methylene-/5 -Heptene 3- one , 5-methyl; Benzaldehyde; β-Farnesene; α-Farnesene; 2,2,4 -trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl, 1,3-pentandiol, 1-monoisobutylate (texanol)); 2,2,4-trimethyl-1,3 -pentanediol 3-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 3-monoisobutylate (texanol isomer)) and 5,9-undecadien-2-ol,6,10-dimethyl ( 5,9-Undecadien-2-ol, 6,10-dimethyl). 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 1-monoisobutylate (texanol)) and 2,2,4- containing at least one compound selected from the group consisting of trimethyl-1,3-pentanediol-3-monoisobutyrate (2,2,4-trimethyl-1,3-pentanediol3-monoisobutyrate (texanol isomer) At least one mammalian urinary depression and/or anxiety biomarker selected from the group consisting of mice, monkeys, pigs, dogs, cats, rabbits, guinea pigs and rats. (1) a collection step of collecting mouse-derived urine as a specimen; (2) methylamine, N, N- dimethyl- ; -penten-2-one); Pentanoic acid, 4-methyl, ethylester (Pentanoicacid, 4-methyl-, ethylester); 3-Heptanone, 6-methyl (3-Heptanone, 6-methyl-) ; Styrene 3-heptanone, 5-methylene (3-Heptanone, 5-methylene-) / 5-heptene3- one , 5-methyl (5-Heptene3-one, 5-methyl); Benzaldehyde; β-farnesene ( beta-Farnesene); α-Farnesene; 2,2,4-trimethyl-1,3-pentanediol 1-monoisobutyrate 1-monoisobutylate (texanol) ; 2,2,4-trimethyl-1,3-pentanediol-3-monoisobutyrate (2,2,4-trimethyl-1,3-pentanediol3-monoisobutyrate (texanol isomer) and comprising a detection step of detecting at least one compound selected from the group consisting of 5,9-Undecadien-2-ol,6,10-dimethyl Method for testing depression and/or anxiety in mice. (1) a collection step of collecting as a sample urine of at least one mammal selected from the group consisting of mice, monkeys, pigs, dogs, cats, rabbits, guinea pigs and rats ; ,4-trimethyl-1,3-pentanediol 1-monoisobutyrate (2,2,4-trimethyl,1,3-pentandiol, 1-monoisobutylate (texanol)) and 2,2,4-trimethyl-1 ,3-pentanediol-3-monoisobutyrate (2,2,4-trimethyl-1,3-pentanediol3-monoisobutyrate (texanol isomer)). A method for testing depression and/or anxiety in a mammal.

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