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


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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.
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Antioxidative effects of sulfurous mineral water:
protection against lipid and protein oxidation
S Benedetti
, F Benvenuti
, G Nappi
, NA Fortunati
, L Marino
, T Aureli
, S De Luca
, S Pagliarani
and F Canestrari
Istituto di Istologia e Analisi di Laboratorio, Universita
di Urbino ‘Carlo Bo’, Urbino, Italy;
Centro di Studi e Ricerche in Medicina
Termale, Universita
di Milano, Milano, Italy and
Terme di Saturnia, Grosseto, Italy
Objectives: To investigate the antioxidative properties of sulfurous drinking water after a standard hydropinic treatment
(500 ml day
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
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, 106112; doi:10.1038/sj.ejcn.1602892; published online 22 August 2007
Keywords: hydropinic treatment; sulfurous mineral water; hydrogen sulfide; thiols; oxidative markers; antioxidant profile
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
4200 mg l
) and/or hydrogen sulfide (H
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,
degli Studi di Urbino ‘Carlo Bo’, Via Ubaldini, 7 Urbino (PU) 61029,
European Journal of Clinical Nutrition (2009) 63, 106112
2009 Macmillan Publishers Limited All rights reserved 0954-3007/09 $
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
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
as reported in Table 1. To avoid H
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
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
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
The linearity range of the test is between 4 and 40 mg
, 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
(Cesarone et al.,
Table 1 Chemical and physical characteristics of the mineral water from
Temperature 1C 36.9
pH (251C) 6.25
Conductivity (251C) mScm
Hardness 1f 204
Fixed residue at 1801Cmgl
Sulfuric degree mg l
mg l
2 þ
mg l
2 þ
mg l
mg l
mg l
mg l
mg l
mg l
mg l
mg l
mg l
mg l
mg l
Iron mg l
mg l
Antioxidative effects of sulfurous mineral water
S Benedetti et al
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
solution and 100 ml 42 mmol l
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
column (4.6 250 mm, 5 mm, from Alltech,
Milan, Italy) equipped with a guard column Alltima C
(4.6 7.5 mm, 5 mm). The eluent phase was methanol/buffer
(40:60, v/v), buffer consisting of 50 mmol l
6.8. The flow rate was 1 ml min
. 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
). 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
phosphate-buffered saline
(PBS), 10 ml 1.16 mmol l
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
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
column (4.6 250 mm, 5 mm, from Alltech)
equipped with a guard column Alltima C
(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
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
-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
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
of vitamin C; the normal value in
healthy subjects is approximately 2200 mmol l
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).
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
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
S-rich water consumption were observed in the study group
throughout the hydropinic treatment.
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
)23.972.6 23.873.0 23.672.7 23.872.9
MDA (mmol l
) 1.8970.25 1.4170.14
1.8770.22 1.8570.21
Carbonyls (nmol/mg proteins) 0.8970.09 0.6370.08
0.9170.09 0.9370.08
AOPP (mmol l
)26.372.0 23.471.2
27.672.1 27.071.9
TAC (mmol l
) 2184749 2284755
2179745 2187751
–SH groups (mmol l
) 533742 570738
525739 532743
Retinol (mmol l
) 3.570.4 3.670.4 3.470.3 3.570.5
a-Tocopherol (mmol l
)23.771.3 23.571.0 24.0771.4 23.871.3
d-Tocopherol (mmol l
) 0.09870.008 0.09570.008 0.09770.008 0.09970.008
g-Tocopherol (mmol l
) 0.4970.05 0.04670.06 0.5270.06 0.4970.05
Lutein (mmol l
) 0.3870.05 0.3770.05 0.4070.05 0.3970.05
Lycopene (mmol l
) 0.7370.14 0.7570.15 0.7270.12 0.7470.13
a-Carotene (mmol l
) 0.08070.011 0.08670.013 0.08470.012 0.08370.011
b-Carotene (mmol l
) 0.4370.07 0.4470.08 0.4570.07 0.4270.06
Abbreviations: AOPP, advanced oxidation protein products; MDA, malondialdehyde; TAC, total antioxidant capacity.
Significantly different from T0 (Po0.05, t-test for paired data).
Significantly different from controls (Po0.05, t-test for unpaired data).
T0 T1
(nmol/mg protein)
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
). Po0.05 T1 vs T0, t-test for paired
Antioxidative effects of sulfurous mineral water
S Benedetti et al
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
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
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
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
S-rich water (500 ml day
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
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
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
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
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
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
S can be
hydrolyzed to hydrosulfide and sulfide ions; however, even if
in aqueous solution, about one-third of H
S remains un-
dissociated at pH 7.4 and can rapidly diffuse through tissues
(Wang, 2002). Indeed, H
S is permeable to plasma membranes
as its solubility in lipophilic solvents is approximately fivefold
Total thiols (µmol/l)
TAC (µmol/l vitamin C)
T0 T0T1 T1
Figure 2 Significant increment in total thiols plasmatic levels (a) and total antioxidant capacity (b) in healthy volunteers receiving 500 ml day
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
European Journal of Clinical Nutrition
greater than in water; for this reason, H
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
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.
We thank Mrs Francesca Baldon (Terme di Saturnia) for
technical assistance in secretarial work.
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sulfurous mineral water
+ SH
Intestinal absorption
Sulfurous mineral water
Diffusion through tissues
Regulation of cell
cystine transport
GSH synthesis
(intracellular pool)
(plasmatic pool)
(disulfide form)
Oxidized thiols
(cysteine, proteins)
Reduced thiols
(cysteine, proteins)
Reduction of plasmatic
Involvement in redox
Circulating H
Figure 3 Possible relationships between orally ingested H
glutathione (GSH) synthesis and plasmatic thiols. (1) H
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
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
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Antioxidative effects of sulfurous mineral water
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European Journal of Clinical Nutrition
... Crenotherapy with sulfureous mineral water exerts beneficial effects in several diseases and also improves cellular redox state in both animal models and humans [2,[14][15][16][17][18][19][20]. Moreover, the sulfureous hydropinotherapy cycle significantly reduces circulating levels of reactive oxygen metabolites (ROMs) in patients with gastrointestinal disorders, likely protecting them from oxidative stress [21]. ...
... Crenotherapy or spa salus per aquam has therapeutic effects on various disorders, especially in those triggered by oxidative stress, and in some cases, they represent an alternative approach to standard pharmacological treatments [14][15][16][17][18][19][20][21][22]. Based on their ionic compositions, mineral waters can be classified as sulfureous, sulfate, bicarbonate, sodium chloride, ferrous arsenic waters, and others [23]. ...
... Mud-bath therapy uses mud, a mixture of solid (organic and inorganic) and liquid (mineral water) components applied on specific skin areas, while hydropinotherapy with sulfureous mineral water relies on drinking mineral water for therapeutic aims [27,28]. Both treatments can reduce oxidative stress, as described in human and animal models [2,[14][15][16][17]19,20]. Therefore, some studies also suggest combining both strategies to ensure long-lasting chondroprotective effects by persistently reducing oxidative stress, inflammation, and degradative stimuli [28]. ...
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Oxidative stress, a condition induced by an excessive amount of free radicals, such as reactive oxygen species (ROS), shows several gender-related differences in basal cellular redox state and antioxidant responses. Crenotherapy with sulfureous mineral water can improve the cellular redox state. In this retrospective observational study, gender-related differences in the efficacy of sulfureous crenotherapy in decreasing oxidant species were investigated. Seventy-eight patients, stratified by sex, with osteoarthritis or degenerative joint disease and Vulgar psoriasis who have received a cycle of sulfureous mud-bath therapy + sulfureous hydropinotherapy were enrolled. Plasma concentration of oxidant species and clinical outcomes were measured at baseline and at the end of treatment. After 2 weeks of sulfureous crenotherapy, a significant amelioration of clinical outcomes and a significant reduction of oxidant species were observed in both sexes, more marked in females than in males (p = 0.0001 and p = 0.04, respectively). For patients with high oxidant species at baseline, females showed a greater reduction in itching compared to males (−95% vs. −50%), while men had a higher amelioration in pain and morning stiffness (−45% vs. −32%, and −50% vs. −37%, respectively). In conclusion, sulfureous crenotherapy can be a valuable strategy to improve cellular redox state in both sexes.
... GYY4137, FW1256 and others) that are currently under development (Gojon 2020; Gojon & Morales 2020) may be used as potential DR mimetics (Ng et al. 2020) and drugs against vascular aging (Arumugam & Kennedy 2018). Levels of both lipid and protein oxidation products, namely, malondialdehyde (the lipid peroxidation product), advanced oxidation protein products, and carbonyls decrease significantly in plasma samples of healthy volunteers subjected to a cycle of hydroponic therapy with H 2 S-rich water (500 ml day -1 for 2 weeks) (Benedetti et al. 2009). Exogenous H 2 S in the form of injected NaHS or inhaled H 2 S also prevents neurodegeneration and neurovascular dysfunction in mouse models of Parkinson's disease induced by intracerebral homocysteine injection or administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, respectively (Kida et al. 2011;Kamat et al. 2013 The second messenger NO induces smooth muscle relaxation via the actions of cGMP (Das et al. 2015;Papapetropoulos et al. 2015;Vannini et al. 2015;Bobin et al. 2016). ...
... 24 In vivo studies demonstrated the beneficial effect of drinking of natural sulphur containing water on animal models of diabetes and glucose metabolism, as well as gastroprotection, antioxidant effects in intestinal physiology and improvement of lipid metabolism and lipid lowering effect. [25][26][27][28][29][30][31] It can be concluded that there is no evidence of a minimum effective concentration of H 2 S in drinking water. Different concentrations of H 2 S in natural waters exhibit different biological effects regardless of H 2 S level. ...
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Background/Aim: Sulphur mineral water is widely used in the treatment of musculoskeletal diseases. Hydrogen sulphide is an important regulator of bone metabolism and its application in the treatment of osteoporosis is intensively researched. The aim of this study was to analyse biochemical and histological effects of H2S containing mineral water of "Mlječanica" spring on ovariectomy-induced experimental osteoporosis in rats. Methods: In this experiment a 14-week-old Wistar female rats were used. The animals undergone bilateral ovariectomy (OVX groups) as an experimental model for oestrogen-deficient osteoporosis. After six weeks, animals were divided into control and the experimental group. Rats from the experimental group treated with H2S (SW group) containing mineral water ad libitum during five weeks. Biochemical parameters for monitoring sulphur water effects were concentration in serum of osteocalcin, alkaline phosphatase, calcium and phosphorus. Histological analyses of the left tibia coloured with haematoxylin-eosin were carried out. Results: Regarding the biochemical parameters, a statistically significant increase was observed in the OVX group for osteocalcin, alkaline phosphatase calcium and phosphorus compared to the sham-operated (CNT) group (p < 0.01). In SW + OVX, alkaline phosphatase was statistically significantly decreased (p < 0.01) and serum osteocalcin and phosphorus increased (p < 0.01). Calcium values were increased without significance. In the OVX + SW group, histological analyses showed numerous osteoblasts along the trabecular endosteum and the growth of young chondrocytes in the central bone zone and their migration to the peripheral parts. Conclusion: Drinking the H2S containing "Mlječanica" mineral water has led to decreased alkaline phosphatase, increased osteocalcin and phosphorus concentration in serum and stimulated the bone reparation in osteoporotic rats.
... In accordance with a few authors, these kinds of water principally act on the subset of T memory cells (Kazandjieva et al., 2008). Mineral waters consisting of sulfur are consumed for drinking as well because of their antioxidant properties (Benedetti et al., 2009). Sulfur water helps in cleansing the skin surface and is beneficial in conditions like chronic ulcers, pannus, furuncles, leprosy and vitiligo (Avicenna & Gruner, 1973). ...
Management of ailments using water, clay and medicinal mineral water is an ancient treatment modality since it includes a collection of processes or practices validated by science. Balneotherapy is different from hydrotherapy. In balneotherapy, the temperature of bathrooms and the use of medicinal mineral clays or water is fundamental. In contrast, hydrotherapy is the internal or external application of water in any condition (ice, steam, liquid) to promote health or manage various disorders at varying durations, temperatures, locations and pressures. Pelotherapy or mud therapy is one of the interventions in balneology that comprises the application of mud externally for healing purposes. As far as people are returning to nature, this therapy is advised and found to be effective for many health issues, including dermatological disorders, chronic inflammations of body systems and fibromyalgia. This chapter will discuss the ancient Greco-Arabic, Roman and Turkish bathhouses, the difference between balneotherapy and hydrotherapy, the effects of peloids and mineral baths on human health, balneotherapy and hydrotherapy in fibromyalgia, balneotherapy and hydrotherapy in dermatological disorders and balneotherapy and hydrotherapy for back pain and rheumatism.
... In the present study, we chose MCD instead of other models because it replicates the inflammatory and fibrogenetic injuries occurring in NASH more consistently than other models, thereby allowing us to better study the potential inflammatory response to LPS in the liver [29,30]. On the other hand, the model is not suitable for evaluating the potential beneficial effects of mineral water on glucose and lipid metabolisms [19,33]; accordingly, we did not observe significant effects on hepatic steatosis, and future studies are necessary to expand the investigations on metabolic features by using other disease models. ...
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The progression of nonalcoholic fatty liver disease (NAFLD) is associated with alterations of the gut–liver axis. The activation of toll-like receptor 4 (TLR4) pathways by endotoxins, such as lipopolysaccharide (LPS), contributes to liver injury. The aim of the present study was to evaluate the possible beneficial effects of a calcium-sulphate-bicarbonate natural mineral water on the gut–liver axis by evaluating liver and terminal ileum histopathology in a murine model of NAFLD. NAFLD was induced in mice by administrating a methionine-choline-deficient (MCD) diet. The following experimental groups were evaluated: controls (N = 10); MCD+Tap water (MCD; N = 10); MCD+Calcium-sulphate-bicarbonate water (MCD/Wcsb; N = 10). Mice were euthanised after 4 and 8 weeks. Liver and terminal ileum samples were collected. Samples were studied by histomorphology, immunohistochemistry, and immunofluorescence. In mice subjected to the MCD diet, treatment with mineral water improved inflammation and fibrosis, and was associated with a reduced number of activated hepatic stellate cells when compared to MCD mice not treated with mineral water. Moreover, MCD/Wcsb mice showed lower liver LPS localization and less activation of TLR4 pathways compared to the MCD. Finally, Wcsb treatment was associated with improved histopathology and higher occludin positivity in intestinal mucosa. In conclusion, calcium-sulphate-bicarbonate water may exert modulatory activity on the gut–liver axis in MCD mice, suggesting potential beneficial effects on NAFLD.
... An improved body redox status in healthy volunteers was observed after a standard hydropinic treatment with 500 mL sulfurous drinking water per day for 2 weeks. In particular, there was a significant decrease of lipid and protein oxidation products and a substantial increment in the total antioxidant capacity and total thiol levels in the plasma samples from the study group as compared to the control group [47]. Some recent reviews of available H 2 S donors that serve as a platform for the release of H 2 S have underlined their considerable promise as favorable drugs in the clinical treatment of many diseases [48][49][50]. ...
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The growing increases in the global life expectancy and the incidence of chronic diseases as a direct consequence have highlighted a demand to develop effective strategies for promoting the health of the aging population. Understanding conserved mechanisms of aging across species is believed helpful for the development of approaches to delay the progression of aging and the onset of age-related diseases. Mitochondrial hormesis (or mitohormesis), which can be defined as an evolutionary-based adaptive response to low-level stress, is emerging as a promising paradigm in the field of anti-aging. Depending on the severity of the perceived stress, there are varying levels of hormetic response existing in the mitochondria called mitochondrial stress response. Hydrogen sulfide (H2S) is a volatile, flammable, and toxic gas, with a characteristic odor of rotten eggs. However, H2S is now recognized an important gaseous signaling molecule to both physiology and pathophysiology in biological systems. Recent studies that elucidate the importance of H2S as a therapeutic molecule has suggested its protective effects beyond the traditional understanding of its antioxidant properties. H2S can also be crucial for the activation of mitochondrial stress response, postulating a potential mechanism for combating aging and age-related diseases. Therefore, this review focuses on highlighting the involvement of H2S and its sulfur-containing derivatives in the induction of mitochondrial stress response, suggesting a novel possibility of mitohormesis through which this gaseous signaling molecule may promote the healthspan and lifespan of an organism.
... Extensive cellular, animal model, and human clinical association studies have focused on the involvement of H 2 S in the prevention of aging, and on its reduction as a risk factor for various age-related diseases [36,37]. Many of these studies measure enzymatic generation capacity and/or protein persulfidation in plasma, serum, or other tissues, rather than measuring endogenous levels [8,30,38,39]. ...
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Background Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. Methods After synthesizing [³⁴S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. Results Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r² > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0–66.7% reduction with antibiotics). Conclusions A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.
... It is a reactive aldehyde that forms adducts with proteins and DNA, leading to alterations in cellular function [19]. MDA levels after HAM thawing were evaluated in HAM-derived supernatants by RP-HPLC as previously described [20]. Briefly, sample derivatization was carried out by adding 50 µL 0.05% butylated hydroxytoluene (BHT), 400 µL 0.44 M H 3 PO 4, and 100 µL 42 mM thiobarbituric acid (TBA) to 50 µL supernatant. ...
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The biological properties of the human amniotic membrane (HAM) and its characteristic ability to be a reservoir of growth factors promoting wound healing make it an ideal biological dressing for the treatment of different clinical conditions, such as burns and non-healing wounds. However, the application of a preservation method on the HAM is required during banking to maintain biological tissue properties and to ensure the release overtime of protein content for its final clinical effectiveness after application on the wound bed. Although cryopreservation and freezing are methods widely used to maintain tissue properties, reactive oxygen species (ROS) are produced within tissue cellular components during their switching from frozen to thawed state. Consequently, these methods can lead to oxidative stress-induced cell injury, affecting tissue regenerative properties and its final clinical effectiveness. Taking advantage of the antioxidant activity of the natural compound quercetin, we used it to improve the antioxidant and regenerative properties of frozen or cryopreserved HAM tissues. In particular, we evaluated the oxidative damage (lipid peroxidation, malondialdehyde) as well as the regenerative/biological properties (bFGF growth factor release, wound healing closure, structure, and viability) of HAM tissue after its application. We identified the effectiveness of quercetin on both preservation methods to reduce oxidative damage, as well as its ability to enhance regenerative properties, while maintaining the unaltered structure and viability of HAM tissue. The use of quercetin described in this study appears able to counteract the side effects of cryopreservation and freezing methods related to oxidative stress, enhancing the regenerative properties of HAM. However, further investigations will need to be performed, starting from these promising results, to identify its beneficial effect when applied on burns or non-healing wounds.
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Hydrogen sulfide (H2S) was recognized as a gaseous signaling molecule, similar to nitric oxide (-NO) and carbon monoxide (CO). The aim of this review is to provide an overview of the formation of hydrogen sulfide (H2S) in the human body. H2S is synthesized by enzymatic processes involving cysteine and several enzymes, including cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (3MST) and D-amino acid oxidase (DAO). The physiological and pathological effects of hydrogen sulfide (H2S) on various systems in the human body have led to extensive research efforts to develop appropriate methods to deliver H2S under conditions that mimic physiological settings and respond to various stimuli. These functions span a wide spectrum, ranging from effects on the endocrine system and cellular lifespan to protection of liver and kidney function. The exact physiological and hazardous thresholds of hydrogen sulfide (H2S) in the human body are currently not well understood and need to be researched in depth. This article provides an overview of the physiological significance of H2S in the human body. It highlights the various sources of H2S production in different situations and examines existing techniques for detecting this gas.
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Evidence suggests an imbalance between antioxidant and oxidant-generating systems resulting in oxidative stress in uremic patients. As plasma proteins are critical targets for oxidants, we developed a novel spectrophotometric assay which allows to detect advanced oxidation protein products (AOPP) in uremic plasma. By size-exclusion chromatography AOPP are retrieved in two distinct peaks at 600 and below 80 kDa in uremic plasma, while no such peaks are found in control plasma. Further biochemical characterization revealed that AOPP are carried by oxidized plasma proteins, especially albumin and do not have oxidant properties. AOPP increased in a dose-dependent manner following in vitro exposure of plasma or purified human serum albumin (HSA) to hypochlorous acid. Advanced glycation end products of human serum albumin (AGE-HSA) also increased AOPP levels. In vivo, plasma level of AOPP was the highest in patients on hemodialysis, followed by those on peritoneal dialysis and by undialyzed patients with advanced chronic renal failure. AOPP levels correlated with plasma concentrations of dityrosine and AGE-pentosidine, as indices of oxidant-mediated protein damage, but not with thiobarbituric reactive substances as lipid peroxidation markers. A close correlation was also found between AOPP and neopterin levels, suggesting that AOPP could be part in the monocyte-mediated inflammatory disorders associated with uremia. In conclusion, we propose the measurement of AOPP as a reliable marker to estimate the degree of oxidant-mediated protein damage in uremic patients and to predict the potential efficacy of therapeutic strategies aimed at reducing such an oxidative stress.
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We previously demonstrated the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of uremic patients receiving maintenance dialysis. The present study in a cohort of 162 uremic patients showed that plasma concentrations of AOPP increased with progression of chronic renal failure and were closely related to advanced glycation end products (AGE)-pentosidine (r = 0.52, p < 0.001), taken as a marker of AGE. In vivo, the relevance of AOPP and AGE-pentosidine in monocyte-mediated inflammatory syndrome associated with uremia was evidenced by close correlations between AOPP or AGE-pentosidine and monocyte activation markers, including neopterin, IL-1R antagonist, TNF-alpha, and TNF soluble receptors (TNF-sR55 and TNF-sR75). To determine the mechanisms by which AOPP and AGE could be directly involved in monocyte activation, AOPP-human serum albumin (HSA) and AGE-HSA were produced in vitro by treating HSA with oxidants or glucose, respectively. Spectroscopic analysis confirmed that AOPP-HSA contains carbonyls and dityrosine. Both AOPP-HSA and AGE-HSA, but not purified dityrosine, were capable of triggering the oxidative burst of human monocytes in cultures. The AOPP-HSA-induced respiratory burst was dependent on the chlorinated nature of the oxidant and on the molar ratio HSA/HOCI. Collectively, these data first demonstrate that AOPP act as a mediator of oxidative stress and monocyte respiratory burst, which points to monocytes as both target and actor in the immune dysregulation associated with chronic uremia.
This book discusses, in relatively simple language, the importance of even minute amounts of certain trace elements for the protection of human health and how insufficiency or excess may produce serious diseases. It also examines the use of metal chelators in the treatment of such diseases.
Publisher Summary All of the plasma sulfhydryl (SH) groups are protein associated. Albumin is the most abundant plasma protein (40-60 mg/ml) and is a powerful extracellular antioxidant. Plasma SH groups are susceptible to oxidative damage and are often low in patients suffering from diseases, such as coronary artery disease and rheumatoid arthritis. Additionally, protein SH (P-SH) groups, plasma contains small amounts of glutathione (GSH). Decreased plasma GSH is reported in human immunodeficiency virus (HIV) infection. A spectrophotometric assay based on 2,2-dithiobisnitrobenzoic acid (DTNB or Ellman's reagent) is commonly used for thiol assay. Most of the procedures are for cellular thiols. This chapter describes convenient assays for P-SH and GSH in plasma using spectrophotometric and spectrofluorometric methods. The P-SH level of plasma is calculated by subtracting the GSH level from the T-SH level. There normally is little difference between T-SH and P-SH because of the low GSH levels in the plasma.
Recent progress in a number of areas of biochemistry and biology has drawn attention to the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. This brief review is intended as a broad overview of this currently rapidly moving field of sulfur biochemistry, for those who are interested or are involved in one or the other aspect of it, a synopsis by one who has stumbled into this field from several directions in the course of time. Only for iron are metal–sulfur relationships discussed in detail, as the iron–sulfur subfield is one of the most active areas.
A simple, automated test measuring the ferric reducing ability of plasma, the FRAP assay, is presented as a novel method for assessing "antioxidant power." Ferric to ferrous ion reduction at low pH causes a colored ferrous-tripyridyltriazine complex to form. FRAP values are obtained by comparing the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions in known concentration. Absorbance changes are linear over a wide concentration range with antioxidant mixtures, including plasma, and with solutions containing one antioxidant in purified form. There is no apparent interaction between antioxidants. Measured stoichiometric factors of Trolox, alpha-tocopherol, ascorbic acid, and uric acid are all 2.0; that of bilirubin is 4.0. Activity of albumin is very low. Within- and between-run CVs are <1.0 and <3.0%, respectively, at 100-1000 micromol/liter. FRAP values of fresh plasma of healthy Chinese adults: 612-1634 micromol/liter (mean, 1017; SD, 206; n = 141). The FRAP assay is inexpensive, reagents are simple to prepare, results are highly reproducible, and the procedure is straightforward and speedy. The FRAP assay offers a putative index of antioxidant, or reducing, potential of biological fluids within the technological reach of every laboratory and researcher interested in oxidative stress and its effects.
Lipid peroxidation results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. The approach to the quantitative assessment of lipid peroxidation depends on whether the samples involve complex biological material obtained in vivo, or whether the samples involve relatively simple mixtures obtained in vitro. Samples obtained in vivo contain a large number of products which themselves may undergo metabolism. The measurement of conjugated diene formation is generally applied as a dynamic quantitation e.g. during the oxidation of LDL, and is not generally applied to samples obtained in vivo. Lipid hydroperoxides readily decompose, but can be measured directly and indirectly by a variety of techniques. The measurement of MDA by the TBAR assay is non-specific, and is generally poor when applied to biological samples. More recent assays based on the measurement of MDA or HNE-lysine adducts are likely to be more applicable to biological samples, since adducts of these reactive aldehydes are relatively stable. The discovery of the isoprostanes as lipid peroxidation products which can be measured by gas chromatography mass spectrometry or immunoassay has opened a new avenue by which to quantify lipid peroxidation in vivo, and will be discussed in detail.