In vivo metabolism of diallyl disulphide in the rat: Identification of two new metabolites

Article abstract of DOI:10.1080/0049825021000017902

1. Diallyl disulphide (DADS), a compound formed from the organosulphur compounds present in garlic, is known for its anticarcinogenic effects in animal models. 2. The aim was to identify and analyse the metabolites produced in vivo after a single oral administration of 200mgkg^-1 DADS to rats. The organic sulphur metabolites present in the stomach, liver, plasma and urine were measured by gas chromatography coupled with mass spectrometry over 15 days. 3. Data indicate that DADS is absorbed and transformed into allyl mercaptan, allyl methyl sulphide, allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO₂), which are detected throughout the excretion period. Overall, the highest amounts of metabolites were measured 48-72 h after the DADS administration. AMSO₂ is the most abundant and persistent of these compounds. The levels of all the sulphur compounds rapidly decline within the first week after administration and disappear during the second week. Only AMSO and AMSO₂ are significantly excreted in urine. 4. These potential metabolites are thought to be active in the target tissues. Our data warrant further studies to check this hypothesis.

Full text of DOI:10.1080/0049825021000017902

xenobiotica,
vol.
no.
32, 12, 1127± 1138
2002,
In vivo m etabolism of diallyl disulphide in the
rat: identi®cation of two new m etabolites
E. GERMAIN , J. AUGER , C. GINIES , M.-H. SIESS and C.
{
}
{
{
TEYSSIER
{
}
Unite Mixte de Recherche de Toxicologie Alimentaire and Unite Mixte de Recherche des
{
Aromes, Institut National de Recherche Agronomique, BP 86510, 17 rue Sully, F-21065
}
Dijon cedex, France
Institut de Recherche sur la Biologie des Insectes, Centre National de Recherche
Scienti®que, Universite F. Rabelais, Parc Grandmont, F-37200 Tours, France
{
Received 14 March 2002
1. Diallyl disulphide (DADS), a compound formed from the organosulphur
compounds present in garlic, is known for its anticarcinogenic e€ects in animal models.
2. The aim was to identify and analyse the metabolites produced in vivo after a single
oral administration of 200 mg kg¡ DADS to rats. The organic sulphur metabolites
1
present in the stomach, liver, plasma and urine were measured by gas chromatography
coupled with mass spectrometry over 15 days.
3. Data indicate that DADS is absorbed and transformed into allyl mercaptan, allyl
methyl sulphide, allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO2),
which are detected throughout the excretion period. Overall, the highest amounts of
metabolites were measured 48±72 h after the DADS administration. AMSO2 is the most
abundant and persistent of these compounds. The levels of all the sulphur compounds
rapidly decline within the ®rst week after administration and disappear during the second
week. Only AMSO and AMSO2 are signi®cantly excreted in urine.
4. These potential metabolites are thought to be active in the target tissues. Our data
warrant further studies to check this hypothesis.
Introduction
Since early times, many bene®cial properties have been ascribed to garlic for
human health. Recent investigations have provided a scienti®c basis to these
properties and have shown that garlic can interfere with di€erent processes
involved in the development of cardiovascular, neoplasic and several other diseases
(Reuter 1995, Amagase et al. 2001). The reported biological e€ects have been
attributed to the organosulphur compounds present in the bulb or formed after
cutting or chopping the bulb.
Among these compounds, diallyl disulphide (DADS) (®gure 1), which
accounts for 40±60% of the essential oil of garlic, has often been reported to
inhibit or reduce chemically induced carcinogenesis in rodents (Le Bon and Siess
2000, Milner 2001). DADS exerts a broad spectrum of e€ects on several stages
involved in the process of carcinogenesis, including modulatory e€ects on drug-
metabolizing enzymes (Yang et al. 2001), prevention of genotoxicity (Guyonnet
* Author for correspondence; e-mail: siess@dijon.inra.fr
Present address: Unite Amelioration, genetique et physiologie forestieres, Institut National de
}
Recherche Agronomique, Avenue de la Pomme de Pin Ð Ardon, BP 20619, F-45166 Olivet cedex,
France.
Xenobiotica ISSN 0049±8254 print/ISSN 1366±5928 online
http://www.tandf.co.uk/journals
2002 Taylor & Francis Ltd
#
DOI: 10.1080/0049825021000017902

1128
E. Germain et al.
SH
Allyl mercaptan, AM
S
Allyl methyl sulphide, AMS
O
S
Allyl methyl sulphoxide, AMSO
O
S
O
Allyl methyl sulphone, AMSO2
S
S
Diallyl disulphide, DADS
O
S
S
Allicin, diallyl thiosulphinate, DADSO
Figure 1. Structure of DADS and its metabolites.
et al. 2001), inhibition of cell proliferation, apoptosis and cell di€erentiation
(Sundaram and Milner 1996, Lea and Ayyala 1997), and an immunostimulating
e€ect (Kuttan 2000). In addition, DADS has also been shown to interact with
cholesterol synthesis (Gebhardt and Beck 1996).
Although there is considerable knowledge about the anticarcinogenic e€ects of
DADS, only one study has been dedicated to its in vivo metabolism and distri-
bution in rodents. Pushpendran et al. (1980) studied the uptake and metabolic fate
in mice of labelled DADS given at a sublethal dosage. They reported that DADS
was rapidly absorbed, with a maximal concentration in the liver 90 min after i.p.
administration. A total of 8% of the radioactivity present in the liver was identi®ed
as DADS. In addition, several ex vivo and in vitro systems have been used to
analyse and identify the metabolites of DADS. In a study using an isolated
perfused rat liver, DADS appeared to be converted to allylmercaptan (AM) (®gure
1) (Egen-Schwind et al. 1992a). In primary rat hepatocytes, the metabolites of
DADS have been analysed (Sheen et al. 1999) and within 120 min the majority of
DADS disappeared from the extracellular ¯uid and was converted to AM and allyl
methyl sulphide (AMS) (®gure 1). The amount of AM was greater than the
amount of AMS. Lawson and Wang (1993) reported that DADS was converted to
AM in presence of blood before reaching the liver or other organs. All these results
suggest that the metabolism of DADS occurred by way of reduction and
methylation. Furthermore, under other experimental conditions, the oxidation
of DADS has been reported (Teyssier et al. 1999). Previous experiments in our
laboratory have demonstrated the oxidation of DADS in allicin in the presence of

In vivo biotranformation of DADS in rat
1129
human liver microsomes (Teyssier et al. 1999). Other naturally occurring orga-
nosulphur compounds such as diallyl sulphide (Brady et al. 1991) and dipropyl
disulphide (Teyssier and Siess 2000) have also been shown to be oxidized.
Similarly, Mitchell (1988) pointed out the importance of the S-oxidation of
sulphides by monooxygenases as a pathway in the biotransformation of several
sulphur-containing compounds.
The aim of this current study was to investigate the in vivo fate of DADS and
its metabolites in rats after a single oral administration. For this purpose, we
analysed xenobiotic distribution in various tissues and determined their pharma-
cokinetic parameters. Special attention was paid to the hypothetical formation of
S-oxidized metabolites. The study was performed in rats with one oral adminis-
tration of 200 mg kg¡ DADS. At distinct time intervals over 15 days, the
1
concentrations of organosulphur compounds were determined in the stomach,
plasma, liver and urine by gas chromatography coupled with mass spectrometry
(GC-MS). The data con®rmed the presence of AM and AMS as in vivo
metabolites of DADS. Two new metabolites were detected and identi®ed as
allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO2) (®gure 1)
by di€erent analytical approaches.
M aterials and m ethods
Chemicals
AM, AMS, sodium metaperiodate, p-cymene, nonane and DADS were purchased from Sigma±
Aldrich Chemical (Strasbourg, France). DADS was puri®ed by distillation under reduced pressure
(95% purity checked by GC-MS), whereas AM (80% purity) and AMS (98% purity) were used as
standards without further puri®cation. Allicin (78% purity) was synthesized by the action of meta-
chloroperbenzoic acid on DADS as described by Mondy et al. (2001). All other reagents and organic
solvents were of HLPC or analytical grades (SDS, Peypin, France). Dichloromethane was distilled
before use for the solvent extractions.
Synthesis of AM SO and AM SO2
AMSO and AMSO2 were obtained by oxidation of AMS with sodium metaperiodate in
a
methanolic solution thawed on ice overnight (Furniss et al. 1989). The compounds were extracted
before injection into gas chromatography±mass spectrometry (GC-MS) and gas chromatography±
Fourier transformed infrared (GC-FTIR) in order to assess their structure. The reaction was total but
gave a mixture of AMSO (85%) and AMSO2 (15%) as estimated by GC-MS.
Animals and drug administration
Male 8-week-old Wistar rats (n 55, average weight 350 g) were purchased from Janvier (Le
ˆ
Genest, Saint Isle, France). Rats were housed in individual stainless-wire cages, maintained at 22 C
8
with a 12-h light/12-h dark cycle. They were fed ad libitum with standard diet from Harlan France (Ref.
20118S Harlan Gannat, France) and water. The animals were fasted for 18 h before and 6 h after drug
administration. DADS, dissolved in corn oil, was orally administered at a dose of 200 mg kg¡ body
1
weight. Negative controls were included for seven animals in order to check that no sulphur compounds
were detectable in their tissues. After oral administration, animals were maintained separately in
metabolic cages. Urine (kept under cold conditions) was collected every 24 h and stored at 20 C until
7
further analysis. Animals were killed by exsanguination under anaesthesia (iso¯urane 2.5% in oxygen)
8
…
ˆ
†
3 , 20
…
ˆ
†
3 , 40 min
…
ˆ
†
3 , 1
…
ˆ
†
3 , 2
…
ˆ
†
3 , 6
…
ˆ
†
3 , 24
…
ˆ
†
3 , 48
…
ˆ
†
and 72 h
during the 12 remaining days of the experiment.
10
…
n
n
n
after oral dosing and then every 2 days
n
n
†
n
n
n
4
ˆ
†
…
ˆ
n
3
n
3
Livers and stomachs were removed, weighed and stored at 20 C until further analysis. Blood and
7
8
urine were immediately centrifuged and were also stored at 20 C before analysis.
7
8
Sample preparation
Sample preparation was performed as described by Teyssier and Siess (2000). Brie¯y, stomachs and
livers were homogenized with a high-speed blender in distilled water. A total of 100 l of an ethanol
m

1130
E. Germain et al.
solution containing p-cymene (0.17 mg ml¡ 1), used as an internal standard (Martin-Lagos et al. 1995),
was added to the homogenate. After adding 10% w/v trichloroacetic acid 30%, the homogenate was
centrifuged and the supernatant extracted three times with dichloromethane. The same protocol
without homogenization was used for the plasma samples whereas urine samples were subjected to
only the dichloromethane extraction. The extracts were then concentrated by evaporation under a
nitrogen stream to a ®nal volume of 500 l. To this volume, 100 l nonane dissolved in dichloromethane
m
m
(0.15 mg ml¡ 1) was added in order to standardize each sample concentration and an aliquot of 2 l was
then injected into the chromatograph under the conditions described below.
m
GC-M S conditions for DADS, AM , AMS, AM SO and AM SO2 analysis
GC-MS was carried out using a Agilent Technologies (Les Ullis, France) 6890 gas chromatograph
coupled to Agilent Technologies 5973 Mass Selective Detector using a capillary DB1701 column
£
(30 m 0.32 mm i.d., 1 m ®lm thickness), a splitless injection and an electronic ionization at 70 eV.
m
The oven was programmed from 35 C (2 min) to 220 C at a rate of 5 C min¡ 1. The other conditions
8
8
8
were: carrier gas, helium (constant velocity ®xed at 35 cm s¡ 1); and temperature of the injection port,
200 C. Peak identi®cations were based on comparison with retention times and spectra of standards.
8
Each sample was analysed twice. For the ®rst acquisition, GC-MS was used in scan mode from m/z 28±
300 amu in order to ascertain the identi®cation of compounds by comparison with standards. For the
second acquisition, GC-MS was used in selected ion monitoring (SIM) mode. The quanti®cation of
AM, AMS, AMSO, AMSO2 and DADS was achieved by monitoring the ions at m/z 74, 88, 104, 120
and 146, respectively. The purity was continuously checked by the control of the ratio between this
quantifying ion and another one, being speci®c for each compound. Chemical ionization was performed
with NH3 as gas reactant.
GC-M S conditions for allicin analysis
For allicin analysis, we applied the recently developed speci®c GC-MS method (Mondy et al. 2001).
Brie¯y, analyses were carried out on a benchtop Perkin Elmer turbomass system with a split±splitless
£
injector and a fused-silica capillary column (10 m 0.32 mm) with 4 m methylsilicon coating. The
m
carrier gas was helium; the oven temperature programme was 5 C min¡ from 70 to 250 C. The
1
8
injection port temperature was 200 C. Total ion chromatograms and mass spectra were recorded in the
8
8
electron impact ionization mode of 70 eV. The transfer line and source temperature were maintained at
150 C. Peak identi®cations were based on comparison with retention times and spectra were obtained
8
with pure allicin. These GC-MS conditions allowed the direct detection of allicin and the detection of
3-vinyl[4]-1,2 dithiin and 2-vinyl[4]-1,3 dithiin, two compounds formed from the allicin degradation
during GC-process (Mondy et al. 2001). The limit of detection of allicin was estimated with a standard
solution at 15 ng.
GC-FTIR conditions for AM SO and AM SO2 analysis
AMSO and AMSO2 from dichloromethane extracts were identi®ed by GC-FTIR analysis using
a Biorad Digilab FTS 60A spectrometer. This equipment was connected by means of a digilab
Tracer1 direct-deposition interface to a Hewlett-Packard HP 5890 Series II (Palo Alto, CA, USA)
gas chromatograph, which was equipped with a splitless±split injector. GC separation was similar to
the above-mentioned method (GC-MS conditions for DADS, AM, AMS, AMSO and AMSO2
analysis).
Quantitative determination of the organosulphur compounds
For each sulphur compound, the sensitivity and linearity of the GC-MS detector used in the SIM
mode was calculated from a series of standard solutions dissolved in dichloromethane. The response
ˆ
‡
coecient (a) was calculated from the curve y ax b, where y is the adjusted area (with the areas of p-
cymene and nonane) of the quanti®ed ion and x is the quantity of the compound (ng). The response
coecient of all other compounds was essentially similar, between 1.24 and 4.19, except for AMSO2 ,
2
…
†
was about 0.995. The limits of detection
which was low (0.32). The lowest correlation coecient
r
varied depending on the sulphur compounds and were 0.04 ng for DADS, 0.64 ng for AM, 0.04 ng for
AMS, 0.03 ng for AMSO and 0.11 ng for AMSO2.
The concentration of each sulphur compound in the biological samples was calculated by the ratio
between the area of the sulphur compound and the product of the p-cymene area and the nonane area.
After excluding every value beneath the linearity region of the detector, the amount of each compound
was obtained by integrating its speci®c response coecient. Results were given as a mean of three
separate experiments ( g g¡ organ).
1
m

In vivo biotranformation of DADS in rat
1131
Pharmacokinetic analysis
The non-compartmental method was used to calculate the pharmacokinetic parameters with the
Kinetica1 software (InnaPhase, Champs sur Marne, France). The area under the plasma concentration±
¡
time curve (AUC) was computed using the trapezoidal log±linear method, when Cn Cn 1. The
>
…
†
ˆ
elimination half-life t1=2 was calculated as t1=2
The plasma clearance (Clp) was calculated as Clp dose AUC¡ 1 . Tmax corresponded to the time for
ln 2L¡z 1, where Lz is the elimination rate constant.
ˆ
…
which the concentration was maximum Cmax
†
.
Results
Identi®cation of volatile metabolites
After DADS administration to rats, the parent molecule was detected in almost
all analysed tissues within the ®rst hours (data not shown). In addition, the DADS
metabolites such as AM, AMS and two unknown compounds were detected in all
the tested organs. Despite the speci®city of the method used, neither allicin nor
vinyl dithiines were detected. Figure 2 shows a gas chromatogram of the volatile
components detected in the stomach. DADS, AM and AMS were identi®ed by
comparing their retention times and mass spectra with those of standard com-
pounds. The molecular weights of the two unknown compounds, 104 and 120,
were determined by chemical ionization in GC-MS. Owing to the absence of the
corresponding standards, their identi®cation was achieved by di€erent approaches
summarized in table 1. Based on GC-MS data (®gure 3), the intensity of the ion at
Abundance
5
7
6500000
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
1
6
3
4
2
4.00
6.00
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time (min)
Figure 2. Total ionic current chromatogram of extracted stomach (second day) obtained on GC-MS.
Peaks 1, AM; 2, AMS; 3, nonane; 4, p-cymene; 5, DADS; 6, AMSO; 7, AMSO2. *Unidenti®ed
compounds detected both in extracts originating from control and DADS-treated animals.

1132
E. Germain et al.
Table 1. Chemical characteristics of the AMSO and AMSO2 metabolites.
MW
104
120
CIa (MH)‡
Isotopic ratio (%)b
Formula
C4H8 SO
C4H8 SO2
AMSO
105
106/105 5.95 (5.36)
107/105 4.71 (4.60)
122/121 5.80 (5.40)
123/121 4.72 (4.80)
AMSO2
121
a Chemical ionization.
b Reported values (relative abundance) correspond to the isotopic ratio of the pseudo-molecular ion
(MH)‡ . The theoretical values are given in parentheses
100
41
(A)
75
104
50
25
64
45
38
48
29
73
67
65
87
85
58 61
60
0
20
25
30
35
40
45
50
55
70
75
80
90
95
100 105 110 115 120 125 130 135 140
100
41
(B)
75
50
25
0
120
38
64
45
45
56
79
105
48
29
30
20
25
35
40
50
55
60
65
70
75
80
85
90
95 100 105 110 115 120 125 130 135 140
m/z
Figure 3. Mass spectra of AMSO (A) and AMSO2 (B).
‡
the m/z ratio of (M 2) indicated the presence of one sulphur atom in each
compound. The observed isotopic ratios were in agreement with the calculated
ratios for molecules containing one atom of sulphur, four atoms of carbon, eight
atoms of hydrogen and one or two atoms of oxygen for the ®rst and the second
metabolites, respectively. Since oxidation of sulphur compounds has been re-
ported in the literature (Williams et al. 1966, Brady et al. 1991, Nickson et al. 1995,
Teyssier and Siess 2000), we hypothesized that these two unknown metabolites
corresponded to AMSO and AMSO2. Subsequently, the chemical synthesis of
these compounds was undertaken. Their chemical structures were ascertained by
GC-MS and GC-FTIR. Indeed, the infrared spectra showed strong absorption
ˆ
bands for the sulphoxide moiety (
998 cm¡ 1), for the sulphone moiety
¸
ˆ
S
O

In vivo biotranformation of DADS in rat
1133
1136 cm¡
;
asymmetric stretching
(
¸
SO2
1
ˆ
ˆ
1
(symmetric stretching
¸
SO2
1289 cm¡ 1) and small absorption bands for the allyl chain (
1641 cm¡
;
ˆ
¸
ˆ
C
C
3088 cm¡ 1) (Socrates 1994). Hence, the in vivo metabolites were then
ˆ
¸
ˆ
¡
H
C
compared with the synthesized standards and were identi®ed as AMSO and
AMSO2.
M etabolite pro®les in stomach, liver, plasma and urine
Figure 4 shows the time-dependent tissue concentrations of DADS and its
metabolites AM, AMS AMSO and AMSO2 after DADS oral administration. In
the stomach, all these compounds were detected with various rates of appearance
stomach
A
100
80
DADS
AM
AMS
60
AMSO
AMSO2
40
20
0
0
5
10
15
dsay
liver
B
60
40
20
AMS
AMSO
AMSO2/10
0
0
5
10
15
day
plasma
C
15
10
AMS
AMSO/10
AMSO2/10
5
0
0
5
10
15
day
urine
D
15
10
AMS
AMSO
AMSO2
5
0
0
5
10
15
dsay
Figure 4. Concentrations of volatile metabolites in the stomach, liver, plasma and urine after one oral
administration of DADS to male rats (200 mg kg¡ 1). Results are the mean of three separate
experiments ( g g¡ organ).
1
m

1134
E. Germain et al.
and di€erent concentration±time pro®les (®gure 4A). The highest levels of DADS
were found during the 24 h. Thereafter, it sharply decreased and was not
detectable 3 days after oral administration. In contrast, AMSO2 reached a maximal
concentration later in the period of follow-up, i.e. on the third day, and was still
detectable for 11 days (®gure 4A). In the plasma and the liver, AMSO2 was the
most abundant and persistent compound. The other compounds were, by rank
order: AMSO and then AMS. In plasma, AM was barely detectable and DADS
appeared transiently at 20 min after administration (data not shown). AMSO2
concentration reached its maximum on the second day and thereafter decreased,
formed a plateau between days 3 and 5 and became undetectable on day 9 (®gure
4C). In liver, DADS and AM were detected only during the ®rst hours (data not
shown). The levels of AMSO2 increased extensively over the ®rst 2 days (®gure
4C). After reaching a maximum, the level of AMSO2 slowly decreased but was still
detected 11 days after DADS administration. In urine, neither AM nor DADS
were detected (®gure 4D). Only trace amounts of AMS were excreted only on the
second day, whereas AMSO and AMSO2 were excreted throughout a longer
period. The AMSO2 concentration±time pro®le was similar to the one observed in
plasma with its highest level in urine corresponding to 15% of the maximal plasma
level (®gure 4C, D).
Pharmacokinetic parameters
The plasma concentration±time curve of the metabolites of DADS after oral
administration to the rat is shown in ®gure 5 and the pharmacokinetic parameters
are summarized in table 2. In the case of DADS, only Cmax and Tmax were
determined since the plasma concentrations were too low to enable an accurate
determination of other pharmacokinetic parameters. The Cmax of the metabolites
were higher than that of DADS (0.001 mmol l¡ 1) as were plasma Tmax. Indeed, the
AM
AMS
1e+0
AMSO
AMSO2
1e-1
1e-2
1e-3
1e-4
1e-5
1e-6
0
25
50
75 100 125 150 175 200 225
hours
Figure 5. Plasma concentration±time curve of metabolites of DADS after one oral administration of
DADS (200 mg kg¡ ) to male rats. Data are the means of three separate experiments SEM.
1
§
After 75 h for AM and AMS, and after 125 h for AMSO, the measured amounts were beneath the
detection limit.

In vivo biotranformation of DADS in rat
1135
Table 2. Pharmacokinetic parameters of DADS and its metabolites after a single oral administration.
DADS
AM
AMS
AMSO
AMSO2
t1=2(h)
n.d.a
0.001
<1b
n.d.
4.39
0.008
24
0.324
1.475
6.78
0.008
24
0.328
1.455
7.16
0.376
48
23.75
0.020
8.64
1.440
48
116.86
0.004
Cmax (mmol l¡
Tmax (h)
)
1
AUC total (h mmol l¡
)
1
Clp (l h¡
n.d.
1
†
Parameters were calculated from three separate experiments.
a Not determined; b estimated value.
DADS Tmax was estimated to be < 1 h whereas this time increased to 24 h for AM
and AMS and to 48 h for AMSO and AMSO2. The AUC for AM of
0.324 h mmol l¡ increased for the other metabolites until the maximal value of
1
116.86 h mmol l¡ was determined for AMSO2.
1
Discussion
The present study was carried out to investigate the in vivo biotransformation
of DADS after oral administration to rats. For this purpose, we applied a
quantitative and sensitive GC-MS method suitable for organosulphur compound
analysis, which has already been validated (Martin-Lagos et al. 1995). We meas-
ured not only the amounts of DADS, but also those of AM, AMS, AMSO and
AMSO2, which were identi®ed as DADS metabolites in the stomach, plasma, liver
and urine. Based upon their plasma concentrations, their pharmacokinetic par-
ameters were determined. We observed large interindividual variations which are
particularly representative in the bioavailability (coecient of variation of DADS,
n.d.; AM, 81%; AMS, 32%; AMSO, 38%; AMSO2, 7%). Our results, reported
herein, show that DADS is absorbed, but owing to its low plasma concentrations,
it was not possible to determine accurately the related pharmacokinetic par-
ameters. However, the uptake of DADS in the liver was observed only during
the ®rst 2 h after dosing (data not shown), and DADS was transiently detected in
plasma and was totally undetectable in the urine. The DADS levels in the liver and
plasma did not exceed 0.5% of the DADS level in the stomach. These data are in
accordance with a previous study in humans in which no DADS was detected in
the blood and urine after ingestion of raw garlic (Amagase et al. 2001). The fact
that DADS was hardly detected in any sample could be linked with its metabo-
lism. Indeed, in an experiment in our laboratory using an isolated rat liver
perfused with DADS, the apparent t1 2 of DADS was estimated as <1 h
=
(unpublished data). In spite of the impossibility of assessing the in vivo pharma-
cokinetic parameters of DADS, all these observations taken together strongly
suggest that once absorbed, DADS is rapidly and extensively metabolized.
Our data show that DADS is transformed into AM, AMS, AMSO and
AMSO2. Among these metabolites, AMSO2 and AMSO are the major and more
persistent volatile metabolites found in all assayed tissues. AMSO2 appears to be
the ®nal metabolite since no new metabolite was detected when AMSO2 levels
decreased. In the same way, Nickson et al. (1995) reported that only dipropyl
sulphone was detected in the urine of rats after oral ingestion of dipropyl
sulphoxide, indicating the absence of subsequent biotransformation of the sul-

1136
E. Germain et al.
phone. AM and AMS were actually detected, but their bioavailabilities (table 2)
were lower than those of the S-oxidized metabolites (table 2). The rapid elim-
ination of AM and AMS in the breath described by Rosen et al. (2001) could
explain their moderate amounts and low bioavailability observed in our study.
However, an alternative hypothesis is that AMS is the precursor of AMSO and
AMSO2. This hypothesis is consistent with the Tmax of the metabolites which are
compatible with sequential DADS transformation into AM to AMS to AMSO and
®nally to AMSO2 (table 2).
Interestingly, as far as pharmacokinetic pro®les were concerned, AMSO and
AMSO2 presented similar patterns. In the liver and the plasma, they were
characterized by an initial sharp rise during the ®rst 2 days followed by a slow
decline lasting 3±9 days suggesting a slow elimination. This phenomenon was
unexpected since the overall hydrophilic nature of S-oxidized metabolites would
favour a rapid and complete urinary excretion. However, owing to the charge
separation of sulphur-oxygen linkages in the S-oxidized metabolites, it is possible
that these metabolites may interact more strongly with proteins and lipids and
thereby be retained in the body, delaying their overall excretion. Similar pharma-
cokinetic results have been described during a study in rats on the bioavailability of
vinyldithiins, other transformation products of allicin (Egen-Schwind et al.
1992b). These latter compounds are additional transformation products of allicin
with elimination half-lives of about 5 h and were detected in many tissues over
24 h, but the maximal serum concentration was observed within the 30 ®rst min
after ingestion. In any case, the poor elimination of the sulphone correlates with
the persistence of these sulphur compounds in plasma enabling their availability to
the organism.
In good agreement with the literature, our study con®rms that the liver plays a
major role in the in vivo fate of DADS not only in terms of metabolism, but also as
a storage site. Indeed, the liver was the organ with the highest concentrations of all
sulphur compounds (®gure 4) and this phenomenon is further highlighted when
the organ weight is taken into account. Moreover, the close quantitative and
qualitative pro®les obtained in the liver, plasma and urine for sulphur compounds
strongly suggest that their output from the liver is performed by the plasma, and
subsequently to the urine. The well-known poor elimination of sulphone by the
kidney before the passing on to urine is also observable in our study by the
persistence of these sulphur compounds in plasma.
This study has clearly established the presence of volatile metabolites in several
organs after DADS ingestion but the formation of other metabolites cannot be
ruled out. In particular, it has been reported that several glutathione conjugates are
formed from diallyl sulphide (Jin and Baillie 1997) and dipropyl disulphide
(Teyssier and Siess 2000), which could also happen during the DADS metabolic
process. Hence, if those conjugates actually exist, it would be of major interest to
compare their levels with those of AMSO and AMSO2.
In conclusion, we con®rmed that AM and AMS are in vivo metabolites of
DADS and identi®ed AMSO and AMSO2 as new and predominant metabolites of
DADS in vivo in rats. Previously, DADS has been assumed to be one of the active
components of garlic in vivo. Nevertheless, owing to the rapid and extensive
hepatic metabolism described herein, there seems to be no systemic bioavailability
of DADS after oral administration. Taking into account the time of exposure and
quantity of the di€erent metabolites of DADS, the oxidized compounds are

In vivo biotranformation of DADS in rat
1137
probably involved in the bene®cial e€ects ascribed to garlic. Further studies are
needed to assess the biological e€ects of these metabolites of DADS.
Acknowledgem ents
The authors thank Etienne Semon and Joelle Chevalier for technical assistance.
This research was carried out within the framework of the EU Garlic & Health
project (QLK1-CT-1999-00498). The authors acknowledge the ®nancial support
of the EU.
References
Amagase, H., Petesch, B. L., Matsuura, H., Kasuga, S.
Itakura
, Y., 2001, Intake of garlic and its
and
bioactive components. Journal of Nutrition, 131, 955S±962S.
Brady, J. F., Ishizaki, H., Fukuto, J. M., Lin, M. C., Fadel, A., Gapac, J. M.
Yang,
and
C. S., 1991,
Inhibition of cytochrome P-450 2E1 by diallyl sul®de and its metabolites. Chemical Research in
Toxicology, 4, 642±647.
Egen Schwind, C., Eckard, R.
Kemper
, F. H., 1992a, Metabolism of garlic constituents in the
-
and
isolated perfused rat liver. Planta Medica, 58, 301±305.
Egen Schwind, C., Eckard, R., Jekat, F. W. Winterhoff
, H., 1992b, Pharmacokinetics of
-
and
vinyldithiins, transformation products of allicin. Planta Medica, 58, 8±13.
Furniss, B. S., Hannaford, A. J., Smith, P. W. G.
Tatchell
, A. R., 1989, Vogel’s Text Book of
and
Practical Organic Chemistry, 5th edn (Harlow: Wiley).
Gebhardt, R.
Beck
, H., 1996, Di€erential inhibitory e€ects of garlic-derived organosulfur
compounds on cholesterol biosynthesis in primary rat hepatocyte cultures. Lipids, 31, 1269±
and
1276.
Guyonnet, D., Belloir, C., Suschetet, M., Siess, M. H.
Le Bon,
A. M., 2001, Antimutagenic
and
activity of organosulfur compounds from Allium is associated with phase II enzyme induction.
Mutation Research, 495, 135±145.
Jin, L.
Baillie
, T. A., 1997, Metabolism of the chemoprotective agent diallyl sul®de to glutathione
and
conjugates in rats. Chemical Research in Toxicology, 10, 318±327.
Kuttan
,
G., 2000, Immunomodulatory e€ect of some naturally occurring sulphur-containing
compounds. Journal of Ethnopharmacology, 72, 93±99.
Lawson, L. D. Wang
and
garlic (Allium sativum). Planta Medica, 59, A688±A689.
Le Bon, A. M. Siess
, Z. J., 1993, Pre-hepatic fate of the organosulfur compounds derived from
and
of cancer. Drug Metabolism and Drug Interaction, 17, 51±79.
Lea, M. A. Ayyala
, M. H., 2000, Organosulfur compounds from Allium and the chemoprevention
and
cancer cells. International Journal of Oncology, 11, 181±185.
Martin Lagos, R. A., Serrano, M. F. Lopez
, M. D. R., 1995, Determination of organic sulphur
, U. S., 1997, Di€erentiating and growth inhibitory e€ects of diallyl disul®de on
-
and
compounds in garlic extracts by gas chromatography and mass spectrometry. Food Chemistry, 53,
91±93.
Milner
, J. A., 2001, A historical perspective on garlic and cancer. Journal of Nutrition, 131, 1027S±
1031S.
Mitchell
,
Disposition , 6, 245±252.
S. C., 1988, Biological consequences of drug sulphoxidation. Drug Metabolism and
Mondy, N., Naudin, A., Christides, J. P., Mandon, N.
Auger,
J., 2001, Comparison in GC-MS
and
and HPLC in Allium volatiles analysis. Chromatographia, 53, S356±S360.
Nickson, R. M., Mitchell, S. C.
Zhang
, A. Q., 1995, Fate of dipropyl sulphone in rat.
and
Xenobiotica , 25, 1391±1398.
Pushpendran, C. K., Devasagayam , T. P., Chintalwar, G. J., Banerji, A.
Eapen, J
., 1980, The
and
metabolic fate of [35S]-diallyl disulphide in mice. Experientia , 36, 1000±1001.
Reuter
, H. D., 1995, Allium sativum and Allium ursinum: Part 2. Pharmacology and medicinal
application. Phytomedicine, 2, 73±91.
Rosen, R. T., Hiserodt, R. D., Fukuda, E. K., Ruiz, R. J., Zhou, Z., Lech, J., Rosen, S. L.
and
, T. G., 2001, Determination of allicin, S-allylcysteine and volatile metabolites of garlic
Hartman
in breath, plasma or simulated gastric ¯uids. Journal of Nutrition, 131, 968S±971S.
Sheen, L. Y., Wu, C. C., Lii, C. K. Tsai,
and
sul®de in primary rat hepatocytes. Food and Chemical Toxicology, 37, 1139±1146.
Socrates
S. J., 1999, Metabolites of diallyl disul®de and diallyl
, G., 1994, Infrared Characteristic Group Frequencies. Tables and Charts, 2nd edn (Chichester:
Wiley).

1138
In vivo biotranformation of DADS in rat
Sundaram, S. G.
Milner
, J. A., 1996, Diallyl disul®de induces apoptosis of human colon tumor
and
cells. Carcinogenesis, 17, 669±673.
Teyssier, C., Guenot, L., Suschetet, M.
Siess
, M. H., 1999, Metabolism of diallyl disul®de by
and
human liver microsomal cytochromes P-450 and ¯avin-containing monooxygenases. Drug
Metabolism and Disposition, 27, 835±841.
Teyssier, C.
Siess
, M. H., 2000, Metabolism of dipropyl disul®de by rat liver phase I and phase II
and
enzymes and by isolated perfused rat liver. Drug Metabolism and Disposition, 28, 648±654.
Williams, K. I., Burstein, S. H. Layne
and
, D. S., 1966, Metabolism of dimethyl sul®de, dimethyl
sulfoxide, and dimethyl sulfone in the rabbit. Archives of Biochemistry and Biophysics, 117, 84±
87.
Yang, C. S., Chhabra, S. K., Hong, J. Y.
Smith
, T. J., 2001, Mechanisms of inhibition of chemical
toxicity and carcinogenesis by diallyl sul®de (DAS) and related compounds from garlic [Review].
and
Journal of Nutrition, 131, 1041S±1045S.