Antioxidative effects of sulfurous mineral water: Protection against lipid and protein oxidation

Abstract

To investigate the antioxidative properties of sulfurous drinking water after a standard hydropinic treatment (500 ml day(-1) for 2 weeks).
Forty apparently healthy adults, 18 men and 22 women, age 41-55 years old. The antioxidant profile and the oxidative condition were evaluated in healthy subjects supplemented for 2 weeks with (study group) or without (controls) sulfurous mineral water both before (T0) and after (T1) treatment.
At T1, a significant decrease (P<0.05) in both lipid and protein oxidation products, namely malondialdehyde, carbonyls and AOPP, was found in plasma samples from subjects drinking sulfurous water with respect to controls. Concomitantly, a significant increment (P<0.05) of the total antioxidant capacity of plasma as well as of total plasmatic thiol levels was evidenced. Tocopherols, carotenoids and retinol remained almost unchanged before and after treatment in both groups.
The improved body redox status in healthy volunteers undergoing a cycle of hydropinic therapy suggests major benefits from sulfurous water consumption in reducing biomolecule oxidation, possibly furnishing valid protection against oxidative damage commonly associated with aging and age-related degenerative diseases.

Full text

ORIGINAL ARTICLE
Antioxidative effects of sulfurous mineral water:
protection against lipid and protein oxidation
S Benedetti
1
, F Benvenuti
1
, G Nappi
2
, NA Fortunati
3
, L Marino
3
, T Aureli
3
, S De Luca
2
, S Pagliarani
1
and F Canestrari
1
1
Istituto di Istologia e Analisi di Laboratorio, Universita
`
di Urbino ‘Carlo Bo’, Urbino, Italy;
2
Centro di Studi e Ricerche in Medicina
Termale, Universita
`
di Milano, Milano, Italy and
3
Terme di Saturnia, Grosseto, Italy
Objectives: To investigate the antioxidative properties of sulfurous drinking water after a standard hydropinic treatment
(500 ml day
1
for 2 weeks).
Subjects/Methods: Forty apparently healthy adults, 18 men and 22 women, age 41–55 years old. The antioxidant profile and
the oxidative condition were evaluated in healthy subjects supplemented for 2 weeks with (study group) or without (controls)
sulfurous mineral water both before (T0) and after (T1) treatment.
Results: At T1, a significant decrease (Po0.05) in both lipid and protein oxidation products, namely malondialdehyde,
carbonyls and AOPP, was found in plasma samples from subjects drinking sulfurous water with respect to controls.
Concomitantly, a significant increment (Po0.05) of the total antioxidant capacity of plasma as well as of total plasmatic thiol
levels was evidenced. Tocopherols, carotenoids and retinol remained almost unchanged before and after treatment in both
groups.
Conclusions: The improved body redox status in healthy volunteers undergoing a cycle of hydropinic therapy suggests major
benefits from sulfurous water consumption in reducing biomolecule oxidation, possibly furnishing valid protection against
oxidative damage commonly associated with aging and age-related degenerative diseases.
European Journal of Clinical Nutrition (2009) 63, 106–112; doi:10.1038/sj.ejcn.1602892; published online 22 August 2007
Keywords: hydropinic treatment; sulfurous mineral water; hydrogen sulfide; thiols; oxidative markers; antioxidant profile
Introduction
Sulfur (S) is an interesting nonmetallic element representing
about 0.25% of our total body weight (Taylor and Williams,
1995; Beinert, 2000). As a part of the amino acids
methionine, cysteine and taurine, S performs a number of
functions in enzyme reactions and protein synthesis. It is
necessary for the formation of collagen, the protein found in
connective tissue, and keratin, which is fundamental for the
maintenance of the skin, hair and nails. Taurine is found in
bile acids used in digestion and mucopolysaccharides may
contain chondroitin sulfate, which is important for joint
tissues. S is part of other important body chemicals such as
insulin, which helps regulate carbohydrate metabolism, and
glutathione (GSH), the principal antioxidant in cells.
For all these reasons, mineral water employed in thermal
medicine, containing S in the format of sulfate
(SO
4
2
4200 mg l
1
) and/or hydrogen sulfide (H
2
S41mgl
1
),
has a long history of use in the treatment of various clinical
conditions, from dermatological to muscle/skeletal disorders
(Sukenik et al., 1999; Gupta and Nicol, 2004). In the past few
years, it has been documented that some S-based therapies
might play an important role in antioxidant strategies
against oxidative damage commonly associated with aging
and age-related degenerative diseases (Casetta et al., 2005;
Sachidanandam et al., 2005). For example, Bellometti et al.
(1996) observed that a cycle of mud therapy with sulfurous
mineral water consumption increased serum antioxidant
defenses in patients suffering from osteoarthritis through the
increment of both ceruloplasmin and transferrin levels, and
the reduction of the lipid peroxidation product, malondial-
dehyde (MDA). Accordingly, Caraglia et al. (2005) evidenced
the antioxidant effect of mud therapy in mice with
osteoarthritis showing a significant decrease in the produc-
tion of endogenous NO, which, on the one hand, reacts with
superoxide anion to form the pro-oxidant agent peroxy-
Received 23 February 2007; revised 16 July 2007; accepted 20 July 2007;
published online 22 August 2007
Correspondence: Dr S Benedetti, Istituto di Istologia e Analisi di Laboratorio,
Universita
`
degli Studi di Urbino ‘Carlo Bo’, Via Ubaldini, 7 Urbino (PU) 61029,
Italy.
E-mail: s.benedetti@uniurb.it
European Journal of Clinical Nutrition (2009) 63, 106–112
&
2009 Macmillan Publishers Limited All rights reserved 0954-3007/09 $
32.00
www.nature.com/ejcn

nitrite, and on the other, inhibits cartilage matrix synthesis
and promotes its degradation (Scher et al., 2007). At the same
time, sulfur bath therapy may cause a significant decline in
peroxide concentrations and superoxide dismutase activities,
as well as a reduction in homocysteine plasma levels in
patients with degenerative osteoarthritis (Ekmekcioglu et al.,
2002; Leibetseder et al,. 2004).
Together with mud and bath therapies, therapies involving
the drinking of water containing S (hydropinic treatments)
are also employed in thermal medicine, especially for their
action on gastroenteric and hepatic functions; until now,
however, the response of the antioxidant defense system to
orally ingested sulfurous waters has been poorly documen-
ted. The aim of the present study is to investigate the
antioxidative properties of a standard cycle of hydropinic
therapy in healthy volunteers receiving sulfurous mineral
water for a period of 2 weeks with respect to a group of
control subjects who regularly drank commercial mineral
water. Participants were evaluated both before (T0) and after
(T1) treatment to monitor their antioxidant profile as well as
their lipid and protein oxidation markers. It is well-known
that the balance between pro-oxidant agents and anti-
oxidant molecules is not perfect even under nonpathological
conditions; thus a certain degree of oxidative damage to
biomolecules also occurs in healthy subjects. From this point
of view, sulfurous water may be valuable in preserving and
enhancing antioxidant status.
Materials and methods
Subjects and study design
Forty subjects (18 men and 22 women, ages 41–55 years)
were recruited to participate in this study after giving
informed consent. All participants were in good health as
determined by a medical history questionnaire, physical
examination and clinical laboratory tests. All subjects
fulfilled the following eligibility criteria: (1) no history of
cardiovascular, hepatic, gastrointestinal or renal disease; (2)
no antibiotic or supplemental vitamin and/or mineral use
for at least 4 weeks before the beginning of the study and (3)
nonsmoker. The study protocol was in accordance with the
Helsinki Declaration of 1975, as revised in 1983.
The subjects were randomly divided into two groups. The
study group (n ¼ 20) received 500 ml day
1
of sulfurous
mineral water from the Thermal Center of Saturnia (Grosseto,
Italy) for 2 weeks, which has a sulfuric degree of 14.5 mg l
1
,
as reported in Table 1. To avoid H
2
S loss, water was consumed
within 1 h after the opening of the bottle. In the control
group (n ¼ 20), subjects drank regularly natural mineral water
from local food markets, which did not contain H
2
S.
Participants were asked to continue their usual diet; therefore,
changes in the serum lipid levels during this short time period
were not taken into account. At the end of the hydropinic
therapy, subjects underwent a final medical examination to
exclude any toxic effect of H
2
S-rich water consumption.
Blood sampling
Blood samples were collected from each subject in hepar-
inized tubes both before (T0) and after (T1) treatment. At T1,
the time interval from the last consumption of thermal water
and the blood sample collection was 24 h. Tubes were
immediately centrifuged at 2500 r.p.m. for 10 min and
plasma aliquots were stored at 801C until assayed. The
following parameters were monitored during the study:
hydroperoxides, MDA, protein carbonyls and advanced
oxidation protein products (AOPPs) as markers of oxidative
stress; total thiols (–SH) and liposoluble vitamins (a-, d- and
g-tocopherol, retinol, lutein, lycopene, a- and b-carotene) as
nonenzymatic antioxidants; and finally, the total anti-
oxidant capacity (TAC) of plasma that takes into account
both lipophilic and hydrophilic antioxidant components.
Hydroperoxide determination
Hydroperoxides were evaluated in plasma samples using a
commercial kit from Diacron s.r.l. (Grosseto, Italy). In this
test, plasmatic hydroperoxides, in the presence of iron (that
is released from plasma proteins by an acidic buffer), are able
to generate alkoxyl and peroxyl radicals, according to
Fenton’s reaction. Such radicals, in turn, are able to oxidize
an alkyl-substituted aromatic amine (that is dissolved in a
chromogenic mixture), thus transforming them to a pink-
colored derivative photometrically quantified at 505 nm.
The intensity of the developed color is directly proportional
to the concentration of hydroperoxides, according to
Lambert–Beer’s law. Results are expressed in mg of H
2
O
2
dl
1
.
The linearity range of the test is between 4 and 40 mg
H
2
O
2
dl
1
, the intra-assay coefficient of variation is 2.1%,
while the inter-assay is 3.1%. Reference values of healthy
subjects are between 20 and 24 mg H
2
O
2
dl
1
(Cesarone et al.,
1999).
Table 1 Chemical and physical characteristics of the mineral water from
Saturnia
Temperature 1C 36.9
pH (251C) — 6.25
Conductivity (251C) mScm
1
2996
Hardness 1f 204
Fixed residue at 1801Cmgl
1
2990
Sulfuric degree mg l
1
14.5
CO
2
mg l
1
674
Ca
2 þ
mg l
1
598
Mg
2 þ
mg l
1
134
Na
þ
mg l
1
63.7
K
þ
mg l
1
9.3
HCO
3
mg l
1
675
F

mg l
1
1.9
Cl

mg l
1
71.4
NO
2

mg l
1
o0.01
P
2
O
5
mg l
1
o0.01
SO
4
2
mg l
1
1469
NO
3

mg l
1
o0.1
NH
4
þ
mg l
1
26.8
Iron mg l
1
o0.01
SiO
2
mg l
1
20.7
Antioxidative effects of sulfurous mineral water
S Benedetti et al
107
European Journal of Clinical Nutrition

Malondialdehyde determination
Malondialdehyde plasmatic levels were evaluated by reverse-
phase high-performance liquid chromatography (HPLC) as
described previously (Agarwal and Chase, 2002). Briefly,
sample derivatization was carried out by adding 50 ml 0.05%
butylated hydroxytoluene solution, 400 ml 0.44 mol l
1
H
3
PO
4
solution and 100 ml 42 mmol l
1
thiobarbituric acid
to 50 ml plasma. Tubes were vortexed and then heated for 1 h
at 1001C. Following derivatization, samples were placed on
ice for 5 min and 250 ml of butanol was added to extract the
MDA–thiobarbituric acid complex. Tubes were vortexed and
then centrifuged at 10 000 g to separate the two phases.
Twenty microliters of the sample was removed from the
butanol layer and placed into an HPLC injector for analysis
without evaporation. The assay was performed using an
Alltima C
18
column (4.6  250 mm, 5 mm, from Alltech,
Milan, Italy) equipped with a guard column Alltima C
18
(4.6  7.5 mm, 5 mm). The eluent phase was methanol/buffer
(40:60, v/v), buffer consisting of 50 mmol l
1
KH
2
PO
4
,pH
6.8. The flow rate was 1 ml min
1
. UV detection was carried
out at 532 nm, and the fluorescence detector was set at an
excitation wavelength of 515 nm and emission wavelength
of 553 nm. All the organic solvents were pure HPLC grade
from Carlo Erba (Milan, Italy). The HPLC instrument was
from Jasco Corporation (Tokyo, Japan).
Carbonyl assay
The spectrophotometric analysis of plasmatic carbonyls was
based on the reaction of dinitrophenylhydrazine with
protein carbonyls to form protein hydrazones (Levine et al.,
2000). Carbonyl content was calculated from the peak
absorbance (355–390 nm) of dinitrophenylhydrazine-treated
samples using the molar extinction coefficient of dinitro-
phenylhydrazine (22 000
M
1
cm
1
). Protein content was
calculated from a bovine serum albumin standard curve
dissolved in guanidine hydrochloride and read at 280 nm.
Advanced oxidation protein product assay
Plasmatic levels of AOPP were measured by spectrophoto-
metry and calibrated with chloramine-T, which in the presence
of potassium iodide (KI), absorbed at 340 nm (Witko-Sarsat
et al., 1996). The reaction mixture was formed by 200 ml
plasma diluted 1:5 in 20 mmoll
1
phosphate-buffered saline
(PBS), 10 ml 1.16 mmol l
1
KI and 20 ml acetic acid; the
absorbance was immediately read at 340 nm on a microplate
reader (Bio-Rad Laboratories, Milan, Italy) against a blank
containing 200 ml of PBS instead of plasma. AOPP concentra-
tion was expressed in mmol l
1
of chloramine-T equivalents.
Determination of tocopherols, retinol and carotenoids
Plasma levels of liposoluble antioxidants were measured by
reversed-phase HPLC following deproteinization with ethanol
and extraction with hexane (Aebischer et al., 1999). After
centrifugation, the organic layer was removed and evaporated;
the residue was dissolved in 400 mlofamixtureofacetonitrile/
tetrahydrofuran/methanol (68:22:7, by vol.) and 100 ml were
injected into the HPLC system. The assay was performed using
an Alltima C
18
column (4.6  250 mm, 5 mm, from Alltech)
equipped with a guard column Alltima C
18
(4.6  7.5 mm,
5 mm). The eluent phase was acetonitrile/tetrahydrofuran/
methanol (68:22:7, by vol.) adjusted to 100 (v/v) with 1%
ammonium acetate; the flow rate was 1.5 ml min
1
.UVand
fluorescent detectors were programmed according to absorp-
tion, excitation and emission wavelengths of each molecule.
All the organic solvents were pure HPLC grade from Carlo
Erba. The HPLC instrument was from Jasco Corporation.
Determination of plasmatic –SH groups
The total thiol groups were evaluated by the use of a
commercial kit distributed by Diacron s.r.l. (Grosseto, Italy).
The method is based on the capacity that plasmatic –SH
groups have to react with 5,5
0
-dithiobis-2-nitrobenzoic acid,
followed by the development of a colored complex that can
be measured photometrically at 405 nm (Hu, 1994). Plas-
matic values range from 400 to 600 mmol l
1
.
Total antioxidant capacity determination
Total antioxidant capacity was evaluated in plasma samples by
using a commercial kit from Diacron s.r.l. The method
measures ability of plasma to reduce ferric ions and is based
on the ability of a solution of ferric ions, which bind to a
particular chromogen, to decolorize when they are reduced
from ferric to ferrous ions (Benzie and Strain, 1996). Values are
obtained by comparing the absorbance change at 505 nm in
the test reaction mixture with mixture containing ferrous ions
in known concentration. Absorbance changes are linear over a
wide concentration range in the antioxidant mixture, includ-
ing plasma, and with solutions containing one antioxidant in
purified form (vitamin C). Intra- and inter-assay coefficients of
variation are less than 5.5%. The plasmatic antioxidant power
is expressed in mmol l
1
of vitamin C; the normal value in
healthy subjects is approximately 2200 mmol l
1
.
Statistics and data processing
Results are expressed as mean7s.d. Statistical analysis was
carried out using the t-test for paired data (to evaluate T0 vs
T1) or unpaired data (to evaluate the study group vs control
group). Probability values of o0.05 were accepted. Statistics
and graphs were obtained using Microcal Origin 6.0 software
(Microcal Software Inc., Northampton, MA, USA).
Results
The oxidative condition of healthy subjects, supplemented
for 2 weeks with (study group) or without (controls)
Antioxidative effects of sulfurous mineral water
S Benedetti et al
108
European Journal of Clinical Nutrition

sulfurous mineral water both before (T0) and after (T1)
treatment, was evaluated. Data are summarized in Table 2.
At T0, no significant differences were found between study
and control groups with regard to the plasmatic levels of both
oxidative stress biomarkers (hydroperoxides, MDA, carbonyls
and AOPP) and nonenzymatic antioxidants (tocopherols,
carotenoids, retinol and total thiols). Accordingly, TAC values
were comparable in the two groups. At T1, these parameters
remained almost unchanged (P ¼ NS) with respect to the
baseline in controls receiving nonsulfurous mineral water; on
the contrary, a significant decrease (Po0.05) in lipid (MDA)
and protein (carbonyls and AOPP) oxidation markers was
observed in the study group after the 2-week treatment with
sulfurous water (Figures 1a–c), while hydroperoxide levels
were not affected. With regard to the antioxidant defense
system, no variations in the plasmatic levels of tocopherols,
carotenoids and retinol were detected in the study group at T1
when compared to the baseline; however, a significant
increment (Po0.05) in total thiol levels was evidenced after
the hydropinic treatment, with a concomitant increase
(Po0.05) in the TAC, as indicated in Figures 2a and b.
Concerning the final medical examination, no toxic effects of
H
2
S-rich water consumption were observed in the study group
throughout the hydropinic treatment.
Discussion
The pivotal importance of S in the biosynthesis of vital
cofactors and biomolecules (Taylor and Williams, 1995;
Beinert, 2000) and its involvement in complex reaction
mechanisms such as the sulfation of glycoproteins in
the lining of the gastrointestinal tract to build mucous
membranes or sulfation of galactosyl ceramides to form
sulfatides needed for myelin formation are well-known;
often, sulfation is a key step in the detoxication of unwanted
or excess metabolites (Mulder and Jakoby, 1990). All these
aspects may have clinical relevance for the interpretation of
Table 2 Oxidative stress biomarkers and antioxidant profile in healthy volunteers before (T0) and after (T1) treatment with (study group) or without
(control group) sulfurous water
Study group Control group
T0 T1 T0 T1
Hydroperoxides (mg H
2
O
2
dl
1
)23.972.6 23.873.0 23.672.7 23.872.9
MDA (mmol l
1
) 1.8970.25 1.4170.14
a,b
1.8770.22 1.8570.21
Carbonyls (nmol/mg proteins) 0.8970.09 0.6370.08
a,b
0.9170.09 0.9370.08
AOPP (mmol l
1
)26.372.0 23.471.2
a,b
27.672.1 27.071.9
TAC (mmol l
1
) 2184749 2284755
a,b
2179745 2187751
–SH groups (mmol l
1
) 533742 570738
a,b
525739 532743
Retinol (mmol l
1
) 3.570.4 3.670.4 3.470.3 3.570.5
a-Tocopherol (mmol l
1
)23.771.3 23.571.0 24.0771.4 23.871.3
d-Tocopherol (mmol l
1
) 0.09870.008 0.09570.008 0.09770.008 0.09970.008
g-Tocopherol (mmol l
1
) 0.4970.05 0.04670.06 0.5270.06 0.4970.05
Lutein (mmol l
1
) 0.3870.05 0.3770.05 0.4070.05 0.3970.05
Lycopene (mmol l
1
) 0.7370.14 0.7570.15 0.7270.12 0.7470.13
a-Carotene (mmol l
1
) 0.08070.011 0.08670.013 0.08470.012 0.08370.011
b-Carotene (mmol l
1
) 0.4370.07 0.4470.08 0.4570.07 0.4270.06
Abbreviations: AOPP, advanced oxidation protein products; MDA, malondialdehyde; TAC, total antioxidant capacity.
a
Significantly different from T0 (Po0.05, t-test for paired data).
b
Significantly different from controls (Po0.05, t-test for unpaired data).
T0 T1
p=0.028
p=0.032
p=0.027
p=0.028
p=0.032
MDACarbonylsAOPP
(µmol/l)
(nmol/mg protein)
(µmol/l)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
30
25
20
15
10
Figure 1 Significant decrease in malondialdehyde (MDA) (a),
carbonyl (b) and advanced oxidation protein product (AOPP) (c)
plasmatic levels in healthy subjects treated for 2 weeks with sulfurous
mineral water (500 ml day
1
). Po0.05 T1 vs T0, t-test for paired
data.
Antioxidative effects of sulfurous mineral water
S Benedetti et al
109
European Journal of Clinical Nutrition

the beneficial effects evidenced during S-based thermal
treatments such as mucolytic, expectorant, antiphlogistic
and antiseptic actions together with the improvement of
gastrointestinal functions.
Less known is the role of S contained in thermal waters in
oxidative stress reactions; in particular, the effects of
drinking therapies involving H
2
S-rich waters on the human
redox status has been poorly investigated. Interestingly,
recent papers have shown that the exogenous administra-
tion of the H
2
S donor, NaHS, to rats with myocardial injury
may reduce the accumulation of plasmatic lipid peroxida-
tion markers such as MDA and conjugated dienes; at the
same time, H
2
S may inhibit the formation of protein
carbonyls induced in vitro by hypochlorous acid (Geng
et al., 2004; Whiteman et al., 2005). In accordance with
these findings, in this study we observed a significant
decrease in both lipid and protein oxidation products,
namely MDA, AOPP and carbonyls, in plasma samples from
healthy volunteers subjected to a cycle of hydropinic therapy
with H
2
S-rich water (500 ml day
1
for 2 weeks). MDA is the
principal and most studied product of polyunsaturated fatty
acid peroxidation (Moore and Roberts, 1998). This aldehyde
is a highly toxic molecule and should be considered more
than just a marker of lipid peroxidation; in fact, its
interaction with nucleic acids and proteins has often been
referred to as potentially mutagenic and atherogenic (Del Rio
et al., 2005). On the other hand, AOPP levels are a measure of
highly oxidized protein concentration (especially albumin)
and correlate with plasma level of both dityrosine and
pentosidine (an advanced glycation end product) as indices
of oxygen-mediated protein damage (Witko-Sarsat et al.,
1998). Finally, carbonyls are early markers of protein
oxidation due to metal-catalyzed oxidative modifications
of amino-acid residues such as lysine, arginine, proline and
histidine (Stadtman and Levine, 2003).
The protective effects of sulfurous water administration
against the free-radical damage of lipids and proteins were
linked to a significant increment in the TAC of plasma that
takes into account both lipophilic and hydrophilic anti-
oxidant components. As the levels of tocopherols, carotenoids
and retinol almost remained unchanged before and after
treatment, the increase in TAC values was possibly related to
the significant increase in total –SH levels, which include
both protein (principally albumin) and nonprotein (cysteine
and GSH) thiol groups.
In this context, a very interesting article was published
during the draft of this paper, showing that endogenously
produced H
2
S can protect neurons from oxidative injury by
increasing the intracellular concentrations of GSH, the
principal antioxidant in cells (Kimura et al., 2006). This
effect depends on the ability of H
2
S to increase the activity of
g-glutamylcysteine synthetase and to upregulate cystine
transport, resulting in an increment in GSH levels. At the
moment, it is not possible to demonstrate the direct
connection between H
2
S-rich water ingestion and the
increase in intracellular GSH levels; in fact, no blood cells
were collected at T0 and T1. Nevertheless, it is possible to
hypothesize (Figure 3) that the increment in endogenous
H
2
S following the ingestion of sulfurous water may lead to
an increase in intracellular GSH levels, which in turn is
released from tissues to maintain plasmatic thiols in their
reduced and functional forms. In addition, H
2
S itself might
be involved in the reduction of thiols, thus being directly
implicated in redox reactions as an antioxidant.
Literature reports that endogenously produced H
2
S can be
hydrolyzed to hydrosulfide and sulfide ions; however, even if
in aqueous solution, about one-third of H
2
S remains un-
dissociated at pH 7.4 and can rapidly diffuse through tissues
(Wang, 2002). Indeed, H
2
S is permeable to plasma membranes
as its solubility in lipophilic solvents is approximately fivefold
700
600
500
400
300
200
100
Total thiols (µmol/l)
TAC (µmol/l vitamin C)
T0 T0T1 T1
2600
2400
2200
2000
1800
1600
1400
p=0.032
p=0.044
ab
Figure 2 Significant increment in total thiols plasmatic levels (a) and total antioxidant capacity (b) in healthy volunteers receiving 500 ml day
1
of sulfurous drinking water for a period of 2 weeks. Po0.05 T1 vs T0, t-test for paired data.
Antioxidative effects of sulfurous mineral water
S Benedetti et al
110
European Journal of Clinical Nutrition

greater than in water; for this reason, H
2
S is now considered
a gasotransmitter able to induce specific cellular responses
(Jeong et al., 2006; Oh et al., 2006; Rinaldi et al., 2006). To
date, the rate of H
2
S absorption in the gastrointestinal tract
after oral ingestion of sulfurous water is not documented,
nor it is clear in which forms this compound is actually
bioavailable. Studies along these lines are currently in
progress; at the same time, it would be of value to investigate
how long the effects of sulfurous water consumption last
after suspension of the therapy and if an acute treatment
(that is, 1 day) generates the same results. In this context,
possible increments in intracellular GSH levels should be
investigated as well as the balance of GSH and oxidized
glutathione (GSSG), which may reflect changes in redox
signaling and control.
In conclusion, our findings of improved body redox status
in healthy volunteers undergoing a cycle of hydropinic
therapy suggest major benefits from sulfurous water con-
sumption in reducing biomolecule oxidation, possibly fur-
nishing valid protection against oxidative damage commonly
associated with aging and age-related degenerative diseases.
Acknowledgements
We thank Mrs Francesca Baldon (Terme di Saturnia) for
technical assistance in secretarial work.
References
Aebischer CP, Schierle J, Schuep W (1999). Simultaneous determina-
tion of retinol, tocopherols, carotene, lycopene and xanthophylls
in plasma by means of reversed-phase high-performance liquid
chromatography. Methods Enzymol 299, 348–362.
Agarwal R, Chase SD (2002). Rapid, fluorimetric-liquid chromato-
graphic determination of malondialdehyde in biological samples.
J Chromatogr B Biomed Appl 775, 121–126.
Beinert H (2000). A tribute to sulfur. Eur J Biochem 267, 5657–5664.
Bellometti S, Cecchettin M, Lalli A, Galzigna L (1996). Mud pack
treatment increases serum antioxidant defences in osteoarthrosic
patients. Biomed Pharmacother 50, 37.
Benzie IF, Strain JJ (1996). The ferric reducing ability of plasma
(FRAP) as a measure of ‘antioxidant power’: the FRAP assay. Anal
Biochem 239, 70–76.
Caraglia M, Beninati S, Giuberti G, D’Alessandro AM, Lentini A,
Abbruzzese A et al. (2005). Alternative therapy of earth elements
increases the chondroprotective effects of chondroitin sulfate in
mice. Exp Mol Med 37, 476–481.
Casetta I, Govoni V, Granieri E (2005). Oxidative stress, antioxidants
and neurodegenerative diseases. Curr Pharm Des 11, 2033–2052.
Cesarone MR, Belcaro G, Caratelli M, Cornelli U, De Sanctis MT,
Incandela L et al. (1999). A simple test to monitor oxidative stress.
Int Angiol 18, 127–130.
Del Rio D, Stewart AJ, Pellegrini N (2005). A review of recent studies
on malondialdehyde as toxic molecule and biological marker of
oxidative stress. Nutr Metab Cardiovasc Dis 15, 316–328.
Ekmekcioglu C, Strauss-Blasche G, Holzer F, Marktl W (2002). Effect of
sulfur baths on antioxidative defense systems, peroxide concentra-
tions and lipid levels in patients with degenerative osteoarthritis.
Forsch Komplementarmed Klass Naturheilkd 9,216–220.
Geng B, Chang L, Pan C, Qi Y, Zhao J, Pang Yet al. (2004). Endogenous
hydrogen sulfide regulation of myocardial injury induced by
isoproterenol. Biochem Biophys Res Commun 318,756–763.
Gupta AK, Nicol K (2004). The use of sulfur in dermatology. J Drugs
Dermatol 3, 427–431.
Hu ML (1994). Measurement of protein thiol groups and glutathione.
Methods Enzymol 233, 380–385.
Jeong SO, Pae HO, Oh GS, Jeong GS, Lee BS, Lee S et al. (2006).
Hydrogen sulfide potentiates interleukin-1beta-induced nitric
oxide production via enhancement of extracellular signal-
regulated kinase activation in rat vascular smooth muscle cells.
Biochem Biophys Res Commun 345, 938–944.
Kimura Y, Dargusch R, Schubert D, Kimura H (2006). Hydrogen
sulfide protects HT22 neuronal cells from oxidative stress. Antioxid
Redox signal 8, 661–670.
Leibetseder V, Strauss-Blasche G, Holzer F, Marktl W, Ekmekcioglu C
(2004). Improving homocysteine levels through balneotherapy:
effects of sulphur baths. Clin Chim Acta 343, 105–111.
Levine RL, Wehr N, Williams JA, Stadtman ER, Shacter E (2000).
Determination of carbonyl groups in oxidized proteins. Methods
Mol Biol 99, 15–24.
Moore K, Roberts II LJ (1998). Measurement of lipid peroxidation.
Free Radic Res 28, 659–671.
Mulder GJ, Jakoby WB (1990). Sulfation. In: GJ Mulder (ed).
Conjugation Reactions in Drug Metabolism. Taylor and Francis:
London pp 107–161.
Oh GS, Pae HO, Lee BS, Kim BN, Kim JM, Kim HR et al. (2006).
Hydrogen sulfide inhibits nitric oxide production and nuclear
factor-kappaB via heme oxygenase-1 expression in RAW264.7
macrophages stimulated with lipopolysaccharide. Free Radic Biol
Med 41, 106–119.
Rinaldi L, Gobbi G, Pambianco M, Micheloni C, Mirandola P, Vitale
M (2006). Hydrogen sulfide prevents apoptosis of human PMN via
inhibition of p38 and caspase 3. Lab Invest 86, 391–397.
Sachidanandam K, Fagan SC, Ergul A (2005). Oxidative stress and
cardiovascular disease: antioxidants and unresolved issues. Cardi-
ovasc Drug Rev 23, 115–132.
sulfurous mineral water
H
2
S H
+
+ SH
−
Intestinal absorption
Sulfurous mineral water
Diffusion through tissues
Regulation of cell
function
cystine transport
γ-glutamylcysteine
synthetase
GSH synthesis
(intracellular pool)
GHS
(plasmatic pool)
GSSG
(disulfide form)
Oxidized thiols
(cysteine, proteins)
Reduced thiols
(cysteine, proteins)
Reduction of plasmatic
thiols
Involvement in redox
reactions
Circulating H
2
S
(2)
(1)
Figure 3 Possible relationships between orally ingested H
2
S,
glutathione (GSH) synthesis and plasmatic thiols. (1) H
2
S may
upregulate cystine transport and increase the activity of g-glutamyl-
cysteine synthetase, resulting in an increment in intracellular GSH
levels which in turn is released from tissues to maintain plasmatic
thiols in their reduced form. (2) H
2
S itself may be involved in the
reduction of plasmatic thiols, thus being directly implicated in redox
reactions as an antioxidant.
Antioxidative effects of sulfurous mineral water
S Benedetti et al
111
European Journal of Clinical Nutrition

Scher JU, Pillinger MH, Abramson SB (2007). Nitric oxide synthases
and osteoarthritis. Curr Rheumatol Rep 9, 9–15.
Stadtman ER, Levine RL (2003). Free radical-mediated oxidation of
free amino acids and amino acid residues in proteins. Amino Acids
25, 207–218.
Sukenik S, Flusser D, Abu-Shakra M (1999). The role of spa therapy
in various rheumatic diseases. Rheum Dis Clin North Am 25,
883–897.
Taylor D, Williams DR (1995). Trace Element Medicine and Chelation
Therapy. The Royal Society of Chemistry Paperbacks: Cambridge.
Wang R (2002). Two’s company, three’s a crowd: can H
2
S be the third
endogenous gaseous transmitter? FASEB J 16, 1792–1798.
Whiteman M, Cheung NS, Zhu YZ, Chu SH, Siau JL, Wong BS et al.
(2005). Hydrogen sulphide: a novel inhibitor of hypochlorous
acid-mediated oxidative damage in the brain? Biochem Biophys Res
Commun 326, 794–798.
Witko-Sarsat V, Friedlander M, Capeillere-Blandin C, Nguyen-Khoa T,
Nguyen AT, Zingraff J et al. (1996). Advanced oxidation protein
products as a novel marker of oxidative stress in uremia. Kidney Int
49, 1304–1313.
Witko-Sarsat V, Friedlander M, Nguyen Khoa T, Capeillere-Blandin C,
Nguyen AT, Canteloup S et al. (1998). Advanced oxidation protein
products as a novel mediators of inflammation and monocyte
activation in chronic renal failure. J Immunol 161, 2524–2532.
Antioxidative effects of sulfurous mineral water
S Benedetti et al
112
European Journal of Clinical Nutrition