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laura.scheffl[email protected] (L.S.); [email protected] (Y.S.); [email protected] (G.Z.); [email protected] (K.H.); [email protected] (A.H.); [email protected] (C.S.) 2 Fraunhofer Institute for Process Engineering and Packaging IVV, Giggenhauser Str. 35, Freising 85354, Germany * Correspondence: [email protected]; Tel.: +49-9131-85-22739; Fax: +49-9131-85-22587 Academic Editor: Per Bruheim Received: 28 April 2016; Accepted: 28 May 2016; Published: 6 June 2016 Abstract: The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography´mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO2 are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences. Keywords: garlic; human milk; gas-chromatography mass-spectrometry; allyl methyl sulfide; allyl methyl sulfoxide; allyl methyl sulfone 1. Introduction Human milk is commonly the primary and often sole food source of a newborn during the early stages of perinatal life. The specific composition of human milk adapts during an infant’s development to satisfy its nutritional requirements; it is thus commonly recommended that mothers breastfeed their children at least during the first six months after birth [1]. Although the nutritional adaptation of the milk proceeds more or less irrespective of the maternal diet, there is evidence that the latter can have an influence on its odor. Several studies report that the suckling behavior and later dietary habits of infants may be influenced by the mothers’ choice of diet during pregnancy and nursing. For example, infants whose mothers consumed carrot during pregnancy and breastfeeding periods accepted carrot-flavored cereals more readily than infants who were exposed to this flavor for the first time [2]. Likewise, after ingestion of garlic [3], alcohol [4], and vanilla flavor [5], the infants’ behavior was altered, displaying intensified suckling and longer breast attachment. These observations have led to the assumption that potential flavor changes of human milk can be detected by the infant during breastfeeding. Other studies have made similar observations of odor changes in human milk after mothers had ingested garlic [3], beer [6] or carrot juice [2], although it was only reported that mothers observed a difference; the exact nature of these sensorial changes was not characterized by an expert Metabolites 2016, 6, 18; doi:10.3390/metabo6020018 www.mdpi.com/journal/metabolites 47 Metabolites 2016, 6,18 2of24 panel, and the underlying molecular processes remained unclear. On the other hand, other studies found no evidence of the transfer of odorous constituents into human milk from foods such as herbal tea [7] or fish oil [8], as determined by sensory evaluations and chemical analysis. In view of this, it is important to note that sensorial changes in human milk readily occur within relatively short periods due to oxidation processes, thereby rendering unclear whether these changes reported by mothers in previous studies relate to such oxidation effects rather than direct odorant transition [9–12]. Accordingly, an expert sensory rating appears indispensable in terms of elucidating a relationship of human milk aroma changes to specific food interventions. The molecular and physiological basis of the transfer of odor compounds into human milk is complex and not yet fully understood. Several factors including the milk fat content or the absorption in the gastrointestinal tract may influence the transmission of odorants. Additionally, further metabolism steps, e.g., oxidation of the compounds, may take place prior to their excretion into human milk. Such functionalization has been demonstrated for 1,8-cineole (eucalyptol) with the formation and secretion of a number of derivatives into human milk after oral administration of this substance [13,14]. Other studies have similarly reported the transfer of specific flavor compounds into human milk. Hausner et al. [15], for example, demonstrated the transfer of D-carvone, trans-anethole and L-menthol to human milk when administering encapsulated odorants. On the other hand, in the same study, 3-methylbutyl acetate was not detected in human milk despite the relatively high dosage, indicating biotransformation of the compound in vivo. Recently, Kirsch et al. [13] confirmed the transfer of 1,8-cineole to human milk, although in this and the latter study relatively high concentrations of the substances were applied. The rationale in the case of the study by Kirsch et al. [13] was to investigate dosages that are common in pharmacological preparations and that are also prescribed to breastfeeding women; in this case this related to capsules containing 100 mg 1,8-cineole. Likewise, Hausner et al. [15] used capsules containing 100 mg of the respective compounds to standardize administration for monitoring the subsequent potential odorant transfer into human milk. Nevertheless, the use of such odorant models does not accurately reflect real-life food consumption whereby the complex food matrix of an everyday meal is commonly composed of different constituents such as meat, vegetables and other sides, thus containing an array of aroma constituents. Accordingly, conclusions regarding everyday food consumption are difficult to draw from the insights obtained by studies on model systems. The potential aroma transfer into human milk is therefore not a conditional effect but must be regarded as a variable process that is dictated by the specific chemical structures as well as quantitative composition of the aromas applied. Obviously, not all aroma compounds that are consumed reach the milk unchanged, but metabolism or general biotransformation may occur. In view of this, it is important to note that thio-containing aromas, such as garlic aroma, have hitherto never been investigated for their potential modification of human milk aroma on a molecular basis. Such analytical investigations present a great challenge due to the typically low concentrations of these substances that exhibit extremely high odor potency. Based on these considerations, the aim of this study was to determine whether ingestion of garlic influences the overall aroma of human milk; the main consideration hereby was to follow a consumption protocol that accurately reflects real-life garlic consumption. In our study, garlic was chosen based on previous studies, which indicated that garlic alters the aroma profile of human milk [3]. Furthermore, garlic is frequently consumed by humans and has not been reported to induce any side effects in the infants when consumed in normal quantities [16]. In contrast to the study by Mennella et al. [3], which investigated human milk after the consumption of capsules containing 1.5 g garlic extract, we aimed at investigating the influence of fresh garlic on human milk when consumed at food-relevant concentrations. In doing so, we aimed at demonstrating if dietary-relevant amounts of garlic have an influence on the aroma of human milk. The second aim of our study was to identify the molecules which are responsible for the potential change of the odor of human milk and to characterize their relative temporal transfer into the milk. 48 Metabolites 2016, 6,18 3of24 An identification of the underlying molecules present in the milk reveals whether these are the original food constituents (in this case odor constituents of garlic) that are indeed directly transferred into the milk, as is commonly believed, or whether these constituents have undergone metabolism before being excreted into the milk. These findings will broaden our understanding of the physiological processes occurring in the maternal body and how infants are influenced by the maternal diet, both on a sensory but also a molecular basis. 2. Results 2.1. Determination of Odor Qualities of Reference Compounds A series of garlic odorant or potential metabolite compounds was freshly synthesized for use as reference compounds; their synthesis was necessary as most were not commercially available (cf. 4.2). Moreover, the odor qualities of some reference compounds had to be determined prior to performing the study due to an absence of data on these particular compounds. The compounds evaluated were diallyl sulfoxide (DASO), diallyl disulfone (DASO2), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). DASO was described by the panel as garlic-like. DASO2 on the other hand was μ not perceivable at all, even at high concentrations of up to 477.5 g/mL. AMSO and AMSO2 were similarly odorless. The analytical and olfactometric data obtained for all compounds, together with literature reports, are presented in Table 1. Table 1 further indicates if these substances were detectable in breast milk in this study, as will be further detailed in Sections 2.3 and 2.4. 49 Metabolites 2016, 6,18 4of24 Table 1. Retention indices on DB-FFAP and DB-5 chromatographic capillaries and odor qualities of all substances investigated in this study. Literature reports on these substances in human or animal studies are provided together with an indication of their presence in breast milk samples in this study. Retention Index (RI) Identified in Milk Substance (Abbreviation) a Odor Quality Previously Detected in/Described as Reference FFAP DB-5 after Garlic Intake Human breath after garlic consumption [17–26] Allyl methyl sulfide (AMS) <1000 715 + b garlic-like c,d Human urine after garlic consumption [24] Garlic [27–31] Identified in rat stomach, liver, plasma and urine Allyl methyl sufloxide (AMSO) 1742 1018 + odorless e after administration of diallyl disulfide (DADS) [32] Garlic metabolite in the human body Identified in rat stomach, liver, plasma and urine e Allyl methyl sulfone (AMSO2) 1983 1061 + odorless after administration of DADS [26,32] Garlic metabolite in the human body Diallyl sulfoxide (DASO) 1889 1163 - garlic-like e Potential garlic metabolite in the human body e Diallyl sulfone (DASO2) 2079 1289 - odorless Potential garlic metabolite in the human body [26] garlic-like c,d Human breath after garlic consumption [17,19–25,33,34] Diallyl disulfide (DADS) 1462 1083 - pungent d Garlic [28–30,35–37] Human breath after garlic consumption [18–20,24,25] Allyl methyl disulfide (AMDS) 1265 921 - garlic-like, cooked garlic-like d Garlic [27–31,38] cabbage-like c cooked garlic-like, Human breath after garlic consumption [18] Dimethyl disulfide (DMDS) 1071 751 - onion-like, rubber-like d Garlic [27–31,36] garlic-like c [27,29–31,36] Dimethyl trisulfide (DMTS) - Garlic 1362 973 burnt garlic-like, diffusive, penetrating, sulfury d garlic-like c Human breath after garlic consumption [18,25] Diallyl trisulfide (DATS) 1771 1308 - garlic-like, onion-like d Garlic [27–31,35–38] Human breath after garlic consumption [18,21–23,25] Diallyl sulfide (DAS) 1138 868 - garlic-like c Garlic [27–31,35–37] 2-Vinyl-4H-1,3-dithiin 1827 1222 - garlic-like c Garlic [27,31,36–38] 3-Vinyl-4H-1,2-dithiin 1720 1194 - Pungent garlic-like c Garlic [27,31,36–38] a + identified in the sample extracts via GC-GC-MS in comparison to the corresponding reference compound; not detected via GC-GC-MS in the sample extract, with relation to the respective reference substance; b detected additionally via GC-O; c odor quality of the substance as described in [ 29]; d odor quality of the substance as described in [ 28]; e odor determination was performed via GC-O using a reference solution. 50 Metabolites 2016, 6,18 5of24 2.2. Aroma Profile Analysis Comparative aroma profile analysis (APA) was performed on milk samples taken before and approx. 2–3 and 4–5 h after garlic consumption (depending on the lactation period of the mother) in order to assess possible odor changes of the respective breast milk samples due to the garlic intervention. The following attributes were found to be adequate descriptors: fishy, fatty, metallic, grassy-green, rancid, sweaty, buttery, sweet, hay-like, egg white-like and lactic, plus garlic- and cabbage-like. The attributes were chosen based on those which were used to describe the odor of human milk in other studies [39], as well as on descriptors selected by our sensory panel in preliminary sensory experiments with breast milk obtained after garlic consumption. All odor attributes associated with the typical milk odor were rated as having very low odor intensities and in most cases were not perceivable (0) or just detectable (1), which is in agreement with previous studies [10,39]. In rare cases, single panelists judged some impressions as being intense. Garlic- and cabbage-like attributes were not perceivable in any of the milk samples taken before the mothers consumed garlic. By comparison, the samples collected after garlic consumption were judged by the majority of the panelists as having a slight to average garlic- or cabbage-like odor (intensities 0.5–2). A selection of aroma profiles of human milk samples that were taken over a period of 5 h are shown in Figure 1. All APA results are available in Figure S1 of the supplementary material. human milk sample human milk sample 5 min before garlic ingestion 3 h after garlic ingestion hay-like hay-like 1.50 1.50 lactic fishy lactic fishy cabbage- 1.00 cabbage- 1.00 fatty fatty like like 0.50 0.50 garlic-like rancid garlic-like rancid 0.00 0.00 buttery sweaty buttery sweaty egg egg metallic metallic white-like white-like grassy- grassy- sweet sweet green green human milk sample 5 h after garlic ingestion hay-like 1.50 lactic fishy cabbage- 1.00 fatty like 0.50 garlic-like rancid 0.00 buttery sweaty egg white- metallic like grassy- sweet green Figure 1. Odor profiles of human milk samples of test person c, as a representative example. The samples were collected at different times before and after ingestion of 3 g raw garlic. Panelists were asked to rate the orthonasal perception on a scale from 0 (no perception) to 3 (strong perception). Values are mean ratings of all panelists. Note: The scale is only presented up to the value of 1.5 for better visualization. 51 Metabolites 2016, 6,18 6of24 2.3. Comparative Aroma Extract Dilution Analysis (cAEDA) of the Milk before and after Garlic Consumption Comparative aroma extract dilution analysis (cAEDA) [40,41] was performed for the extracts of the milk samples taken before and after garlic consumption to characterize the odor-active compounds which are responsible for the garlic-/cabbage-like odor in the milk samples after garlic consumption. cAEDA showed that only one substance with a garlic-like odor and retention index (RI) < 1000 on the FFAP capillary and RI 715 on the DB-5 capillary was detectable as an additional substance in those samples that were collected after garlic consumption compared to the controls before garlic consumption. This substance was identified as AMS based on the comparison of the odor quality and the respective RIs with reference substances (see Table 1). All remaining substances that were detected are common odor substances in human milk, including several fatty acids and lactones, as reported in previous studies [10,42]. These compounds are therefore not reported here in further detail. 2.4. Identification of Garlic-Derived Metabolites in Human Milk A targeted search of the potential presence of odorless or less potent garlic-derived metabolites in human milk was performed via high-resolution gas chromatography´mass spectrometry (HRGC-MS) and two-dimensional high-resolution gas chromatography (HRGC-GC-MS). Chromatograms recorded for the extracts of the milk samples taken before and after garlic consumption were thereby compared to establish whether additional peaks were present in the latter extracts. The samples were also screened for the presence of substances that had already been reported in literature either in human breath after garlic consumption, in garlic, or that had previously been suspected to be metabolites being formed in the human body after garlic consumption (see Table 1); these screening assays were carried out based on the respective original reference substances using both HRGC-MS and HRGC-GC-MS to ensure highest sensitivity in the detection of these compounds. Use of the latter system enabled the successful separation of AMS from other co-eluting substances, which was not possible via HRGC-MS. This was additionally confirmed by HRGC-O analysis (see above). Using this approach it was further possible to detect a total of three metabolites, namely AMS, AMSO and AMSO2. Example chromatograms are displayed in Figure 2. The presence of AMS in the milk sample prior to garlic ingestion could similarly be excluded (see Figure 2b). The oxidized garlic derivatives AMSO and AMSO2 after ingestion of raw garlic were determined in human milk by HRGC-MS (see Figure 2c,d). The other substances which were monitored as possible garlic metabolites were not detectable in human milk either before or after garlic ingestion (see Table 1): these substances were DASO, DASO2, DADS, AMDS, DMDS, DMTS, DATS, DAS, 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin. Accordingly, we can exclude their presence in human milk, at least at any relevant concentrations. The structures of the investigated molecules are shown in Figure 3. 52 Metabolites 2016, 6,18 7of24 Figure 2. Identification and relative quantification of garlic-derived components in human milk samples of test person c.(a): Total ion chromatogram of human milk extract from HRGC-MS analysis (FFAP); (b): AMS, measured with HRGC-GC-MS; m/z 73 and 88 are extracted for relative quantification; (c): AMSO, measured with HRGC-MS (FFAP); m/z 104 is extracted for relative quantification; (d): AMSO2, measured with HRGC-MS (FFAP); m/z 120 is extracted for relative quantification. The human milk extract is shown 5 min before (a-1) and 3 h after (b-1) garlic ingestion. The compounds AMS, AMSO and AMSO2 are shown in human milk 5 min before (x-1) and 3 h after (x-2) garlic ingestion as well as the standard compound (x-3), which was used for identification. In x-4 to x-6 the respective mass spectra to x-1 to x-3 are shown. The mass spectra are shown at the time when the standard compound eluted. 53 Metabolites 2016, 6,18 8of24 Figure 3. Garlic-associated compounds. (A) Detected in human milk after garlic ingestion; (B) Not detected in human milk after garlic ingestion. Abbreviations: refer to Table 1. 2.5. Time Dependency of Appearance of the Garlic-Derived Metabolites in the Human Milk after Consumption of Garlic The area/kg human milk ratios for the different substances were calculated in order to obtain a relative semi-quantitative estimation of the appearance and the temporal profiles of the identified garlic metabolites in the milk samples. Values were recorded for the milk samples that were taken before and after garlic consumption at different times, and for six individual mothers. The corresponding metabolite profiles for all six mothers are shown in Figure 4. a 3.0E+043.0 x 104 2.02.0E+06 x 106 2.5E+042.5 x 104 1.51.5E+06 x 106 2.0E+042.0 x 104 1.5E+041.5 x 104 1.01.0E+06 x 106 1.0E+041.0 x 104 5.05.0E+05 x 105 AMS Area/kg 5.0E+035.0 x 103 0.0E+00 0 00.0E+00 AMSO or AMSO2 Area/kg 024 time (h) b 5.0E+035.0 x 103 6.06.0E+06 x 106 5.0E+066 4.0E+034.0 x 103 5.0 x 10 4.04.0E+06 x 106 3.0E+033.0 x 103 3.03.0E+06 x 106 2.0E+032.0 x 103 2.02.0E+06 x 106 AMS Area/kg 3 1.0E+031.0 x 10 1.01.0E+06 x 106 0.0E+00 0 00.0E+00 AMSO or AMSO2 Area/kg 024 time (h) Figure 4. Cont. 54 Metabolites 2016, 6,18 9of24 c 6 5.05.0E+04 x 104 7.0E+067.0 x 10 6.0E+066.0 x 106 4.04.0E+04 x 104 5.0E+065.0 x 106 3.03.0E+04 x 104 4.0E+064.0 x 106 3.0 x 106 2.02.0E+04 x 104 3.0E+06 2.0E+062.0 x 106 AMS Area/kg 1.01.0E+04 x 104 1.0E+061.0 x 106 0.0E+000 0.0E+000 AMSO or AMSO2 Area/kg 024 time (h) d 3.0E+043.0 x 104 3.0E+073.0 x 107 2.5E+042.5 x 104 2.5E+072.5 x 107 2.0E+042.0 x 104 2.0E+072.0 x 107 1.5E+041.5 x 104 1.5E+071.5 x 107 1.0E+041.0 x 104 1.0E+071.0 x 107 AMS Area/kg 5.0E+035.0 x 103 5.0E+065.0 x 106 0.0E+00 0 0.0E+000 AMSO or AMSO2 Area/kg 024 time (h) e 2.02.0E+04 x 104 3.03.0E+06 x 106 2.52.5E+06 x 106 1.51.5E+04 x 104 2.02.0E+06 x 106 1.01.0E+04 x 104 1.51.5E+06 x 106 1.01.0E+06 x 106 AMS Area/kg 5.05.0E+03 x 103 5.05.0E+05 x 105 0.0E+00 0 00.0E+00 AMSO or AMSO2 Area/kg 024time (h) f 6.0E+036.0 x 103 3.53.5E+06 x 106 6 5.0E+035.0 x 103 3.03.0E+06 x 10 2.52.5E+06 x 106 4.0E+034.0 x 103 2.02.0E+06 x 106 3.0E+033.0 x 103 1.51.5E+06 x 106 3 2.02.0E+03 x 10 6 AMS Area/kg 1.01.0E+06 x 10 1.0E+031.0 x 103 5.05.0E+05 x 105 0.0E+00 0 00.0E+00 AMSO or AMSO2 Area/kg 024time (h) Figure 4. Time-resolved metabolite profiles of AMS, AMSO and AMSO2, a–f: Milk samples taken from six different mothers. AMS ( ), AMSO ( ), AMSO2 ( ), time 0 h represents the milk sample collected prior to garlic consumption, following times represent milk samples after garlic consumption. Garlic was consumed 5 to 15 min after the first milk sample was given. Although the milk samples were gathered at similar times, the profiles indicate that there are high inter-individual differences in the metabolism and/or excretion rate of the respective garlic constituents. In particular, AMS formation and excretion appeared to be highly variable between different individuals. The relative values obtained for the milk samples from mothers a and b showed a continuous increase in AMS content over the whole sampling period. Conversely, the highest values of AMS in the milk of mothers c, d, e and f were recorded in the second milk sample, which was taken between 2 to 3 h after garlic consumption; thereafter, the content of AMS decreased. In the case of AMSO, the highest value was observed in the second milk sample. Only in the case of mother 55 Metabolites 2016, 6,18 10 of 24 b did the AMSO content increase over the whole sampling time; however, the second increase was noticeably slower than the increase between the first sample (before garlic consumption) and the second sample (2–3 h after garlic consumption). Likewise, AMSO2 displayed a continuous increase in the milk of mother a as well as in the milk of mother b for the respective sampling events. During the collection period, no clear maximum was observed. In contrast to this, however, a decline in AMSO2 was observed in the milk samples from mothers c, d, e and f, which were taken 4–5 h after garlic consumption. In the case of the milk of mother f it should be noted that traces of garlic metabolites were also found in the milk that was taken prior to garlic consumption, despite the fact that the sensory profile did not reveal any related smell characteristics. This might be due to the fact that the respective mother reported that she consumed some tomato sauce in a restaurant one day prior to the sampling day (see supplementary material Table S6). According to the staff of the restaurant that was consulted regarding this matter, no garlic was used but a broth powder, potentially containing ingredients from onion, leek and garlic. Despite this background effect, we observed a clear increase in the second milk sample taken 3 h after the garlic consumption. In relation to this increase, the amount of AMS in the blank sample was very low, so that it was even not sensorially detectable during GC-O analysis. Interestingly, another mother reported that she was unsure if she potentially consumed some garlic during the wash-out phase with a potato meal that had been prepared for her the day before. However, neither in sensory nor in analytical analysis any indications for garlic smell or metabolite transmission was found in the control milk, so that any relevant impact could be excluded. 3. Discussion 3.1. Aroma Profile Analysis The aroma profile analyses showed that the odor of the breast milk changed about 2.5 h after the consumption of raw fresh garlic, which is in agreement with the study by Mennella and Beauchamp [3]. In the latter study [3], the mothers consumed either placebo capsules or capsules containing 1.5 g of garlic extract and the milk samples were collected at hourly intervals, one hour before and up to 3 h after consumption of the capsules. Sensory evaluation of the milk samples showed that samples that were taken 2 h after ingestion of the garlic capsules had higher odor intensities in comparison to the placebo group; these intensities peaked after 2 h and then decreased again. However, the nature of these sensory quality changes was not examined. The present study demonstrates that a clear garlic-/cabbage-like odor appears in human milk samples 2.5 h after consumption of 3 g of raw garlic. The rationale for using3gofgarlic was that this represents a realistic amount of garlic that may be consumed by a person during a normal diet. On the other hand, no clear difference in the intensities of these descriptors for milk samples taken either 2–3 or 4–5 h after garlic consumption, respectively, were detected. In the case of the milk samples that are shown in Figure 1, the garlic-like attribute was rated with intensities of 1 and 0.33 (mean of all panelists) in the milk samples taken 3 and 5 h after garlic consumption, respectively. For other milk samples, the garlic-like attribute was rated to have the highest in the last milk sample (4–5 h) after garlic-consumption, as can be seen in supplementary material Figure S1. Although this observation might suggest major changes, it nevertheless should be kept in mind that in either case the overall odor intensities were quite low in orthonasal evaluation. Retronasal evaluation (tasting) might have revealed higher sensory intensities, as has been demonstrated before in the case of freeze-stored human milk evaluation [9]. However, retronasal tasting was not carried out by the expert panel in the present study due to work safety considerations. In general, the milk samples were always evaluated directly after expression as our previous work had shown that human milk aroma can undergo quite pronounced sensory changes even after relatively short storage periods [12]. Accordingly, a direct comparison of the samples taken after 2–3 and 4–5 h was not achievable. 56 Metabolites 2016, 6,18 11 of 24 3.2. Identification of Garlic-Derived Metabolites in Human Milk In this study, we identified three different garlic-derived metabolites, namely AMS, AMSO and AMSO2 after the intake of 3 g raw garlic. Previous studies revealed that food supplements [14]or compounds in high dosages [15] can influence the composition of human milk. During this study we have shown for the first time that dietary-relevant amounts of a food can also have an impact on human milk. In contrast to previous assumptions, however, this study shows that the original aroma constituents are not directly transferred but rather three metabolites, namely AMS, AMSO and AMSO2. Accordingly, this is the first report of the excretion of an aroma metabolite into human breast milk in food-relevant conditions. While AMS has previously been found as a metabolite in human breath and in urine after garlic consumption [23–26], this is the first report of AMS appearance as an odorous garlic-derived metabolite in human milk. Likewise, AMSO and AMSO2 were detected here for the first time in human milk after garlic consumption. Both substances are odorless, which explains why neither AMSO nor AMSO2 were detected in cAEDA. It is interesting to note that neither of these substances has been found previously in any human bodily fluids or breath after garlic consumption. However, their formation related to garlic consumption has been reported in rat, where both substances were detected in stomach, liver, plasma and urine after administration of diallyl disulfide (DADS) [32]. DADS is a decomposition product of allicin that is formed through the interaction of alliin with allinase after disruption of the cell structure of garlic. It represents 40% to 60% of the essential oil of garlic [32]. Besides providing proof of the appearance of AMS, AMSO and AMSO2 in milk after garlic ingestion, we could further exclude the presence of these substances in the milk prior to garlic consumption, with the sole exception of the milk of mother f. In this case we observed traces of garlic metabolites that likely relate to prior dietary exposure (see above). This case demonstrates that a wash-out time of at least 24 h prior to the testing time should be adhered to for investigation of such odorant and metabolite transmission effects. In addition, we could further exclude the appearance of a series of other garlic-derived substances in human milk after garlic ingestion, at least for the samples investigated within this study. Future studies are required to explain this preferential appearance of AMS, AMSO and AMSO2, which might be related to diverse factors such as (in)stability in the gastro-intestinal tract, biodegradation and bioformation, resorption as well as excretion processes, and the respective quantities of the compounds and their precursors. 3.3. Metabolism of Garlic We observed the excretion of different garlic-derived metabolites over a period of up to 5.2 h. On a semi-quantitative basis, we further observed different excretion profiles with differences in the relative maximum intensities of the monitored metabolites in milk samples from the individual mothers. Temporal differences might relate to variations in milk sampling times, owing to the necessity to adhere to lactation intervals. The metabolism rates might also be influenced by the age of the infant, since the composition of human milk changes according to the lactation period. There are several more potentially influencing factors that might impact the composition of human milk, such as the age or physiological status of the mother, e.g., body mass index [43]. Potential links of these differences with individual metabolism rates or different contents of metabolite precursors in the garlic bulb are thus not discernable from this data. This would require further investigation to first elaborate their potential precursors and to quantify these in relation to the monitored derivatives, not only in breast milk but also taking into consideration other excretion pathways such as urine and breath. Nevertheless, the main aim of this study was to elucidate the chemical nature of substances and metabolites of garlic in breast milk. In view of this it is important to discuss how the typical garlic-odor of a garlic bulb arises. The release of the garlic smell is caused by the interaction of the enzyme alliinase and the odorless, non-proteinogenic amino acid alliin (S-allyl-L-cysteine sulfoxide). These two compounds are located in different cell compartments that only come into contact with each other from cutting or disrupting the cell structure, e.g., by chewing. This then leads to the production of dehydroalanine and allyl sulfenic acid. In a further reaction, two molecules of allyl sulfenic acid 57 Metabolites 2016, 6,18 12 of 24 may condensate to form allicin [44]. Allicin itself is not stable and can degrade to secondary substances such as ajoenes, vinyldithiins and different sulfides [45]. The exact nature of which metabolites are formed from which precursors, or if and who the different pathways are linked with each other is not presently known. Regarding the metabolism of garlic constituents in the human body, the different pathways are likewise not fully understood. A series of studies addressed this issue, primarily in animal studies involving rats, or in tissue model studies. However, only a few investigations were carried out regarding the metabolism of garlic constituents in human subjects. Lawson and Wang [26] investigated the effect of garlic and garlic-derived compounds on breath composition. Specifically, the authors recorded the acetone and AMS levels in breath after consumption of allicin, allicin-derived compounds (DATS, DADS, DAS, ajoene, distilled garlic oil), allicin metabolites (allyl mercaptan, AMS) and S-allyl cysteine. Administration of these compounds resulted in an increase in acetone and AMS in breath in each case, with the sole exception of DAS and S-allyl cysteine; DAS therefore had no influence on the AMS level but still led to an increase in acetone. Both compounds are the only ones lacking a dithioallyl group. Accordingly, Lawson and Wang [26] concluded that a dithioallyl group is a necessary requirement for the respective precursor substances in the formation of AMS. They also proposed allyl mercaptan as a precursor of AMS. However, as they could show that transformation of allicin to AMS is extremely fast, they did not expect any accumulation of allyl mercaptan under physiological conditions. These observations are in good agreement with our findings as only AMS and its metabolism products AMSO and AMSO2 were detected. There were no further compounds detectable that might be considered as precursors of AMS. Apart from that, Lawson and Wang [26] observed that AMS was exhaled sooner than acetone, which indicated that AMS or a metabolite of AMS might be responsible for the increase in breath acetone levels after garlic consumption. Referring to the work of Germain et al. [32], the authors further suggested AMSO and AMSO2 to be additional metabolites of AMS (see Figure 5), but did not confirm this assumption by experimental data. The present study may be regarded as further support for the proposed formation and occurrence of AMSO and AMSO2 as metabolites of AMS in humans. In view of this, it is interesting to note that Lawson and Wang [26] also suggested that DAS is metabolized differently than AMS, since after consumption it is only measureable in trace amounts, but still has an impact on the acetone level in breath. Based on experiments performed by Brady et al. [46] and Jin and Baillie [47], they proposed DASO2 as a metabolite of DAS (see Figure 5). The results of our study cannot support this assumption, since neither DASO2 nor its supposed precursor DASO was present in measurable amounts. Other studies on garlic breath confirmed the assumption that AMS is a garlic-derived metabolite; they all confirmed exhalation of AMS after ingestion of garlic [17–25]. Nevertheless, deeper insights into metabolism of garlic constituents may be obtained from animal and tissue studies. In the study by Germain et al. [32], the metabolism of DADS, a decomposition product of allicin, was investigated in rats. The authors administered 200 mg/kg DADS to rats, and the metabolites present in stomach, liver, plasma and urine were monitored over a period of 15 days. DADS and allyl mercaptan in plasma were only detected at 20 min after oral administration of DADS, whereas the other metabolites, namely AMS, AMSO and AMSO2, were detectable at this time until 7 days after oral administration, which is in good agreement with the results of the present study. DADS and allyl mercaptan were not detectable at all in urine and the only metabolites were AMS, AMSO and AMSO2. Accordingly, it is possible that no allyl mercaptan is excreted into human milk, but this has to be confirmed in further experiments. In view of temporal profiles, Germain et al. [32] reported that all metabolites (allyl mercaptan, AMS, AMSO and AMSO2) were most abundant 48 to 72 h after DADS administration. In our study, we only monitored up to 5.2 h after garlic consumption; during this time it appears as if we already observed a maximum of some of the metabolites. However, in our human study, continued sampling was limited due to constraints regarding the natural breastfeeding regimes. Moreover, the rationale for the sampling period used in our study was that excretion maxima in humans for odorants and their metabolites have been reported to occur commonly around two 58 Metabolites 2016, 6,18 13 of 24 to three hours after intake [7,13–15,48]. Regarding the administered concentrations, it has also to be mentioned that Germain et al. [32] applied 200 mg/kg of pure DADS to the rats whereas the test persons in our study only consumed 3 g of raw garlic. The allicin content of raw garlic lies between 0.35% and 0.53% [49,50]; under the assumption that the total amount of allicin is converted to DADS, 3 g of garlic accordingly relate to about 120 mg DADS (calculated with an allicin content of 0.4%). If one considers a human weight of about 60 kg, this amount represents just a fraction of the amount administered in the study by Germain et al. [32]. Accordingly, the experimental conditions are not directly comparable. Moreover, other garlic constituents might also influence the metabolic fate of the substances, and it is further possible that humans metabolize garlic differently, i.e., faster than rodents, as garlic is a common part of their diet. Figure 5. Metabolic fate and metabolic effect of allicin and allicin transformation compounds. GSH: glutathione (γ-Glu-Cys Gly); GSSA: S-allylmercaptoglutathione (γ-Glu-Cys-(S-allyl)-Gly); GSSG: oxidized glutathione, SAM: S-adenosylmethionine; and SAH: S-adenosylhomocysteine. Grey areas mark the components which were found in the present study as human milk metabolites after garlic ingestion. Adapted with permission from Lawson, L.D.; Wang, Z.J. Allicin and allicin-derived garlic compounds increase breath acetone through allyl methyl sulfide: Use in measuring allicin bloavailability. J. Agric. Food Chem. 2005, 53, 1974–1983 [26]. Copyright (2005) American Chemical Society. Apart from the in vivo study by Germain et al. [32], other authors studied the metabolism of garlic constituents based on rat tissues, cells or body fluids. Brady et al. [46] utilized liver, blood and urine samples from rats which were treated with 200 mg/kg DAS. In their studies they detected DASO as well as DASO2 in each of the samples. Egen-Schwind et al. [51,52] identified DADS and allyl mercaptan as metabolites of allicin in isolated perfused rat liver, and reported DADS as a precursor for allyl mercaptan. Sheen et al. [53] detected allyl mercaptan and AMS as metabolites of DADS in primary rat hepatocytes when treating the cells with 1 mM DADS or DAS dissolved in propylene glycol for different periods. DADS was almost completely converted to allyl mercaptan and AMS within 120 min. 59 Metabolites 2016, 6,18 14 of 24 DAS, on the other hand, only led to the formation of AMS. This observation is in contrast to the results of Brady et al. [46], and contradicts the assumption of Lawson and Wang [26] who proposed DASO and DASO2 as metabolites of DAS. In our study, neither allyl mercaptan nor DADS were detected, which is not in line with the findings of Egen-Schwind et al. [51,52] and Germain et al. [32]. An explanation for this observation might be that DADS and allyl mercaptan are rapidly converted to other metabolites, as proposed by Lawson and Wang [26], or that both substances are not excreted into human milk. On the other hand it is possible that allyl mercaptan is not detectable by the method used within this study. Allyl mercaptan is a very volatile compound and could be lost during the work-up procedure of the human milk samples. This aspect should be addressed in future quantification studies, ideally involving stable isotope dilution assays [8,11,12,54]. DADS is possibly completely converted to other metabolites in the human body, leading to the formation of compounds such as allyl mercaptan, AMS, AMSO and AMSO2, which could be the reason why it was not detected in our study. Likewise, DASO and DASO2 were not detected in our study, indicating that DAS might be metabolized differently in humans than in rats. However, it might also be that not all substances are transferred from blood to human milk; their presence in blood would also need to be investigated in further studies. On the other hand, there are studies reporting glutathione conjugates of DAS and its putative metabolites DASO and DASO2 [47], as well as N-acetyl conjugates. In view of this, it is important to note that De Rooij et al. [55], as well as Jandke and Spiteller [56], identified N-acetyl-S-allyl-L-cysteine in human urine after garlic consumption. This metabolite may be formed from the water-soluble S-allyl cysteine catalyzed by N-acetyl transferase. Even if excretion of these conjugates occurs via urine, the potential presence of such conjugates or less volatile metabolites in human milk would need to be regarded in future studies involving other techniques such as HPLC. To conclude our findings, this study clearly shows that garlic consumption leads to a distinct sensory impact on the human milk aroma profile, even eliciting odor impressions that are related to the original aroma profile of the ingested food. At first sight, this might lead to the assumption that aroma transmission into milk occurs with a direct representation of the odorous substances of the garlic. Nevertheless, our study clearly shows that this sensory impression is misleading, and that other derivatives are found in the milk, whereby some are even odorless and only one bears the characteristic garlic smell, namely AMS. Accordingly, this study clearly demonstrates that the odor composition after transmission into human milk does not necessarily coincide with what is found in the original food, and that metabolism and other transmission or resorption processes may play a major role in human milk odor formation. This is in agreement with other studies of our group where it was either shown that nutritional odorants did not translate into human milk [7,8], or that substances were heavily metabolized and showed up as their derivatives [14]. More studies will be required in the future to elucidate which odorants or substance classes, or their respective precursors, bear the potential of influencing the odor profiles of human milk, and at which concentration levels. Only then will it be possible to gauge if babies can be influenced by such substance transmission, and if such influence would be only with sensory impact or if there is even the potential of other physiological effects. Especially metabolites are worth considering with regard to these aspects. 4. Materials and Methods 4.1. Chemicals/Materials The following reference compounds were obtained from the suppliers shown: dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) (Sigma-Aldrich, Steinheim, Germany), allyl methyl disulfide (AMDS) (abcr, Karlsruhe, Germany). Allyl methyl sulfide (AMS), allyl methyl sulfone (AMSO2), allyl methyl sulfoxide (AMSO), diallyl disulfide (DADS), diallyl sulfide (DAS), diallyl sulfone (DASO2), diallyl sulfoxide (DASO), 3-vinyl-4H-1,2-dithiin and 2-vinyl-4H-1,3-dithiin, and diallyl trisulfide (DATS) were synthesized as described below. 60 Metabolites 2016, 6,18 15 of 24 Chemicals used for the syntheses of reference substances were obtained from the suppliers shown: bis(tri-n-butyltin)sulfide ((Bu3Sn)2S), Triton X 100 (abcr, Karlsruhe, Germany); acrolein diethyl ě acetal 96%, allyl bromide 97%, meta-chloroperoxybenzoic acid 99%, cobalt-(II)-chloride (CoCl2) 97%, 4,4-cyclohexylidenebis[N,N-bis(4-methylphenyl)benzenamine] (TAPC) 97%, ethanol absolute, hydrogen peroxide 30 wt % in H2O, magnesium sulfate (MgSO4) anhydrous, manganese-(IV)-oxide ě ě (MnO2) 99%, methyl iodide 99%, oxalic acid 98%, petroleum ether, polyethylene glycol 300 (PEG 300), 2-propen-1-thiol ~60%, pyridine anhydrous 99.8%, sodium bicarbonate (NaHCO3), sodium bisulfite (NaHSO3) solution ~40%, sodium carbonate (Na2CO3) anhydrous, sodium iodate ě ě ě (NaIO3) 99%, sodium thiosulfate pentahydrate 99.5%, tetrahydrofuran anhydrous, thiourea 99%, toluene anhydrous 99.8%, trimethylaluminum solution (2.0 M in toluene) (Sigma-Aldrich, Steinheim, Germany); dichloromethane (DCM) high performance liquid chromatography (HPLC) grade, ethyl acetate (EtOAc), hexane, silica gel 60 (SiO2), sodium hydroxide (NaOH), sodium sulfate anhydrous (Na2SO4), sodium sulfide anhydrous (Na2S) (VWR, Darmstadt, Germany). The solvents were freshly distilled prior to analysis. 4.2. Syntheses of Reference Substances AMS was obtained from 2-propen-1-thiol by using the method described by [57] for the synthesis of unsymmetrical sulfides via a CoCl2 catalyzed reaction of thiols with allyl iodide under visible irradiation in the presence of pyridine. This was achieved by mixing 2-propen-1-thiol with methyl iodide, pyridine and CoCl2 and irradiating the mixture with a 500 W lamp for 15 min. After the mixture had cooled down to room temperature, the solution was extracted with diethyl ether. The deep red organic phase was separated, washed with water, dried over MgSO4, and the solvent evaporated under reduced pressure. The compound was further purified via column chromatography using 1 hexane as solvent and SiO2 as stationary phase. Yield: 63 mg, 15.5 mmol, 39.65%. H-NMR (600 MHz, δ CDCl3): H = 5.78 (1H, m, 2), 5.12 (1H, m, HC=CHH trans, 1), 5.09 (1H, m, HC=CHH cis, 1), 3.12 (2H, 13 δ d, 3), 2.03 (3H, s, 4). C-NMR (150 MHz, CDCl3): C = 134.23 (2), 117.08 (1), 33.30 (3), 14.05 (4). MS-EI: m/z (%) = 89 (5), 88 (100), 73 (69), 72 (6), 71 (6), 61 (15), 47 (13), 46 (12), 45 (34), 41 (26), 39 (25). AMS was then further oxidized to AMSO as described by Oae, et al. [58]. The oxidizing agent MCPBA was used for the reaction with the difference that the powdered MCPBA was not added directly to the reaction mixture as described in the literature, but first dissolved in DCM and then added dropwise. The reaction mixture was then allowed to stir for 30 min instead of 4.5 h. An additional drying step with Na2SO4 was applied after neutralizing with NaHCO3 and washing with water. ˝ To obtain the crude product, the solvent was removed under reduced pressure at 30 C. Further purification of the product was then accomplished by column chromatography with EtOAc. Yield: 0.206 g, 2.0 mmol, 70.64%. 1H-NMR (600 MHz, CDCl3): δH = 5.75 (1H, m, 2), 5.45 (1H, m, 1), 3.12 (2H, 13 δ d, 3), 2.03 (3H, s, 4). C-NMR (150 MHz, CDCl3): C = 125.52 (2), 128.81 (1), 53.41 (3), 14.21 (4). MS-EI: m/z (%) =104 (100), 103 (8), 89 (41), 87 (16), 76 (24), 75 (10), 73 (12), 72 (7), 71 (10), 61 (22), 59(14), 58 (17), 57 (50), 55(8), 49 (10), 48 (56), 46 (7), 45 (50), 41 (10). Oxidation of AMS leads to the generation of AMSO2 when applying different conditions. The method described by Bland and Stammer [59] was used to achieve this, albeit with the reaction mixture left to stand for 4 days instead of 30 h. Yield: 0.421 g, 3.5 mmol, 89.44%. 1H-NMR (600 MHz, δ CDCl3): H = 5.99 (1H, m, 2), 5.53 (1H, m, HC=CHH trans, 1), 5.49 (1H, m, HC=CHH cis, 1), 3.74 (2H, d, 3), 2.87 (3H, s, 4). 13C-NMR (600 MHz, CDCl3): δC = 125.40 (2), 124.69 (1), 59.53 (3), 39.05 (4). MS-EI: m/z (%) = 120 (19), 105 (5), 79 (7), 65 (6), 64 (10), 63 (8), 57 (6), 48 (5), 45 (6) 42 (7), 41 (100), 40 (8), 39 (86), 38 (10). DADS was synthesized according to Firouzabadi, et al. [60]. However, the extraction and purification of the desired product was changed and performed as follows: Water was added and the mixture was subsequently extracted five times with EtOAc to extract the product from the reaction mixture. The combined organic phases were then dried over MgSO4 and the solvent was evaporated under reduced pressure. DADS was recovered as a colorless liquid after column chromatography 61 Metabolites 2016, 6,18 16 of 24 1 over SiO2 with petroleum and evaporation of the solvent. Yield: 3.84 g, 26.25 mmol, 79%. H-NMR δ 13 (600 MHz, CDCl3, RT): (ppm) = 5.86 (t, 2H, 2); 5.22–5.15 (dd, 4H, 1); 3.36 (t, 4H, 1). C-NMR δ + (150 MHz, CDCl3, RT): (ppm) = 133.46 (2); 118.41 (1); 42.28 (3). MS-EI: m/z (%) = 146 (M , 53), 113 (54), 105 (45), 103 (25), 85 (33), 81 (100), 79 (36), 73 (38), 71 (27), 45 (85). Synthesis of DAS was carried out according to the method described by Lu and Cai [61], albeit 1 using Trition X 100 rather than Trition X 10. Yield: 1.27 g, 11.12 mmol, 90%. H-NMR (600 MHz, CDCl3, δ 13 RT): (ppm) = 5.76 (t, 2H, 2); 5.09–5.06 (dd, 4H, 1); 3.08 (t, 4H, 1). C-NMR (150 MHz, CDCl3, RT): δ (ppm) = 134.23 (2); 117.15 (1); 33.32 (3).MS-EI: m/z (%) = 114 (67), 99 (56), 81 (30), 80 (25), 73 (95), 72 (63), 71 (34), 45 (100), 41 (62), 39 (74). The synthesis of DASO was carried out as described by Mokhtary et al. [62] with only minor changes applied: ethanol was used as solvent instead of methanol, and 20 mL of water was added for better separation of the phases. Finally, the extraction with DCM was performed twice instead of once. A mixture of hexane/ EtOAc (7/3) was used as solvent for the column chromatography. Yield: 3.87 g, 1 δ 29.72 mmol, 83%. H-NMR (600 MHz, CDCl3, RT): (ppm) = 5.89 (m, 2H, 2); 5.41–5.50 (m, 4H, 1); 13 δ 3.41–3.53 (m, 4H, 3). C-NMR (150 MHz, CDCl3, RT): (ppm) = 125.71 (2); 123.61 (1); 54.22 (3). MS-EI: m/z (%) = 100 (7), 82 (7), 81 (45), 80 (10), 79 (6), 73 (6), 68 (13), 67 (5), 45 (9), 41 (100). DASO2 was additionally obtained from DAS. Synthesis was performed according to Bahrami, et al. [63]. Column chromatography over SiO2 with hexane/EtOAc (v/v 7/3) was applied for 1 δ purification. Yield: 2.30 g, 15.73 mmol, 90%. H-NMR (600 MHz, CDCl3, RT): (ppm) = 5.83–5.95 (m, 13 δ 2H, 2); 5.37–5.49 (dd, 4H, 1); 3.66–3.68 (d, 4H, 3). C-NMR (150 MHz, CDCl3, RT): (ppm) = 124.91 (2); 124.65 (1); 55.93 (3). MS-EI: m/z (%) = 81 (8), 67 (40), 54 (17), 41 (100). 3-Vinyl-4H-1,2-dithiiin was synthesized according to Li, et al. [64], albeit with a nitrogen instead of an argon atmosphere. Further deviations from the previously reported protocol were carried out after the addition of acrolein diethyl acetal: The reaction mixture was not poured into ice-water but stirred overnight at room temperature. The mixture was then poured into water and the organic layer was separated and treated as described by Li et al.[64] but with a hexane/DCM ratio of 4/1 (v/v) instead of 5/1 (v/v) for column chromatography. Yield: 0.021 g, 0.15 mmol, 5%. 1H NMR (600 MHz, δ CDCl3, RT): (ppm) = 5.95–6.08 (m, 2H, 3/4/5); 5.27–5.31 (m, 2H, 6); 4.71–3.75 (m, 1H, 1); 2.45–2.63 (m, 13 δ 2H, 2). C NMR (150 MHz, CDCl3, RT): (ppm) = 135.90 (4); 125.47 (3); 120.06 (5); 117.01 (6); 43.60 (1); 30.41 (2). MS-EI: m/z (%) = 144 (91), 111 (100), 103 (55), 97 (62), 79 (34), 77 (40), 72 (33), 71 (41), 45 (30), 39 (21). 2-Vinyl-4H-1,3-dithiin was synthesized using the same procedure as for 3-vinyl-4H-1,2-dithiin. 1 δ Yield: 0.22 g, 1.53 mmol, 47%. H-NMR (600 MHz, CDCl3, RT): (ppm) = 6.31 (d, 1H, 4); 5.99 (m, 13 2H, 3/5); 5.27–5.42 (dt, 2H, 6); 4.74 (d, 1H, 1); 3.27-3.40 (dd, 2H, 2). C-NMR (150 MHz, CDCl3, RT): δ (ppm) = 134.13 (4); 122.07 (3); 118.45 (5); 117.17 (6); 45.16 (1); 25.12 (2). MS-EI: m/z (%) = 144 (M+, 99), 111 (77), 103 (22), 97 (28), 85 (10), 79 (13), 73 (14), 72 (99), 71 (100), 45 (39). The synthesis of DATS was performed according to the method described by Ren, et al. [65]. Small modifications on the synthesis protocol were made: After separation of the two layers, the aqueous layer was washed twice with DCM. The combined organic layers were washed with H2O and dried over MgSO4. After evaporation of the solvent the product was recovered as colorless liquid. No further column chromatography was performed as the substance had already the desired purity of 89%. Yield: 1 δ 2.61 g, 14.64 mmol, 89%. H-NMR (600 MHz, CDCl3, RT): (ppm) = 5.89 (m, 2H, 2); 5.26 (t, 4H, 1); 13 δ 3.52 (d, 4H, 3). C-NMR (150 MHz, CDCl3, RT): (ppm) = 132.70 (2); 119.09 (1); 41.67 (3). MS-EI: m/z (%) = 178 (M+, 8), 114 (10), 113 (100), 74 (7), 73 (91), 72 (9), 71 (10), 47 (7), 45 (30), 41 (53). 4.3. Human Milk Samples Human milk samples were obtained from six volunteer donors. The volunteers (age range 25–40 years (mean 31)) had no known illnesses at the time of examination, their breast milk production was normal, and they produced milk in excess of their infants’ needs. The sampling took place in the lactation period from 22 to 51 (mean 30) weeks postpartum using a mechanical breast pump (Medela 62 Metabolites 2016, 6,18 17 of 24 Harmony™, Medela AG, Baar, Switzerland). To reduce the amount of sulfurous substances in the human milk on the sampling day, each subject was asked not to eat food containing high amounts of sulfur compounds for two days prior to the intervention and also on the sampling day; foods to be avoided were garlic, onion, wild garlic, chives, cabbage and leek. Furthermore, mothers were instructed to keep a record of all foods and beverages they consumed during this three-day period. The respective dietary records can be found in the online supplementary material Tables S1–S6. On the sampling day, donors were asked to ingest approx. 3 g raw garlic which was obtained from a local supermarket (Aldi-Süd, Erlangen, Germany and Aldi-Süd, Freising, Germany) and peeled and cut into approx. 3 mm cubes by using a garlic cutter (Genius GmbH, Germany). Three human milk samples (9.5 to 50 g) were provided by each mother: one prior to garlic consumption and two afterwards according to the normal lactation period of each woman (usually with two to three-hour intervals between sampling). Minor deviations in sampling time were accepted, to minimize disruption to the nursing intervals. The milk samples obtained were evaluated and analyzed immediately. A table with the exact sampling time and the amount of the milk samples is given in the online supplementary material Table S7. 4.4. Study Design The study was conducted in agreement with the Declaration of Helsinki. Written consent was provided by all four volunteers before sampling and analysis after a full explanation of the nature and purpose of the study. Resignation from the study was possible at any time. The study (registration number 49_13B) was approved by the Ethical Committee of the Medical Faculty, Friedrich-Alexander-Universität Erlangen-Nürnberg. 4.5. Aroma Profile Analysis The sensory analyses of the human milk samples were performed by trained volunteers from the University of Erlangen-Nürnberg (Erlangen, Germany) who exhibited no known illness at the time of examination and with audited olfactory and gustatory function. Panelists were trained at recognizing about 90 selected odorants and different odorant concentrations according to their odor qualities, and in naming these according to an in-house developed flavor language in weekly training sessions over at least four months prior to performing the sensory analyses of this study. Orthonasal evaluation (smelling) of the human milk samples was performed by presenting the ˝ samples to the sensory panel in a brown glass bottle (capacity 50 mL) in a sensory room at 21 ˘ 1 C. No information about the purpose of the experiment was given. The panelists were asked to score the intensities of different sensory attributes on a scale from 0 (no perception) to 3 (strong perception). Attributes used were as follows and were the same as already established for the odor of human milk by Sandgruber et al. [39]: hay-like, fishy, fatty, rancid, sweaty, metallic, grassy-green, sweet, egg white-like, and buttery; only the attribute lactic was added, as required by our sensory panel. Panelists were additionally asked to score the intensities of the two attributes garlic- and cabbage-like. 4.6. Solvent-Assisted Flavor Evaporation (SAFE) of Volatiles from Human Milk Solvent-assisted flavor evaporation (SAFE) [66] was used for the isolation of the volatile compounds from the breast milk samples. DCM was added at a ratio of 1:2 (v/v) to 12–50 g milk samples. The solution was then stirred for 30 min and thereafter immediately applied for SAFE ˝ distillation at 50 C. The distillate obtained was then extracted three times with 25 mL DCM and the combined DCM phases were dried over anhydrous Na2SO4 and concentrated to a total volume of ˝ 100 μLat50 C by means of Vigreux distillation and micro-distillation [67]. The extracts were stored ˝ at ´20 C until analysis. A blank sample comprised of 25 mL DCM that was worked up instead of a milk sample. 63 Metabolites 2016, 6,18 18 of 24 4.7. High-Resolution Gas Chromatography-Olfactometry (HRGC-O) High-resolution gas chromatography´olfactometry (HRGC-O) was performed with a Trace Ultra GC (Thermo Finnigan, Dreieich, Germany) using the following capillaries: DB-FFAP (30 m ˆ 0.32 mm, film thickness 0.25 μm; J&W Scientific, Fisons Instruments, Mainz-Kastel, Germany) and DB-5 (30 m ˆ 0.32 mm, film thickness 0.25 μm; J&W Scientific).The effluent was split at the end of the capillaries between a sniffing port and a flame ionization detector (FID) using two deactivated, ˝ uncoated fused silica capillaries (i.d. 0.32 mm). The FID and the sniffing port were held at 250 C ˝ and 270 C, respectively. The flow rate of helium carrier gas was 2.0 mL/min. Administration of the samples was performed by the cold on-column technique, whereby 2.0 μL of the extract were injected ˝ manually into a cold-on-column injector at 40 C directly on a pre-column of uncoated, deactivated fused silica capillary (2–3 m ˆ 0.32 mm). The pre-column was changed regularly to avoid accumulation of contaminants. 4.8. Determination of Odor Qualities of Reference Compounds HRGC-O was applied for determining the odor qualities of reference compounds according to the following instrument parameters: The helium carrier gas flow rate was set to 2.5 mL/min. ˝ ˝ The temperature program of the oven started at 40 C and was raised to 240 C (DB-FFAP) or ˝ ˝ 300 C (DB-5) at a rate of 8 C/min. DB-5 and DB-FFAP columns were used for DASO and DASO2, and AMSO and AMSO2, respectively. The reference compounds were diluted in DCM μ at the following concentrations: The concentration of AMSO and AMSO2 were 47.1 g/mL and 49.0 μg/mL, respectively. The concentration of DASO was 50.2 μg/mL. Concentrations of 47.8 μg/mL μ μ and 477.5 g/mL were applied for DASO2. The reference solutions comprised 2 L, which were injected manually. The odor qualities of all substances were determined by 10–12 panelists by sniffing the effluent at the sniffing port. 4.9. Comparative Aroma Extract Dilution Analysis (cAEDA) Comparative aroma extract dilution analysis (cAEDA) was used to determine the flavor dilution (FD) factors of the odor compounds in human milk before and after garlic consumption [40,41]. The original extracts of 100 μL were thereby diluted stepwise (1 + 1, v/v) in DCM. HRGC-O was then performed on 2 μL of the original extracts (FD = 1) and the respective dilutions on a DB-5 column. The temperature program for the GC oven was as follows: After holding an initial temperature of ˝ ˝ ˝ 40 C for 7 min the temperature of the GC oven was raised to 250 Cat8 C/min and then held for 5 min. The odorants were screened by two panelists who sniffed the effluent after gas chromatographic separation. Linear retention indices (RIs) of the compounds were calculated as described by Van den Dool and Kratz [68]. 4.10. High-Resolution Gas Chromatography´Mass Spectrometry (HRGC-MS) The characteristic mass spectra (EI, CI) of eluting compounds were obtained on an Agilent MSD quadrupole system (GC 7890A and MSD 5975C, Agilent Technologies, Waldbronn, Germany) equipped with a GERSTEL CIS 4 injection system and GERSTEL MPS 2 autosampler (GERSTEL, Duisburg, Germany). The software used to record the mass spectra and perform the data analysis was MSD ChemStation E.02.00.493 (Agilent Technologies). DB-FFAP and DB-5 (30 m ˆ 0.25 mm, film thickness 0.25 μm, Agilent J&W Scientific, Santa Clara, USA) were used in the GC. An uncoated, deactivated fused silica capillary was used as a pre-column (2–3 m ˆ 0.53 mm) and changed regularly to avoid accumulation of impurities. Helium was used as carrier gas and the total flow of the system was 1.0 mL/min, which was transferred into the MS using an uncoated, deactivated fused silica capillary (0.3–0.6 m ˆ 0.25 mm) transfer line. EI mass spectra were generated in full scan mode ˝ ˝ ˝ (m/z 30–350) at 70 eV. The GC oven was held at 40 C for 7 min, then raised to 240 C and 250 C for ˝ FFAP and DB-5, respectively, at a rate of 8 C/min and held for 7 min. 64 Metabolites 2016, 6,18 19 of 24 4.11. Two-Dimensional High-Resolution Gas Chromatography´Mass Spectrometry/Olfactometry (HRGC-GC-MS/O) (Heart-Cut) A two-dimensional gas chromatographic system was used for mass spectrometric identification of trace constituents. It consisted of two Varian 450 GCs in combination with a Varian 220 MS ion trap mass spectrometer (Varian, Darmstadt, Germany). The first GC was equipped with a GERSTEL MCS 2 multi-column switching system and was connected to the second GC by a GERSTEL CTS 1 cryo-trap system (GERSTEL, Duisburg, Germany). A DB-FFAP column (30 m ˆ 0.32 mm, film thickness 0.25 mm (Agilent J&W Scientific, Santa Clara, CA, USA); first oven) and a Rxi-5HT column (30 m ˆ 0.25 mm, film thickness 0.25 mm (Restek, Bad Homburg, Germany); second oven) were used. An uncoated, deactivated fused silica capillary was used as pre-column (2–3 m ˆ 0.53 mm). The flow rate of helium carrier gas was 2.5 mL/min. The effluent was split in the first oven between an olfactory detection port (ODP, GERSTEL) and an FID, as well as a cryo-trap during the cut interval. In the second oven, the effluent was split toward a second ODP and the mass spectrometer. All split capillaries were made of uncoated, deactivated fused silica material. The FID and the sniffing ports were held at 250 ˝ and 260 C, respectively. Mass spectra (m/z 35–300) in EI mode were generated at 70 eV ionization energy. The cut time intervals on the main column were determined by injection of the respective ˝ reference substances. Application of the samples to the GC system was performed at 40 C using the cold-on-column technique. The temperature programs were as follows: For AMS: The first oven ˝ ˝ ˝ at 40 C was held for 7 min and then raised to 240 C at a rate of 8 C/min, then kept for 5 min. ˝ ˝ The second oven started at a temperature of 40 C, was held for 7 min and then raised to 250 Cat ˝ ˝ ˝ a rate of 8 C/min. Afterwards, it was further raised to 300 C at a rate of 25 C/min and held for ˝ 5 min. For AMSO/AMSO2: The first oven started at a temperature of 40 C, which was then raised ˝ ˝ ˝ ˝ ˝ to 150 C at a rate of 20 C/min and further to 240 C at a rate of 8 C/min. The oven at 240 C was ˝ ˝ then held for 5 min. The second oven started at a temperature of 40 C and was raised to 250 C ˝ at a rate of 8 C/min. For DAS, DATS, DMTS, DMDS, AMDS, DADS; 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin: The first oven was heated as described above for AMSO/AMSO2. The second ˝ ˝ oven started at a temperature of 40 C, which was held for 2 min. It was then raised to 250 C at a rate ˝ of 8 C/min. 4.12. Identification of Metabolites and Calculation of Metabolite Profiles Metabolites of garlic constituents in human milk were identified by comparing retention indices according to Van den Dool and Kratz [68]. Their odors were subsequently perceived at the sniffing port via GC-O, and by comparison of EI mass spectra generated by either HRGC-MS or HRGC-GC-MS/O with those of synthesized references. Comparison of the mass spectra of the analyte with the reference standard was performed using the NIST Mass Spectral Search Program (Version 2.0 d, National Institute of Standards and Technology, Gaithersburg, MD, USA). The identification was ranked positive if reverse match values were above 900. For calculation of retention indices, two analytical capillaries of different polarities were used (DB-FFAP and DB-5). The time dependency of metabolite formation and excretion, specifically in relation to potential inter-individual differences, was assessed based on the relative concentration of the metabolites in different human milk samples, as follows: AMS was determined by 2D-HRGC-MS, with m/z 73 and 88 extracted from the total ion chromatogram and the area of the resulting peak then determined. The peak area was then normalized to the amount of the human milk (in kg) in order to express the concentration in units of area/kg milk. AMSO and AMSO2 were determined by HRGC-MS, with m/z 104 and 120 extracted, respectively. The peak areas were determined and the concentrations were calculated as described for AMS. 5. Conclusions Several novel aspects could be successfully demonstrated in this study: First, sensory evaluation by an expert panel showed that the intake of a dietary-relevant amount of raw garlic changed the odor 65 Metabolites 2016, 6,18 20 of 24 of breast milk, leading to garlic/cabbage-like odor notes being detectable in the milk. Accordingly, the expert panel confirmed previous observations made by naïve mothers and further specified the nature of the sensory changes observed. Second, garlic-derived metabolites were successfully identified in the milk by chemo-analytical means for the first time. These were allyl methyl disulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2), whereby AMS was found to be the only odor-active substance eliciting a garlic-like odor. It is important to note that despite its garlic odor attribute, the substance present in the milk does not chemically represent the characteristic odorant profile of garlic itself [27–31,35–38]. Accordingly, the transmission of an odorous substance into human milk does not necessarily reflect the chemical composition of the original aroma in the food consumed, as has been commonly proposed in previous studies. The metabolites detected were monitored for up to 5.2 h after garlic consumption. It could thereby be shown that metabolite profiles differed between individuals, resulting in different temporal profiles and the occurrence of different maximum concentration levels of the substances monitored. AMSO and AMSO2 are thus assumed to be oxidation products of AMS. Diallyl sulfoxide (DASO) and diallyl sulfone (DASO2), which were assumed to be oxidation products of diallyl sulfide (DAS), were not detectable. Future studies are needed to elucidate the potential occurrence of other less volatile metabolites in breast milk. Likewise, the potential physiological impact of garlic metabolites in human milk on the infant should be addressed in future studies. Supplementary Materials: The following are available online at www.mdpi.com/ 2218-1989/6/2/18/s1, Figure S1: Aroma profiles of human milk samples of test persons a–f. The samples were collected at different times before and after ingestion of 3 g of raw garlic, Tables S1–S6: Dietary records of test persons a–f, Table S7: Time of sampling, garlic consumption and the respective amounts of the gathered samples. Acknowledgments: This work was supported by the German Research Foundation (DFG) in the frame of grant BU 1351/15-1. The authors are exclusively responsible for the contents of this publication. We are grateful to the mothers for voluntarily providing samples of their milk. Author Contributions: A.B., C.S., L.S. and A.H. conceived and designed the experiments; L.S., Y.S. and C.S. performed the experiments; L.S. and C.S. analyzed the data; G.Z. and K.H performed the syntheses, A.B. contributed reagents/materials/analysis tools; L.S. and C.S. wrote the publication. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations The following abbreviations are used in this manuscript: AMDS Allyl methyl disulfide AMS Allyl methyl sulfide AMSO Allyl methyl sulfoxide AMSO2 Allyl methyl sulfone APA Aroma profile analysis cAEDA Comparative aroma extract dilution analysis DADS Diallyl disulfide DAS Diallyl sulfide DASO Diallyl sulfoxide DASO2 Diallyl sulfone DATS Diallyl trisulfide DMDS Dimethyl disulfide DMTS Dimethyl trisulfide FD-factor Flavor dilution factor FID Flame ionization detector Two-dimensional high-resolution gas chromatography´mass HRGC-GC-MS/O spectrometry/olfactometry HRGC-MS High-resolution gas chromatography´mass spectrometry RI Linear retention indices SAFE Solvent-assisted flavor evaporation 66 Metabolites 2016, 6,18 21 of 24 References 1. Peterson, R.; Cheah, W.Y.; Grinyer, J.; Packer, N. Glycoconjugates in human milk: Protecting infants from disease. Glycobiology 2013, 23, 1425–1438. [CrossRef][PubMed] 2. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 70 'HWHFWLRQRIYRODWLOHPHWDEROLWHVRIJDUOLFLQKXPDQEUHDVWPLON 6XSSRUWLQJPDWHULDO /DXUD 6FKHIIOHU 'HSDUWPHQWRI&KHPLVWU\DQG3KDUPDF\(PLO)LVFKHU&HQWHU)ULHGULFK$OH[DQGHU8QLYHUVLWlW (UODQJHQ1UQEHUJ )$8 +HQNHVWU (UODQJHQ *HUPDQ\ )UDXQKRIHU,QVWLWXWHIRU3URFHVV(QJLQHHULQJDQG3DFNDJLQJ,99*LJJHQKDXVHU6WU)UHLVLQJ *HUPDQ\ &RUUHVSRQGHQFH DQGUHDEXHWWQHU#IDXGH 71 Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S1 of S7 Supplementary Materials: Detection of Volatile Metabolites of Garlic in Human Breast Milk Laura Scheffler, Yvonne Sauermann, Gina Zeh, Katharina Hauf, Anja Heinlein, Constanze Sharapa and Andrea Buettner Figure S1. Cont. 73 Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S2 of S7 Figure S1. Aroma profiles of human milk samples of test persons (a–f). Table S1. Dietary record-test person a. Day of Dietary Record Type of Meal Food Beverages break-fast cereal coffee S1 apple fennel-anis-caraway-tea lunch pasta with cheese sauce coffee, water Day 1 S2 apple, banana dinner bread with cheese water S3 break-fast cereal coffee, fennel-anis-caraway-tea S1 apple lunch semolina porridge with berries coffee, water Day 2 S2 apple, chocolate cake dinner bread, pretzel with cheese, aspic water S3 break-fast croissant, apple, banana S1 lunch Sampling Day S2 dinner S3 S1, S2, S3: Snacks. 74 Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S3 of S7 Table S2. Dietary record-test person b. Day of Dietary Record Type of Meal Food Beverages cereal with fruits, amaranth and milk, break-fast cappuccino ΅-Thyroxin 50 mg S1 water tofu-stir-fried vegetables with rice and curry Day 1 lunch water sauce S2 chocolate croissant cappuccino dinner sugared pancake with raisins with apple sauce water S3 ½ banana water cereal with fruits, amaranth and milk, break-fast cappuccino ΅-Thyroxin 50 mg S1 1 banana water mozzarella-tomato with balsamic vinegar and lunch water pretzel Day 2 S2 ½ Bavarian doughnut cappuccino pasta with soy Bolognese sauce and salad dinner (cucumber, tomato, carrot, sunflower seeds, water pumpkin seeds) S3 water whole-grain bread with quark, currant-jelly, break-fast cacao honey and butter S1 Sampling Day lunch turkish spinach cake, endive salad with carrots water S2 dinner S3 S1, S2, S3: Snacks. Table S3. Dietary record-test person c. Day of Dietary Record Type of Meal Food Beverages break-fast cheese roll coffee S1 lunch roll with chicken, salad and sauce (without garlic) thyme tea, water Day 1 S2 dinner pasta with tomato sauce (mushrooms, tomatoes) water, apple juice S3 chocolate break-fast cereal (grains, apple, milk) coffee S1 lunch cake with nuts, roll with herbs thyme tea, water Day 2 S2 rice pudding with cherries coconut milk dinner pasta with tomato sauce (mushrooms, tomatoes) water S3 potato chips break-fast cereal (grains, apple, milk) coffee, water S1 lunch cheese roll Sampling Day S2 chocolate dinner S3 S1, S2, S3: Snacks. 75 Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S4 of S7 Table S4. Dietary record-test person d. Day of Dietary Type of Meal Food Beverages Record break-fast cereal with wild berries blood orange juice S1 lunch bread and ham Day 1 S2 chocolate dinner gratin S3 nursing tea break-fast cereal with wild berries blood orange juice S1 lunch gratin Day 2 S2 oriental, red tea dinner gratin, mandarins S3 cookies good-night tea break-fast cereal with wild berries blood orange juice S1 lunch gratin: zucchini, tomato, feta cheese, egg Sampling Day S2 chocolate dinner S3 S1, S2, S3: Snacks. 76 Metabolites 2016, 6, x; doi: S5 of S6 Table S5. Dietary record-test person e. Day of Dietary Type of Meal Food Beverages Record 3 slices rye-wheat bread, butter, Nutella, break-fast 1 tall cup of coffee with whole milk, 1 glass of ACE-spritzer 2 apricots S1 2 glasses of water polenta with stir-fried vegetables (zucchini, lunch 1 glass of water Day 1 pepper, kohlrabi) S2 carrot, nectarine and banana 2 glasses of water 5 slices rye-wheat bread (1x with butter, 2x dinner 1 glass of water with liverwurst, 2x with cheese S3 2 glasses of water, 1 glass of currant spritzer 3 slices rye-wheat bread, 1x liverwurst, 2x break-fast 1 tall cup of coffee with whole milk, 1 glass of currant spritzer butter and marmalade, ½ apricot, ½ nectarine S1 1 glass of water, 1 glass of currant spritzer lunch 3 slices rye-wheat bread with cheese, carrot 1 glass of water Day 2 S2 nectarine, apricot, Hanuta 2 glasses of water whole-grain pasta with minced meat sauce dinner 1 glass of water (beef mince, tomato, pepper, peas), cheese S3 Hanuta, 2 Kinder-Schoko-Bons 1 glass of water, 1 glass of tonic 3 slices rye-wheat bread with Nutella, 1 break-fast 1 tall cup of coffee with whole milk, 2 glasses of water apricot, ½ nectarine S1 2 glasses of water, 1 glass of apple spritzer whole-grain pasta with minced meat sauce Sampling Day lunch 1 glass of water (beef mince, tomato, pepper, peas), cheese S2 Hanuta 1 small cup of coffee with milk, 1 glass of water dinner S3 77 S1, S2, S3: Snacks. Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S6 of S7 Table S6. Dietary record- test person f. Day of Dietary Type of Meal Food Beverages Record break-fast 3 slices rye-wheat bread coffee with soy milk S1 noodles with olive-tomato sauce (tomato purée, olives, basil, lunch Day 1 provençal herbs, olive oil, pizza soft cheese) S2 trail mix, gummi bears dinner hash browns with apple sauce S3 break-fast Alnatura breakfast puree with rice milk nursing tea (fenugreek, fennel, anise, caraway, lemon verbena) S1 eggplants with tomato sauce (according to the restaurant lunch Day 2 without onions and garlic, but with broth from Maggi) S2 dinner bread with margarine and ham, cucumber S3 dark chocolate break-fast vegan marble cake coffee with soy milk, Fanta S1 lunch bread bun with butter, ham, tomato, cucumber water Sampling Day S2 water dinner S3 S1, S2, S3: Snacks. 78 Metabolites 2016, 6, 18; doi: 10.3390/metabo6020018 S7 of S7 Table S7. Time of sampling, garlic consumption and the respective amounts of the gathered samples. Subject Event Time Amount of Human Milk (g) 1st sample 9:00 437,953 garlic consumption 9:10 test person a 2nd sample 11:10 118,489 3rd sample 13:30 490,093 1st sample 9:20 337,798 garlic consumption 9:25 test person b 2nd sample 11:45 277,028 3rd sample 14:30 154,362 1st sample 9:05 159,959 garlic consumption 09:10 test person c 2nd sample 12:10 185,397 3rd sample 14:00 125,154 1st sample 9:20 152,956 garlic consumption 9:25 test person d 2nd sample 12:10 131,801 3rd sample 14:20 261,274 1st sample 9:40 436,317 garlic consumption 9:55 test person e 2nd sample 12:35 446,195 3rd sample 14:15 231,551 1st sample 9:35 326,611 garlic consumption 9:45 test person f 2nd sample 12:45 95,967 3rd sample 14:50 112,297 79 'HWHFWLRQ RI YRODWLOH PHWDEROLWHV GHULYHG IURP JDUOLF Allium sativum LQKXPDQXULQH Metabolites 2016, 6, 43; doi: 10.3390/metabo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metabolites OH Article Detection of Volatile Metabolites Derived from Garlic (Allium sativum) in Human Urine Laura Scheffler 1, Yvonne Sauermann 1, Anja Heinlein 1, Constanze Sharapa 2 and Andrea Buettner 1,2,* 1 Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Henkestr. 9, 91054 Erlangen, Germany; laura.scheffl[email protected] (L.S.); [email protected] (Y.S.); [email protected] (A.H.) 2 Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Str. 35, 85354 Freising, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-9131-85-22739; Fax: +49-8161-491-777 Academic Editor: Peter Meikle Received: 13 October 2016; Accepted: 28 November 2016; Published: 1 December 2016 Abstract: The metabolism and excretion of flavor constituents of garlic, a common plant used in flavoring foods and attributed with several health benefits, in humans is not fully understood. Likewise, the physiologically active principles of garlic have not been fully clarified to date. It is possible that not only the parent compounds present in garlic but also its metabolites are responsible for the specific physiological properties of garlic, including its influence on the characteristic body odor signature of humans after garlic consumption. Accordingly, the aim of this study was to investigate potential garlic-derived metabolites in human urine. To this aim, 14 sets of urine samples were obtained from 12 volunteers, whereby each set comprised one sample that was collected prior to consumption of food-relevant concentrations of garlic, followed by five to eight subsequent samples after garlic consumption that covered a time interval of up to 26 h. The samples were analyzed chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O), as well as sensorially by a trained human panel. The analyses revealed three different garlic-derived metabolites in urine, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2), confirming our previous findings on human milk metabolite composition. The excretion rates of these metabolites into urine were strongly time-dependent with distinct inter-individual differences. These findings indicate that the volatile odorant fraction of garlic is heavily biotransformed in humans, opening up a window into substance circulation within the human body with potential wider ramifications in view of physiological effects of this aromatic plant that is appreciated by humans in their daily diet. Keywords: garlic; human urine; gas-chromatography mass-spectrometry/olfactometry; allyl methyl sulfide; allyl methyl sulfoxide; allyl methyl sulfone 1. Introduction Garlic (Allium sativum) is a well-known aromatic plant used in everyday cuisine. According to the Agricultural Marketing Resource Center the annual per capita retail consumption of garlic was 1.2 pounds (~0.54 kg) in 1991 in the USA, whereas in 2001 the retail consumption raised to 2 pounds (~0.9 kg), i.e., almost doubling over a 10-year period [1]. This increase might relate to increased awareness of the beneficial health properties of garlic. To the best of our knowledge, research on garlic flavor started as early as the 1930s [2]; since then garlic has become one of the most researched plant food materials. In the course of these research activities, several health effects have been associated with garlic consumption, e.g., reduction of blood pressure, improvement of cholesterol levels, and decrease Metabolites 2016, 6, 43; doi:10.3390/metabo6040043 www.mdpi.com/journal/metabolites 83 Metabolites 2016, 6,43 2of23 in blood triacylglyceride levels. Since high blood pressure, a high total cholesterol (TC) level and a low high density lipoprotein (HDL)/low density lipoprotein (LDL)-ratio are the main risk factors for cardiovascular diseases (CVD), garlic is believed to have a protective effect against such impacting factors [3–7]. Most of the garlic constituents responsible for these effects are not yet known, although the active compounds saponins and flavonoids of the garlic bulb have been proposed [8–10]. Another compound that has been reported to be responsible for the effects of garlic is allicin and its (unspecified) degradation products [11]. Allicin itself is not present in the intact garlic bulb but is generated by the enzyme alliinase in the course of cell disruption, e.g., by chewing or cutting; the precursor substance is the non-proteinogenic amino acid alliin (S-allyl-L-cysteine sulfoxide) [12]. Allicin is then further degraded, thereby generating a multitude of compounds including disulfides, trisulfides, dithiins and ajoenes [13–16]. These compounds constitute the typical aroma of garlic but may also contribute to its beneficial health effects. Recent research has demonstrated that volatiles may cause physiological effects. Aroma compounds have been shown to modulate different types of receptors beyond the olfactory system, such as the γ -aminobutanoic acid (GABAA)-receptor [17,18] and the vanilloid transient receptor potential cation channel (TRPV1)-receptor [19]. Potential physiological effects in relation to food or beverages are often investigated on substances that occur naturally in the product, but potential metabolism within the body is rarely considered. In view of this, recent research has shown that volatile substances can undergo major transformation steps within the body or can be released from non-volatile precursors, e.g., in the human gastrointestinal tract [20] or during absorption processes [21], leading to compounds that were not present in the original product [22–25]. Regarding garlic aroma, the metabolism of its constituents in the human body is not fully understood. This issue has previously been addressed in animal or tissue model studies, leading to the discovery of allyl mercaptan, allyl methyl sulfide (AMS), diallyl disulfide (DADS) and diallyl sulfone (DASO2) as possible metabolites [13,26–31]. In a recent study, we were able to identify three garlic metabolites in human milk, namely AMS, allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). To complement these findings, the present study aimed to investigate the volatile garlic-derived metabolites in human urine over time after consumption of garlic at dietary relevant concentration. 2. Results 2.1. Sensory Analysis Changes in the odor of bodily fluids may indicate the presence of volatile constituents or metabolites of food components [22,23,25]. Pre-trials indicated that slight odor changes, relating to the attribute “garlic-/cabbage-like”, were observable in human urine after garlic consumption; accordingly, this attribute was rated by the panelists in the different urine samples on a scale from 0 (no perception) to 3 (strong perception). In the case of the high dosage test (Urine a), where the test person consumed about 30 g of raw garlic, a “garlic-/cabbage-like” odor was rated only as weak, with an average value of 0.6 in the sample that was obtained about 2 h after garlic consumption, subsequently decreasing to 0.5 and 0.4 in the third and fourth urine sample of the set. The first urine sample of this set, which was obtained prior to garlic consumption, was also rated as having a very slight “garlic-/cabbage-like” odor (0.3). However, these results are only from a single test subject. Sensory analysis of the other urine sets where each test person consumed about 3 g of raw garlic indicated that a slight “garlic-/cabbage-like” odor was perceivable during the first hours after garlic consumption, sometimes being perceivable up to 24 h later. The intensities of this odor ranged from 0 to 0.7, corresponding to a very weak smell. The perceived intensities of a “garlic-/cabbage-like” odor in urine at different time intervals before and after ingestion of raw garlic are shown in supplementary Figure S1. 84 Metabolites 2016, 6,43 3of23 2.2. High Resolution Gas Chromatography-Olfactometry (HRGC-O) and Comparative Aroma Extract Dilution Analysis (cAEDA) of Urine before and after Garlic Consumption For identification of potentially odor active compounds in urine deriving from garlic consumption, the solvent extracts of the urine samples of Urine a were analyzed by HRGC-O using a capillary DB-5 as well as a capillary DB-FFAP. For all odor active substances, that could be perceived at the sniffing port, the retention indices (RI) according to Van den Dool and Kratz [32] were calculated. Using this approach two substances with a garlic-like odor were detected via HRGC-O. One compound had RIs of 715 on the DB-5 capillary and <1000 on the DB-FFAP capillary, whereas the other had RIs of 973 and 1362 on the DB-5 and DB-FFAP capillaries, respectively. The first compound was identified as AMS and the second as dimethyl trisulfide (DMTS), as based on a comparison of odor qualities and RIs of pure reference compounds. No additional substances that could directly be related to garlic consumption were identified. All remaining odorous molecules detected were also present in the control samples, indicating that these were common urine odor constituents (data not shown). The cAEDA confirmed the presence of AMS and DMTS in the urine sample set b to f, as summarized in Table 1. No additional odorous substances that could be attributed to the garlic intervention were detected, with all remaining odorous molecules also being present in the control samples (data not shown). These substances were different lactones such as γ-nonalactone or δ-decalactone, and several guaiacol-derivatives, as have been previously reported to be present in urine [33–35]. Accordingly, these compounds are not described in further detail here. AMS was only detected in samples after garlic consumption, but DMTS was also detected in some samples prior to garlic consumption, and with comparable flavor dilution (FD) factors. Furthermore, DMTS was not always detected over the entire sampling period, in some cases disappearing and reappearing at later times. Moreover, in some cases the urine sample collected 24 h after garlic ingestion had a higher FD factor for DMTS. Table 1. Flavor dilution (FD) factors obtained for allyl methyl sulfide (AMS) and dimethyl trisulfide (DMTS) in five different urine sample sets as determined via High Resolution Gas Chromatography-Olfactometry (HRGC-O) using a DB-5 capillary. Eight samples per set were collected at different time intervals before and after ingestion of 3 g of raw garlic from different volunteers, whereby urine sets b and f were provided by the same volunteer, albeit at different days. Pre relates to the urine sample that was collected prior to garlic consumption. 0.5 h post to 24 h post relate to the urine samples that were obtained after garlic consumption. Urine b Urine c Urine d Urine e Urine f Sample FD FD FD FD FD AMS DMTS AMS DMTS AMS DMTS AMS DMTS AMS DMTS pre n.d. n.d. n.d. n.d. n.d. n.d. n.d. 4 n.d. 8 0.5 h post - - n.d. n.d. 32 n.d. 1 1 16 2 1 h post - - n.d. 1 16 1 8 8 8 1 2 h post 2 1 4 1 128 1 16 n.d. 4 4 4 h post 8 2 8 2 4 2 1 1 4 16 6 h post 32 32 16 16 8 4 4 2 64 32 8 h post 8 32 8 4 4 1 4 2 16 4 12 h post 2 4 ------24 h post n.d. 4 n.d. 32 n.d. 1 2 16 n.d. 4 n.d. not detected; - no sample was provided at this time point. Based on the finding that only AMS and DMTS were detectable as additional odorous compounds after garlic consumption and that the appearance of DMTS was not only related to garlic consumption, a shortened cAEDA was performed on the extracts of urine samples of the sets g to n, whereby only the first five minutes of each GC-run were analyzed. The FD factors obtained for AMS for the sample sets Urine g to Urine n are provided in the supplementary Table S1. 85 Metabolites 2016, 6,43 4of23 2.3. Identification of Garlic-Derived Metabolites in Urine 2.3.1. Identification Using HRGC-MS Subsequently, targeted analyses via HRGC-MS were performed to further confirm the olfactometric identification of AMS and DMTS, and to screen for potential further garlic-derived metabolites that are odorless or less odor active and therefore not detectable by HRGC-O. To this aim, the extracts of the sample series of Urine a and b were analyzed both on capillary DB-5 and DB-FFAP. The obtained chromatograms were compared and matched with their respective control samples, so that additional peaks in the urine extracts resulting from garlic intervention could be elaborated. Additionally, a targeted search was carried out for those substances that have either been reported to be present in garlic itself or have been previously proposed as metabolites [22,29,36–40]. This target substance selection is shown in Table 2, together with the RIs and m/z ratios used for data extraction. Table 2. Compilation of investigated substances, their structure, retention indices (RIs) on DB-FFAP and DB-5 chromatographic capillaries, m/z ratios used for extracted ion chromatograms/identification via GC-MS, and respective odor qualities. Confirmed presence of the target compounds by means of HRGC-GC-MS analysis is listed for urine sampled after consumption of 30 g (U a 4 h post)and3g(Uf 2 h post) of raw garlic. U a was collected at 4.1 h and U f at 2 h after garlic consumption. The urine samples were chosen because they contained the highest concentrations of the metabolites AMSO and AMSO2, as determined via HRGC-MS. RI Detected in Substance Structure m/z Odor Quality a4h f2h (Abbreviation) U U FFAP DB-5 post post Allyl methyl sulfide <1000 715 73 + 88 Garlic-like a,b Yes Yes (AMS) Allyl methyl sulfoxide 1742 1018 104 Odorless c Yes Yes d (AMSO) Allyl methyl sulfone 1983 1061 107 Odorless c Yes Yes d (AMSO2) Diallyl sulfoxide 1889 1163 81 Garlic-like c n.d. n.a. (DASO) Diallyl sulfone 2079 1289 67 Odorless c n.d. n.a. (DASO2) Diallyl disulfide Garlic-like a 1462 1083 146 n.d. n.a. (DADS) Garlic-like, pungent b Allyl methyl disulfide Garlic-like a 1265 921 120 Yes n.d. (AMDS) Cooked garlic-like b a Dimethyl disulfide Cabbage-like 1071 751 94 Yes n.d. e (DMDS) Cooked garlic-like, onion-like, rubber-like b Garlic-like a Dimethyl trisulfide Burnt garlic-like, 1362 973 126 Yes Yes d (DMTS) diffusive, penetrating, sulfury b Garlic-like a Diallyl trisulfide (DATS) 1771 1308 113 n.d. n.a. Garlic-like, onion-like b Diallyl sulfide (DAS) 1138 868 114 Garlic-like a n.d. n.a. 2-Vinyl-4H-1,3-dithiin 1827 1222 144 Garlic-like a n.d. n.a. 3-Vinyl-4H-1,2-dithiin 1720 1194 144 Pungent-garlic-like a n.d. n.a. n.d.: not detected. n.a.: not analyzed. Yes: detectable; a odor quality as described by Tokarska and Karwowska [41]; b odor quality as described by Ma et al. [42]; c odor quality as described by Scheffler et al. [22]; d identified in the sample extracts via HRGC-MS; e identified in other samples via HRGC-MS. The presence of DMTS was confirmed in the majority of samples (84 of 105 urine samples; 80.0%), although this substance was often present in only trace amounts. Additionally, DMDS was identified in a series of samples, as shown in Figure 1 for two samples of urine set b. In this case, DMDS was 86 Metabolites 2016, 6,43 5of23 detected in almost all samples collected after garlic consumption, as well as in trace amounts in the sample obtained prior to garlic consumption (89 of 105 urine samples; 84.8%). The detected amounts of DMDS and DMTS in all urine samples and the corresponding sampling times of the respective samples are given in the supplementary Table S2. Figure 1. Identification of dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) in urine samples was performed via HRGC-MS. (a): Extracted ion chromatogram (m/z 94) of DMDS in an urine sample that was collected prior to garlic consumption (pre) and in an urine sample that was obtained after garlic consumption (6 h post); (b): Extracted ion chromatogramm (m/z 126) of DMTS in an urine sample that was collected prior to garlic consumption (pre) and in an urine sample that was obtained after garlic consumption (6 h post); (c): Mass spectra of DMDS in urine sample that was collected prior garlic consumption (pre) and after garlic consumption (6 h post) and a DMDS-standard (standard); (d): mass spectra of DMTS in urine sample that was collected prior garlic consumption (pre) and after garlic consumption (6 h post) and a DMTS-standard (standard). Exemplarily urine sample b is displayed. By comparison, AMS could not be determined with sufficient resolution by one-dimensional GC-MS analysis due to co-elution of substances; accordingly, identification of this compound was based on two-dimensional GC-MS analyses, as described in Section 2.3.2. Two additional substances that were not perceivable via GC-O were detected using HRGC-MS: Allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). In the high dosage experiment (Urine a) both compounds were detectable as two additional peaks in the total ion chromatogram (TIC). Figure 2 displays the TIC of the urine sample prior to garlic consumption (Figure 2a-pre) together with the TIC of the urine sample that was collected 2.1 h after garlic consumption (Figure 2a-2 h post). In addition, the chromatograms of the reference substances AMSO and AMSO2 together with their respective mass spectra (Figure 2b,c) are displayed, confirming unambiguous identification of either compound. The presence of AMSO and AMSO2 in the sample collected prior to garlic consumption could thereby be excluded (cf. Figure 2a pre, 2b pre, 2c pre). 87 Metabolites 2016, 6,43 6of23 Figure 2. Identification of garlic-derived compounds in urine samples was performed via HRGC-MS. (a): Total ion chromatogram (TIC) of urine samples before (pre) and 2.2 h after garlic consumption (2 h post), AMSO reference standard (AMSO standard) and AMSO2 reference standard ◦ (AMSO2-standard). The volunteer consumed about 30 g of raw garlic. Temperature program: 40 C, ◦ ◦ held for 7 min, raised with 8 C/min to 240 C and held for 8 min; (b): mass spectra of AMSO in urine sample before garlic consumption (pre), after garlic consumption (2 h post) and AMSO reference standard (AMSO-standard); (c): mass spectra of AMSO2 in urine sample before garlic consumption (pre), after garlic consumption (2 h post) and AMSO2-reference standard (AMSO2-standard). The mass spectra are shown at the time when the standard compound eluted. 2.3.2. Identification Using HRGC-GC-MS As previously discussed, an unambiguous detection of AMS by means of HRGC-MS was not achievable due to interference with co-eluting substances. Accordingly, a two dimensional HRGC-GC-MS system was applied for increased selectivity and sensitivity in the detection of the target compound. Using this approach, AMS was successfully confirmed in the urine samples that were obtained both after consumption of 30 g as well as 3 g of raw garlic. Thereby, no extraction of specific m/z was necessary for the high dosage experiment, whereas for the lower dosage experiments m/z 73 + 88 were extracted from the TIC for targeted detection of AMS. Moreover, the presence of AMS in urine samples collected prior to garlic consumption could be excluded (cf. Figure 3). 88 Metabolites 2016, 6,43 7of23 Figure 3. Identification of AMS in urine was performed via HRGC-GC-MS using capillary DB-5 as well as capillary DB-FFAP. 4 μL were applied for analyses. (a): TIC of urine samples before (pre) and 2.2 h after garlic consumption (2 h post) as well as an AMS-Standard (conc. 5 μg/mL) (standard). Test person consumed about 30 g of raw garlic; (b): Extracted ion chromatogram (m/z 73 + 88) of urine sample before (pre) and 2.1 h after garlic consumption (2 h post) as well as an AMS-Standard (conc. 0.25 μg/mL) (standard). Test person consumed about 3 g of raw garlic. Exemplarily urine sample b is displayed; (c) Respective mass spectra of AMS for the samples shown in (a); (d) Respective mass spectra of AMS for the samples shown in (b). The mass spectra are shown at the time when the standard compound eluted. In addition to AMS, the urine samples were screened for compounds that have previously been identified in garlic or proposed as potential garlic metabolites. These substances were diallyl sulfoxide (DASO), DASO2, DADS, allyl methyl disulfide (AMDS), dimethyl disulfide (DMDS), DMTS, diallyl trisulfide (DATS), diallyl sulfide (DAS), 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin (see Table 2). Urine sample U a 4 h post was used to search for the presence of these compounds, since the highest concentrations of excreted substances and potential metabolites could be expected in this sample. A targeted search was conducted in comparison to the respective reference standards by parallel analyses applying the same analytical conditions. As compiled in Table 2, most of the substances were not detectable in the urine sample whereas the presence of AMDS, DMDS and DMTS could be confirmed. DMTS was also detected via HRGC-MS, as discussed above. In a second experiment, it was screened for the presence of AMDS and DMDS in urine sample f obtained 2 h after garlic consumption; this choice was based on the consideration that the excretion of the garlic metabolites AMS, AMSO and AMSO2 reached a maximum at this time for this sample set (see Section 2.5.1). Neither AMDS nor DMDS were detectable in U f 2 h post, whereas DMDS was successfully detected in urine set b over the course of the identification experiments, as described in Section 2.3.1. 89 Metabolites 2016, 6,43 8of23 2.4. Difference between Concentrations of the Garlic-Derived Metabolites Depending on Calculation Method The concentrations of constituents in urine are highly dependent on the water intake of the volunteer. This also applies to garlic-derived metabolites. Accordingly, the concentrations of AMS, AMSO and AMSO2 were calculated in two different ways, as area/kg and normalized to the creatinine content of the urine sample. Two urine sets of test persons c and d are shown in Figure 4. Figure 4. Difference between calculation methods of the concentrations of the garlic-derived metabolites. (a) and (c): concentrations of garlic-derived metabolites in Urine c and Urine d, calculated as area/kg; (b) and (d): concentrations of garlic-derived metabolites in Urine c and Urine d, calculated as area/mmol creatinine. Areas are based on the m/z-ratios 73 + 88, 104 and 120 for AMS, AMSO and AMSO2, respectively. AMS (ƹ), AMSO ( ), AMSO2 ( ). A distinct difference between the two calculation methods is observable in Figure 4a,c, in which the amount of the garlic-derived metabolites was calculated as area/kg. AMS and AMSO show their maxima about 1 h after garlic consumption and a second distinct increase 2 to 4 h after garlic consumption. Likewise, a maximum at about 1 h after garlic intervention can be observed for AMSO2 for sample set c. For sample set d there was a continuous increase until 6 h after garlic consumption; AMSO2 decreased only in the last two samples, collected about 8 and 24 h after garlic consumption. The creatinine-normalized values showed distinct maxima in AMS, AMSO and AMSO2 1 to 2 h after garlic consumption. A second increase of the metabolites was observed for Figure 4d, but this was not as distinct as in Figure 4c. No second increase was observable in Figure 4b. A table listing the creatinine content, amount of urine, areas of the respective metabolites and calculated amounts of metabolites (in area/kg as well as area/mmol creatinine) for all urine samples is provided in the supplementary Table S2 (DMTS and DMDS) and Table S3 (AMS, AMSO and AMSO2). 2.5. Time Dependency of Appearance of Specific Garlic-Derived Metabolites in Urine after Consumption of Garlic 2.5.1. AMS, AMSO and AMSO2 In order to monitor the temporal profiles of the identified garlic-derived metabolites in human urine in more detail, a relative semi-quantitative estimation was performed. For this aim the area/mmol creatinine ratios were calculated and plotted versus the sampling time of the respective urine sample. 90 Metabolites 2016, 6,43 9of23 The amounts of AMS, AMSO and AMSO2 in urine sets b to n are shown in Figure 5. A table with the exact values of the metabolites is provided in the supplementary Table S3. In the case of urine set a, the metabolites were calculated as area/kg, since creatinine levels were not measured during this high-dosage test. This set differed from the others (3 g garlic consumption), in so far as it was characterized by a continuous increase over the entire sampling period (5.8 h); only AMSO decreased slightly at the last point of determination. A figure displaying the metabolite profile of the high dosage test is provided in supplementary Figure S2. Figure 5. Cont. 91 Metabolites 2016, 6,43 10 of 23 Figure 5. Time-Resolved metabolite profiles of AMS, AMSO and AMSO2. 12 urine sample sets; only samples b and e were provided by the same test person, albeit at different days. AMS (ƹ), AMSO (), AMSO2 ( ), time 0 h represents the urine sample collected prior to garlic consumption, subsequent time intervals represent urine samples after garlic consumption. Garlic was consumed between 2 to 38 min after the first urine sample was collected. (a) set b;(b) set c; (c) set d;(d) set e;(e) set f (f) set g; (g) set h; (h) set i;(i) set j;(j) set k;(k) set l;(l) set m;(m) set n. • Urine series b–l: For most of the urine sets (Urine f, g, h, j, l) the maximum concentration of all three garlic-derived metabolites, AMS, AMSO and AMSO2, approximately coincided, namely at about 1 h after garlic consumption. This maximum was observed at about 2.4 h after garlic consumption in sample set h only. The maxima in AMS and AMSO for sets b, d, e, i and k were reached before AMSO2. AMS and AMSO showed the highest values at about 1 to 1.5 h after garlic consumption (at 2 h for sample set b), whereas AMSO2 was observed to peak at about 2 to 3 h (4.5 h for sample set i). AMSO reached its maximum before AMS and AMSO2 only in sample set c. Whereas AMSO was excreted mostly at about 0.9 h after garlic consumption, AMS and AMSO2 reached their highest values at about 1.8 h after garlic consumption. Apart from the first maximum, a second increase in the metabolites was observed in several cases, which differed in time as well as intensity between the sample sets, indicating large inter-individual differences in the metabolism and excretion rate of garlic constituents. In most cases the second increase was smaller than the first; this was the case for AMS in sets d, f, g, i, j, k and l, for AMSO in sets b, j and l, and for AMSO2 in sets d, e, g, j, k and l. On the other hand, the second increase was more distinct than the first for AMS in two sample sets (b, i) and for AMSO2 in set l. No second increase was observed for all three 92 Metabolites 2016, 6,43 11 of 23 garlic-derived metabolites in set c, for AMS in sets e and h, for AMSO in sets d, e, f, g, h and k, and for AMSO2 in sets b, f and h. In some cases the formation of a “shoulder” was observed for AMS (sets e and h), AMSO (sets g, h and k), and AMSO2 (sets b and h) at time intervals of 3 h (set l) to 7.8 h (set k) after garlic consumption. • Urine series m–n: Urine sample sets m and n were investigated over a period of about 6 h, comprising six different samples. Only one maximum was measured for each metabolite during the sampling period. For sample set m all three metabolites reached their maximum at about 1.5 h after garlic ingestion, whereas for sample set n maximum concentrations of both AMSO and AMSO2 were excreted at about 1.5 h but AMS reached its maximum at about 2.5 h after garlic ingestion. 2.5.2. DMTS and DMDS The excretion profiles of DMTS and DMDS were monitored in order to determine whether their excretion directly related to garlic consumption. Example profiles are shown in Figure 6. Further profiles are provided in the supplementary Figure S3 (DMTS) and S4 (DMDS). Figure 6. Time-resolved metabolite profiles of DMTS and DMDS. Urine sample sets from three volunteers (set c, d and e). (a)–(c): DMTS; (d)–(f): DMDS, time 0 h represents the urine sample collected prior to garlic consumption, subsequent time points represent urine samples after garlic consumption. Garlic was consumed 10 to 22 min after the first urine sample was collected. Large inter-individual differences were observed in the excretion profiles of both DMTS and DMS. A distinct maximum of these two compounds appeared about 1 and 1.8 h after garlic consumption in sample set c. For sample set d both compounds were mostly excreted at about 5.7 h after garlic consumption. In contrast to Urine c and d, an increase in DMTS and DMDS was observed in sample set e that did not decrease over the period of observation. DMTS and DMDS were also measurable in most of the urine samples collected prior to garlic consumption (see supplementary Table S2). 3. Discussion 3.1. Sensory Analysis Sensory analyses revealed that the smell of urine changes slightly after garlic consumption, exhibiting a weak garlic-like and cabbage-like odor. This change was distinct in urine samples collected after consumption of 30 g raw garlic, but in other cases (3 g garlic consumption) the change 93 Metabolites 2016, 6,43 12 of 23 was not as clear, and a faint garlic-like impression was perceivable even in several samples collected prior to garlic consumption. This indicates that the odor is not only related to the consumption of garlic but also other substances that are excreted into urine. The lack of a pronounced garlic odor in urine might be due to its very low perceived intensity (always rated below 1), making it difficult to detect unambiguously. Accordingly, this odor impression was potentially covered by other smells of the urine. Additionally, some substances exhibiting a garlic-like, sulfury smell have previously been described as odorous constituents of human urine, irrespective of garlic consumption. Wagenstaller and Buettner [33], for example, identified DMTS as a common odorous constituent in several urine samples, and Wahl et al. [43] described several sulfur-containing compounds, such as methyl propyl disulfide, methyl-2-propenyl disulfide, DMDS and DMTS in the urine of healthy subjects without garlic intervention. These findings are confirmed by Mills et al. [44], who additionally identified methanethiol in human urine, and by Smith et al. [45]. DMDS and DMTS specifically exert a cabbage- or garlic-like smell [41,42] and their presence in urine does not necessarily depend on garlic-consumption. Contrary to our findings in urine, in our previous study we perceived a clear garlic-/cabbage-like odor in human milk after garlic ingestion. This was related to the excretion of AMS, whereas such a clear relationship could not be established for urine. The likely reason is the high inherent smell of urine compared to human milk, which exhibits a very low overall smell intensity. AMSO and AMSO2 are odorless and do not contribute to the odor of either urine or milk. 3.2. Identification of Garlic-Derived Metabolites in Human Urine In the present study, the investigation of urinary constituents that are associated with garlic consumption led to the successful identification of three garlic-derived metabolites in human urine, namely AMS, AMSO and AMSO2. AMS has previously been reported as a compound in human breath and urine after garlic consumption [26,37,38,46], as well as a constituent of garlic itself [41,42,47–49]. In contrast, the derivatives AMSO and AMSO2 are reported here for the first time as garlic-derived compounds in human urine, demonstrating that constituents of garlic are heavily metabolized prior to their excretion via urine. It is also interesting to note that AMS, AMSO and AMSO2 have previously been reported as garlic-derived constituents in human milk by our group [22], indicating that there are similar pathways for the excretion of such substances via different bodily fluids. The presence of relevant amounts of AMS, AMSO and AMSO2 in urine samples collected prior to garlic consumption could be generally ruled out, with these garlic-metabolites being detectable at only trace amounts in the blank urine samples of sets g, h and n. It is interesting to note that, according to the dietary records, these volunteers consumed olives during the wash-out days (see supplementary Tables S9 and S10). Like garlic, olives are characterized by a relatively high sulfur content; Fleming et al. [50] identified DMS as a major odorous compound in olives, and Collin et al. [51] detected several sulfur-containing compounds in olives. Accordingly, it is possible that olive constituents are metabolized in a similar way as garlic compounds, potentially explaining the presence of trace amounts of sulfury metabolites in the blank urine samples. As reported in our previous study on human milk, volunteer n exhibited traces of garlic substances in both milk and urine samples, which is suspected to relate to this subject consuming some tomato sauce of unknown composition in a restaurant on the day prior to sampling (see supplementary Table S16), thus the ingestion of garlic could not be fully excluded. Nevertheless, in relation to the trace amounts that were detected in the blank samples, a major increase of the metabolites in the following samples was observed, confirming a clear correlation between garlic consumption and the excretion of AMS, AMSO and AMSO2. The present study further focused on those compounds that have been previously reported as being constituents of garlic or potential metabolites. With regards to the compounds that are present in garlic itself (DADS, AMDS, DMDS, DMTS, DATS, DAS, 2-vinyl-4H-1,3-dithiin, 94 Metabolites 2016, 6,43 13 of 23 3-vinyl-4H-1,2-dithiin) [39–42,47–49,52,53] it was found that solely DMDS and DMTS were detectable in human urine after garlic consumption, albeit with some cases of detection of these compounds prior to garlic consumption. DMDS and DMTS have been described earlier as compounds that can naturally occur in urine [33,43–45]. Regarding the temporal profiles of DMDS and DMTS monitored in the present study, no clear pattern was found. In most sets of urine a maximum in the temporal excretion profiles of both compounds was detected over the sampling period, with the time of the maximum ranging from 1 to 8 h after garlic consumption. In one case DMDS and DMTS reached their maxima at about 24 h after garlic consumption (Urine e). Accordingly, other sulfur-containing compounds from other dietary sources are likely to be metabolized and excreted in urine in the form of DMDS and DMTS. It is interesting to note here that DMTS has recently been reported to be present in cranberries [54], indicating that this compound is not restricted to Allium species. Nevertheless, an additional influence of garlic consumption cannot be fully ruled out. Other compounds that have been previously detected in human breath after administration of garlic are methyl mercaptan, allyl mercaptan, dimethylsulfide, DADS, AMDS, DMDS, DATS, and DAS [36–38,46,55–61]. However, studies by Taucher et al. [46], Suarez et al. [38] and Buhr et al. [55] indicate that solely AMS and dimethylsulfide are metabolites of garlic constituents whereas the other compounds might originate from garlic residues in the oral cavity. In the present study neither DADS, DATS nor DAS were detectable in urine whereas AMDS was only detectable in the urine samples that were collected after consumption of large amounts (30 g) of garlic. It is possible that this high dosage caused the direct excretion of unmetabolized garlic constituents or that other metabolic pathways are recruited in cases of such high concentrations. On the other hand, the presence of methylmercaptan, allylmercaptan and dimethylsulfide in urine cannot be fully excluded since monitoring of these compounds would require adapted procedures for such highly volatile compounds. In future analyses those compounds should be addressed with methods, that are more specific for highly volatile compounds, like headspace analysis or on-line mass spectrometric volatile monitoring such as proton-transfer reaction mass spectrometry (PTR-MS) [62]. DASO and DASO2, compounds which have previously been reported as metabolites of DAS in rat liver, blood and urine [27] were not detectable in any relevant concentration in urine, neither after consumption of 3 g nor of 30 g of raw garlic, in these trials. 3.3. Metabolism of Garlic The main aim of the current study was to identify garlic-derived metabolites in human urine to further elucidate the metabolism of garlic constituents in the human body and to monitor the excretion of different garlic-derived metabolites over a period of up to 26 h. The semi-quantitative analysis revealed high inter-individual variation in the excretion profiles from different volunteers, both with regards to differences in relative substance concentrations and temporal appearance of the compounds’ maxima. The observed differences are related to uptake, distribution, metabolism and excretion of the respective precursors or metabolites, which are strongly individual processes. The metabolism of garlic components has been addressed in previous studies, although primarily in animal or tissue studies and only few investigations were performed with human subjects. The majority of the human studies have targeted the influence of garlic on human breath composition as mentioned in Section 3.2. To further clarify the metabolism of garlic compounds, Lawson and Wang [26] investigated the effect of garlic and garlic-derived compounds on breath composition and concluded that garlic compounds that have a dithioallyl group, e.g., DADS and DATS, lead to the formation of AMS, whereas compounds lacking the dithioallyl group have to be metabolized in a different way. As a precursor of AMS they suggested allyl mercaptan, but due to the fast metabolism to AMS they did not expect any accumulation of this compound, AMS itself should be then further oxidized to AMSO and AMSO2. Furthermore, they suggested that DAS is metabolized in a different way compared to AMS, due to the absence of the dithioallyl group and proposed DASO2 as its metabolite. In our previous study 95 Metabolites 2016, 6,43 14 of 23 we found AMS, AMSO and AMSO2 in human milk [22]. Now, we can confirm the presence of these metabolites in human urine, showing that lactating women and non-lactating people metabolize garlic similarly. However, the presence of DASO2 or its presumed precursor DASO could not be confirmed to be present. Studies on rat tissues, cells or body fluids offer only disparate data. For example, Sheen et al. [31] treated primary rat hepatocytes with DADS or DAS and observed a conversion of DADS to allyl mercaptan and AMS within 120 min, whereas DAS was only converted to AMS. In the study of Germain et al. [29] DADS was administered directly to rats and the metabolites in the stomach, liver, plasma and urine were monitored over a period of 15 days. Depending on the investigated medium, they observed differences in the metabolite profile. DADS and allyl mercaptan were found in plasma within the first 20 min after oral administration; and AMS, AMSO and AMSO2 were detected up to 7 days after oral administration. In the case of rat urine, neither allyl mercaptan nor DADS have been detected, the only metabolites detected were AMS, AMSO and AMSO2. In comparison, Brady et al. [27] treated rats with DAS and identified DASO and DASO2 as metabolites in liver tissue, blood and urine. In the present study neither of these compounds were detected which is in agreement with the study by Germain et al. [29], who did not observe those metabolites. Instead, they reported AMS, AMSO and AMSO2, three metabolites which were now confirmed in human urine after garlic consumption. A possible explanation for these different results is that Brady et al. [27] administered DAS, whereas Germain et al. [29] administered DADS to the rats. It is also possible that DAS is converted to other compounds at a very early stage of digestion, e.g., orally, and can then not be further oxidized to DASO and DASO2, whereas DADS may reach the blood and liver and can then be metabolized to AMS, AMSO and AMSO2. This mechanism would be in line with the findings of Germain et al. [29], indicating that DADS and allyl mercaptan are precursors of AMS, AMSO and AMSO2, at least in rats. However, the authors only observed these compounds in plasma for a short time after administration of DADS and not in urine. Accordingly, it would be necessary to investigate their potential presence in humans not only via urine but also in serum samples. Regarding the time dependency of the appearance of the metabolites Germain et al. [29] reported the highest concentrations of garlic metabolites (AMS, AMSO and AMSO2) in rat plasma and urine 3 days after oral administration of DADS. However, as is discussed in our previous publication on garlic metabolites in human milk [22], the applied amount of this compound is not comparable with the garlic amount consumed by the volunteers in this study. Nevertheless, the temporal appearance of the garlic-derived metabolites in urine between this study and that of Germain et al. is comparable [29]: First, a maximum of the metabolites is observable. In the study by Germain et al. this maximum is followed by a decrease and the formation of a plateau, before it decreased further until the metabolites were no longer measurable. In the present study, a plateau and even a second increase in some cases were observed. According to Germain et al. [29], this pattern is surprising since AMSO and AMSO2 are hydrophilic and therefore can be expected to be excreted rapidly in urine. The authors assumed that these metabolites interact with proteins and lipids, leading to a retardation effect in the body, although this would not explain the second increase that we observed for some individuals (e.g., Urine g, (Figure 5f)). Another possible explanation could be that garlic constituents are absorbed at different locations throughout the gastrointestinal path, starting from the oral cavity, via the stomach, small intestine and colon; even an interaction with the microbiota of the colon cannot be excluded, as has been reported for other substances such as short-chain fatty acids [63]. Moreover, it is possible that a potential second increase is less pronounced when urine is kept in the bladder for a longer period. Such an effect might further explain the formation of a plateau. With regard to potential physiological effects, neither AMS, AMSO nor AMSO2 have, to the best of our knowledge, previously been considered as potential active compounds. As such, these should be addressed in future studies. Equally, the identification of conjugated garlic-derived compounds should be subject to further investigation. For these less volatile compounds it is necessary to involve other techniques such as high performance liquid chromatography (HPLC), or to release 96 Metabolites 2016, 6,43 15 of 23 the respective compounds prior to GC analysis. Possible conjugates are glutathion-conjugates, like S-allylmercaptoglutathion [64], or acetyl-conjugates, like N-acetyl-S-allyl-L-cysteine [65,66]. 4. Materials and Methods 4.1. Chemicals/Materials The following reference compounds were obtained from the suppliers shown: DMDS, DMTS (Sigma-Aldrich, Steinheim, Germany), AMDS (abcr, Karlsruhe, Germany). The remaining reference substances AMS, AMSO2, AMSO, DADS, DAS, DASO2, DASO, 3-vinyl-4H-1,2-dithiin and 2-vinyl-4H-1,3-dithiin as well as DATS were synthesized as described in [22]. Dichloromethane (DCM, HPLC grade) and anhydrous sodium sulfate (Na2SO4) were purchased from VWR (Darmstadt, Germany). The solvent was freshly distilled prior to analysis. 4.2. Human Urine Samples Urine samples were obtained from 12 volunteers, with one volunteer providing urine on three different days. All volunteers gave written, informed consent to participate in the study and were able to withdraw from participation at any time. The volunteers (age 25–33 years (mean 29); nine females, three males) had no known illnesses that might potentially influence the urine excretion or metabolism at the time of examination. There was no control with regard to the test persons for hormonal contraceptives. The test persons did not take any medication, with the sole exception of one woman who had to use some eye drops because of high intraocular pressure. As the eye drops were applied locally and the expected uptake in the body is very low, this test person was not excluded from the study. Each subject was told to refrain from eating food containing high amounts of sulfur compounds for two days prior to the intervention, as well as on the sampling day, in order to eliminate potential sulfurous artefacts in the urine from other dietary sources; foods to be avoided were garlic, onion, ramson, chives, cabbage and leek. Additionally, donors were instructed to keep a record of all foods and beverages consumed during this three-day period. The dietary records are provided in Table S4–S16 of the supplementary material. The procedure followed that of our previous study [22]. On the sampling day volunteers were asked to ingest approx. 3 g raw garlic (equals 1–2 garlic cloves) obtained from a local supermarket. The garlic cloves were peeled and cut into approx. 3 mm cubes using a garlic cutter (Genius GmbH, Limburg/Lahn, Germany). For the high dosage test the volunteer ingested approx. 30 g (1 garlic bulb) of raw garlic. The sampling time differed according to the purpose of the test: • High dosage test: One urine sample was provided 4 min prior to garlic consumption (30 g) and three samples were collected afterwards at approx. 2 h intervals. The urine samples were immediately evaluated and analyzed. This sample set was termed Urine a. • Pre-trials: One urine sample was provided 5 min prior to garlic consumption (3 g) and seven samples were collected afterwards, at 2.1 h, 3.7 h, 6.6 h, 7.8 h, 12.1 h, and 24.1 h after garlic consumption. The urine samples were immediately evaluated and analyzed. This sample set was termed Urine b. • Main tests: In each test set, one urine sample was provided immediately prior to garlic consumption (3 g) and seven samples were collected afterwards, at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after garlic consumption. Two volunteers did not provide a sample at 24 h as they were nursing mothers. Another two volunteers each provided an additional sample between 2 h and 4 h after garlic consumption. The samples were immediately evaluated and analyzed or kept ◦ frozen at −80 C until further analysis. This protocol was applied to a set of urine samples from 12 different volunteers that were termed Urine c to Urine n. 97 Metabolites 2016, 6,43 16 of 23 The samples of each set have been termed according to their sampling time. The following list provides the time intervals that are related to the respective sample name: - Pre: prior garlic consumption - 0.5 h post: 0.3 h to 0.74 h after garlic consumption - 1 h post: 0.75 h to 1.49 h after garlic consumption - 2 h post: 1.5 h to 2.49 h after garlic consumption - 3 h post: 2.5 h to 3.49 h after garlic consumption - 4 h post: 3.5 h to 5 h after garlic consumption - 6 h post: 5 h to 7 h after garlic consumption - 8 h post: 7 h to 9 h after garlic consumption - 24 h post: 23 h to 26 h after garlic consumption A scheme of the sampling together with the respective methods applied for analysis of the respective samples is provided in the supporting Figure S5. The exact sampling times and the respective volumes of the urine samples are provided in the supplementary Table S3. 4.3. Study Design and Ethics Approval The study was conducted in agreement with the Declaration of Helsinki. Written, informed consent was provided by all 12 volunteers prior to sampling. Withdrawal from the study was possible at any time. The study (registration number 49_13B) was approved by the Ethical Committee of the Medical Faculty, Friedrich-Alexander Universität Erlangen-Nürnberg. 4.4. Sensory Analysis Sensory analyses of the urine samples were carried out by a trained panel (5 to 7 panelists) of the University of Erlangen-Nuremberg (Erlangen, Germany) who exhibited no known illness at the time of examination and with audited olfactory and gustatory function. The panel was trained at recognizing approx. 140 selected odorants at different odorant concentrations according to their odor qualities in preceding weekly training sessions, and in naming these according to an in-house developed flavor language. The panelists were trained for at least 3 months. For the orthonasal evaluation (smelling) of the urine samples, the samples were presented to ◦ the sensory panel in brown glass bottles (capacity 50 mL) in a sensory assessment room at 21 ± 1 C. No information about the purpose of the experiment was given. The panelists were asked to score the intensities of the attribute “garlic-/cabbage-like” on a scale from 0 (no perception) to 3 (strong perception). The sensory analyses were performed on sample sets Urine a to Urine e. 4.5. Determination of the Creatinine Concentration in Urine Samples The creatinine content of each urine sample was determined using a creatinine kit (Labor + Technik Eberhard Lehmann GmbH, Berlin, Germany). This method is based on the Jaffe reaction [67] with creatinine and picric acid forming a yellow complex in an alkaline milieu. The color intensity of the complex is determined photometrically at a wavelength of 492 nm, and is directly proportional to the creatinine concentration. 4.6. Solvent-Assisted Flavor Evaporation (SAFE) of Volatiles from Human Urine SAFE [68] was used to isolate the volatile compounds from the urine samples. To this aim, 3 to 25 mL DCM were added to the respective volumes of the urine samples, which ranged between 6 to 50 g, respectively, resulting in a ratio of 0.5 mL DCM/1 g urine in each case. Each solution was then ◦ stirred for 30 min and immediately underwent SAFE distillation at 50 C. The resulting distillate was then extracted three times with 25 mL DCM in each case and the combined DCM phases obtained μ from each sample were dried over anhydrous Na2SO4 and enriched to a total volume of 100 Lat 98 Metabolites 2016, 6,43 17 of 23 ◦ 50 C by means of Vigreux distillation and micro-distillation [69]. The final extracts were stored at ◦ −80 C until further analysis. Generally, the urine samples were worked up and measured within a few weeks after collection. However, AMS was determined as mentioned below using GC-GC-MS with a specific column configuration of the instrument. This meant that the setup of the instrument had to be changed exclusively for this measurement, so that the samples needed to be analyzed for this specific analyte as ◦ a sample set. Because of these analytical requirements, a few samples needed to be stored at −80 C for a period of up to 12 months for these measurements. To confirm the stability of AMS during storage, a stability test for 1 month was performed. The concentration of AMS remained constant during this period of time confirming that AMS is not degraded in the course of storage. Apart from that, previous studies of our group on the storage of different bodily fluids revealed stability against oxidation at ◦ −80 C for a time period of more than one year [70]. 4.7. High Resolution Gas Chromatography-Olfactometry (HRGC-O) To identify odor active substances being associated with the garlic/cabbage- like odor of the urine after garlic consumption and to identify other trace metabolites that are odor-active HRGC-O was performed with a Trace Ultra GC (Thermo Finnigan, Dreieich, Germany) using the following capillaries: DB-FFAP (30 m × 0.32 mm, film thickness 0.25 μm; J&W Scientific, Fisons Instruments, Mainz-Kastel, Germany) and DB-5 (30 m × 0.32 mm, film thickness 0.25 μm; J&W Scientific). At the end of the capillaries, the effluent was split between a sniffing port and a flame ionization detector (FID) using two deactivated, uncoated fused silica capillaries (i.D. 0.32 mm). The FID and the sniffing port ◦ ◦ were held at 250 C and 270 C, respectively. The flow rate of the helium carrier gas was 2.0 mL/min. ◦ The extract (2 μL) was injected manually into a cold-on-column injector at 40 C[71], directly on a pre-column of uncoated, deactivated fused silica capillary (2–3 m × 0.32 mm). After 7 min the oven ◦ ◦ ◦ temperature was raised at 8 C/min to 240 C (DB-FFAP) or 250 C (DB-5), which was held for 5 min. ◦ ◦ In the case of the DB-5 capillary, the oven temperature was further raised to 300 Cat25 C/min and held for 5 min. The pre-column was changed regularly to avoid accumulation of any contaminants. 4.8. Comparative Aroma Extract Dilution Analysis (cAEDA) The FD factors of the odorants in human urine before and after garlic consumption were determined by cAEDA [72,73]. The original extracts comprising a total volume of 100 μL were diluted stepwise (1 + 1, v/v) with DCM. HRGC-O analysis was then performed on 2 μL of the original extracts (FD = 1) and the respective dilutions using the DB-5 column until no odor was detectable. The FD factor of each odorant relates to the highest dilution in which this specific odorant could still be perceived. The temperature program for the GC oven was as described above. The odorants were screened by sniffing the effluent after gas chromatographic separation. Linear retention indices (RIs) of the compounds were calculated as described by Van den Dool and Kratz [32]. Complete cAEDAs were performed for Urine b to f. For Urine g to n the cAEDA was shortened and only the first 5 min were analyzed by the panelist. 4.9. High Resolution Gas Chromatography-Mass Spectrometry (HRGC-MS) Identification of the metabolites and odor-active derivatives was made based on the mass spectra of the target compounds in the respective urine samples and reference compounds obtained on an Agilent MSD quadrupole system (GC 7890A and MSD 5975C, Agilent Technologies, Waldbronn, Germany) equipped with a Gerstel CIS 4 injection system and GERSTEL MPS 2 autosampler (GERSTEL, Duisburg, Germany). The software used for mass spectral recording and data analysis was MSD ChemStation E.02.00.493 (Agilent Technologies). Analytical capillaries were DB-FFAP and DB-5 (30 m × 0.25 mm, film thickness 0.25 μm, Agilent J&W Scientific, Santa Clara, CA, USA). An uncoated, deactivated fused silica capillary was used as a pre-column (2–3 m × 0.53 mm) and changed regularly to avoid accumulation of impurities. Carrier gas was helium at a total flow of 1.0 mL/min, which was 99 Metabolites 2016, 6,43 18 of 23 then transferred in un-split mode into the MS using an uncoated, deactivated fused silica capillary (0.3–0.6 m × 0.25 mm) as a transfer line. Identification of garlic-derived metabolites in the urine samples was based on EI mass spectra that were generated in full scan mode (m/z range 30–350) at 70 eV, whereby representative m/z-ratios that corresponded to the investigated compounds were extracted (see Table 2). For analysis of AMS, AMSO and AMSO2 single ion monitoring (SIM) mode was applied using m/z 73 + 88, 104 and 120, respectively. The initial temperature of the GC oven was ◦ ◦ ◦ 40 C, which was held for 7 min and then raised to 240 C and 250 C for FFAP and DB-5, respectively, ◦ at a rate of 8 C/min, and held for 7 min. In the case of the DB-5 capillary, the oven temperature was ◦ ◦ further raised to 300 Cat25 C/min and held for 5 min. 4.10. Two-Dimensional High Resolution Gas Chromatography-Mass Spectrometry/Olfactometry (HRGC-GC-MS/O) (Heart-Cut) A two-dimensional gas chromatographic system was used for the mass spectrometric identification of trace constituents. The system consisted of two Varian 450 GCs in combination with a Varian 220 ion trap MS (Varian, Darmstadt, Germany). The first GC was equipped with a multi-column switching system MCS 2 and was connected to the second GC by a cryo-trap system CTS 1 (both: GERSTEL, Duisburg, Germany). A DB-5 column (30 m × 0.32 mm, film thickness 0.25 mm (Agilent J&W Scientific, Santa Clara, CA, USA); first oven) and a DB-FFAP column (30 m × 0.25 mm, film thickness 0.25 mm (Agilent J&W Scientific, Santa Clara, CA, USA); second oven) were used. An uncoated, deactivated fused silica capillary was used as pre-column (2–3 m × 0.53 mm). The flow rate of the helium carrier gas was 2.5 mL/min. In the first oven the effluent was split between an olfaction detection port (ODP, Gerstel) and an FID, as well as a cryo-trap during the cut interval. In the second oven the effluent was transferred to the MS. All split capillaries were made of uncoated, deactivated fused silica material. The FID and the sniffing port were held at ◦ ◦ 250 C and 260 C, respectively. Mass spectra in EI mode were generated at 70 eV ionization energy. The m/z ranges measured for each compound were as follows: AMS, m/z 30–100; AMSO and AMSO2, m/z 30–150, AMDS, DADS, DAS, DASO, DASO2, DATS, DMDS, DMTS, 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin, m/z 30–350. The cut time intervals on the main column were determined by injection of the respective reference substances. Application of the samples to the GC system was ◦ performed at 40 C using the cold-on-column technique. The used temperature programs were as ◦ ◦ follows: For AMS: In the first oven, 40 C were held for 7 min and then raised to 300 C at a rate of ◦ 20 C/min. The final temperature was then held for 5 min. The second oven started at a temperature ◦ ◦ ◦ of 40 C that was held for 7 min, and then raised to 240 C at a rate of 20 C/min; the final temperature was held for 5 min. For AMSO/ AMSO2 and AMDS, DADS, DAS, DASO, DASO2, DATS, DMDS, DMTS, 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin: The first oven started at a temperature of ◦ ◦ ◦ ◦ 40 C. Then, the temperature was raised at a rate of 8 C/min to 200 C, and further to 300 C at a rate ◦ of 20 C/min. The final temperature was held for 5 min. The second oven started at a temperature of ◦ ◦ ◦ 40 C, and was then heated up to 140 C at a rate of 20 C/min. The temperature was further raised to ◦ ◦ 240 Catarateof8 C/min, and finally held for 5 min. The transfer line between first and second oven ◦ ◦ was held at 250 C and was cooled down to −100 C for trapping of the respective substances. In the case of DASO, DASO2, 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin the transfer line temperature ◦ ◦ was set to 100 C and cooled down to −100 C during trapping. 4.11. Identification of Metabolites and Calculation of Metabolite Profiles Garlic metabolites in human urine were identified by comparing RIs according to Van den Dool and Kratz [32], their odor perceived at the sniffing port via GC-O, and by comparing the EI mass spectra generated by either HRGC-MS or HRGC-GC-MS/O with those of purchased and/or synthesized reference compounds. Comparison of the mass spectra of the analytes with the reference standards was performed with the aid of the NIST Mass Spectral Search Program (Version 2.0 d). The identification 100 Metabolites 2016, 6,43 19 of 23 was ranked positive if reverse match values were above 900. Two analytical capillaries of different polarities (DB-FFAP and DB-5) were used for calculating the RIs. To obtain first insights into the time dependency of the metabolite formation and excretion, specifically taking into account potential inter-individual differences, the relative concentration of the metabolites in different urine samples were determined as follows: AMS was determined by HRGC-GC-MS, whereby m/z 73 and 88 were extracted from the total ion chromatogram and the area of the resulting peak was determined. The concentration was calculated in two different ways. First, the amount was expressed as area/kg. In order to do this, the peak area of AMS was determined, which was then divided by the amount of the investigated urine (in kg). Second, the determined peak area was divided by the amount of the applied human urine (in L), which led to a concentration expressed as area/L. This was then further divided by the creatinine concentration (in mmol/L), so that the final concentration could be expressed as area/mmol creatinine. AMSO and AMSO2 were determined by HRGC-MS. For relative quantification a DB-FFAP column was used. For AMSO and AMSO2 the SIM mode with m/z 104 and 120 were applied, respectively. The peak areas were determined and the concentrations were calculated as described for AMS. 5. Conclusions This study shows that garlic consumption has an effect on the composition of urine. The garlic constituents were shown to be strongly metabolized before being excreted into the urine, with identification of allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these compounds only AMS is odorous, but due to the strong inherent smell of urine, the influence of AMS on the overall odor of urine is negligible. The metabolites detected were monitored up to 26 h after garlic consumption. The metabolite profiles differed between individuals, resulting in different temporal profiles. In most cases the metabolites reached a maximum concentration between1hto2hafter garlic consumption. Furthermore, a second increase was occasionally observable, about 3 h to 7.8 h after garlic consumption, potentially relating to absorption of garlic derivatives at different locations within the gastrointestinal tract. A range of garlic constituents and metabolites were not detected in urine, indicating that these are metabolized or not excreted in urine, namely DASO, DASO2, DADS, AMDS, DMDS, DMTS, DATS, DAS, 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin. In view of the high inter-individual differences in the metabolite profiles, future studies should address if subject-specific parameters such as gender or hormonal status of the test person have an influence on the metabolite profiles. However, this aspect will require a representative cohort study and quantification of the respective metabolites. Supplementary Materials: The following supplementary material is available online at www.mdpi.com/2218- 1989/6/4/43/s1, Table S1: Determined FD factors of AMS in urine sample sets g to n, Table S2: Compilation of investigated urine samples: time of urine sampling, as well as mass (g), volume (mL) and creatinine content (mmol/L) of the investigated sample; DMTS and DMDS concentrations are given in area/kg and area/mmol creatinine, Table S3: Compilation of investigated urine samples: time of urine sampling, as well as the time of garlic intake, mass (g), volume (mL) and creatinine content (mmol/L) of the investigated sample; AMS, AMSO and AMSO2 concentrations are given in area/kg and area/mmol creatinine, Tables S4–S16: dietary records—test person b to n, Figure S1: Perceived intensity of a “garlic-/cabbage-like” odor in urine at different time intervals before and after ingestion of raw garlic, Figure S2: Time-resolved metabolite profiles of AMS, AMSO and AMSO2 for urine set a, Figure S3: Time-resolved profiles of DMTS excretion in urine for sets b to n, Figure S4: Time-resolved profiles of DMDS excretion in urine for sets b to n, Figure S5: Scheme of urine sampling and analytical methods. Acknowledgments: This work was financed by the German Research Foundation (DFG) in the frame of grant BU 1351/15-1. The authors are exclusively responsible for the contents of this publication. We are grateful to the volunteers for providing urine samples. Author Contributions: Andrea Buettner, Constanze Sharapa, Laura Scheffler and Anja Heinlein conceived and designed the experiments; Laura Scheffler and Yvonne Sauermann performed the experiments; Laura Scheffler analyzed the data; Andrea Buettner contributed reagents/materials/analysis tools; Laura Scheffler conceived the publication that was approved by Constanze Sharapa and Andrea Buettner. All authors have read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest. 101 Metabolites 2016, 6,43 20 of 23 Abbreviations The following abbreviations are used in this manuscript: AMDS: Allyl methyl disulfide AMS: Allyl methyl sulfide AMSO: Allyl methyl sulfoxide AMSO2: Allyl methyl sulfone cAEDA: Comparative aroma extract dilution analysis CVD: Cardiovascular disease DADS: Diallyl disulfide DAS: Diallyl sulfide DASO: Diallyl sulfoxide DASO2: Diallyl sulfone DATS: Diallyl trisulfide DMDS: Dimethyl disulfide DMTS: Dimethyl trisulfide FD factor: Flavor dilution factor FID: Flame ionization detector HDL: High density lipoprotein HRGC-GC-MS: Two dimensional high-resolution gas chromatography-mass spectrometry HRGC-MS: High-resolution gas chromatography-mass spectrometry HRGC-O: High-resolution gas chromatography-olfactometry LDL: Low density lipoprotein RI: (Linear) retention indices SAFE: Solvent-assisted flavor evaporation TC: Total cholesterol TIC: Total ion chromatogram References 1. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 105 'HWHFWLRQ RI YRODWLOH PHWDEROLWHV GHULYHG IURP JDUOLF $OOLXP VDWLYXP LQ KXPDQXULQH 6XSSRUWLQJPDWHULDO /DXUD6FKHIIOHU 'HSDUWPHQWRI&KHPLVWU\DQG3KDUPDF\(PLO)LVFKHU&HQWHU)ULHGULFK$OH[DQGHU8QLYHUVLWlW (UODQJHQ1UQEHUJ )$8 +HQNHVWU (UODQJHQ*HUPDQ\ )UDXQKRIHU,QVWLWXWHIRU3URFHVV(QJLQHHULQJDQG3DFNDJLQJ ,99 *LJJHQKDXVHU6WU )UHLVLQJ*HUPDQ\ &RUUHVSRQGHQFH DQGUHDEXHWWQHU#IDXGH 107 Metabolites 2016, 6, 43; doi: 10.3390/metabo6040043 6RI6 Supplementary Materials: Detection of Volatile Metabolites Derived from Garlic (Allium sativum) in Human Urine Laura Scheffler, Yvonne Sauermann, Anja Heinlein, Constanze Sharapa and Andrea Buettner Table S1. Determined flavor dilution (FD) factors of AMS in different urine sample sets g to n as determined via HRGC-O using a DB-5 capillary. Six to nine samples per set were collected at different time intervals before and after ingestion of 3 g of raw garlic from different volunteers. “Pre” relates to the urine sample that was collected prior to garlic consumption. “0.5 h post” to “24 h post” relate to the urine samples that were obtained after garlic consumption. sample Urine g Urine h Urine i Urine j Urine k Urine l Urine m Urine n pre n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.5 h post84848888 1 h post 32 8 32 16 8 8 8 8 2 h post 8 8 16 8 16 8 - 16 3 h post - - - - 4 8 32 - 4 h post 8 8 16 4 16 8 32 16 6 h post 8 8 16 2 8 8 32 n.d. 8 h post n.d. 8 16 n.d. 16 4 - - 24 h post n.d. 2 n.d. n.d. n.d. n.d. - - n.d. not detected; - no sample was provided at this time point. Table S2. Compilation of investigated urine samples: time of urine sampling, as well as mass (g), volume (mL) and creatinine content (mmol/L) of the investigated sample; DMTS and DMDS-concentrations are given in area/kg and area/mmol creatinine. DMTS DMDS Volume Creatinine Sample Time (h) Mass (g) (area/mmol (area/mmol (mL) (mmol/L) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 53.1486 5926.8 0.0 2 h post 2.17 50.1150 5547.2 18497.5 4 h post 4.08 49.5297 9408.5 1221872.9 urine a 6 h post 5.83 55.6357 21874.4 73100.5 pre 0.00 18.2110 1.67 0.0 0.0 * 110043.4 65894.2 * 2 h post 2.08 26.0446 1.75 0.0 0.0 * 68689.9 39251.4 * 4 h post 3.67 37.1800 1.47 28294.8 19248.2 * 467670.8 318143.4 * 6 h post 6.58 54.3923 2.62 315154.9 120288.1 * 3538791.3 1350683.7 * 8 h post 7.75 49.5103 1.47 82346.5 56018.0 * 251624.4 171173.1 * urine b 12 h post 12.12 61.7643 3.91 9973.4 2550.7 * 326078.3 83396.0 * 21 h post 21.08 38.1209 9.89 106214.7 10739.6 * 723461.4 73150.8 * 24 h post 24.08 50.4667 3.08 19577.3 6356.3 * 671868.8 218139.2 * pre 0.00 39.6450 39.2 5.96 30319.1 5143.4 198713.6 33710.1 0.5 h post 0.63 49.9958 50.0 1.11 7280.6 6544.4 110969.3 99747.5 1 h post 1.08 19.9021 50.0 0.57 158224.5 110309.7 284844.3 198585.4 2 h post 1.95 49.9436 50.0 0.66 11873.4 18038.0 179282.2 272365.1 4 h post 3.78 50.3893 50.0 2.60 21492.7 8340.9 149496.0 58016.5 urine c 6 h post 5.75 50.5265 50.0 3.88 79582.0 20723.9 304652.0 79334.4 8 h post 8.13 50.2804 50.0 3.13 45007.6 14438.7 199501.2 64001.1 24 h post 24.15 50.743 50.0 7.31 122066.1 16955.5 479711.5 66634.1 109 Metabolites 2016, 6, 43 6RI6 Table S2. Cont. DMTS DMDS Volume Creatinine Sample Time (h) Mass (g) (area/mmol (area/mmol (mL) (mmol/L) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 50.1570 50.0 3.81 15331.9 4034.1 45596.8 11997.5 0.5 h post 0.47 49.8599 50.0 1.13 7380.7 6498.8 36141.3 31823.0 1 h post 1.05 49.9479 50.0 1.63 11532.0 7067.9 42364.1 25964.7 2 h post 2.13 49.9834 50.0 1.74 6082.0 3493.4 21607.2 12410.7 4 h post 4.00 50.5099 50.0 5.64 30964.2 5550.7 276322.1 49533.6 urine d d urine 6 h post 5.75 50.5739 50.0 5.17 92498.3 18110.6 270495.3 52961.3 8 h post 7.52 50.0155 50.0 1.82 15475.2 8492.0 78395.7 43019.4 24 h post 24.92 50.019 50.0 2.40 19052.8 7929.7 113396.9 47195.5 pre 0.00 50.0938 50.0 0.93 15251.4 16346.5 38607.6 41379.8 0.5 h post 0.77 49.9007 50.0 0.85 17154.1 20161.1 7815.5 9185.5 1 h post 1.45 49.8642 50.0 0.74 17627.9 23887.8 11531.3 15626.3 2 h post 2.25 49.9592 50.0 1.39 0.0 0.0 6585.4 4746.5 4 h post 3.88 50.0838 50.0 2.04 2775.3 1364.1 20665.4 10157.1 urine e e urine 6 h post 5.72 50.0233 50.0 1.81 5017.7 2773.2 42900.0 23710.3 8 h post 7.88 50.2255 50.0 3.09 5077.1 1652.6 45494.8 14809.0 24 h post 24.68 50.0227 50.0 1.78 40801.5 22917.1 121504.8 68246.1 pre 0.00 32.5189 32.0 14.31 24693.3 1753.0 202005.6 14340.7 0.5 h post 0.55 49.9647 50.0 2.99 0.0 0.0 48534.3 16193.9 1 h post 1.03 49.8430 50.0 0.95 0.0 0.0 0.0 0.0 2 h post 2.03 49.9875 50.0 1.74 0.0 0.0 0.0 0.0 urine f 4 h post 4.07 50.0798 50.0 3.92 57528.2 14690.2 94848.6 24220.3 6 h post 6.02 50.3908 50.0 6.78 146296.5 21756.1 379533.6 56441.2 8 h post 8.05 50.503 50.0 6.94 0.0 0.0 0.0 0.0 24 h post 24.68 39.7167 39.0 14.10 0.0 0.0 33512.4 2421.0 pre 0.00 50.1354 50.0 4.31 19128.2 4450.3 124363.2 28934.1 0.5 h post 1.12 49.8514 50.0 1.18 5917.6 4989.2 33860.6 28548.6 1 h post 1.73 49.9335 50.0 1.22 4846.4 3951.9 24272.3 19792.2 2 h post 2.92 50.0587 50.0 3.96 60988.4 15433.2 0.0 0.0 4 h post 4.67 49.9454 50.0 2.47 33296.4 13480.8 0.0 0.0 urine g g urine 6 h post 6.92 50.8932 50.0 12.82 289331.4 22977.5 686751.9 54539.0 8 h post 7.83 6.8311 6.0 15.86 0.0 0.0 0.0 0.0 24 h post 24.53 18.411 18.0 18.94 0.0 0.0 0.0 0.0 pre 0.00 10.1392 10.0 15.17 44086.3 2947.1 890405.6 59522.8 0.5 h post 1.02 49.9072 50.0 1.05 44342.3 42111.3 0.0 0.0 1 h post 1.73 49.9145 50.0 0.75 67555.5 90030.4 0.0 0.0 2 h post 2.92 49.8837 50.0 0.84 0.0 0.0 0.0 0.0 4 h post 4.82 49.7932 50.0 0.94 0.0 0.0 0.0 0.0 urine h h urine 6 h post 6.92 49.798 50.0 1.49 7630.8 5112.9 0.0 0.0 8 h post 7.83 50.1232 50.0 6.10 16479.4 2709.7 0.0 0.0 24 h post 23.63 50.024 50.0 2.08 32784.3 15778.2 0.0 0.0 pre 0.00 19.3317 19.0 22.94 19656.8 871.8 87524.6 3881.8 0.5 h post 0.92 34.5982 34.0 19.15 22631.2 1202.3 130324.7 6923.5 1 h post 1.50 26.2297 25.8 14.51 53069.6 3717.4 204729.8 14341.0 2 h post 2.73 35.5222 35.0 12.07 43775.4 3680.6 107285.0 9020.5 urine i 4 h post 4.62 33.5814 33.0 12.31 29480.6 2436.4 120870.5 9989.4 6 h post 6.67 50.6976 50.0 9.10 19843.1 2209.9 95507.5 10636.4 8 h post 7.45 43.5456 43.0 10.10 16810.0 1685.0 38442.5 3853.5 24 h post 23.88 15.5162 15.2 37.11 63546.5 1748.0 163313.2 4492.4 110 Metabolites 2016, 6, 43 6RI6 Table S2. Cont. DMTS DMDS Volume Creatinine Sample Time (h) Mass (g) (area/mmol (area/mmol (mL) (mmol/L) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 50.7676 50.0 11.79 12409.5 1068.8 76564.6 6594.2 0.5 h post 0.53 49.9728 50.0 0.80 0.0 0.0 9465.1 11755.4 1 h post 1.03 49.9724 50.0 0.66 16088.9 24442.0 10265.7 15595.4 2 h post 2.07 50.0155 50.0 1.37 0.0 0.0 13535.8 9850.8 urine j 4 h post 4.12 40.2985 40.0 7.45 15509.3 2096.2 33524.8 4531.1 6 h post 6.03 50.1150 50.0 2.76 0.0 0.0 0.0 0.0 8 h post 7.78 50.0429 50.0 1.17 0.0 0.0 8952.3 7626.9 24 h post 24.12 45.8566 45.0 20.84 35087.6 1715.6 135683.8 6634.4 pre 0.00 50.0446 50.0 5.75 5475.1 952.3 41083.4 7146.0 0.5 h post 0.77 50.0027 50.0 2.29 2319.9 1013.1 7739.6 3379.8 1 h post 1.30 49.9764 50.0 3.07 3421.6 1113.2 4202.0 1367.1 2 h post 2.33 49.9929 50.0 2.96 3220.5 1086.5 17262.5 5823.8 3 h post 3.35 49.9729 50.0 2.90 0.0 0.0 32117.4 11054.2 urine k k urine 4 h post 4.38 50.0049 50.0 5.03 0.0 0.0 47935.3 9536.3 6 h post 6.22 49.9742 50.0 5.08 0.0 0.0 5422.8 1066.1 8 h post 7.92 50.2461 50.0 11.01 17653.1 1611.8 49794.9 4546.4 24 h post 23.80 50.2049 50.0 7.43 0.0 0.0 22069.6 2982.3 pre 0.00 51.2038 50.0 41.00 17811.2 444.9 151883.3 3793.7 0.5 h post 0.75 39.7135 39.0 25.03 51393.1 2090.8 224759.8 9143.7 1 h post 1.30 50.0618 50.0 4.09 0.0 0.0 57828.5 14159.8 2 h post 2.33 50.0743 50.0 5.47 12860.9 2354.6 92262.9 16891.6 3 h post 3.12 50.0046 50.0 3.32 2819.7 850.6 30177.2 9103.6 urine l 4 h post 4.33 50.2867 50.0 12.32 15590.6 1272.5 212123.7 17314.0 6 h post 6.20 50.8602 50.0 27.19 36531.5 1366.9 175382.7 6562.1 8 h post 7.90 50.9033 50.0 23.54 22513.3 973.8 126593.0 5475.5 24 h post 26.05 30.5201 30.0 28.57 21821.7 777.1 36664.4 1305.7 pre 0.00 50.0039 50.0 2.45 11839.1 4841.5 75174.1 30741.6 0.5 h post 0.77 49.9662 50.0 1.80 10166.9 5636.6 132809.8 73630.6 1 h post 1.42 49.8828 50.0 1.53 6856.1 4479.5 126416.3 82595.7 3 h post 2.67 50.3904 50.0 5.22 53204.6 10264.4 479456.4 92497.9 urine m urine 4 h post 3.75 40.5086 40.0 7.81 79785.5 10342.1 375821.4 48715.5 6 h post 5.92 50.7212 50.0 10.27 24644.5 2434.4 181502.0 17929.1 pre 0.00 35.7715 35.0 16.70 39388.9 2410.3 71537.4 4377.6 0.5 h post 1.00 30.6561 30.0 13.53 22997.1 1736.8 51148.1 3862.9 1 h post 1.50 30.0953 30.0 2.60 22927.2 8838.1 41136.0 15857.3 2 h post 2.67 50.1476 50.0 3.68 7218.7 1969.0 22732.9 6200.9 urine n n urine 4 h post 4.37 45.8559 45.0 16.86 27193.9 1643.2 139022.5 8400.5 6 h post 6.50 37.8167 37.0 19.77 55055.0 2846.6 90832.9 4696.5 * Calculated under the assumption 1 mL urine equals 1 g urine. 111 Metabolites 2016, 6, 43 6RI6 Table S3. Compilation of investigated urine samples: time of urine sampling, as well as the time of garlic intake, mass (g), volume (mL) and creatinine content (mmol/L) of the investigated sample; AMS, AMSO and AMSO2-concentrations are given in area/kg and area/mmol creatinine. AMS AMSO AMSO2 Creatinine Sample Time (h) Mass (g) Volume (mL) (area/mmol (area/mmol (area/mmol (mmol/L) (area/kg) (area/kg) (area/kg) creatinine) creatinine) creatinine) pre 0.00 53.1486 2709 0 0 2 h post 2.17 50.1150 551851 147868143 25609638 4 h post 4.08 49.5297 731844 198612731 45229428 urine a urine 6 h post 5.83 55.6357 829449 196025448 67629166 garlic intake 0.07 pre 0.00 18.2110 1.67 0 0.0 * 0 0.0 * 0 0.0 * 2 h post 2.08 26.0446 1.75 25264 14436.8 * 61396451 35083686.1 * 11712793 6693024.4 * 4 h post 3.67 37.1800 1.47 19392 13191.9 * 27941366 19007732.2 * 14414578 9805835.2 * 6 h post 6.58 54.3923 2.62 93910 35843.6 * 8602118 3283251.3 * 9027068 3445445.9 * 8 h post 7.75 49.5103 1.47 13997 9521.8 * 4866078 3310257.4 * 6382248 4341665.1 * urine b b urine 12 h post 12.12 61.7643 3.91 10265 2625.3 * 5232359 1338199.3 * 6228614 1592996.0 * 21 h post 21.08 38.1209 9.89 12880 1302.3 * 5027846 508376.7 * 3408944 344685.9 * 24 h post 24.08 50.4667 3.08 3864 1254.5 * 2295236 745206.6 * 1835547 595956.8 * garlic intake 0.08 pre 0.00 39.6450 39.2 5.96 0 0.0 0 0.0 0 0.0 0.5 h post 0.63 49.9958 50.0 1.11 8841 7946.7 13939734 12794397.3 1028266 924282.6 1 h post 1.08 19.9021 50.0 0.57 54768 38182.8 78210046 54717046.1 13358942 9313476.9 2 h post 1.95 49.9436 50.0 0.66 37242 56578.0 33506577 51392272.9 8811780 13386832.4 4 h post 3.78 50.3893 50.0 2.60 35642 13832.2 30865036 12057294.2 8560686 3322235.2 urine c c urine 6 h post 5.75 50.5265 50.0 3.88 28777 7493.8 14106376 3706338.4 8790892 2289234.0 8 h post 8.13 50.2804 50.0 3.13 14678 4708.7 6165683 2032776.7 5572370 1787646.7 24 h post 24.15 50.743 50.0 7.31 788 109.5 246031 23033.7 249236 34620.1 garlic intake 0.17 pre 0.00 50.1570 50.0 3.81 0 0.0 0 0.0 0 0.0 0.5 h post 0.47 49.8599 50.0 1.13 24388 21474.4 13266880 11850649.6 1336284 1176622.7 1 h post 1.05 49.9479 50.0 1.63 88272 54101.4 32913660 20268289.9 5356822 3283165.1 2 h post 2.13 49.9834 50.0 1.74 71864 41277.1 29935489 17279463.7 7224679 4149700.6 4 h post 4.00 50.5099 50.0 5.64 84597 15165.0 32655112 5830828.4 12151281 2178245.2 urine d urine 6 h post 5.75 50.5739 50.0 5.17 79764 15617.4 20506070 3996540.9 12698922 2486370.4 8 h post 7.52 50.0155 50.0 1.82 26292 14427.6 5440388 3063540.1 6122842 3359887.0 24 h post 24.92 50.019 50.0 2.40 1799 748.9 380770 200357.2 929667 386924.6 garlic intake 0.10 112 Metabolites 2016, 6, 43 6RI6 Table S3. Cont. AMS AMSO AMSO2 Creatinine Sample Time (h) Mass (g) Volume (mL) (area/mmol (area/mmol (mmol/L) (area/kg) (area/kg) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 50.0938 50.0 0.93 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.77 49.9007 50.0 0.85 8236 9680.1 5889981 6930051.2 648978.4 767283.4 1 h post 1.45 49.8642 50.0 0.74 35196 47694.1 12127876 16455469.6 2488620.7 3384807.7 2 h post 2.25 49.9592 50.0 1.39 47138 33975.7 21495418 15467377.4 5969869.6 4287459.3 4 h post 3.88 50.0838 50.0 2.04 34782 17095.3 9634387 4692755.1 4455042.1 2164191.3 urine e e urine 6 h post 5.72 50.0233 50.0 1.81 24309 13435.1 5627221 3072008.9 4064367.9 2223535.7 8 h post 7.88 50.2255 50.0 3.09 15291 4977.4 3488713 1080858.1 3690340.9 1168479.4 24 h post 24.68 50.0227 50.0 1.78 3498 1965.0 806598 415620.8 723893.8 384198.8 garlic intake 0.22 pre 0.00 32.5189 32.0 14.31 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.55 49.9647 50.0 2.99 30802 10277.3 14706735 4921190.4 1701715.7 583577.4 1 h post 1.03 49.8430 50.0 0.95 49696 52032.3 16496262 17324329.0 3297595.0 3511266.9 2 h post 2.03 49.9875 50.0 1.74 63936 36717.9 17517307 10087145.2 4892169.9 2839775.1 4 h post 4.07 50.0798 50.0 3.92 48523 12390.6 13514300 3460899.1 5476275.8 1409483.9 urine f 6 h post 6.02 50.3908 50.0 6.78 91386 13590.2 9812221 1463380.8 5838786.3 872968.2 8 h post 8.05 50.503 50.0 6.94 27880 4058.4 4026823 590201.1 3572983.2 524601.1 24 h post 24.68 39.7167 39.0 14.10 1586 114.6 408252 29560.2 323730.3 23462.0 garlic intake 0.05 pre 0.00 50.1354 50.0 4.31 1636 380.5 14410 15880.1 63747.4 14831.3 0.5 h post 1.12 49.8514 50.0 1.18 23149 19517.2 3683963 3151426.1 885531.8 746610.7 1 h post 1.73 49.9335 50.0 1.22 34306 27973.6 11226718 9198438.4 3420148.8 2788870.3 2 h post 2.92 50.0587 50.0 3.96 47924 12127.1 11784496 2995700.1 4307922.5 1090122.6 4 h post 4.67 49.9454 50.0 2.47 51616 20898.1 6075067 2481436.6 4576797.9 1853025.9 urine g g urine 6 h post 6.92 50.8932 50.0 12.82 28412 2256.4 3910436 314827.2 3503945.5 278269.1 8 h post 7.83 6.8311 6.0 15.86 7905 567.4 2665482 195202.3 2105224.6 151119.9 24 h post 24.53 18.411 18.0 18.94 4508 243.5 520974 31044.2 341915.2 18465.7 garlic intake 0.63 pre 0.00 10.1392 10.0 15.17 2564 171.4 232568 19146.5 176345.3 11788.5 0.5 h post 1.02 49.9072 50.0 1.05 6071 5765.8 3604725 3474499.9 415731.6 394815.3 1 h post 1.73 49.9145 50.0 0.75 14865 19811.0 9682283 12975223.1 1332378.4 1775644.8 2 h post 2.92 49.8837 50.0 0.84 28787 34112.2 11975876 14255060.4 2540328.8 3010256.0 4 h post 4.82 49.7932 50.0 0.94 19722 20878.8 7976307 8501330.9 2588184.7 2740049.4 urine h h urine 6 h post 6.92 49.798 50.0 1.49 14378 9633.8 6580397 4445166.9 3132274.4 2098729.9 113 8 h post 7.83 50.1232 50.0 6.10 18614 3060.7 6264646 1038932.4 3524535.5 579529.8 24 h post 23.63 50.024 50.0 2.08 2539 1221.8 297366 169028.6 330961.1 159282.7 garlic intake 0.50 Metabolites 2016, 6, 43 6RI6 Table S3. Cont. AMS AMSO AMSO2 Creatinine Sample Time (h) Mass (g) Volume (mL) (area/mmol (area/mmol (mmol/L) (area/kg) (area/kg) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 19.3317 19.0 22.94 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.92 34.5982 34.0 19.15 9740 517.5 21225792 1128422.2 2443216.7 130683.6 1 h post 1.50 26.2297 25.8 14.51 49982 3501.1 31358773 2198958.6 6279361.5 442456.5 2 h post 2.73 35.5222 35.0 12.07 40425 3399.0 24635876 2074977.7 6867832.1 581461.5 4 h post 4.62 33.5814 33.0 12.31 52350 4326.5 20143541 1668248.5 8155278.7 677870.6 urine i urine 6 h post 6.67 50.6976 50.0 9.10 41501 4621.8 9715785 1088086.4 5762099.5 648480.6 8 h post 7.45 43.5456 43.0 10.10 11069 1109.5 4642071 470382.6 4112459.6 417881.3 24 h post 23.88 15.5162 15.2 37.11 1418 39.0 1092252 28519.9 881379.5 22542.4 garlic intake 0.08 pre 0.00 50.7676 50.0 11.79 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.53 49.9728 50.0 0.80 11206 13917.6 4088646 5198776.8 1153147.3 1432173.8 1 h post 1.03 49.9724 50.0 0.66 10466 15899.4 5989328 9248660.7 2506143.4 3807289.8 2 h post 2.07 50.0155 50.0 1.37 13576 9879.9 4824676 3578254.0 2489448.3 1811714.2 4 h post 4.12 40.2985 40.0 7.45 3499 472.9 2115344 291025.6 1717830.7 232175.0 urine j 6 h post 6.03 50.1150 50.0 2.76 3033 1101.1 801620 319972.0 1089214.8 395440.6 8 h post 7.78 50.0429 50.0 1.17 1099 936.3 367799 393383.5 548749.2 467502.8 24 h post 24.12 45.8566 45.0 20.84 785 38.4 91073 561.9 53056.7 2594.3 garlic intake 0.03 pre 0.00 50.0446 50.0 5.75 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.77 50.0027 50.0 2.29 25779 11257.4 5505849 2412621.0 820255.7 358201.0 1 h post 1.30 49.9764 50.0 3.07 39779 12941.5 14055977 4577664.8 2847003.8 926232.2 2 h post 2.33 49.9929 50.0 2.96 37525 12659.7 12987337 4386606.2 3915536.0 1320965.7 3 h post 3.35 49.9729 50.0 2.90 22612 7782.7 7711865 2659616.2 4080411.6 1404397.3 4 h post 4.38 50.0049 50.0 5.03 29777 5923.9 6405217 1275045.9 3757291.8 747479.8 urine k urine 6 h post 6.22 49.9742 50.0 5.08 15528 3052.8 5117965 1006917.4 4173993.8 820618.6 8 h post 7.92 50.2461 50.0 11.01 37416 3416.2 3547811 321331.4 3367962.9 307504.9 24 h post 23.80 50.2049 50.0 7.43 2530 341.8 391873 51736.4 327916.2 44312.0 garlic intake 0.13 pre 0.00 51.2038 50.0 41.00 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.75 39.7135 39.0 25.03 18457 750.9 10443854 424876.1 1156231.5 47037.7 1 h post 1.30 50.0618 50.0 4.09 16200 3966.7 6633361 1624234.7 1687354.4 413163.0 2 h post 2.33 50.0743 50.0 5.47 13540 2478.9 5214431 954662.8 2008335.6 367687.9 3 h post 3.12 50.0046 50.0 3.32 8779 2648.4 3347492 1009846.4 1837171.0 554224.0 urine l 4 h post 4.33 50.2867 50.0 12.32 20264 1654.0 2805553 228994.8 1668572.4 136192.2 6 h post 6.20 50.8602 50.0 27.19 31616 1182.9 2349892 87923.5 1737822.5 65022.3 114 8 h post 7.90 50.9033 50.0 23.54 8035 347.5 1483460 64163.7 1323057.6 57225.8 24 h post 26.05 30.5201 30.0 28.57 0 0.0 144298 5138.7 126506.8 4505.1 garlic intake 0.13 Metabolites 2016, 6, 43 6RI6 Table S3. Cont. AMS AMSO AMSO2 Creatinine Sample Time (h) Mass (g) Volume (mL) (area/mmol (area/mmol (mmol/L) (area/kg) (area/kg) (area/kg) (area/kg) creatinine) creatinine) pre 0.00 50.0039 50.0 2.45 0 0.0 0 0.0 0.0 0.0 0.5 h post 0.77 49.9662 50.0 1.80 26798 14857.1 9463304 5251620.4 1499653.8 831417.9 1 h post 1.42 49.8828 50.0 1.53 26983 17629.8 9136842 5978242.4 2256268.7 1474161.1 3 h post 2.67 50.3904 50.0 5.22 31315 6041.5 9744504 1872355.3 2856119.4 551009.3 urine m m urine 4 h post 3.75 40.5086 40.0 7.81 19280 2499.1 7362093 944506.8 3156391.5 409144.3 6 h post 5.92 50.7212 50.0 10.27 28450 2810.3 4316870 415547.9 2930037.1 289433.7 garlic intake 0.08 pre 0.00 35.7715 35.0 16.70 4389 268.6 958473 44307.0 1004402.9 61462.0 0.5 h post 1.00 30.6561 30.0 13.53 9231 697.2 7076406 534442.5 1336471.4 100936.4 1 h post 1.50 30.0953 30.0 2.60 23027 8876.5 26998435 10407496.1 4058241.7 1564391.9 2 h post 2.67 50.1476 50.0 3.68 54399 14838.6 18852767 5142488.3 4426971.6 1207549.5 urine n n urine 4 h post 4.37 45.8559 45.0 16.86 77177 4663.4 12180875 736032.5 4719392.7 285170.5 6 h post 6.50 37.8167 37.0 19.77 21525 1112.9 5720991 295801.4 3612240.1 186769.3 garlic intake 0.50 * calculated under the assumption 1 mL urine equals 1 g urine. 115 Metabolites 2016, 6, 43 6RI6 Table S4. Dietary record—test person b. Food Beverages st porridge with coconut blossom sugar coffee, juice break-fa S1 pizza with salami and ham water, cola, coffee lunch DAY 1 DAY S2 croissants juice dinner S3 st coffee, juice break-fa S1 pizza with salami and ham water lunch DAY 2 DAY S2 pasta with pepper, pesto, cream tea with honey dinner S3 “Alnatura Frühstücksbrei” (porridge, spelt, rice st coffee, water (puffed), linseed, different nuts) break-fa S1 panini with tomato and mozzarella tea lunch S2 SAMPLING DAY pasta with cream sauce water, tea dinner S3 S1, S2, S3: Snacks. 116 Metabolites 2016, 6, 43 6 RI6 Table S5. Dietary record—test person c. Food Beverages ast breakfast cereals milk break-f S1 cookies tea pretzel, roll with turkey, cheese and salad lunch lunch DAY 1 S2 apple, cake rice with tomato, egg dinner dinner S3 ast toast tea break-f S1 cookies roll with ham, cucumber, orange tea lunch lunch DAY 2 S2 apple, cookies salad, cutlet, orange dinner dinner S3 ast toast milky coffee break-f S1 cookies salad (ewe’s cheese, olives), bread lunch lunch S2 muffin SAMPLING DAY DAY SAMPLING cutlet, egg, orange dinner dinner S3 S1, S2, S3: Snacks. 117 Metabolites 2016, 6, 43 6RI6 Table S6. Dietary record—test person d. Food Beverages ast water break-f S1 roll with mild, full-fat cheese, tomato water lunch lunch DAY 1 S2 2 truffels scrampled eggs with pepper and basil, water, orange spritzer, Fanta classic 2 slices of sunflower-bread dinner dinner S3 apple cake ast water break-f S1 bagel with cream cheese and rocket, orange, chocolate bar with milk-filling water lunch lunch (Kinderriegel) DAY 2 S2 spelt-chocolate-cookie pizza water, orange spritzer dinner dinner S3 ast chocolate-cereal water, apple spritzer break-f S1 turkey-egg-round flat bread water, apple spritzer lunch lunch S2 rhubarb-pie SAMPLING DAY DAY SAMPLING pasta with ham water, lemonade dinner dinner S3 S1, S2, S3: Snacks. 118 Metabolites 2016, 6, 43 6RI6 Table S7. Dietary record—test person e. Food Beverages ast cereal coffee break-f S1 bratwurst, mustard, horseradish water lunch lunch DAY 1 S2 pasta with salmon, tomato and escallion water dinner dinner S3 pancake with chocolate, banana ast chocolate muffin, half and half cookie coffee break-f S1 water, cola lunch lunch DAY 2 S2 pancake, omelet with pepper cacao dinner dinner S3 ast peach, cereal coffee break-f S1 panini with tomato and mozzarella, water lunch lunch granary bread with cream cheese S2 SAMPLING DAY DAY SAMPLING pasta with vegetables, soy cream water dinner dinner S3 S1, S2, S3: Snacks. 119 Metabolites 2016, 6, 43 6RI6 Table S8. Dietary record—test person f. Food Beverages bread with Nutella, tea sausage spread st chocolate-cream-dessert cappuccino, water, orange juice break-fa rockmelon S1 naan bread au naturel with cheese, ham and rocket orange spritzer DAY 1 lunch lunch bread with sausage, meat salad S2 nut roll coffee with milk bread with meat salad, sausage, cheese Iso grapefruit (lemonade), water dinner dinner S3 chocolate-oat-cookie ast toast with cheese, ham, jam, Nutella coffee, orange spritzer break-f S1 chocolate-oat-cookie water - lunch lunch DAY 2 S2 sushi (artichoke, salmon, cucumber, carrot, orange spritzer sprouts, peanut), wasabi, ginger, soy sauce dinner dinner S3 brownie with vanilla ice-cream cider, Guiness, water (after garlic-intake) st prezel with pumpkin seeds, coffee with milk, water break-fa croissante with nut-nougat croissante S1 water sandwich tomato/mozzarella coffee with milk, water lunch lunch sushi with smoked salmon S2 donuts (raspberry, chocolate, nut) coffee SAMPLING DAY DAY SAMPLING sandwich with cheese, rocket, bacon water, passion fruit spritzer dinner dinner S3 cheeseburger, chickenburger (McDonalds) water S1, S2, S3: Snacks. 120 Metabolites 2016, 6, 43 6RI6 Table S9. Dietary record—test person g. Food Beverages ast - coffee break-f S1 roll with poppy seed water lunch lunch DAY 1 S2 yoghurt (mango, passion fruit) water dinner dinner S3 vanilla ice cream with red fruit jelly water ast - coffee break-f S1 Wrap with olives and feta cheese (no fruit-buttermilk, water lunch lunch garlic!) DAY 2 S2 buffalo mozzarella with tomato and olive white wine, water oil tea dinner dinner baguette with olives S3 ast yoghurt coffee, water break-f S1 champignon, parmesan cheese, olive oil, water lunch lunch ham, baguette with butter S2 Cornetto lemon (ice cream) Fanta champignon, parmesan cheese, olive oil, SAMPLING DAY DAY SAMPLING mozzarella, tomato, ham, baguette with water butter; dinner dinner yoghurt S3 S1, S2, S3: Snacks. 121 Metabolites 2016, 6, 43 6RI6 Table S10. Dietary record—test person h. Food Beverages ast -water break-f S1 bread (with butter) water lunch lunch DAY 1 S2 salad (tomato, cucumber, pepper, ewe’s water, alcohol-free wheat beer cheese, olives), buttered bread dinner dinner S3 Raffaelo ast - lemon tea (instant) break-f S1 tomato/ mozzarella with balsamic vinegar water lunch lunch and oil, bread DAY 2 S2 coffee with milk flasky pastries with tomato, feta, olives and beer basil dinner dinner S3 ast bread with cream cheese and cucumber water break-f S1 water flasky pastries with tomato, feta, olives and water lunch lunch basil S2 water salad with cucumber, tomato and SAMPLING DAY DAY SAMPLING mozzarella, bread with cream cheese and water dinner dinner cheese S3 S1, S2, S3: Snacks. 122 Metabolites 2016, 6, 43 6RI6 Table S11. Dietary record—test person i. Food Beverages ast peanut butter, Nutella and bread coffee break-f S1 tea pasta with mushroom sauce, tea lunch lunch fruits DAY 1 S2 fruits goulash soup, fruits tea dinner dinner S3 tea ast peanut butter and bread, fruits coffee break-f S1 pasta witch vegetables and tomato sauce tea, coffee lunch lunch DAY 2 S2 fruits chicken breast and eggs, fruits tea dinner dinner S3 ast bread and cheese, fruits coffee break-f S1 bread and cheese, fruits tea, coffee lunch lunch S2 SAMPLING DAY DAY SAMPLING pasta with tomato sauce, fruits tea dinner dinner S3 S1, S2, S3: Snacks. 123 Metabolites 2016, 6, 43 6RI6 Table S12. Dietary record—test person j. Food Beverages ast chocolate crisped rice with milk coffee break-f S1 pasta with cherry sauce water lunch lunch DAY 1 S2 ice cream with chocolate and nuts coffee cucumber, pepper, tomato, cheese, sausage water and roll dinner dinner S3 ast chocolate croissante coffee break-f S1 2 Leberkäse roll with pickled cucumber, 1 water lunch lunch ½ blueberry muffin DAY 2 S2 1 butter biscuit pasta with paprika pesto and tomatoes, 1 water berry-smoothie dinner dinner S3 ast 1 banana coffee, water with juice break-f S1 water with juice pasta with paprika pesto and tomatoes, 1 water with juice lunch lunch blueberry muffin S2 1 piece of cake water with juice SAMPLING DAY DAY SAMPLING gummi bears, potatoes with pepper cream cheese dinner dinner S3 S1, S2, S3: Snacks. 124 Metabolites 2016, 6, 43 6RI6 Table S13. Dietary record—test person k. Food Beverages ast soy yoghurt, pretzel breadstick with cheese coffee break-f S1 casserole with potatoes, carrots, paprika, Spezi (cola and lemonade mix) lunch lunch mincemeat DAY 1 S2 poppy seed strudel water with lime juice dinner dinner S3 ast poppy seed strudel coffee break-f S1 casserole with potatoes, carrots, paprika, water lunch lunch mincemeat DAY 2 S2 risotto with paprika, tomato and peas water dinner dinner S3 ast soy yoghurt coffee break-f S1 risotto with paprika, tomato and peas water lunch lunch S2 gummi bears Spezi (cola and lemonade mix) SAMPLING DAY DAY SAMPLING pizza margherita Punica, water dinner dinner S3 salt pretzel S1, S2, S3: Snacks. 125 Metabolites 2016, 6, 43 6RI6 Table S14. Dietary record—test person l. Food Beverages ast chocolate-vanilla pudding break-f S1 bread roll with bratwurst and mustard Spezi (cola and lemonade mix) casserole with potatoes, carrots, paprika, Spezi (cola and lemonade mix) lunch lunch mincemeat DAY 1 S2 roll with ham and cheese apple spritzer dinner dinner S3 ast poppy seed strudel break-f S1 casserole with potatoes, carrots, paprika, apple spritzer lunch lunch mincemeat DAY 2 S2 coffee risotto with paprika, tomato and peas apple spritzer dinner dinner S3 ast granary bread with ham break-f S1 risotto with paprika, tomato and peas Spezi (cola and lemonade mix) lunch lunch S2 gummi bears Spezi (cola and lemonade mix) SAMPLING DAY DAY SAMPLING pizza margherita Punica dinner dinner S3 S1, S2, S3: Snacks. 126 Metabolites 2016, 6, 43 6 RI6 Table S15. Dietary record—test person m. Food Beverages 3 slices rye-wheat bread, butter, Nutella, 2 1 tall cup of coffee with whole milk, 1 ast apricots glass of ACE-spritzer break-f S1 2 glasses of water polenta with stir-fried vegetables (zucchini, 1 glass of water lunch lunch pepper, kohlrabi) DAY 1 S2 carrot, nectarine and banana 2 glasses of water 5 slices rye-wheat bread (1x with butter, 2x 1 glass of water with liverwurst, 2x with cheese dinner dinner 2 glasses of water, 1 glas of currant S3 spritzer 3 slices rye-wheat bread, 1x liverwurst, 2x 1 tall cup of coffee with whole milk, 1 st butter and marmalade, glass of currant spritzer break-fa ½ apricot, ½ nectarine 1 glass of water, 1 glas of currant S1 spritzer 3 slices rye-wheat bread with cheese, carrot 1 glass of water DAY 2 lunch lunch S2 nectarine, apricot, Hanuta 2 glasses of water whole-grain pasta with minced meat sauce 1 glass of water (beef mince, tomato, pepper, peas), cheese dinner dinner S3 Hanuta, 2 Kinder-Schoko-Bons 1 glass of water, 1 glass of tonic 3 slices rye-wheat bread with Nutella, 1 tall cup of coffee with whole milk, 2 ast 1 apricot, ½ nectarine glasses of water break-f 2 glasses of water, 1 glass of apple S1 spritzer whole-grain pasta with minced meat sauce 1 glass of water lunch lunch (beef mince, tomato, pepper, peas), cheese 1 small cup of coffee with milk, 1 glass S2 Hanuta of water SAMPLING DAY DAY SAMPLING dinner dinner S3 S1, S2, S3: Snacks. 127 Metabolites 2016, 6, 43 6RI6 Table S16. Dietary record—test person n. Food Beverages ast 3 slices rye-wheat bread coffee with soy milk break-f S1 noodles with olive-tomato sauce (tomato purée, olives, basil, provençal herbs, olive lunch lunch DAY 1 oil, pizza soft cheese) S2 trail mix, gummi bears hash browns with apple sauce dinner dinner S3 nursing tea (fenugreek, fennel, anise, ast Alnatura breakfast puree with rice milk caraway, lemon verbena) break-f S1 eggplants with tomato sauce (according to the restaurant without onions and garlic, lunch lunch but with broth from Maggi) DAY 2 S2 bread with margarine and ham, cucumber dinner dinner S3 dark chocolate coffee with soy milk, ast vegan marble cake Fanta break-f S1 bread bun with butter, ham, tomato, water lunch lunch cucumber S2 water SAMPLING DAY DAY SAMPLING dinner dinner S3 S1, S2, S3: Snacks. 128 Metabolites 2016, 6, x 6RI6 Figure S1. Perceived intensity of a “garlic-/cabbage-like” odor in urine at different intervals before and after ingestion of raw garlic. Up to eight samples were collected before and after ingestion of 30 g (Urine a) or 3 g (Urine b–e) of raw garlic from different volunteers, whereby sets a, b and e were provided by the same subject but on different days. The first time interval (0 h) corresponds with the urine sampled prior to garlic consumption. Panelists were asked to rate the attribute “garlic-/cabbage-like” on a scale from 0 (no perception) to 3 (strong perception). The values shown represent the mean intensity ratings from all panelists. (a): odor intensity on a scale for 0 to 3; (b): for better visualization the intensity is presented up to 0.7. Figure S2. Time-resolved metabolite profiles of AMS, AMSO and AMSO2 for urine set a. AMS (¡), AMSO ( ), AMSO2 (c), time 0 h represents the urine sample collected prior to garlic consumption, subsequent time intervals represent urine samples after garlic consumption. Garlic was consumed 4 min after the first urine sample was collected. 129 Metabolites 2016, 6, x 6RI6 Figure S3. Time-resolved profiles of DMTS-excretion in urine for sets b to n. Time 0 h represents the urine sample collected prior the garlic consumption, following times represent urine samples after garlic consumption. Garlic was consumed 2 to 38 min after the first urine sample was given. (a) set b; (b)set c; (c) set d; (d) set e; (e) set f (f) set g; (g) set h; (h) set i; (i) set j; (j) set k; (k) set l; (l) set m; (m) set n. 130 Metabolites 2016, 6, x 6RI6 Figure S4. Time-resolved profiles of DMDS-excretion in urine for sets b to n. Time 0 h represents the urine sample collected prior the garlic consumption, following times represent urine samples after garlic consumption. Garlic was consumed 2 to 38 min after the first urine sample was given. (a) set b; (b)set c; (c) set d; (d) set e; (e) set f (f) set g; (g) set h; (h) set i; (i) set j; (j) set k; (k) set l; (l) set m; (m) set n. 131 Metabolites 2016, 6, x 6RI6 Figure S5. Scheme of urine sampling and analytical methods applied for analysis of the respective samples (a) for the high dosage test, (b) for the trial test (c) for the main test. 132 4XDQWLILFDWLRQ RI YRODWLOH PHWDEROLWHV GHULYHG IURP JDUOLF LQ KXPDQEUHDVWPLON Food Chemistry 2019, 274, 603-610;doi: 10.1016/j.foodchem.2018.09.039 /DXUD6FKHIIOHU&RQVWDQ]H6KDUDSD$QGUHD%XHWWQHU 'HSDUWPHQW RI &KHPLVWU\ DQG 3KDUPDF\ (PLO )LVFKHU &HQWHU )ULHGULFK$OH[DQGHU 8QLYHUVLWl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² Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Quantification of volatile metabolites derived from garlic in human breast milk ⁎ Laura Schefflera, Constanze Sharapab, Andrea Buettnera,b, a Chair of Aroma and Smell Research, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Henkestr. 9, 91054 Erlangen, Germany b Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauserstr. 35, 85354 Freising, Germany ARTICLE INFO ABSTRACT Keywords: Maternal garlic intake during pregnancy and the breastfeeding period has been reported to be associated with Gas chromatography-mass spectrometry the potential of modulating later garlic acceptance in infants. However, the metabolism of garlic constituents in Stable isotope dilution analysis (SIDA) humans and their elimination and potential excretion into human milk are not yet fully understood. In previous fi Ally methyl sul de studies, we identified volatile garlic-derived metabolites in human milk as well as in human urine, namely allyl Allyl methyl sulfoxide methyl sulfide, allyl methyl sulfoxide and allyl methyl sulfone. To monitor the excretion of these garlic meta- Allyl methyl sulfone bolites in a larger cohort, we quantified these metabolites in a total of 18 human milk sets, whereby each set comprised of one sample collected before and three samples after garlic consumption. The analyses revealed that the concentrations of the metabolites were most abundant 1–3.5 h after garlic consumption, with distinct dif- ferences between test persons regarding metabolite concentrations as well as temporal excretion. 1. Introduction alcohol consumption during breastfeeding time evidently compromises the child’s development (May et al., 2016), there is, however, no sci- Human milk is commonly the only food source of a neonate during entific evidence for any detrimental side-effects of garlic consumption the early stages of postnatal life. With its specific composition, human to the infant. Likewise, the common belief that garlic consumption of milk meets best the nutritional requirements of the neonate, in most the nursing mother leads to flatulence in the babies could not be con- cases irrespective of the maternal diet. However, there is evidence that firmed (Mennella & Beauchamp, 1993). Garlic consumption has rather intake of foods and beverages may have an influence on the aroma been associated with several beneficial health effects, at least for adults. profile of human milk, and several studies report sensorial changes of Among others, garlic has been described to exert anticarcinogenic, human milk in relation to the maternal diet. For example, Mennella and antimicrobial and antifungal effects (Rose, Whiteman, Moore, & Zhu, Beauchamp (1991) observed an odor change of human milk after mo- 2005). Furthermore, garlic has been reported to reduce the risk of thers ingested garlic. Although these changes were only observed by the cardiovascular diseases by lowering the blood pressure, reducing the mothers themselves, there is evidence that the potential flavor changes risk for arteriosclerosis, inhibiting platelet aggregation and improving of human milk may be detected by the infants. For example, breastfed cholesterol levels (Adler & Holub, 1997; Bayan, Koulivand, & Gorji, children remained for a longer time attached to the mother’s breast, and 2014; Ried, Frank, & Stocks, 2013). While allicin has been proposed as displayed more suckling behavior after their mothers had consumed an active compound (Cho, Rhee, & Pyo, 2006), the active compounds garlic (Mennella & Beauchamp, 1991). and modes of action that are responsible for the various effects are not In view of potential transmission processes into the milk, alcohol yet fully understood. Allicin is generated from the non-proteinogenic and garlic are commonly not recommended for nursing mothers. While amino acid alliin, being catalyzed by the enzyme alliinase (Ellmore & Abbreviations: AMS, allyl methyl sulfide; AMSO, allyl methyl sulfoxide; AMSO2, allyl methyl sulfone; APA, aroma profile analysis; DCM, dichloromethane; HRGC- GC-MS/O, two-dimensional high-resolution gas chromatography-mass spectrometry/olfactometry; HRGC-MS, high-resolution gas chromatography-mass spectro- metry; FID, flame ionization detector; LOD, limit of detection; LOQ, limit of quantification; m/z ratio, mass-to-charge ratio; ODP, olfactory detection port; ODV, odor detection value; ORV, odor recognition value; OTV, odor threshold value; SAFE, solvent-assisted flavor evaporation; SIDA, stable isotope dilution analysis; SIM, selected ion monitoring ⁎ Corresponding author at: Chair of Aroma and Smell Research, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Henkestr. 9, 91054 Erlangen, Germany. E-mail addresses: laura.scheffl[email protected] (L. Scheffler), [email protected] (C. Sharapa), [email protected] (A. Buettner). https://doi.org/10.1016/j.foodchem.2018.09.039 Received 28 May 2018; Received in revised form 31 August 2018; Accepted 4 September 2018 $YDLODEOHRQOLQH6HSWHPEHU (OVHYLHU/WG$OOULJKWVUHVHUYHG 135 L. Scheffler et al. )RRG&KHPLVWU\ ² Feldberg, 1994). Since alliin and alliinase are located in different cell 2.2. Human milk samples compartments, allicin is not present in the intact garlic bulb. Only when the cell structure is destroyed, e.g., by chewing or cutting, allicin is Human milk samples were obtained from 12 different mothers (age formed, which in turn is rapidly further decomposed, yielding diverse range 26–43 years, mean 34), whereby four mothers participated twice products, namely disulfides, trisulfides, dithiins and ajoenes. Several of and one mother participated three times in the experiments. Between these constituents contribute to the typical aroma of garlic (Egen- every repetition were time intervals of about four to five weeks. In total, Schwind, Eckard, Jekat, & Winterhoff, 1992; Freeman & Kodera, 1995; 18 milk sets were investigated. The sampling took place in the lactation Iberl, Winkler, & Knobloch, 1990), but may also explain the biological period from 9 to 41 weeks (mean 22 weeks) postpartum. All mothers activity of garlic (Bayan et al., 2014). However, to the best of our gave written consent to the analysis of their milk samples after a full knowledge, there are no data available on bioconversion rates and explanation of the purpose and nature of the study. Withdrawing from further bioavailability of these substances within the human body. the study was possible at any time. The Ethical Committee of the Moreover, compounds are often heavily metabolized after ingestion. Medical Faculty of the University Erlangen-Nuremberg approved the Apart from that there might be further metabolization steps before and/ study design (registration number 49_13B). The volunteers had no or during the transmission into breast milk (Buettner, 2013). Such a known illnesses and their breast milk production was normal and in functionalization has been demonstrated with the example of 1,8-ci- excess of their infantś need. For sampling, a mechanical breast milk neole, showing that a number of derivatives was secreted into human pump (Medela™ Harmony, Medela AG, Baar, Switzerland) was used. milk after oral administration of this compound (Kirsch, Beauchamp, & The participants were asked to list all food and beverages they Buettner, 2012; Kirsch, Horst, Röhrig, Rychlik, & Buettner, 2012). On consumed two days prior to sampling as well as on the test day. They the other hand, a transfer of odorous constituents of herbal tea (Denzer, were further instructed not to consume foods containing high amounts Kirsch, & Buettner, 2015) as well as of fish oil (Sandgruber, Much, of sulfur substances like garlic, onion, wild garlic, chives, cabbage and Amann-Gassner, Hauner, & Buettner, 2011) into human milk was ruled leek, over the time course of these three days. On the sampling day they out by means of expert sensory evaluations and chemo-analytical were asked to ingest approx. 3 g raw garlic, which was from a local odorant determination, leading to the conclusion that odorant trans- supermarket (Aldi-Sued, Erlangen, Germany), peeled and cut using a mission into human milk is likely to be related to not only the chemical garlic cutter (Genius GmbH, Limburg/Lahn, Germany), resulting in structures of the respective substances and their susceptibility to (bio) approx. 3-mm cubes. Each mother provided four consecutive milk chemical transformation, but also their relative concentration in the samples. One sample was collected prior to garlic intake whereas three consumed food. The higher the ingested dose the higher the potential of additional samples were taken afterwards, with two to four hours in- a breakthrough effect into the milk. Accordingly, the transfer process of tervals in each case and according to the natural lactation intervals of odorants into human milk is complex and not yet fully resolved. For each mother. other substances, this process has been shown to be influenced by several factors including the pH difference between plasma and milk, 2.3. Aroma profile analysis (APA) the lipophilicity of the respective compound and the mammary phy- siology of the individual mother (Agatonovic-Kustrin, Tucker, Zecevic, Sensory evaluations of the human milk samples were carried out by & Zivanovic, 2000; Fleishaker, 2003). a trained panel (2–8 participants, age range 23–34 years, mean 27) of With regard to garlic consumption we were recently able to identify the University Erlangen-Nürnberg (Erlangen, Germany). The panel was three garlic derivatives in human milk and urine, namely allyl methyl trained for at least three months in recognizing approx. 140 selected sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone odorants in different concentrations according to their odor qualities (AMSO2)(Scheffler, Sauermann, Heinlein, Sharapa, & Buettner, 2016; and in naming these based on an in-house developed flavor language. Scheffler, Sauermann, Zeh, et al., 2016). We could further demonstrate The training was performed weekly and included food-related (e.g., that these substances were only present in samples collected after garlic fruity, sweet or cheese-like odorants) as well as non-food related consumption. Of these compounds, AMS has been solely described as odorants (e.g., plastic-like or solvent-like odorants). For each com- constituent of garlic itself (Molina-Calle, Priego-Capote, & de Castro, pound a stock solution in ethanol was prepared. For the training the 2016; Yu, Wu, & Liou, 1989). However, further quantitative studies stock solution was diluted with water, considering that the resulting were required for a better assessment of the potential impact of such concentration was above the odor threshold value (OTV). Thereby the derivatives on the neonates. For this reason, the aim of the present training instructor decided which odorants were tested. The panelists study was to determine the concentrations of garlic-derived metabolites should learn the odor description according to the substance to estab- in human milk, and specifically to monitor their temporal excretion. As lish a joint flavor language. For orthonasal evaluation, samples were in our previous studies (Scheffler, Sauermann, Heinlein, et al., 2016; presented in covered glass vessels (capacity 140 mL). The panelists Scheffler, Sauermann, Zeh, et al., 2016) the main consideration was to were asked to score the intensities of different attributes on a scale from define a consumption protocol that was well in line with a real-life 0 (no perception) to 3 (strong perception). This scale was adapted from garlic consumption scenario. earlier studies and is well established in our research group (Kirsch,et al., 2012; Scheffler, Sauermann, Zeh, et al., 2016; Spitzer, 2. Materials and methods Klos, & Buettner, 2013). However, no information about the purpose of the experiment was given. The attributes were the same as already 2.1. Chemicals described in Scheffler, Sauermann, Zeh, et al. (2016) and were as fol- lows: hay-like, fishy, fatty, rancid, sweaty, metallic, grassy-green, Dichloromethane (DCM) and anhydrous sodium sulfate (Na2SO4) sweet, egg white-like, lactic, buttery and garlic/ cabbage-like. were purchased from VWR (Darmstadt, Germany). DCM was freshly distilled. The reference substances AMS and AMSO2 were obtained from 2.4. Determination of the odor threshold value (OTV) of AMS Aldrich (Steinheim, Germany), the reference standard AMSO from ar- omalab (Freising, Germany). The isotopically labelled standards 2.4.1. Determination of the OTV of AMS in water and goat milk 2 2 2 H3–AMS, H3-AMSO and H3-AMSO2 were supplied by aromalab The OTVs in water and milk were determined by performing a tri- (Freising, Germany). Goat milk (3.0% fat; Andechser Natur, Andechs, angle test, following the method described by Ramaekers, Verhoef, Germany) was purchased from a local supermarket (REWE, Erlangen, Gort, Luning, and Boesveldt (2016). As human milk is a very valuable Germany), and garlic (white, origin: Spain) was from a local super- and limited resource, goat milk was used as a substitute. Goat milk has market (Aldi-Süd, Erlangen, Germany). a similar composition compared to human milk, including, amongst 136 L. Scheffler et al. )RRG&KHPLVWU\ ² others, a complex range of nucleotides and high quantities of oligo- 2.6. High-resolution gas chromatography-mass spectrometry (HRGC-MS) saccharides (Prosser, McLaren, Frost, Agnew, & Lowry, 2008; Kumar et al., 2012). The test consisted of three covered glass vessels, one For GC–MS analyses an Agilent MSD quadrupole system (GC 7890A containing an AMS solution and two containing controls (water or goat and MSD 5975C; Agilent Technologies, Waldbronn, Germany) equipped milk, respectively). For determination of the threshold value, a stock with a GERSTEL MPS 2 autosampler and a GERSTEL CIS 4 injection solution of AMS of 1144 μg/mL ethanol was prepared. Then, 1 mL of system (GERSTEL, Duisburg, Germany) was applied. The analytical this solution was diluted in 50 mL of water or goat milk, and stepwise capillary was a DB-FFAP (30 m × 0.25 mm, film thickness 0.25 μm; diluted further in a ratio 1:3. Volunteers (trained panel: 7–9 partici- Agilent J&W Scientific, Santa Clara, CA). Uncoated, deactivated fused pants, age range 22–36 years, mean 28; untrained panel: 4–9 partici- silica capillaries (2–3 m × 0.53 mm) were used as pre-columns and pants, age range 26–31 years, mean 27) from the Friedrich-Alexander changed regularly to avoid accumulation of impurities. Another un- University Erlangen-Nürnberg, Department of Chemistry and Phar- coated fused silica capillary (0.3–0.6 m × 0.25 μm) was connected to macy, participated in these experiments. the end of the analytical capillary as a transfer line into the MS. Helium For determination of the OTV in water 42 vessels and for the de- was used as a carrier gas at a flow rate of 1.0 mL/min. Mass spectra termination of the OTV in goat milk 30 vessels were presented to the were recorded at 70 eV in full scan mode (mass-to-charge ratio (m/z) panelists, corresponding to 14 and 10 triangle tests, respectively. The range 30–350) as well as in selected ion monitoring (SIM) mode. For AMS concentration in the different testing levels differed by a factor of SIM conditions refer to section 2.9 Quantification by stable isotope dilu- 3 and tests were conducted in ascending order. All vessels were labeled tion assay (SIDA). The temperature program of the oven was as follows: with randomized three-digit numbers and the position of the vessels, The initial temperature of the oven (40° C) was held for 7 min and then containing the AMS solution, was randomized as well. The samples raised at 8 °C/min to 240 °C and held for 8 min. Injection volume was were presented at room temperature. The panelists were instructed to 2 μL. These parameters were adapted from Scheffler, Sauermann, Zeh, uncap and smell the samples in the vessels and choose the sample that et al. (2016). differed from the other two samples. Samples where odor association was possible (odor recognition value, ORV) were indicated separately, 2.7. Two-dimensional high-resolution gas chromatography-mass in comparison to those where identification of difference only, without spectrometry/olfactometry (HRGC-GC–MS/O) (heart-cut) any further smell specification, was achieved (odor detection value, ODV). The sensory determinations were performed three times on three A two-dimensional gas chromatographic system in combination different days. with a mass spectrometer was applied for the mass spectrometric The ODV was calculated as geometric mean in each case taking into quantification of AMS. The method used within this study was adapted consideration the first concentration detected (without the requirement from Scheffler, Sauermann, Zeh, et al. (2016). The system consisted of to identify the odor) and the last concentration that was missed. The two Varian 450 GCs in combination with a Varian 220 ion trap MS ORV was calculated as geometric mean between the first concentration (Varian, Darmstadt, Germany). The first GC was equipped with a multi- where the odor could be identified and the preceding concentration. column switching system MCS 2 and both GCs were connected via a The group threshold of AMS in each medium was obtained by calcu- cryo-trap system CTS 1 (both GERSTEL, Duisburg, Germany). The lating the geometric mean of the individual thresholds in each case. analytical capillaries were a DB-5 column (30 m × 0.32 mm, film thickness 0.25 μm (Agilent J&W Scientific, Santa Clara, CA); first oven) and a DB-FFAP column (30 m × 0.25 mm, film thickness 0.25 μm 2.4.2. Determination of the OTV of AMS in air (Agilent J&W Scientific, Santa Clara, CA); second oven). An uncoated, The OTV of AMS in air was determined by gas chromatography- deactivated fused silica capillary was used as pre-column olfactometry (GC-O) with (E)-2-decenal as internal standard (Czerny, (2–3 m × 0.53 mm) as described above. Carrier gas was helium at a Brueckner, Kirchhoff, Schmitt, & Buettner, 2011). Two microliters of flow rate of 2.5 mL/min. In the first oven the effluent was split between every dilution were applied for injection into the GC system. The ana- a flame ionization detector (FID) and olfactory detection port (ODP, lyses were performed using a DB-5 capillary column (30 m × 0.32 mm, GERSTEL), as well as a cryo-trap during the cut interval. In the second film thickness 0.25 μm; Agilent J&W Scientific, Santa Clara, CA) with oven the effluent was transferred directly to the MS. All split capillaries the following temperature program: starting temperature was 40 °C, were made of uncoated, deactivated fused silica material. The FID and which was held for 7 min, then the temperature was raised to 250 °C at the sniffing port were held at 250 °C and 260 °C, respectively. Mass a rate of 15 °C/min. This temperature was held for 5 min. Helium was spectra were recorded at 70 eV in full scan mode (m/z range 30–100). used as a carrier gas with a flow rate of 2.0 mL/min. The thresholds Application of the sample (4 μL) was performed at 40 °C using the cold- were determined by ten panelists (age range 23–36, mean age 28), with on-column technique. The oven was held at this temperature for 7 min, each experiment being conducted once. The group threshold was ob- then the temperature was raised at 20 °C/min to 300 °C for the first tained as the geometric mean of the individual thresholds. oven and to 240 °C for the second oven. The final temperature was held for 5 min. 2.5. Solvent-assisted flavor evaporation (SAFE) of volatiles from human 2.8. Determination of the limits of detection (LOD) and quantification milk (LOQ) Solvent-assisted flavor evaporation (SAFE) (Engel, Bahr, & LOD and LOQ were determined according to DIN 32645. DIN 32645 Schieberle, 1999) was applied for the isolation of the volatile fraction of describes the calibration line method as one way to determine LOD and the milk samples. After addition of the respective deuterated standards LOQ: A calibration line of the respective compound is determined in the and stirring for 10 min, DCM was added to the milk samples in a ratio range of the LOD (from about 0 up to 10 × the LOD) by adding the 1:2 (DCM: human milk; v/v). The solution was stirred for another compound in different concentrations to the blank samples. 30 min and then distilled at 50 °C using the SAFE apparatus. The ob- Subsequently, LOD and LOQ can be calculated via different formulas, tained distillate was then extracted three times with 25 mL DCM and which are provided in the DIN. To achieve this aim, goat milk was used the combined solvent phases were dried over anhydrous Na2SO4 and as a substitute for human milk. Unlabeled and labeled reference com- concentrated to a total volume of 100 μL by means of Vigreux distilla- pounds were added to the goat milk in the concentration range of the tion and micro-distillation at 50 °C. LOD and LOQ. Subsequently the samples were worked up as described above (cf. 2.5 Solvent assisted flavor evaporation (SAFE) of volatiles from 137 L. Scheffler et al. )RRG&KHPLVWU\ ² human milk) and analyzed by means of GC–MS and GC–GC–MS analyses Table 1. − as described in 2.6 and 2.7. As the provided human milk samples were In air, the OTV was found to be lowest with 9.83 × 10 4 μg/L air. highly variable with regard to their volume and weight, the respective However, this OTV does not reflect the influence of the matrix on the LOD and LOQ values were calculated as absolute amounts (in ng). disposability of the respective aroma compound. For a realistic re- presentation of the smell situation in an aqueous or lipophilic matrix, respectively, we determined the OTVs of AMS in water and in goat 2.9. Quantification by stable isotope dilution assay (SIDA) milk. The ORVs of AMS in water and goat milk were 1.22 μg/L and 4.22 μg/L, respectively. At these concentrations the panelists were able Quantification was performed using the concept of SIDA (Schieberle & Grosch, 1987). The isotopically labelled standards were dissolved in to recognize the characteristic garlic/cabbage-like odor quality of AMS. The ODVs, the concentrations at which the panelists noted a difference DCM and added to the milk samples containing the respective analytes at similar concentrations as determined in preliminary experiments. but could not determine the odor quality, were slightly lower than the ORVs, namely 0.54 μg/L (water) and 1.52 μg/L (goat milk). The mixture was worked up as described above. For AMSO and AMSO2, the quantification was performed by GC–MS in SIM mode. The m/z- It is interesting to note that the determined amounts of AMS in the human milk samples were in the same range as the OTVs of AMS in ratios 104 and 107 were selected for analyses of AMSO and the re- spective isotopically labelled standard and the m/z-ratios 120 and 123 water and goat milk, but that the ORV of AMS in goat milk was higher than the AMS contents in human milk after garlic consumption. were used for the determination of AMSO2 and its respective labelled analogue. The quantification experiments for AMS were carried out by However, the garlic/cabbage-like odor of human milk after garlic consumption was verified in several milk sets. These findings indicate GC-GC–MS, where the selected ions of the analyte and the labeled standard, m/z 88 and 91, were analyzed in full scan mode. that the ORV of AMS in goat milk does not fully represent the ORV of AMS in human milk. This could be due to the higher protein content of The concentrations were calculated and corrected using the MS response factors obtained by measuring defined mixtures of the labeled goat milk compared to human milk, resulting in more interactions be- tween AMS and proteins. Furthermore, goat milk has a slightly higher compound and the analyte. overall aroma than human milk, with a characteristic note. Therefore, the slight aroma change caused by AMS is likely to be less detectable in 3. Results and discussion goat milk compared to human milk. Nevertheless, we were able to show that the determined AMS con- 3.1. APA and OTV of AMS centrations in human milk are in the range of the ORV and ODV of AMS. This coincides with the findings of the APAs, where the garlic/ APA was performed on all human milk samples. The milk samples cabbage-like attribute was rated as 1 (or lower), equaling a weak per- collected after garlic intervention were found to be characterized by a ception. Only in single cases was this attribute was rated higher. In slight garlic/cabbage-like aroma, which was not present in the milk agreement with that, the AMS content of these samples exceeded the samples taken before garlic consumption. However, all milk samples ORV of AMS in water. There are also indications that infants are able to were rated with an overall odor intensity of 1.5 or lower, indicating that detect garlic-derived odor in human milk. In a study by Mennella and the milk samples had only a slight overall aroma. These results are in Beauchamp (1991) infants spent more time on the breast and showed fi ffl line with the ndings of our previous study (Sche er, Sauermann, Zeh, an intensive suckling behaviour after their mothers consumed garlic. et al., 2016), where samples were also found to exert just slight odor Likewise, the sensory panel rated these milk samples with an increased fi notes. The respective odor pro les are provided in the Supplementary intensity (Mennella & Beauchamp, 1991). Although one cannot directly material S1. In our previous study we were able to identify AMS as the conclude that infants and adults perceive AMS in the same concentra- aroma active compound that was responsible for this aroma change. tion, meaning they have the same OTV for AMS, it seems reasonable To further characterize the impact of AMS on the aroma change of that in the present study infants were also able to detect AMS in milk milk after garlic intervention, the OTV of AMS was investigated. The samples provided after garlic consumption. OTV of a compound is of special interest, as the compound can only be perceived orthonasally when its concentration in the matrix of interest 3.2. Quantitative analysis of garlic-derived metabolites in human milk exceeds its specific OTV (Belitz, Grosch, & Schieberle, 2008). The OTV ff depends on several factors, including matrix e ects as well as the age of The excretion of garlic-derived metabolites, namely AMS, AMSO the test person (Murphy, 1983). In the current study, we separately and AMSO2, in human milk was observed over a time-period of up to regarded the OTV of AMS in air, water and goat milk, respectively. 11 h. The metabolites were quantified by SIDA. Values were recorded Moreover, the ODV and the ORV values were additionally determined for milk samples that were taken before and after garlic consumption both for water and for goat milk. The determined OTVs are shown in from 12 individual mothers. Four of those mothers repeated the ex- periment once, and one mother repeated the experiment twice. The Table 1 obtained results are given in Table 2. Further information for each Odor threshold values (OTV) of AMS in air, water and milk. The average, the sample, e.g., exact sampling time, volume and weight of the sample, are minimum and the maximum OTV for each medium is presented. The average provided in Supplementary material S2. values were calculated as geometric means of all individual determinations. The In most cases AMS, AMSO and AMSO were only detectable in OTV in air was determined by GC-O, whereas the OTVs in water and goat milk 2 were determined by triangle tests. ODV: Odor detection value, ORV: Odor re- human milk samples that were collected after garlic consumption. Only cognition value. in the case of three milk sets (HM XII, HM XIII and HM XVIII) small amounts of the investigated metabolites were detected in samples that OTV in Average (μg/L) Minimum (μg/L) Maximum (μg/L) were collected prior to garlic consumption. According to the food re- − − − Air(a) 9.83 × 10 4 3.02 × 10 4 2.42 × 10 3 cords that were provided by the mothers it could be deduced that they Water(b) ODV 0.54 0.07 16.31 had consumed foods like bratwurst, liver sausage or Asian noodles, ORV 1.22 0.07 16.31 namely foods that often contain onions and, accordingly, sulfurous (c) Goat milk ODV 1.52 0.20 48.93 substances (Keusgen et al., 2002). This might serve as an explanation ORV 4.22 0.20 48.93 for the metabolites found in the samples prior to garlic consumption. (a) OTV determined by 10 panelists. However, despite the presence of these compounds in the first milk (b) OTV determined by 19 panelists (in 53 experiments). sample, a distinct increase in the metabolite concentrations in the fol- (c) OTV determined by 15 panelists (in 37 experiments). lowing samples after garlic consumption was observed, confirming the 138 L. Scheffler et al. )RRG&KHPLVWU\ ² Table 2 Table 2 (continued) Concentrations of garlic-derived metabolites (AMS, AMSO and AMSO2)in human milk samples (in μg/kg human milk). HM: Human milk. HM x-1 cor- Sample AMS μg/kg human AMSO μg/kg AMSO2 μg/kg responds to the sample gathered prior to garlic consumption. HM x-2 to HM x-4 milk human milk human milk correspond to the samples gathered after garlic consumption. HM XVId 1 < LOD1 < LOD2 < LOD3 2 1.47 61.02 99.30 Sample AMS μg/kg human AMSO μg/kg AMSO2 μg/kg milk human milk human milk 3 0.38 24.34 87.66 4 0.56 14.20 68.62 1 2 3 HM I 1 < LOD < LOD < LOD HM XVIIb 1 < LOD1 < LOD2 < LOD3 1 2 traces 30.01 48.68 2 2.22 124.01 141.38 3 0.62 14.79 46.33 3 1.12 53.85 137.38 1 4 < LOD 7.71 33.68 4 1.00 20.56 91.10 1 2 3 HM II 1 < LOD < LOD < LOD HM XVIIIe 1 traces1 < LOD2 5.13 2 0.65 40.28 31.66 2 4.06 110.39 154.30 3 0.62 30.25 51.65 3 3.10 50.60 128.86 4 0.49 13.42 45.12 4 1.23 23.34 93.84 HM III 1 < LOD1 < LOD2 < LOD3 a,b,c,d,e 2 2.75 144.85 120.33 Same letters represent milk sets provided by the same mother. 3 4.01 70.91 134.38 traces: > LOD and < LOQ. 4 2.92 42.61 124.36 1 LOD (AMS): 2.4 ng, LOQ (AMS): 7.4 ng. 2 HM IV 1 < LOD1 < LOD2 < LOD3 LOD (AMSO): 27.7 ng, LOQ (AMSO): 95.3 ng. 3 2 2.32 132.63 144.04 LOD (AMSO2): 25.6 ng, LOQ (AMSO2): 89.4 ng. 3 1.44 57.79 72.34 4 0.86 19.66 98.76 additional contribution of the garlic intervention to the overall meta- HM Va 1 < LOD1 < LOD2 < LOD3 bolite profile. Moreover, the presence of these metabolites in milk 2 1.66 78.05 123.62 samples after consumption of foods like bratwurst confirms that they 3 0.47 17.22 79.85 are not restricted to consumption of 3 g of raw garlic, but can also be 4 0.30 8.87 54.73 present after consumption of processed garlic. b 1 2 3 HM VI 1 < LOD < LOD < LOD The determined amounts of the respective metabolites were ad- 2 1.62 72.62 99.07 3 0.61 33.37 88.24 ditionally plotted, for better visualization, in a scatter plot (see Fig. 1). 4 0.50 15.50 64.16 It further became evident that, following the initial onset, all three HM VII 1 < LOD1 < LOD2 < LOD3 metabolites showed a steady decline in concentration during the time 2 3.39 141.11 129.15 period of metabolite monitoring. Apart from that, large inter-individual 3 2.09 87.63 142.08 differences could be observed when comparing milk sets obtained from 1 4 < LOD 39.00 88.66 different mothers. These differences involved the concentrations as well HM VIIIc 1 < LOD1 < LOD2 < LOD3 as the time-dependent excretion of the metabolites. Whereas all mo- 2 3.96 121.28 200.26 thers consumed about 3 g of raw garlic, the highest observed AMS 3 1.30 35.13 128.99 concentrations within a sample set ranged between 0.62 and 4.01 μg/kg 4 1.13 33.50 113.84 human milk, and the highest concentrations of AMSO and AMSO2 be- d 1 2 3 HM IX 1 < LOD < LOD < LOD tween 30.0 and 145.0 μg/kg human milk, and between 48.7 and 2 1.18 48.32 77.85 3 0.48 20.27 64.98 200.3 μg/kg human milk, respectively. That means, the individual 4 0.43 11.11 43.59 maxima in the milk sets differed by a factor of up to 6.5 (AMS), 4.8 (AMSO) and 4.1 (AMSO ). However, even if the concentrations varied HM X 1 < LOD1 < LOD2 < LOD3 2 2 0.69 41.78 45.70 considerably between milk sets, a high concentration of AMSO2 was 3 0.55 37.61 65.49 commonly associated with a high concentration of AMSO and AMS, and 4 0.51 23.68 61.65 vice versa. Regarding the temporal excretion, we commonly observed HM XI 1 < LOD1 < LOD2 < LOD3 the highest concentrations of AMS and AMSO within each milk set in 2 2.07 81.83 56.76 the first sample gathered after garlic intervention, which corresponded 3 0.73 55.88 75.48 to a time of about 2–3.5 h after garlic consumption. Only the milk sets 4 0.64 20.99 59.37 HM I and HM III revealed the highest AMS concentration in the second e HM XII 1 0.32 3.15 14.21 sample collected after garlic consumption (about 5 h after garlic con- 2 1.96 37.13 56.56 sumption). In the case of AMSO , however, the highest concentration 3 1.23 17.74 51.14 2 fi 4 0.54 10.36 37.18 was commonly found either in the rst (applicable for 12 milk sets) or in the second sample (applicable for 6 milk sets) after garlic con- HM XIIIb 1 traces1 traces2 4.83 2 1.56 112.25 123.77 sumption. Thereby, the latter corresponded to a time of about 5 h after 3 1.26 51.96 124.45 garlic consumption. 4 0.58 21.16 86.12 For better visualization, the generally high inter-individual differ- HM XIVa 1 < LOD1 < LOD2 < LOD3 ences in the excretion profiles are displayed with the examples of HM II, 2 1.08 43.85 116.72 HM VIII and HM XI in Fig. 2. These cases highlight the different me- 3 0.63 28.67 93.76 tabolite concentration ranges, with the substance concentrations in HM 4 0.33 12.36 66.73 II being in a low concentration range of about 40–50 μg/kg in case of HM XVc 1 < LOD1 < LOD2 < LOD3 AMSO and AMSO2 and below 1 μg/kg in case of AMS and HM VIII in a 2 2.57 56.79 101.04 comparatively high concentration range of 100–200 μg/kg in case of 3 traces1 19.99 70.34 4 traces1 12.66 59.18 AMSO and AMSO2 and about 4 μg/kg in case of AMS. Furthermore, different temporal profiles of excretion of the respective metabolites were observed: On the one hand, the highest concentrations of the three garlic-derived metabolites were detected at the same time point in 12 139 L. Scheffler et al. )RRG&KHPLVWU\ ² Fig. 2. Time resolved metabolite-profiles of AMS, AMSO and AMSO2 for HM II, Fig. 1. Concentrations of garlic-derived metabolites in human milk samples (in HM VIII and HM XI (milk sets provided by different test persons). AMS (♦), μg/kg human milk). Milk samples of 18 sets are plotted, whereby each set AMSO (■), AMSO2 (●). Time 0 h represents the time of garlic consumption. consisted of four milk samples (one collected prior to and three collected after The displayed time points represent the milk samples HM x-1 to HM x-4 ac- ♦ ■ garlic consumption). Time 0 h represents the time of garlic intake. ( ) AMS; ( ) cording to Table 2. AMSO; (●) AMSO2. et al., 2017). Accordingly, it can be assumed that the interconnected ff di erent milk sets (e.g. HM VIII). On the other hand, there were also network of diverse secretion and elimination pathways, let alone milk sets where the maximum of AMSO2 occurred with temporal delay elimination via breath and smell, are complex and intertwined, as are after appearance of the maximum of AMS and AMSO (applicable for six the respective inter-individual influencing factors guiding these pro- milk sets, e.g. HM II and HM XI). cesses. Potential reasons for these variations might be: the physiological In view of this, it is interesting to note that we could even observe status of the mother. For example, body mass index has been shown to differences in the excreted metabolite profiles in different milk sets that fl in uence the metabolism rate in other metabolic processes, which had been provided by the same mother. The metabolite profiles of three might be the case here as well (Bachour, Yafawi, Jaber, Choueiri, & milk sets that were provided by the same test person and comprise Abdel-Razzak, 2012). Also lactation period has been shown to impact comparative sampling times are provided in Fig. 3. Although the tem- fl the composition of human milk and might therefore in uence meta- poral progress of metabolite excretion was comparable between the bolite content and excretion. Apart from that, metabolism and (pri- testing days, we observed differences in the excreted metabolite con- marily renal) excretion commonly also involves conjugated metabo- centrations. In the case of the represented milk sets, a later lactation lites, being coupled to, e.g., glutathione, acetyl or cysteine conjugates. week was associated with an increased metabolite concentration. This Ratios of metabolite formation yielding either the conjugated or free trend was confirmed by other repetitions, such as HM IX and HM XVI as form is highly dependent on the individual physiology as is the re- well as HM XII and HM XVIII. However, in the case of milk sets HM VIII spective route of excretion, as has been demonstrated for other aroma and HM XV a reversed trend was monitored. Due to these observations substances in our previous studies (Kirsch et al., 2012; Kirsch et al., as well as the limited data available (only five mothers repeated the 2012; Wagenstaller & Buettner, 2013a, 2013b). As an example, N- experiment), the differences in the metabolite profiles could not be acetyl-S-allyl-L-cysteine has previously been reported in human urine linked to the lactation period alone. It is more reasonable that the ex- after garlic intervention (de Rooij, Boogaard, Rijksen, Commandeur, & cretion process into human milk is subject to a variety of parameters. Vermeulen, 1996). Moreover, S-allyl-L-cysteine sulfoxide, N-acetyl-S- Next to the lactation period also the composition of the maternal diet, allyl-L-cysteine and N-acetyl-S-allyl-L-cysteine sulfoxide have been de- especially its lipophilicity/hydrophilicity, might affect these processes. tected in rat plasma after oral administration of S-allyl-L-cysteine (Park Apart from that, we observed that the decrease in metabolite 140 L. Scheffler et al. )RRG&KHPLVWU\ ² the gastrointestinal tract, as has been proposed in other studies, which might account for different excretion times. Gastric absorption has been reported for different drugs in rat and humans (Hogben, Schanker, Tocco, & Brodie, 1957; Schanker, Shore, Brodie, & Hogben, 1957). Microbial processes in the colon might further influence resorption processes and composition of metabolites as well as excretion processes, as is already well established for other substances, such as short-chain fatty acids (Bergman, 1990). Summarizing the above-said, the metabolite profiles observed in this study are characterized by a rapid onset of the excretion of the respective substance during the first 1–3.5 h after garlic consumption, which was followed by a decrease that was at first distinct but slowed down in the last observed milk sample (equals about 7–10 h). Generally, the excretion of AMSO2 was found to be highest and the most persistent. Interestingly, Germain, Auger, Ginies, Siess, and Teyssier (2002) observed similar effects in an animal study, but on a much larger scale: They administered 200 mg/kg diallyl disulfide, a decomposition product of allicin, and observed the metabolite profiles in stomach, liver, plasma and urine for 15 days. Compared to the pre- sent study the administered amount of diallyl disulfide exceeded by far the amount of garlic consumed by the mothers. In detail, 3 g of garlic would equal a diallyl disulfide amount of about 0.25 mg/kg, assuming an average body weight of the test persons of 60 kg and an allicin content of about 0.5% (Iberl, Winkler, Muller, & Knobloch, 1990) whereby allicin and diallyl disulfide result in the same metabolites. Nevertheless, they recorded similar excretion courses for the garlic- derived metabolites in different media. In the case of AMSO2, for ex- ample, they observed a maximum within the first days after adminis- tration, after which the levels of AMSO2 declined. However, the decline was not linear but formed a plateau between the third and the fifth day after administration. The authors attributed this observation to an in- teraction of the metabolites with proteins and fats, resulting in the formation of the plateau, but resorption processes at different locations within the gastrointestinal tract may also account for this effect, as discussed above. Overall, however, the concentration of AMSO2 was higher in their study in comparison to AMS and AMSO, and was also the most persistent, which is in agreement with our findings. In view of potential physiological effects of the metabolites de- scribed in our study as excretion products via human milk, it is inter- esting to note that a recent study reported the influence of garlic me- tabolites on cardiovascular disease (Khatua et al., 2017). However, no data are available concerning the required concentrations of the re- spective compounds for a factual physiological effect. In view of this it is important to keep in mind the relatively low concentrations of the monitored metabolites that were detected in human milk. Accordingly, at present, no assumptions can be made regarding possible physiolo- gical effects of these metabolites, besides their sensory impact, either on mother or infant. 4. Conclusion Fig. 3. Metabolite-concentrations of AMS, AMSO and AMSO2 for HM VI, HM This study presents the impact of maternal garlic consumption on XIII and HM XVII according to Table 2 (milk sets provided by the same test human milk composition. person at different testing days). HM x-1 represents the milk sample collected The excretion of the odor-active AMS and two further garlic-derived before garlic consumption, whereas HM x-2 to HM x-4 represent the milk samples obtained after garlic consumption. All milk sets were provided by the metabolites, namely AMSO and AMSO2, was monitored in 18 milk sets. fi same mother with time periods of about 5 weeks between the different testing Quanti cation revealed that the lactic excretion of these metabolites days. was highest 1–3.5 h after garlic consumption. These findings are the basis for further investigations into this topic, thereby monitoring the excretion of metabolites under more defined parameters, e.g. a specific concentrations slowed in the last sample of a milk set. This was parti- lactation week. Furthermore, other potentially less volatile metabolites, cularly noticeable in the case of AMS excretion (cf. Fig. 2, HM VIII and such as acetyl- or glutathione-conjugates, should be considered in fu- HM XI). In the case of HM XVI even a slight increase in the con- ture studies. centration of AMS in the last sample compared to the previous sample Based on these results future studies could investigate the effect of was observed. This could be an indication that garlic constituents are garlic-derived metabolites on the infant, thereby addressing not only not only resorbed in the small intestine but at different locations within possible positive health effects but also negative side effects. 141 L. Scheffler et al. )RRG&KHPLVWU\ ² Acknowledgments and dithiins, characterization and their determination by hplc. Planta Medica, 56(2), 202–211. Iberl, B., Winkler, G., Muller, B., & Knobloch, K. (1990). Quantitative-determination of This work was supported by the German Research Foundation allicin and alliin from garlic by HPLC. Planta Medica, 56(3), 320–326. (DFG) [BU 1351/15-1]. We are grateful to the mothers for providing Keusgen, M., Schulz, H., Glodek, J., Krest, I., Kruger, H., Herchert, N., & Keller, J. (2002). samples of their milk Furthermore, we are grateful to Yvonne Characterization of some Allium hybrids by aroma precursors, aroma profiles, and aillinase activity. Journal of Agriculture and Food Chemistry, 50(10), 2884–2890. Sauermann for skillful technical assistance. Khatua, T. N., Borkar, R. M., Mohammed, S. A., Dinda, A. K., Srinivas, R., & Banerjee, S. K. (2017). 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published: 16 April 2019 doi: 10.3389/fnut.2019.00043 Quantification of Volatile Metabolites Derived From Garlic (Allium sativum) in Human Urine Laura Scheffler 1, Constanze Sharapa 1 and Andrea Buettner 1,2* 1 Chair of Aroma and Smell Research, Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, 2 Department Sensorical Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Freising, Germany The consumption of garlic (Allium sativum) is widely known to (negatively) impact body odor, in particular breath and sweat, but also urine. Despite this common phenomenon, the underlying processes in the body that lead to the malodor are not yet fully understood. In previous studies we identified three volatile garlic-derived metabolites in human milk and urine, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO), and allyl methyl sulfone (AMSO2). In the present study, we monitored the excretion processes of these metabolites via human urine after consumption of garlic over time, whereby 19 sets of eight urine samples (one sample pre-ingestion and seven samples post-ingestion) were analyzed using two-dimensional high resolution Edited by: gas chromatography-mass spectrometry/olfactometry (HRGC-GC-MS/O). The highest Dejian Huang, concentrations of these metabolites were detected in urine ∼1–2 h after garlic ingestion, National University of Singapore, Singapore with a second increase observed after 6–8 h in the urine of some participants. Moreover, Reviewed by: the highest observed concentrations differed between the individual participants or test Michael Erich Netzel, series by up to one order of magnitude. University of Queensland, Australia Restituto Tocmo, Keywords: gas chromatography-mass spectrometry, stable isotope dilution assay (SIDA), allyl methyl sulfide, allyl University of Wisconsin-Madison, methyl sulfoxide, allyl methyl sulfone, human urine United States *Correspondence: Andrea Buettner INTRODUCTION [email protected] Garlic (Allium sativum) is used since centuries as a condiment to refine the taste of dishes. Specialty section: However, garlic intake is often followed by a change in body odor, being commonly recognized This article was submitted to in breath (1), sweat (2) but also in human milk (3–5). Besides its typical aroma, garlic is also Food Chemistry, known for its beneficial health properties, including antimicrobial and immunostimulating effects, a section of the journal protective effects against cancer and reduction of the risk of cardiovascular diseases (6). The Frontiers in Nutrition health promoting effects as well as the impact on the body odor are mostly associated with the Received: 23 August 2018 organosulfur compounds being major constituents of garlic, like the thiosulfinate allicin and its Accepted: 26 March 2019 various degradation products (e.g., diallyl disulfide, diallyl trisulfide, ajoene) (7–10). However, food Published: 16 April 2019 components, including volatiles, can be strongly modified by metabolism and biotransformation Citation: processes within the human body (11–13). Recent research has shown, that such transformations Scheffler L, Sharapa C and Buettner A of aroma substances are not only limited to the liver but can already take place within the (2019) Quantification of Volatile Metabolites Derived From Garlic gastrointestinal tract (14) or during absorption processes (15). This can either lead to a loss of the (Allium sativum) in Human Urine. respective bioactivity but can also result in the formation of newly generated bioactive compounds. Front. Nutr. 6:43. In our previous studies, we identified three garlic-derived metabolites, namely allyl methyl sulfide doi: 10.3389/fnut.2019.00043 (AMS), allyl methyl sulfoxide (AMSO), and allyl methyl sulfone (AMSO2) in human milk as well as Frontiers in Nutrition | www.frontiersin.org 1 April 2019 | Volume 6 | Article 43 157 Scheffler et al. Garlic-Derived Metabolites in Human Urine in urine (5, 16). Based on these observations, the aim of the of 3 g. In total, eight consecutive urine samples were provided by present study was to quantify these metabolites in human urine, each test person, comprising one whole urine set. The samples specifically to characterize differences in the metabolite excretion were collected in sterile brown glass bottles, with sampling times between different test persons, and to monitor their temporal as follows: one sample was provided directly prior to garlic development over the course of excretion within a time period of consumption. The series of the second to eighth sample was up to 24 h. To allow for better comparison between individuals, then obtained at about 0.5, 1, 2, 4, 6, 8, and 24 h after garlic test persons consumed portions of one garlic bulb in each case, consumption, respectively. Between the first urine sample (prior and urine profiles were tested in parallel. to garlic consumption) and the seventh urine sample (8 h after garlic consumption) test persons only spent urine at the specific time points mentioned before. Another sample was provided at MATERIALS AND METHODS the next morning (24 h after garlic consumption). Between the Chemicals samples provided 8 and 24 h after garlic consumption no urine was collected. All samples were immediately placed into a −80◦C Ammonium chloride (NH Cl), dichloromethane (DCM), 4 freezer and were kept frozen until further analysis. sodium chloride (NaCl), and anhydrous sodium sulfate (Na SO ) 2 4 The samples of each set were termed according to their were purchased from VWR (Darmstadt, Germany). DCM was sampling time. The following list provides the time intervals that freshly distilled prior to use. The reference substances AMS and are related to the respective sample name: AMSO2, creatinine, disodium hydrogen phosphate (Na2HPO4), sodium sulfite (NaSO3) and urea were obtained from Aldrich - pre: before garlic consumption (Steinheim, Germany). The reference standard AMSO as well as - 0.5 h post: 0.4–0.8 h after garlic consumption 2 2 the isotopically labeled standards H3- AMS, H3-AMSO, and - 1hpost: 0.9–1.3 h after garlic consumption 2 H3-AMSO2 were supplied by Aromalab (Freising, Germany). - 2hpost: 1.8 h−2.2 h after garlic consumption Garlic (white garlic, origin: Spain) was purchased from local - 4hpost: 3.8–4.4 h after garlic consumption supermarkets (Aldi-Süd, Erlangen, Germany or Rewe, Freising, - 6hpost: 5.8–6.4 h after garlic consumption Germany), whereas the creatinine kit was from Labor+Technik - 8hpost: 7.8–8.4 h after garlic consumption Eberhard Lehmann GmbH (Berlin, Germany). - 24 h post: 23.8–25.6 h after garlic consumption The first sample of each set was tested with a dipstick-test, Human Urine Samples the multiple test stripes “Combi-Screen PLUS” (Analyticon Human urine samples were obtained from 18 volunteers (age Biotechnologies AG, Lichtenfels, Germany), to check the health 22–38 years, mean 27 years; 11 females, 7 males); thereby, one status of each test person, allowing for simultaneous testing volunteer conducted the whole experimental series twice. At of ascorbic acid, bilirubin, blood, glucose, ketones, leucocytes, the time of examination, volunteers reported no known illness nitrite, pH, protein, specific gravity/density, and urobilinogen in that might have potentially influenced the metabolism or the the respective urine samples. excretion of urine. The test persons were instructed to avoid foods that were rich in sulfur-containing substances at the testing day Determination of the Creatinine as well as 2 days preceding the testing day, namely garlic, onion, wild garlic, chives, cabbage, and leek, and were only allowed Concentration in Urine Samples + to consume the garlic sample provided in the course of the A creatinine kit (Labor Technik Eberhard Lehmann GmbH, experiments. Additionally, subjects were asked to keep records Berlin, Germany) was used to determine the creatinine content of the food (including food supplements) and beverages they in each urine sample. The determination principle of this kit is consumed during these 3 testing days. Generally, the overall based on the reaction of creatinine with picric acid under basic panelists’ requirements were in accordance with our previous conditions (Jaffé reaction). The formed complex was detected studies (5, 16). photometrically at a wavelength of 492 nm. On the sampling day, the outer garlic cloves of a whole garlic bulb were peeled and cut into ∼3 mm cubes by using a garlic Solvent Assisted Flavor Evaporation cutter (Genius GmbH, Limburg/Lahn, Germany). Subsequently (SAFE) of Volatiles From Human Urine 2 these cubes were thoroughly mixed and aliquoted into 3 g For quantification purposes, the deuterated standards H3-AMS, 2 2 portions. Three to four test persons conducted the experiment at H3-AMSO, and H3-AMSO2 were added to the sample and the same day. Each test person was asked to ingest a garlic portion stirred for 10 min. Subsequently, DCM was added in a ratio of 1:2 (DCM/human urine; v/v) and this solution was stirred for further Abbreviations: AMS, Allyl methyl sulfide; AMSO, Allyl methyl sulfoxide; 30 min at room temperature. To isolate the volatile fraction from AMSO2, Allyl methyl sulfone; DCM, Dichloromethane; HRGC-GC- the urine samples we then applied SAFE-distillation (17), thereby MS/O, Two-dimensional high-resolution gas chromatography-mass distilling the respective samples at 50◦C. After completion of the spectrometry/olfactometry; HRGC-MS, High-resolution gas chromatography- distillation process, 10 mL of DCM were applied in each case mass spectrometry; FID, Flame ionization detector; LOD, Limit of detection; LOQ, Limit of quantification; m/z ratio, Mass-to-charge ratio; ODP, Olfactory detection to rinse the SAFE apparatus and to ensure complete transition port; SAFE, Solvent-assisted flavor evaporation; SIDA, Stable isotope dilution of the volatile compounds. This process was repeated with assay; SIM, Selected ion monitoring. another 10 mL of DCM. After thawing, the obtained aqueous Frontiers in Nutrition | www.frontiersin.org 2 April 2019 | Volume 6 | Article 43 158 Scheffler et al. Garlic-Derived Metabolites in Human Urine distillate phase was extracted three times with 25 mL DCM. The silica material. The FID and both ODPs were held at 250◦ and ◦ organic phases were combined, dried over anhydrous Na2SO4 270 C, respectively. Mass spectra were recorded at 70 eV in full and concentrated to a total volume of 100 μLbymeansof scan mode (m/z range 30–100) as well as in SIM-mode (cf. Vigreux distillation and subsequent micro-distillation at 50◦C. Table 1). 2 μL of the sample were injected on-column using the auto sampler. The temperature programs were as follows: High Resolution Gas starting temperatures were 40◦C for both GCs. In the first GC Chromatography-Mass this temperature was held for 8 min, in the second oven for 7 min. Thereafter it was raised to 300◦C in the first oven and to 240◦C Spectrometry (HRGC-MS) ◦ GC-MS analysis was performed with an Agilent MSD quadrupole in the second oven at a rate of 20 C/min. The final temperatures system (GC 7890A and MSD 5975C, Agilent Technologies, were held for 5 min. The transfer line between first and second oven was kept at 250◦C, and during cut intervals it was cooled Waldbronn, Germany) equipped with a GERSTEL MPS 2 auto − ◦ sampler and a GERSTEL CIS 4 injection system (GERSTEL, down to 100 C with liquid nitrogen. Duisburg, Germany). A DB-FFAP (30 m × 0.25 mm, film thickness 0.25 μm, Agilent J&W Scientific, Santa Clara, CA, Quantification by Stable Isotope Dilution USA) was used as analytical capillary. An uncoated, deactivated Assay (SIDA) × fused silica capillary (2–3 m 0.53 mm) was used as pre-column. In pretest studies the amounts of AMS, AMSO and AMSO2 This capillary was changed regularly to avoid accumulation of in urine samples after garlic ingestion were evaluated (data × impurities. A further uncoated fused silica capillary (0.3–0.6 m not shown). The respective isotopically labeled standards were 0.25 mm) was used as a transfer line into the MS. Carrier gas was dissolved in DCM and added to the urine samples collected Helium at a flow rate of 1.0 mL/min. Mass spectra were recorded before and after garlic ingestion according to the expected at 70 eV in Selected Ion Monitoring (SIM)-mode (cf. Table 1) amounts based on these precedent trials. The mixture was for quantification. To verify the identity of the compounds, their worked up as described above. Quantification of AMSO and mass spectra were additionally recorded in full scan mode (mass- AMSO2 was performed by GC-MS analyses in SIM mode. The to-charge ratio (m/z) range 30–350). The following temperature m/z-ratios 104 + 107 and 120 + 123 were selected for analysis program for the oven was used: 40◦C was held for 7 min. This ◦ ◦ of AMSO and AMSO2 and their respective labeled analogs. temperature was raised to 240 Catarateof8 C/min. The final The quantification of AMS was carried out by GC-GC-MS μ temperature was held for 8 min. Injection volume was 2 L. The analysis in SIM mode. The selected m/z-ratios for AMS and its sample was applied with the auto sampler using the cold-on- labeled standard were 88 and 91. For quantification of the garlic- column technique (18) derived metabolites calibration curves were prepared consisting of defined mixtures of analyte standards and their respective Two-Dimensional High Resolution Gas 2 isotopic labeled standards (w/w; AMS/ H3-AMS: 1:10, 1:5, 1:3, 2 2 Chromatography-Mass 1:2, 1:1, 2:1; AMSO/ H3-AMSO and AMSO2/ H3-AMSO2: 1:10, Spectrometry/Olfactometry 1:5, 1:2, 1:1, 2:1, 3:1, 5:1). Calibration curves were calculated as (HRGC-GC-MS/O) (Heart-Cut) functions of the intensity ratios of standard to labeled standard For quantification of AMS a two-dimensional GC-MS system and their respective mass ratios (cf. Table 1). Calibration curves was applied. The system consisted of two Agilent 7890 B GCs were prepared in triplicates, and at three different days. For coupled with an Agilent 5977 B MS (Agilent, Waldbronn, quantification the average of these calibration curves was used. Germany). The system was equipped with a GERSTEL MPS 2 With the resulting calibration function, the known amount of auto sampler and a GERSTEL CIS 4 injection system (GERSTEL, isotopic labeled standard that was added to the sample, and the Duisburg, Germany). A multi-column switching system μMCS intensity ratio of analyte to isotopic labeled standard, the amount was installed in the first GC and both GCs were connected of the analyte in the sample was calculated. via a cryogenic-trap system CTS 1 (both: GERSTEL, Duisburg, Germany). DB-5 (30 m × 0.32 mm, film thickness 0.25 mm; Calculation of Metabolite Profiles Agilent J&W Scientific, Santa Clara, CA, USA; first oven) and The concentrations of the metabolites were calculated as μg/kg DB-FFAP (30 m × 0.32 mm, film thickness 0.25 mm; Agilent urine by dividing the calculated amount of the metabolites J&W Scientific, Santa Clara, CA, USA; second oven) were [in μg, cf. section Quantification by Stable Isotope Dilution used as analytical capillaries. An uncoated, deactivated fused Assay (SIDA)] by the amount of the respective urine sample silica capillary was used as pre-column (2–3 m × 0.53 mm) as (in kg). Additionally, the concentrations were normalized to the described previously. Helium was used as carrier gas at a constant creatinine content of the respective urine sample by dividing flow rate of 2.5 mL/min in the first GC and 1.0 mL/min in the the amount of the analyte by the volume of the respective second GC. With the help of the μMCS, the effluent was split urine sample (in L), which was further divided by the creatinine in the first oven between a flame ionization detector (FID) and concentration of the respective sample (in mmol/L). As a result, an olfactory detection port (ODP 3, GERSTEL), as well as a the dilution effects attributed to different water intake of the test cryotrap during cut intervals. In the second oven the effluent persons does not influence the absolute obtained excretion profile was split between an ODP and the MS by using a Y-splitter. of the metabolites so that the excretion profiles of different test All split capillaries were made of uncoated, deactivated fused persons can be directly compared. Frontiers in Nutrition | www.frontiersin.org 3 April 2019 | Volume 6 | Article 43 159 Scheffler et al. Garlic-Derived Metabolites in Human Urine TABLE 1 | Parameters of the quantification method including the instrument which was used for measurement, selected ions of analytes, and isotopically labeled standards and calibration factors. Instrument Analyte Standard Calibration function R² Compound m/z Compound m/z 2 GC-GC-MS AMS 88 H3-AMS 91 y= 0.9386x−0.0153 0.9994 2 GC AMSO 104 H3- AMSO 107 y = 0.9883x+0.0062 0.9999 2 GC AMSO2 120 H3-AMSO2 123 y = 0.9935x+0.0264 0.9999 Determination of the Limits of Detection from restaurants, like roasted pork leg, lamb meat, doner kebab, (LOD) and Quantification (LOQ) filled pepper, stir-fried vegetable, or an arabic soup, but no food LOD and LOQ were determined according to the calibration supplements. These foods can be assumed to have contained line method described in DIN 32645. To ensure consistent onion or garlic ingredients for seasoning, which might explain conditions for the analyses of LOD and LOQ, synthetic the presence of the metabolites in the respective control urine urine instead of human urine was used. The formula of the samples. However, as the test persons stayed at the institute synthetic urine was described by Mayrovitz and Sims (19). at the sampling day, the consumed food of the test persons Unlabeled and labeled reference compounds were added to was controlled in this period of time, ensuring no additional the synthetic urine in the concentration range of LOD and entry of garlic sources, besides the garlic administered within LOQ. Subsequently the samples were worked up as described the frame of the intervention. Despite the presence of AMS, above (cf. section Solvent Assisted Flavor Evaporation (SAFE) of AMSO, and AMSO2 in the control urine samples, we observed Volatiles From Human Urine) and analyzed by means of GC- distinct increases in the subsequent samples collected after MS and GC-GC-MS as described in sections High Resolution garlic intake. Gas Chromatography-Mass Spectrometry (HRGC-MS) and The concentrations of the metabolites were calculated μ μ Two-Dimensional High Resolution Gas Chromatography-Mass in g/kg urine and in g/mmol creatinine. The highest Spectrometry/Olfactometry (HRGC-GC-MS/O) (Heart-Cut). As concentrations of AMS, AMSO and AMSO2 in a series of urine theprovidedurinesampleswerehighlyvariablewithregardto samples after garlic consumption varied in a range from 0.5 to μ μ their volume (19–50 mL), LOD, and LOQ were calculated as 2.5 g/kg urine, from 77.0 to 423.9 g/kg urine, and from 75.1 to μ absolute amounts (in ng). 367.0 g/kg urine, respectively. As the metabolite concentration in urine depends on the water intake of the test person, the concentrations of AMS, AMSO, and AMSO2 were normalized to RESULTS the creatinine content of the respective urine samples. Thereby, the normalized concentrations of the metabolite maxima ranged The volatile garlic-derived metabolites were quantified in human between 0.3 and 2.4 μg/mmol creatinine (AMS), 27.6 and urine after test persons consumed a defined amount of raw 344.1 μg/mmol creatinine (AMSO), and between 32.1 and 284.7 garlic (3 g) in order to characterize the metabolism and excretion μg/mmol creatinine (AMSO2), roughly comprising one order processes of volatile garlic components in humans. The target of magnitude in each case. Apart from these variations in the metabolites, namely AMS, AMSO, and AMSO2, had been concentration range, the temporal appearance of the highest identified in our previous study when comparing urine samples metabolite concentration differed to some extent between the collected before garlic consumption with urine samples collected individual urine sets. In most cases (17 out of 19 urine sets) after garlic consumption (16). In the current study, these garlic- the highest normalized AMS concentration was detected 1 or derived metabolites were quantified in urine samples from 2 h after garlic consumption. Only in one case (Urine VIII) different subjects collected over a time period of about 24 h. the highest AMS amount was already detected 0.5 h after garlic In the majority of cases these metabolites were only detected ingestion, whereas in Urine XVI the highest AMS concentration in samples that were gathered after garlic consumption. Only was only detected 4 h after garlic consumption. The highest in a few cases (Urine III, VII, VIII, XI, XII, XVI, and XVII) determined amounts of AMSO and AMSO2 were observed they were also present in samples collected prior to garlic between 1 and 2 h after garlic ingestion in the urine samples intervention, however only small amounts were detected in these with the sole exception of Urine XVI where the maximum ≤ μ ≤ μ cases (AMS 0.2 g/mmol creatinine, AMSO 1.4 g/mmol of AMSO2 was observed as late as 4 h after garlic intake. In ≤ μ creatinine, AMSO2 2.6 g/mmol creatinine). Presumably, relation to the maximum of AMSO, the maximum of AMSO2 these test persons consumed foods during the wash out phase either appeared correspondingly at the same time point (as was that contained sulfur-bearing constituents such as garlic or the case for 14 urine sets), or with a temporal delay, namely onion. Based on these observations, the food protocols of all in the subsequent sample (as was the case for 5 urine sets). participating test persons were checked. According to the food Even 8 h after garlic consumption all metabolites were still protocols of the test persons providing Urine III, VII, VIII, XI, present in quantifiable amounts in all samples, which means XII, XVI, and XVII, they reported that they had consumed foods that all metabolites were above their respective LOQ (LOQ Frontiers in Nutrition | www.frontiersin.org 4 April 2019 | Volume 6 | Article 43 160 Scheffler et al. Garlic-Derived Metabolites in Human Urine (AMS): 7.6 ng, LOQ (AMSO): 77.4 ng, LOQ (AMSO2): 78.6 DISCUSSION ng). Only about 24 h after garlic consumption the metabolite concentrations in urine were reduced to traces in most of the In our previous study (20) we quantified volatile garlic-derived urine sets. In the case of these samples the amounts ranged metabolites in human milk; there, mothers consumed 3 g of roughly between the LOQ and the LOD [LOD (AMS): 1.6 raw garlic, which is in correspondence with the present study. ng, LOD (AMSO): 22.0 ng, LOD (AMSO2): 22.4 ng]. The However, the concentrations determined in the current study results are summarized in Figure 1 (metabolite concentration in for urine were about twice as high in case of AMSO2 and μg/kg urine: Figure 1A; metabolite concentration in μg/mmol about three times as high in case of AMSO. Only AMS revealed creatinine: Figure 1B). comparable excretion levels in both media. These findings For better visualization representative time-resolved generally agree with the fact that water soluble compounds are metabolite profiles of AMS, AMSO and AMSO2 are displayed primarily excreted via urine (21)asAMSOandAMSO2 are more in Figure 2 for four different test persons who conducted the polar than AMS. Accordingly, it is likely that urinary excretion experiment at the same day (Urine XIII, Urine XIV, Urine XV, is preferred to the excretion via human milk. Regarding the and Urine XVI). Despite the fact that all test persons consumed temporal progress of the excretion only a limited comparison 3 g of garlic that was taken from the same chopped and mixed is possible. Since milk samples need to be provided according garlic bulb, thereby ensuring that the composition of the garlic to the established lactation routine of the nursing mothers in sample was comparable, we observed distinct differences in the order to not disturb the lactation rhythm, the sampling times are metabolite profiles between samples. In these cases, the highest often not comparable. Yet, the highest concentrations of garlic- detected AMS concentrations of each sample series were 0.3 derived metabolites were usually detected in the first milk sample μg/mmol creatinine (Urine XIII), 0.4 μg/mmol creatinine (Urine after garlic consumption, which corresponds to a time window UIV), 1.0 μg/mmol creatinine (Urine XV), and 0.3 μg/mmol of about 1–3 h after garlic intake. Likewise, a maximum was creatinine (Urine XVI). Accordingly, the concentrations of observed in case of the urine samples about 1–2 h after garlic the AMS maxima showed a variation by a factor of up to 3.2 consumption, demonstrating corresponding kinetics. between the individual test persons. Regarding AMSO the As described in our previous studies on the identification highest concentrations were 87.7 μg/mmol creatinine (Urine of garlic-derived compounds in human milk and urine, the XIII), 84.7 μg/mmol creatinine (Urine XIV), 150.4 μg/mmol detected thio compounds are likely to originate from allicin creatinine (Urine XV), and 50.1 μg/mmol creatinine (Urine (5, 16). Allicin itself is formed from alliin, an odorless S- XVI), yielding a variation factor of up to 3.0 between urine allyl-L-cysteine sulfoxide, due to the action of the enzyme sets. Finally, in case of AMSO2, the concentrations were 58.5 alliinase after disruption of the cell structure (22). Allicin can be μg/mmol creatinine (Urine XIII), 60.1 μg/mmol creatinine further converted into various compounds, e.g., dithiins, sulfides, (Urine XIV), 83.1 μg/mmol creatinine (Urine U XV), and polysulfides, and ajoenes (23, 24). As shown by Lawson and 37.19 μg/mmol creatinine (Urine XVI) corresponding to a Wang (25) they can react with cysteine that is present in the factor of 2.2 between the highest and lowest concentration. intestinal tract and form S-allylmercaptocysteine. However, there Furthermore, differences in the temporal profiles of metabolite is also evidence that S-allylmercaptoglutathione is formed on excretion were observed in these cases. In the cases of Urine the basis of glutathione, either due to an enzymatic conjugation XIII and Urine XV the concentration of each metabolite with diallyl disulfide or due to a non-enzymatic reaction with increased rapidly after garlic consumption and after reaching allicin (26). In both cases allyl mercaptan is formed subsequently. the maximum about 1 h (Urine XIII) or 2–4 h (Urine XV) Allyl mercaptan can be methylated which leads to the formation after ingestion of garlic, the excreted metabolite concentration of AMS. decreased continuously. In contrast to this, the sample sets Studies performed by Lawson and Wang (27) confirmed that of Urine XIV and Urine XV exhibited two maxima for each compounds comprising a dithioallyl-group (e.g., allicin, diallyl metabolite. The first maximum was observed in each case disulfide, and ajoene) as well as AMS itself are transformed within between 1 and 2 h after garlic consumption, whereas the second the human body to AMS with allyl mercaptan as a precursor. maximum was observed about 6 h after garlic consumption. However, an accumulation of this precursor was not expected due With regard to the other samples, that are not visualized here, to a rapid methylation. In the current study we could confirm similar differences were observed for all test persons, regardless the formation of AMS. Furthermore, we could demonstrate that whether they conducted the experiment on the same day AMS is accompanied by AMSO and AMSO2. These findings are with garlic samples obtained from the same garlic bulb or also in agreement with a previous study where authors identified on different test days. Consequently, the observed differences AMSO and AMSO2 in rat urine and plasma after administration in metabolite profiles cannot be attributed to variations in of diallyl disulfide (28). the garlic samples only, but are obviously additionally linked The fate of garlic compounds that do not comprise a to the individual physiological processes of the subjects. A dithioallyl group, which is the case for diallyl sulfide or dithiins, compilation of all investigated urine samples, including time of is not yet fully resolved. Lawson and Wang (27) suggested diallyl urine sampling, mass (g), volume (mL), and creatinine content sulfone as a metabolite for diallyl sulfide. This compound has (mmol/L) as well as the concentrations of AMS, AMSO, and been identified in rats after administration of diallyl sulfide (29). AMSO2 in μg/mL and μg/mmol creatinine are provided in However, in our previous studies we could exclude the presence the Table S1. of this metabolite in human urine and human milk (5, 16), and Frontiers in Nutrition | www.frontiersin.org 5 April 2019 | Volume 6 | Article 43 161 Scheffler et al. Garlic-Derived Metabolites in Human Urine FIGURE 1 | Box plot (mean value, markers at minimum and maximum metabolite concentration, box perc. 25–75%) of garlic-metabolite (AMS, AMSO, and AMSO2) concentrations in human urine, expressed as μg/kg (A) and μg/mmol creatinine (B). Data compiles 18 urine sets. The term “pre” relates to the urine samples that were collected before garlic consumption. “0.5 h post” – “24 h post” relate to the urine samples that were collected after garlic intake. again confirmed this finding in the present study. It is possible persons originated from different garlic composition. Possible that diallyl sulfide is transformed to less volatile metabolites, reasons for the observed differences in excreted metabolite such as glutathione or acetyl-conjugates (30, 31) and hence not concentrations could comprise, amongst others, differences in detectable with the methods used in this study. Likewise, this the absorption processes that can be influenced by e.g., the could be the metabolic fate of the dithiins. individual resorptive area and the blood circulation and transport The current study aimed at quantifying volatile garlic-derived mechanisms therein. Amongst others, previous studies of our metabolites in human urine over a time interval of up to about group could show that chemosensorially active compounds can 24 h. As a result it could be concluded that the impact of garlic be strongly biotransformed in the gastrointestinal tract, and even on human urine composition differed between test persons. at the resorptive sites (14, 15, 32). Enzymes that are involved The variations included differences in the concentrations as in the metabolism of garlic constituents could further exhibit well as differences in the temporal excretion pattern of the genetic polymorphisms or differential expression rates which excreted metabolites. These findings are shown exemplarily could lead to different plasma concentrations and therefore in the time-resolved metabolite profiles of four test persons also urine concentrations; this has been reported for several (Urine XIII - XVI)(cf.Figure 2). Although all test persons enzymes responsible in drug metabolism (33). Moreover, we consumed garlic samples of the same garlic bulb, distinct observed different temporal excretion pattern. In particular we differences in their metabolite profiles were observed. In this observed maxima for AMS, AMSO, and AMSO2 that commonly way it could be ruled out, that the variations between the test occurred about 1–2 h after garlic ingestion. However, in some Frontiers in Nutrition | www.frontiersin.org 6 April 2019 | Volume 6 | Article 43 162 Scheffler et al. Garlic-Derived Metabolites in Human Urine FIGURE 2 | Time-resolved metabolite profiles of AMS, AMSO, and AMSO2 of four test persons (Urine XIII, Urine XIV, Urine XV, Urine XVI). All four test persons consumed a garlic sample from the same garlic bulb. Time 0 h corresponds to the time of garlic intake. ( )AMS;( ) AMSO; ( )AMSO2. Broken line: time interval, when no sample was collected. cases there was a second maximum additionally about 6–8 h neither AMSO nor AMSO2 are odor-active components (5), after garlic consumption. This indicates that absorption and and only AMS comprises a garlic-like aroma (5). Therefore, metabolism processes may take place at different sites in the AMS might contribute to the changed body odor which is often human body. The rapid detection of garlic-derived metabolites in recognized after garlic consumption. Furthermore, Khatua et al. the urine samples already 0.5–1 h after garlic ingestion suggests (40) recently showed a correlation between AMS and AMSO and an early uptake of garlic constituents. Although foods are cardiac hypertrophy in rats. By increasing the Na+/K+-ATPase primarily crushed in the oral cavity, which is the first step these metabolites attenuated cardiac hypertrophy. Therefore, in digestion, the oral cavity might already be the first stage they could influence the progression of cardiovascular diseases along the gastro-intestinal tract to absorb some of the food (41). However, potential effects in humans still remain to components. Such effects have been observed in case of drugs be proven. (34, 35). In case of volatiles, which are addressed in the present study, an absorption via the nasal epithelial cells could also CONCLUSION lead to an early metabolite appearance in the urine samples (36, 37). Absorption processes have further been reported in The impact of garlic consumption on human urine composition the stomach for different drugs and plant pigments (38, 39). was investigated. Garlic-derived metabolites, namely AMS, Accordingly, they are not restricted to the small intestine, but AMSO, and AMSO2 were quantified via SIDA over a period of can take place throughout the whole gastro-intestinal tract. 24 h. Large inter-individual differences were observed, both with Apart from that, the storage time of urine in the bladder may regard to the metabolite concentrations and ratios in the urine further influence the excretion profiles of the compounds. In samples, as well as their excretion kinetics. The highest observed case of the observed second maximum, for example, it might be concentrations of AMS, AMSO and AMSO2 at the time point the case that due to mixing effects the second increase might when the excretion maximum was reached ranged from 0.3 to become less pronounced in some urine samples compared to 2.4 μg/mmol creatinine, 28 to 344 μg/mmol creatinine, and 32 others or may even coincide with the first maximum. Regarding to 285 μg/mmol creatinine, respectively. Commonly, the highest the physiological effects of garlic, it is interesting to note, that concentrations were found to occur about 1–2 h after garlic Frontiers in Nutrition | www.frontiersin.org 7 April 2019 | Volume 6 | Article 43 163 Scheffler et al. Garlic-Derived Metabolites in Human Urine consumption, however for some urine sets a second increase was conceived the publication that was approved by AB. All authors observed about 6–8 h after garlic consumption. These findings have read and approved the final manuscript. suggest differences in the absorption, distribution, metabolism, and excretion processes of volatile garlic constituents in the FUNDING human body. This work was supported by the German Research Foundation ETHICS STATEMENT (DFG) in the grant [BU 1351/15-1] and the grant [INST 90/979- 1 FUGG]. The study was conducted in agreement with the Declaration of Helsinki. Written, informed consent was given by all 18 ACKNOWLEDGMENTS volunteers prior to the testing day. Withdrawal from the study was possible at any time. The study (registration number 49_13B) We are grateful to the volunteers for participating in our was approved by the Ethical Committee of the Medical Faculty, study, we are grateful to Yvonne Sauermann for skillfull Friedrich-Alexander Universität Erlangen-Nürnberg. technical assistance. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL LS, CS, and AB conceived and designed the experiments. 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Both ramson and garlic belong to the genus allium which is characterized by a high content of sulfurous compounds. However, in contrast to garlic, ramson is in general not associated with an unpleasant breath following consumption. While there is data available regarding Edited by: the metabolism of volatile garlic constituents in the human body, the metabolism of Mirko Bunzel, Karlsruher Institut für Technologie ramson was not yet addressed. To elucidate if ramson has an impact on the body (KIT), Germany odor, this study aimed at identifying volatile ramson-derived metabolites in human milk Reviewed by: and urine. Therefore, milk and urine samples were gathered before and after ramson Jorry Dharmawan, consumption, and were analyzed sensorially by a trained human sensory panel as well Singapore Institute of Technology, Singapore as chemo-analytically applying gas chromatography-mass spectrometry/olfactometry Susana P. Alves, (GC-MS/O). Sensory evaluation revealed a garlic-/cabbage like odor in milk samples Faculdade de Medicina Veterinária, Universidade de Lisboa, Portugal obtained after ramson consumption, demonstrating that ramson consumption affected Zuobing Xiao, the milk aroma. Analyzes by means of GC-MS/O further confirmed excretion of Shanghai Institute of Technology, three ramson-derived metabolites in milk and urine samples collected after ramson China *Correspondence: consumption, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl Andrea Buettner methyl sulfone (AMSO2). Of these metabolites only AMS had a garlic-/cabbage-like odor, [email protected] while the other two were odorless. These metabolites were subsequently quantified using stable isotope dilution assays. Nine urine sets, each comprising eight urine samples, Specialty section: This article was submitted to and nine milk sets, each comprising four samples, were analyzed. In case of the urine Food Chemistry, sets a time interval of about 24 h was monitored, in case of the milk sets a time interval a section of the journal Frontiers in Chemistry of up to 9 h. Despite the fact that all samples contained the same metabolites there were relevant differences found between individual subjects, especially with regard to Received: 03 June 2018 Accepted: 20 August 2018 the temporal rate of metabolite excretion. Generally, the maxima of metabolite excretion Published: 11 September 2018 were observed in milk sets within 3 h after ramson consumption. In urine the highest AMS Citation: and AMSO amounts were observed within 2 h whereas the maximum concentration of Scheffler L, Sharapa C, Amar T and Buettner A (2018) Identification and AMSO2 was reached about 2 to 4 h after ramson ingestion. This study suggests that Quantification of Volatile ramson constituents are heavily metabolized in the human body. Ramson-Derived Metabolites in Humans. Front. Chem. 6:410. Keywords: human milk, human urine, gas chromatography mass-spectrometry/olfactometry, allyl methyl sulfide, doi: 10.3389/fchem.2018.00410 allyl methyl sulfoxide, allyl methyl sulfone, stable isotope dilution analysis (SIDA) Frontiers in Chemistry | www.frontiersin.org 1 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans INTRODUCTION Furthermore, the odorous compounds in urine have also been used as indicators for diseases or metabolic syndromes, e.g., Spice plants and condiments such as garlic are used world- trimethylaminuria, in which case the persons concerned excreted wide to modulate the taste of dishes. However, this is associated increased concentrations of trimethylamine via urine leading to with the side-effect that some people emit a distinct smell after afishyodor(Humbert et al., 1970). consumption of garlic. The typical aroma of garlic is due to its In case of garlic ingestion our group already identified three pronounced content of sulfur-containing compounds. In general, metabolites excreted via human milk and urine, namely allyl a high sulfur substance content is typical for the genus Allium, methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl which includes garlic. However, another representative of the methyl sulfone (AMSO2) with only AMS present in garlic itself genus Allium is ramson (Allium ursinum) which is commonly whereas the other two compounds are only formed within not associated with aversive effects like unpleasant breath despite the human body (Scheffler et al., 2016a,b). In case of ramson the fact that the volatile oil of ramson comprises compounds there is no data available concerning its metabolism within that have also been identified in garlic, namely sulfides, ajoenes, the human body. To close this gap the present study aimed and dithiins (Sendl et al., 1992; Benkeblia and Lanzotti, 2007; at elucidating volatile ramson-derived metabolites in humans. Godevac et al., 2008; Sabha et al., 2012). In relation to body Therefore, human milk and urine samples gathered before odor and excretion processes of ramson-derived substances, and after ramson consumption were analyzed. Additionally, biotransformation processes in the body need to be considered the identified compounds were quantified and their temporal as these processes might lead to neoformation of compounds, excretion was monitored over a time period of about 9 h (milk) whereas others might be degraded. Potential flavor effects of or 24 h (urine). The main consideration during all investigations human nutrition on the sensory properties of human milk are was thereby to obey a consumption protocol that is well in line in this context of special interest as human milk is commonly the with a real-life ramson consumption scenario. sole food source of a newborn, at least during the first months of life. Besides possible effects on the infants’ health, both positive MATERIALS AND METHODS and negative, it has been reported that the maternal diet can influence the suckling behavior and later dietary habits of the The materials and methods that were used in this study are in infant (Mennella et al., 2001). These behavioral effects have been most parts in line with those described in our previous studies attributed to a changed flavor profile of the milk, implicating which addressed the identification and quantification of garlic- that these flavor changes were supposed to be attractive to the derived metabolites in human milk and urine (Scheffler et al., children. However, final evidence in terms of sensory and chemo- 2016a,b; Scheffler et al., submitted). For this reason only a short analytical data, as well as proof of attractiveness of such flavor overview is provided here. Detailed descriptions are given in the impressions to the infants was not provided. In this context respective studies and in the Supplementary Material provided it is interesting to note that intake of specific foods might for this study. impact diverse physiological processes and parameters such as blood pressure and secretory processes. There are several studies Study Design and Ethics Approval which demonstrate that the consumption of garlic can lead The study was conducted in agreement with the Declaration of to reduction in blood pressure which is particularly distinctive Helsinki. All participants gave written, informed consent prior to in case of hypertensive patients (Reinhart et al., 2008; Ried, the testing day. They were able to withdraw from the study at any 2016; Choudhary et al., 2017). Such processes might also impact time. The study (registration no. 163_16 Bc) was approved by the lactation processes, e.g., with elevated milk secretion, which Ethical Committee of the Medical Faculty, Friedrich-Alexander- might directly impact suckling behavior and milk intake of the Universität Erlangen-Nürnberg. children. On the other hand, the analysis of urine opens the Human Milk Samples possibility to investigate low-molecular weight compounds. The Human milk samples were obtained from 13 different mothers dietary influence on the urine composition was demonstrated (age range 27–39 years, mean 33). The volunteers had no known for instance for asparagus: people who consumed asparagus illnesses and their milk production exceeded their infants’ need. excreted urine with a sulfurous odor (Pelchat et al., 2011). The sampling took place 9 to 37 seven weeks postpartum (mean 19 weeks). To avoid sulfurous compounds in the milk samples Abbreviations: AMDS, allyl methyl disulfide; AMS, allyl methyl sulfide; AMSO, that were not associated with ramson consumption, the test allyl methyl sulfoxide; AMSO2, allyl methyl sulfone; APDS, allyl propyl persons were asked to avoid food containing high amounts of disulfide; APS, allyl propyl sulfide; DADS, diallyl disulfide; DAS, diallyl sulfide; sulfur substances (e.g., garlic, onion, ramson, chives, cabbage, DASO, diallyl sulfoxide; DASO2, diallyl sulfone; DATS, diallyl trisulfide; DCM, dichloromethane; DMDS, dimethyl disulfide; DMTS, dimethyl trisulfide; DPDS, and leek) on the testing day and 2 preceding days. Additionally, dipropyl disulfide; DPTS, dipropyl trisulfide; FID, flame ionization detector; they were asked to keep record of their food during these 3 HRGC-GC-MS/O, two-dimensional high-resolution gas chromatography-mass testing days. Each mother provided one milk sample before spectrometry/olfactometry; HRGC-MS, high-resolution gas chromatography- ramson consumption and three further samples after ramson mass spectrometry; HRGC-O, high-resolution gas chromatography-olfactometry; MPDS, methyl propyl disulfide; MPTS, methyl propyl trisulfide; RI, (linear) ingestion. The samples were collected according to the normal retention index; SAFE, solvent assisted flavor evaporation; SIDA, stable isotope lactation period of each mother and immediately analyzed. Four dilution assay; SIM, selected ion monitoring. consecutive milk samples formed a milk set in each case. In a Frontiers in Chemistry | www.frontiersin.org 2 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans pre-test one mother consumed about 3 g of ramson. All other test Isolation, Identification and Quantification persons consumed about 10 g of ramson. of Ramson-Derived Metabolites Four milk sets were collected for identification experiments. The volatile fraction of the urine and milk samples was isolated by They were labeled “M” with Arabic numerals, e.g., M2-1to M2- means of Solvent-assisted flavor evaporation (SAFE)-distillation 4. Quantification experiments were performed after completion (Engel et al., 1999). The obtained distillate was analyzed by of identification experiments. In case of the quantification means of high resolution gas chromatography-olfactometry experiments nine milk sets were gathered and labeled “M”with (HRGC-O) as well as high resolution gas chromatography- Roman numerals, e.g., M II-1 to M II-4. mass spectrometry (HRGC-MS) and two-dimensional HRGC- MS/O (heart-cut). Ramson-derived metabolites were identified Human Urine Samples by comparing their retention indices (RI) of two analytical Human urine samples were obtained from four volunteers (age capillaries with different polarities (DB-5 and DB-FFAP), their 24–28 years, mean 26, two females, two males). One volunteer respective odor as well as their mass spectra with those of conducted the whole experimental series four times, the other reference standards. RI values were calculated according to three volunteers participated twice. Urine samples were collected van Den Dool and Kratz (1963). A relative concentration in sterile brown glass bottles. At the testing day as well as 2 days of the identified metabolites was calculated by normalizing preceding the testing day, the test persons were instructed to respective peak areas to the amount of investigated milk avoid sulfurous compounds and to record consumed foods and or urine (in kg) in order to express the concentration in beverages as described for the milk sampling (cf. chapter 2.2). On units of area/kg milk or urine. In case of the urine samples, the testing day ramson was freshly washed, ground and aliquoted the concentration was additionally expressed as area/mmol into portions of 10 g. The volunteers provided one urine sample creatinine. Therefore, the creatinine levels in each urine before and seven samples after ingestion of 10 g of ramson at sample were determined using a creatinine kit (Labor+Technik about the following times: 0.5, 1, 2, 4, 6, 8, and 24 h after ramson Eberhard Lehmann GmbH, Berlin, Germany). The peak area consumption. Eight consecutive urine samples comprised one was divided by the amount of investigated urine (in L) − ◦ urine set. The samples were kept frozen at 80 C until further and the creatinine content (in mmol/L) of the respective analysis. urine sample. In subsequent studies the respective metabolites To rule out illnesses of the test persons, the first sample were quantified by means of stable isotope dilution analysis of each set was tested with a dipstick test. With the multiple (SIDA). Isotopically labeled standards were added to the test stripes (Combi-Screen Plus, Analyticon Biotechnologies AG, samples prior to sample work up and solutions consisting Lichtenfels, Germany) simultaneous testing of following urine of defined mixtures of analyte standards and their respective parameters was possible: ascorbic acid, bilirubin, blood, glucose, isotopically labeled standards were analyzed. Calibration curves ketones, leucocytes, nitrite, pH, protein, specific gravity/density, were calculated as functions between the intensity ratios of and urobilinogen. standard to labeled standard and their respective mass ratios For identification experiments one urine set was collected. The (cf. Table 1). Calibration curves were prepared in triplicates, respective samples were labeled U1-1to U1-8. For quantification and at 3 different days. For quantification the average of these analyses nine urine sets were analyzed. The urine samples of each calibration curves was used. With the resulting calibration set were labeled “U” with Roman numerals (e.g., U II-1 to U II-8) function, the known amount of isotopic labeled standard added and according to their sampling time: to the sample and the intensity ratio of analyte to isotopic - Pre: 3 to 7 min before ramson consumption labeled standard the amount of the analyte in the sample was calculated. - 0.5 h post: 0.45 h to 0.55 h after ramson consumption In order to express the concentration of ramson-derived metabolites as μg/kg milk or urine or μg/mmol creatinine, the - 1 h post: 1.00 h to 1.05 h after ramson consumption calculated amounts of AMS, AMSO and AMSO2 were divided by the amount of investigated sample (in kg) or normalized to - 2 h post: 1.95 h to 2.10 h after ramson consumption creatinine content as described above. - 4 h post: 4.00 h to 4.10 h after ramson consumption Aroma Profile Analysis of Human Milk Samples - 6 h post: 6.00 h to 6.10 h after ramson consumption Milk samples were additionally evaluated sensorially (orthonasally) prior to the sample work up. Aroma profile - 8 h post: 8.00 h to 8.15 h after ramson consumption analysis was performed based on sensory pre-evaluations and the intensity of the following attributes were rated on a scale - 24 h post: 23.95 h to 24.20 h after ramson consumption from 0 (no perception) to 3 (strong perception): hay-like, fishy, fatty, rancid, sweaty, metallic, grassy-green, sweet, egg UIIto UVwere obtained at the same testing day with each test white-like, buttery, lactic, and garlic/cabbage-like. The evaluated person consuming ramson portions of about 10 g. Likewise, UVI attributes corresponded to those described in Scheffler et al. to UIXwere provided at the same testing days. (2016b). Frontiers in Chemistry | www.frontiersin.org 3 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans TABLE 1 | Parameters for quantification including the instrument which was used for measurement, selected m/z-ratios of analytes and isotopically labeled standards and calibration factors. Instrument Analyte Labeled standard Calibration function R2 Compound m/z Compound m/z 2 Milk GC-GC-MS AMS 88 H3-AMS 91 y= 0.9171x – 0.0492 0.9981 2 GC AMSO 104 H3- AMSO 107 y = 1.0106x + 0.0045 1.0000 2 GC AMSO2 120 H3-AMSO2 123 y = 0.9727x + 0.0058 0.9999 2 Urine GC-GC-MS AMS 88 H3-AMS 91 y= 0.9289x – 0.029 0.9996 2 GC AMSO 104 H3- AMSO 107 y = 1.0034x + 0.0081 1.0000 2 GC AMSO2 120 H3-AMSO2 123 y = 0.9746x + 0.008 0.9999 RESULTS Aroma Profile Analysis of Human Milk Samples in Relation to Ramson Ingestion Aroma profile analyses were performed on all human milk samples that had been obtained before and after ramson consumption in the course of identification experiments. Samples were rated according to the attributes fishy, fatty, metallic, grassy-green, rancid, sweaty, buttery, sweet, hay-like, egg white- like, lactic, and garlic-/cabbage-like. The overall intensity of the milk samples as well as the odor attributes were mostly rated as being just detectable (intensity 1) or even not perceivable (intensity 0). Overall, the attribute garlic-/cabbage-like was not perceivable in all samples obtained before ramson intervention. After consumption of 10 g of fresh ramson the milk samples were rated as having a slight (0.5–1.0) garlic-/cabbage-like odor by most of the panelists. The odor change was most pronounced in milk samples that were provided 2 to 5 h after ramson consumption. Exemplary aroma profiles of a sample series of one milk set are displayed in Figure 1. Nevertheless, this does not rule out a potential sensory detection via tasting that was, however, not possible due to work safety considerations. All other aroma profiles of the milk samples that were also collected FIGURE 1 | Odor profiles of human milk samples of set M2,asa for the identification experiments are provided in Figure S1 of representative sample. The samples were collected at different times before the Supplementary Material. Moreover, aroma profile analyses and after ingestion of 10 g of ramson: 10 min before, 2 h 25 min after, 4 h were repeated for those samples that were collected during the 25 min after and 6 h 40 min after ramson intervention. Panelists were asked to quantification experiments. Also in these cases a slight garlic- rate the orthonasal perception on a scale from 0 (no perception) to 3 (strong /cabbage-like odor was perceived in the milk samples that were perception). Values are mean ratings of all panelists. Note: The scale is only presented up to the value of 1 for better visualization. obtained after ramson consumption (data not shown). Hence, the results from the identification experiments could be confirmed. Identification of Ramson-Derived quality and the respective RI-values with reference substances Metabolites (cf. Table 2), this compound was identified as AMS. All other Comparative GC-O Analysis odors were perceived in all samples, irrespective of ramson Comparative GC-O analyses were performed for the consumption, hence they cannot be associated with ramson corresponding human milk and urine samples collected consumption but rather contribute to the typical smell of human before and after ramson consumption in order to identify milk and urine. odor-active compounds that derived from ramson consumption. In both, urine and milk samples, the analyses revealed one Identification of Ramson-Derived Volatiles in Milk and additional odor-active substance that was only detectable in Urine Samples Applying HRGC-MS and samples obtained after ramson intervention. This substance had HRGC-GC-MS a garlic-/cabbage-like odor, a RI < 1000 on the FFAP capillary In order to further identify less odor-active or odorless and RI 702 on the DB-5 capillary. By comparison of the odor ramson-derived compounds in the milk and urine samples, Frontiers in Chemistry | www.frontiersin.org 4 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans TABLE 2 | Compilation of investigated substances, their chemical structures, retention indices (RI) on two different chromatographic capillaries (DB-FFAP and DB-5), their odor qualities and literature reports on these substances. Substance Structure RI Odor quality Detected in Previously (abbreviation) detected in FFAP DB-5 M pre M after U pre U after 2-Vinyl-4H-1,3-dithiin 1767 1225 Garlic-like n.d. n.d. n.d. n.d. Garlica−g Ramsonh 3-Vinyl-4H-1,2-dithiin 1671 1198 Pungent, n.d. n.d. n.d. n.d. Garlica−c,e,g,i,j garlic-like Ramsonh Allyl methyl disulfide 1206 918 Cooked n.d. n.d. n.d. n.d. Garlica−c,f,g,i,j (AMDS) garlic-like Ramsonh,k−n Human breath after garlic consumptiono−s Allyl methyl sulfide <1,000 702 Garlic- n.d. yes n.d. Yes Garlica,d,f,i (AMS) /cabbage-like Ramsonk,m Human breatho−v, human milkw, human urinex after garlic consumption Allyl methyl sulfone 1917 1058 Odorless n.d. Yes n.d. Yes Human milkw, x (AMSO2) human urine after garlic consumption Allyl methyl sulfoxide 1717 1003 Odorless n.d. Yes n.d. Yes Human milkw, (AMSO) human urinex after garlic consumption Allyl propyl disulfide 1360 1097 Garlic-like n.d. n.d. n.d. n.d. Garlica−c,i (APDS) Ramsonh,k−m Allyl propyl sulfide 1051 876 Garlic-/onion- n.d. n.d. n.d. n.d. Ramsonm (APS) like Diallyl disulfide 1404 1083 Garlic-like n.d. n.d. n.d. n.d. Garlica−g,i,j (DADS) Ramsonh,k−n Human breath after garlic consumptiono−v Diallyl sulfide (DAS) 1085 863 Garlic-like n.d. n.d. n.d. n.d. Garlica,b,d,f,g,i,j Ramsonk,m,n Human breath after garlic consumptiono,u,s (Continued) Frontiers in Chemistry | www.frontiersin.org 5 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans TABLE 2 | Continued Substance Structure RI Odor quality Detected in Previously (abbreviation) detected in FFAP DB-5 M pre M after U pre U after Diallyl sulfone 2018 1193 Odorless n.d. n.d. n.d. n.d. Rat liver, blood and (DASO2) urine after treatment with DASy Diallyl sulfoxide 1858 1159 Garlic-like n.d. n.d. n.d. n.d. Rat liver, blood and (DASO) urine after treatment with DASy Diallyl trisulfide 1712 1310 Garlic-like n.d. n.d. n.d. n.d. Garlica−g,i,j (DATS) Ramsonh,l−n Human breath after garlic consumptiono,s Dimethyl disulfide 1018 739 Cabbage-like n.d. n.d. n.d. n.d. Garlica−d,f,g,i,j (DMDS) Ramsonh,k,m,n Human breath after garlic consumptiono Dimethyl trisulfide 1295 972 Cabbage-like n.d. n.d. n.d. n.d. Garlica,b,f,g,i (DMTS) Ramsonh,k−n Dipropyl disulfide 1312 1111 Garlic-like n.d. n.d. n.d. n.d. Ramsonk,m (DPDS) Dipropyl trisulfide 1603 1337 Cooked n.d. n.d. n.d. n.d. Ramsonh,k,m (DPTS) garlic- /cabbage-like Methyl propyl 1168 935 Garlic- n.d. n.d. n.d. n.d. Garlica,c,d,f,g,j disulfide (MPDS) /cabbage-like Ramsonk−n Methyl propyl 1457 972 Garlic- n.d. n.d. n.d. n.d. Ramsonk−m trisulfide (MPTS) /cabbage-like The presence of the target compounds in milk and urine samples collected before (M 2-1 and U 1-1) and after (M 2-2 and U 1-4) consumption of 10 g of raw ramson is provided. Confirmation/ identification or exclusion of the compounds was conducted by means of HRGC-GC-MS. M 2-2 (2.4 h after ramson consumption) and U 1-4 (2 h after ramson consumption) are displayed as representative examples of data obtained for the samples provided after ramson consumption. a(Yu et al., 1989), b(Mazza et al., 1992), c(Mondy et al., 2001), d(Lee et al., 2003), e(Abu-Lafi et al., 2004), f (Calvo-Gómez et al., 2004), g(Kimbaris et al., 2006), h(Sobolewska et al., 2013), i(Tokarska and Karwowska, 1983), j(Galoburda et al., 2013), k (Schmitt et al., 2005), l(Godevac et al., 2008), m(Radulovic´ et al., 2015), n(Blazewicz-Wozniak and Michowska, 2011), o(Cai et al., 1995), p(Hansanugrum and Barringer, 2010), q(Munch and Barringer, 2014), r (Suarez et al., 1999), s(Taucher et al., 1996), t(Buhr et al., 2009), u(Rosen et al., 2000), v(Lawson and Wang, 2005), w(Scheffler et al., 2016b), x(Scheffler et al., 2016a), y (Brady et al., 1991), n.d.: not detected. the chromatograms obtained from GC-MS analyses of control reported in garlic, or in breath after garlic consumption, samples prior to ramson intervention were compared to those respectively, or that had been proposed as potential metabolites obtained after intervention in full scan-mode. Additionally, the in humans based on animal studies (cf. Table 2). To this samples were screened for potential ramson-derived metabolites aim, a targeted search by means of GC-MS and GC-GC-MS via targeted search, taking into consideration compounds that was conducted utilizing standard solutions of the respective had previously been reported in literature as constituents of compounds. GC-GC-MS analyses thereby allowed screening ramson. Moreover, we screened for substances that had been for minor quantities of the target substances in the low Frontiers in Chemistry | www.frontiersin.org 6 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans μg/mL-range. The concentrations of the respective reference Afterwards, a decline in metabolite concentration was observed compounds thereby ranged between 0.5 and 12.7 μg/mL DCM. which was pronounced in case of AMSO but slow in case of These investigations revealed two additional ramson-derived AMSO2. This trend is visualized in Figure 3. metabolites, namely AMSO and AMSO2. Moreover, GC-GC-MS The metabolite concentrations for each milk sample, arranged analysis confirmed the identification of AMS in urine and milk according to milk sets, are provided in Table S1. samples collected after ramson consumption. Representative The temporal monitoring of the excreted metabolite chromatograms are displayed in Figure 2. The other compounds concentrations revealed some inter-individual variation of the targeted search were not detectable in any urine or milk between test persons. For better visualization, representative samples, even at concentrations as low as about 1–10 μg/mL, time-resolved metabolite profiles are displayed in Figure 4. irrespective if sampling took place prior to or after ramson Generally, differences were observed both in the excreted ingestion. These compounds were: 2-vinyl-4H-1,3-dithiin, 3- metabolite concentrations, as described above, as well as in the vinyl-4H-1,2-dithiin, allyl methyl disulfide (AMDS), allyl propyl time dependency of excretion. It is important to note that, disulfide (APDS), allyl propyl (APS), diallyl disulfide (DADS), however, all test persons consumed the same amount of fresh diallyl sulfide (DAS), diallyl sulfoxide (DASO2), diallyl sulfone ramson following the same consumption protocol. The time- (DASO), diallyl trisulfide (DATS), dimethyl disulfide (DMDS), resolved metabolite profiles revealed that for some milk samples dimethyl trisulfide (DMTS), dipropyl disulfide (DPDS), dipropyl the maxima of all three metabolites coincided in the first milk trisulfide (DPTS), methyl propyl disulfide (MPDS), and methyl sample gathered after ramson consumption (e.g., MI). On the propyl sulfide (MPTS). otherhand,therewerealsomilksetswherethemaximumof AMSO2 was detected after the maximum of AMSO (e.g., M VII), and other cases, where the maximum of AMS appeared Quantification and Time Dependency of in the milk sample following the maximum of AMSO (e.g., M Excretion of Ramson-Derived Metabolites IV). To further clarify whether the maxima of the metabolites Quantitative Analysis of Ramson-Derived Metabolites coincide or are excreted with a time delay, a larger sample in Human Milk set should be analyzed with shorter time intervals between the The quantification of ramson-derived metabolites in human samples. However, the present study aimed to investigate the milk was carried out on nine milk sets, each comprising concentrations of ramson-derived metabolites in human milk in four milk samples. The exact sampling times as well a real-life situation which included that the mothers kept their as volume and weight of the samples are provided in normal feeding habits. Table S1. In the majority of cases the ramson-derived metabolites AMS, Quantitative Analysis of Ramson-Derived Metabolites AMSO, and AMSO2 were only detected in those milk samples in Human Urine that were obtained after ramson consumption. Only in case of The quantification of ramson-derived metabolites in human milk set V and VI small amounts of these metabolites were urine was likewise carried out on nine different urine sets, each observed in the samples gathered prior to ramson consumption. comprising eight urine samples. The exact sampling times as According to the recorded food protocols, the respective test well as volume, weight and creatinine content of each sample are persons consumed bratwurst and kohlrabi 1 day prior to provided in Table S2. the respective testing days. Bratwurst can comprise multiple The metabolites AMS, AMSO, and AMSO2 were only detected spices, amongst others garlic. Garlic, as well as kohlrabi are in urine samples gathered after ramson consumption with the rich in sulfur-containing compounds, which might explain the sole exception of set UIX. In this urine set small amounts of detected metabolites in the samples collected prior to ramson the respective metabolites were detected in the sample collected consumption. Despite these small amounts in the control before ramson intervention. According to the food protocol the samples, distinct increases in the metabolite concentrations were respective test person consumed chicken escalope with mustard observed in the subsequently collected milk samples. The highest the day prior to the experiment. Mustard is rich in glucosinolates observed AMS concentrations in milk samples obtained after (Belitz et al., 2008), compounds that are sulfur-containing and ramson consumption were found to be quite consistent, with could therefore be responsible for the respective derivatives in the values in the range between 1.7 and 2.0 μg/kg milk. AMSO first urine sample. Although the presence of AMS, AMSO, and and AMSO2 reached values between 38.4 and 89.6 μg/kg milk AMSO2 could not be excluded in this case, distinct increases were and 53.0 and 98.6 μg/kg milk, respectively. Commonly, the observed after ramson consumption. Likewise, AMS, AMSO, maxima were either detected in the first or in the second milk and AMSO2 showed a rapid onset in all other urine sets after sample after ramson consumption which corresponds to a time ramson consumption. The maximum AMS concentrations in interval between 1.5 to 5 h after ramson ingestion. Thereby, the respective urine sets were found to be in the range between the AMSO maximum tended to appear slightly earlier than the 0.4 and 0.9 μg/kg urine, whereas the maximum AMSO and AMSO2 maximum. In most cases the highest concentrations of AMSO2 concentrations ranged between 46.3 and 81.2 μg/kg the metabolites AMS and AMSO were reached within 3 h after urine and 40.8 and 87.1 μg/kg urine, respectively. However, ramson consumption (as was the case for seven out of nine relative metabolite concentrations in urine strongly depend milk sets). The highest amount of AMSO2 was mostly observed on the water intake of the test persons. This discontinuous between 3 and 5 h (as was the case for six out of nine milk sets). variability can be taken account of by normalizing the metabolite Frontiers in Chemistry | www.frontiersin.org 7 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans FIGURE 2 | Identification of ramson-derived components in human milk and human urine. The milk samples were collected 10 min before (M2-1)and2h25min (M2-2) after ramson consumption. Urine samples were gathered 5 min before (U1-1)and2h(U1-4) after ramson consumption. (A) AMS, (B) AMSO, (C) AMSO2. The respective mass spectra are shown on the right side; they correspond to the elution time of the respective standard compound. FIGURE 3 | Box plot (mean value, markers at minimum and maximum metabolite concentration, box perc. 25–75%) of concentrations of ramson metabolites (AMS, AMSO, and AMSO2) in human milk, expressed as μg/kg human milk. Data comprise nine milk sets each being composed of four milk samples: one sample was collected before (“1st sample”) and three samples after (“2nd sample” to “4th sample”) ramson ingestion. Frontiers in Chemistry | www.frontiersin.org 8 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans FIGURE 4 | Time-resolved metabolite profiles of AMS, AMSO and AMSO2. Profiles are exemplarily shown for milk sets MI, MIV,andMVII.•AMS, AMSO, AMSO2. Time 0 h corresponds to the time of ramson consumption. concentrations to the creatinine content of the respective urine and 48.3 μg/mmol creatinine) and in U VIII amaximum sample. As a result, urine samples within one urine set as well concentration of 76.6 μg/mmol creatinine AMSO and 69.1 as urine sets from different test persons can be compared with μg/mmol creatinine AMSO2 was detected. Likewise, the each other, and allow a better comparison of the temporal maximum concentration of AMS differed between the urine sets, excretion processes in different subjects. Overall, the highest albeit to a lesser extent: AMS concentrations of 0.7, 0.4 and 0.6 normalized AMS and AMSO concentrations were observed μg/mmol creatinine were observed in UI, UIV,andU VIII, between 1 and 2 h after ramson consumption, and varied, in respectively. case of AMS, between 0.3 and 0.7 μg/mmol creatinine, and, in To exclude that these differences were due to different ramson case of AMSO between 32.9 and 106.9 μg/mmol creatinine. The samples and correspondingly natural differences in the respective highest amounts of AMSO2 in urine generally varied between plant material, additional experiments were conducted testing 27.5 and 97.7 μg/mmol creatinine. The AMSO2 maximum was a total of four subjects on the same day. Panelists were asked either detected temporally coinciding with the AMSO maximum to consume the same aliquots of the same ramson sample. The (as was the case for four urine sets) or with a temporal delay time-resolved metabolite profiles of the respective test persons of about 1 to 2 h (as was the case for five urine sets). In most obtained during this testing day were recorded and data are cases the AMSO2 maxima were observed about 2 h after ramson shown in Figure 7. consumption. Only in case of UIX, a maximum was observed The analyses revealed that there were still differences in the at 1 h, and in case of UVIIat 4 h after ramson consumption. respective metabolite profiles, although all four test persons A high concentration of AMSO2 commonly correlated with consumed 10 g aliquots from the same ramson sample. All high amount of AMSO and AMS in either case. In some urine profiles revealed a metabolite increase within 2 h after ramson sets a second increase in metabolite concentration could be consumption. However, in case of test person V asecond observed about 6 to 8 h after ramson consumption (e.g., U III increase was observed after about 6 h for all three ramson- and UV). However, this second increase was not as distinct derived metabolites. Likewise, a second increase was monitored as the first one, commonly reaching about 10–70% of the total in urine set U III for AMS. In all other cases we observed intensity of the first maximum. In Figure 5 the quantitative only one maximum. Furthermore, there were differences in the analyses of all urine samples are summarized as a box plot metabolite concentrations excreted after ramson consumption diagram. The metabolite concentrations for each urine sample, comparable to those that were observed when subjects were arranged according to urine sets, are additionally provided in tested at different days. While the maximum AMS concentration Table S2. showed only minor differences, with values between 0.3 and We additionally observed some variance in the metabolite 0.4 μg/mmol creatinine, AMSO maxima varied between 36.9 excretion patterns for the same individuals when test persons and 68.8 μg/mmol creatinine and in case of AMSO2 between provided urine sets at different days. In Figure 6, time-resolved 27.5 and 48.3 μg/mmol creatinine. These observations are metabolite profiles of three urine sets, (UI, UIV,and limited to four test persons. It provides a first insight into U VIII) provided by the same test person, are exemplarily the metabolism and excretion processes of ramson-derived displayed. The temporal excretion pattern between the testing metabolites. However, the differences between test persons days was comparable; in each case the highest metabolites might be more or less pronounced if a larger test group is concentrations were observed between 1 and 2 h for each tested. of the investigated metabolites. However, the amounts of excreted metabolites differed between the testing days. In U DISCUSSION I the maximal concentration of AMSO and AMSO2 was about 100 μg/mmol creatinine (106.9 and 97.7 μg/mmol Aroma profile analyses, conducted via orthonasal evaluation, creatinine, respectively). In UIVthe highest amount of revealed a change in the human milk odor after consumption these metabolites was about 50 μg/mmol creatinine (56.2 of about 10 g of ramson which was most pronounced about 2 Frontiers in Chemistry | www.frontiersin.org 9 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans FIGURE 5 | Box plot (mean value, markers at minimum and maximum metabolite concentration, box perc. 25–75%) of concentrations of ramson metabolites (AMS, AMSO, and AMSO2) in human urine, expressed as μg/mmol creatinine. Data comprise nine urine sets each being composed of eight urine samples: one sample was collected before (“pre”) and seven samples after (“0.5 h post” to “24 h post”) ramson ingestion. to 5 h after ramson consumption. As confirmed by our expert consumption. Such change was already described in milk samples sensory panel this change comprised a garlic-/cabbage-like odor that were obtained after garlic consumption (Scheffler et al., which was only perceived in milk samples collected after ramson 2016b). However, whereas a clear aroma change was already Frontiers in Chemistry | www.frontiersin.org 10 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans FIGURE 6 | Time-resolved metabolite profiles of ramson-derived metabolites (AMS, AMSO, and AMSO2) of three urine sets (UI, UIV,and U VIII) provided by the same test person at different days. Time 0 h corresponds to the time of ramson consumption. • AMS, AMSO, AMSO2. Broken line: time interval, when no sample was collected. FIGURE 7 | Time-resolved metabolite profiles of ramson-derived metabolites (AMS, AMSO, and AMSO2) of four urine sets (UII, U III, UIV, UV). All four test persons consumed ramson aliquots from the same ramson sample. Time 0 h corresponds to the time of ramson consumption. • AMS, AMSO, AMSO2. Broken line: time interval, when no sample was collected. observed after consumption of 3 g of raw garlic, in case of the effect might have been more pronounced during tasting, i.e., ramson about 10 g had to be consumed. Hence, garlic seems retronasal evaluation. However, due to work safety consideration to have a higher impact on the human body odor compared this type of evaluation was not carried out by the expert to ramson. This is in line with the general observation that panel. garlic consumption influences the body odor, e.g., breath, to a In relation to ramson consumption three metabolites were higher extent than ramson consumption (Borrelli et al., 2007). identified in human urine and milk, namely AMS, AMSO, and However, it still needs to be kept in mind, that all observed AMSO2. Of these, AMS had a garlic-/cabbage-like odor whereas sensory attributes, including the attribute garlic-/cabbage-like, the other two compounds were odorless. We found that AMS is were rated in an orthonasal evaluation as being just detectable related to the aroma change in the milk samples after ramson (intensity 1 or lower). Nevertheless, this does not rule out that consumption. Frontiers in Chemistry | www.frontiersin.org 11 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans Generally, the transfer of food components originating from assumed that AMS, AMSO, and AMSO2 are not ramson- the maternal diet into breast milk are not yet fully understood specific metabolites but may be rather linked to several sulfur but it has been postulated that compounds that are relevant constituents-bearing foods. On the other hand, further sulfury to the specific flavors of foods and beverages may affect the and/or odorous compounds of ramson such as 2-vinyl-4H-1,3- sensory quality of breast milk, as has been reported in the dithiin, 3-vinyl-4H-1,2-dithiin, AMDS, APDS, APS, DADS, DAS, case of carrots or alcohol (Mennella and Beauchamp, 1991, DATS, DMDS, DMS, DPDS, DPTS, MPDS, and MPTS were 1999). On the other hand, studies on the potential influence proven to be absent at levels as low as 1–10 μg/mL, both in of herbal tea or encapsulated fish oil products contradict samples collected before as well as after ramson consumption. these findings, as they ruled out any sensory or chemical This leads to the conclusion that ramson constituents are changes in human milk composition (Sandgruber et al., 2011; heavily metabolized and eliminated via other physiological Denzer et al., 2015). Here, we report a condiment that has routes. the potential of affecting the milk composition of nursing The highest observed AMSO concentration in human mothers. In most cases, irrespective of the consumed amount milk ranged between 38 and 90 μg/mLhumanmilk;the of ramson and the investigated excretion product, AMS, AMSO, highest observed AMSO2 concentration ranged between 53 and AMSO2, were only detected in samples after ramson and 99 μg/mL human milk. Similar concentrations of the consumption, demonstrating that they clearly derived from respective metabolites were observed in human milk after ramson consumption. In previous studies we were able to garlic consumption. However, the amount of consumed ramson identify the same metabolites in urine and milk samples obtained was more than three times as high as the consumed garlic after garlic consumption (Scheffler et al., 2016a,b). It can be amount (10 vs. 3 g) (Scheffler et al., 2018). The same applies FIGURE 8 | Metabolic fate of alliin. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine. Frontiers in Chemistry | www.frontiersin.org 12 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans when ramson- and garlic-derived metabolites in urine are ramson consumption and were investigated in comparison to compared. These differences are likely related to the different control samples obtained prior to ramson consumption by compositions of garlic and ramson. Both plants contain high means of GC-MS/O and GC-GC-MS/O. The analyses revealed amounts of S-alk(en)ylcysteine sulfoxides. The quantitatively three ramson-derived metabolites, namely AMS, AMSO, and dominating S-alk(en)ylcystein sulfoxide in garlic is alliin [S- AMSO2. Furthermore, the excretion profiles of these metabolites allylcysteine sulfoxide, 481–1,140 mg/100 g fresh weight (fw)] in human milk and urine were investigated. Therefore samples followed by methiin (S-allylcysteine sulfoxide 50–126 mg/100 g were collected over a time interval of about 24 h in case of fw) (Kubec et al., 1999) whereas in ramson it is methiin urine samples and up to 9 h in case of milk samples. In the (60 mg/100 g fw) followed by alliin (40 mg/100 g fw) (Kubec milk sets we observed the maximum concentrations of the et al., 2000). Accordingly, garlic and ramson comprise the metabolites within 3 h after ramson consumption. Likewise, the same sulfurous compounds that are odorless themselves, but highest AMS and AMSO concentrations in urine sets were are precursors for odor-active compounds. These precursors observed within 2 h after ramson consumption. The maximum are present at different ratios and concentrations, with garlic concentration of AMSO2 was reached about 2 to 4 h after containing about ten times the quantity of S-alk(en)ylcysteine ramson ingestion. Overall, the present study revealed that sulfoxides compared to ramson. Upon rapture various aroma ramson constituents are excreted correspondingly to garlic active compounds are formed. The degradation process of the constituents albeit at lower concentration levels, correspondingly S-alk(en)ylcysteine sulfoxides is exemplarily shown for alliin to lower content of the respective precursor substances in in Figure 8. Upon ingestion a mixture of sulfur-containing ramson. compounds is taken in. To the best of our knowledge, there have been no studies regarding the metabolism of ramson in DATA AVAILABILITY STATEMENT the human body. However, consumption of ramson and garlic leads to excretion of the same volatile compounds, both via All datasets generated for this study are included in the urine and milk. It is conceivable that the metabolism of ramson manuscript and the supplementary files. is related to the metabolism of garlic and follows the same routes. AUTHOR CONTRIBUTIONS As discussed in our previous publication the sulfurous compounds containing a dithioallyl-group, e.g. DADS, are LS, CS, and AB conceived and designed the experiments. LS transformed to allyl mercaptan which is subsequently methylated performed the experiments. TA contributed with analyses of to AMS (Germain et al., 2002; Lawson and Wang, 2005). The ramson samples. LS analyzed the data. AB contributed reagents formation of AMS after garlic consumption was previously materials analysis tools. LS and CS conceived the publication that confirmed by our group (Scheffler et al., 2016a,b). Now, we was approved by AB. All authors have read and approved the final can also confirm the presence of AMS in human milk and manuscript. urine after ramson consumption. Likewise, we demonstrated that AMSO and AMSO2 are metabolites of garlic as well as of FUNDING ramson constituents. Importantly, the pronounced differences in the amounts of S-alk(en)yl sulfoxides in the garlic and The authors gratefully acknowledge funding from the German ramson plant were reflected in the detected quantities of Academic Scholarship Foundation (Studienstiftung des AMS, AMSO, and AMSO2. Moreover, we observed differences Deutschen Volkes) for Laura Scheffler, and funding of between test persons that consumed aliquots of the same ramson instrumentation used in this study granted by the German sample. Accordingly, these inter-individual differences are most research foundation (DFG) in the frame of grant INST 90/979-1 likely related to the individual metabolic conversion steps and FUGG. respective enzyme activities due to genetic polymorphisms of enzymes, as has already been reported for drugs (Evans ACKNOWLEDGMENTS and Relling, 1999). Furthermore, the physiological status of each individual such as body mass index may influence the We are grateful to Yvonne Sauermann for skillfull technical metabolism of ramson components (Bachour et al., 2012). assistance. We thank Prof. Pischetsrieder for providing In case of the milk samples, lactation period might be access to the photometer used within this study. We are another influencing factor since the milk composition changes also grateful to all volunteers who participated in this continuously throughout lactation and is adapted to the age of study. the infant. SUPPLEMENTARY MATERIAL CONCLUSION The Supplementary Material for this article can be found The impact of ramson consumption on human milk and urine online at: https://www.frontiersin.org/articles/10.3389/fchem. composition was investigated. Samples were collected after 2018.00410/full#supplementary-material Frontiers in Chemistry | www.frontiersin.org 13 September 2018 | Volume 6 | Article 410 Scheffler et al. Ramson-Derived Metabolites in Humans REFERENCES Kubec, R., Svobodová, M., and Velišek, J. (1999). Gas chromatographic determination of S-alk(en)ylcysteine sulfoxides. J. Chromatogr. A 862, 85–94. Abu-Lafi, S., Dembicki, J. W., Goldshlag, P., Hanus, L. O., and Dembitsky, V. M. doi: 10.1016/S0021-9673(99)00902-4 (2004). The use of the ’Cryogenic’ GC/MS and on-column injection for study Kubec, R., Svobodova, M., and Velísek, J. (2000). Distribution of S- of organosulfur compounds of the Allium sativum. J. Food Composition Anal. alk(en)ylcysteine sulfoxides in some Allium species. 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Liver Physiol. 276, G425–G430. be construed as a potential conflict of interest. doi: 10.1152/ajpgi.1999.276.2.G425 Taucher, J., Hansel, A., Jordan, A., and Lindinger, W. (1996). Analysis Copyright © 2018 Scheffler, Sharapa, Amar and Buettner. This is an open-access of compounds in human breath after ingestion of garlic using proton- article distributed under the terms of the Creative Commons Attribution License (CC transfer-reaction mass spectrometry. J. Agric. Food Chem. 44, 3778–3782. BY). The use, distribution or reproduction in other forums is permitted, provided doi: 10.1021/jf960640e the original author(s) and the copyright owner(s) are credited and that the original Tokarska, B., and Karwowska, K. (1983). The role of sulfur-compounds in publication in this journal is cited, in accordance with accepted academic practice. evaluation of flavoring value of some plant raw-materials. Nahrung Food 27, No use, distribution or reproduction is permitted which does not comply with these 443–447. doi: 10.1002/food.19830270514 terms. 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