Hydrogen sulfide mediates the vasoactivity of garlic.
ABSTRACT The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.
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ABSTRACT: Cardiovascular disease remains the leading cause of death worldwide with hypertension being a major contributing factor to cardiovascular disease-associated mortality. On a population level, non-pharmacological approaches, such as alternative/complementary medicine, including phytochemicals, have the potential to ameliorate cardiovascular risk factors, including high blood pressure. Several epidemiological studies suggest an antihypertensive effect of garlic (Allium sativum) and of many its bioactive components. The aim of this review is to present an in-depth discussion regarding the molecular, biochemical and cellular rationale underlying the antihypertensive properties of garlic and its bioactive constituents with a primary focus on S-allyl cysteine and allicin. Key studies, largely from PubMed, were selected and screened to develop a comprehensive understanding of the specific role of garlic and its bioactive constituents in the management of hypertension. We also reviewed recent advances focusing on the role of garlic bioactives, S-allyl cysteine and allicin, in modulating various parameters implicated in the pathogenesis of hypertension. These parameters include oxidative stress, nitric oxide bioavailability, hydrogen sulfide production, angiotensin converting enzyme activity, expression of nuclear factor-κB and the proliferation of vascular smooth muscle cells. This review suggests that garlic and garlic derived bioactives have significant medicinal properties with the potential for ameliorating hypertension and associated morbidity; however, further clinical and epidemiological studies are required to determine completely the specific physiological and biochemical mechanisms involved in disease prevention and management.Nutrition research 02/2014; 34(2):106-15. · 2.59 Impact Factor
- Folia Pharmacologica Japonica 12/2010; 136(6):335-9.
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ABSTRACT: Sulfur is the seventh most abundant element measurable in the human body and is supplied mainly by the intake of methionine (Met), an indispensable amino acid found in plant and animal proteins. Met controls the initiation of protein synthesis, governs major metabolic and catalytic activities, and may undergo reversible redox processes safeguarding protein integrity. Withdrawal of Met from customary diets causes the greatest downsizing of lean body mass following either unachieved replenishment (malnutrition) or excessive losses (inflammation). These physiopathologically unrelated morbidities nevertheless stimulate comparable remethylation reactions from homocysteine, indicating that Met homeostasis benefits from high metabolic priority. Inhibition of cystathionine-β-synthase activity causes the upstream sequestration of homocysteine and the downstream drop in cysteine and glutathione. Consequently, the enzymatic production of hydrogen sulfide and the nonenzymatic reduction of elemental sulfur to hydrogen sulfide are impaired. Sulfur operates as cofactor of several enzymes critically involved in the regulation of oxidative processes. A combination of malnutrition and nutritional deprivation of sulfur maximizes the risk of cardiovascular disorders and stroke, constituting a novel clinical entity that threatens plant-eating population groups.Nutrition Reviews 07/2013; 71(7):413-32. · 4.60 Impact Factor
Hydrogen sulfide mediates the vasoactivity of garlic
Gloria A. Benavides*†, Giuseppe L. Squadrito*†, Robert W. Mills*, Hetal D. Patel‡, T. Scott Isbell†§, Rakesh P. Patel†§,
Victor M. Darley-Usmar†§, Jeannette E. Doeller*†, and David W. Kraus*†‡¶
Departments of *Environmental Health Sciences,‡Biology, and§Pathology and†Center for Free Radical Biology, University of Alabama at Birmingham,
Birmingham, AL 35294
Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 12, 2007 (received for review
June 18, 2007)
The consumption of garlic is inversely correlated with the
progression of cardiovascular disease, although the responsible
mechanisms remain unclear. Here we show that human RBCs
convert garlic-derived organic polysulfides into hydrogen sul-
fide (H2S), an endogenous cardioprotective vascular cell signal-
ing molecule. This H2S production, measured in real time by
a novel polarographic H2S sensor, is supported by glucose-
maintained cytosolic glutathione levels and is to a large extent
reliant on reduced thiols in or on the RBC membrane. H2S
production from organic polysulfides is facilitated by allyl sub-
stituents and by increasing numbers of tethering sulfur atoms.
Allyl-substituted polysulfides undergo nucleophilic substitution
at the ? carbon of the allyl substituent, thereby forming a
hydropolysulfide (RSnH), a key intermediate during the forma-
tion of H2S. Organic polysulfides (R-Sn-R?; n > 2) also undergo
nucleophilic substitution at a sulfur atom, yielding RSnH and H2S.
Intact aorta rings, under physiologically relevant oxygen levels,
also metabolize garlic-derived organic polysulfides to liberate
H2S. The vasoactivity of garlic compounds is synchronous with
H2S production, and their potency to mediate relaxation in-
creases with H2S yield, strongly supporting our hypothesis that
H2S mediates the vasoactivity of garlic. Our results also suggest
that the capacity to produce H2S can be used to standardize
garlic dietary supplements.
Allium ? aorta ? polysulfides ? red blood cells ? vasorelaxation
factors associated with cardiovascular diseases such as increased
reactive oxygen species, high blood pressure, high cholesterol,
platelet aggregation, and blood coagulation (1), but the active
principles and mechanisms of action remain elusive. Garlic is rich
in organosulfur compounds considered responsible for most of its
pharmacological activities. Allicin (diallyl thiosulfinate), the main
organosulfur compound, is produced from the amino acid alliin by
action of the enzyme alliinase when garlic is crushed. Allicin,
unstable in aqueous solution, rapidly decomposes mainly to diallyl
sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS),
and ajoene (2). After consumption, neither allicin nor its metab-
olites have been found in blood or urine (3), indicating that these
compounds are rapidly metabolized.
a cell signaling role similar to NO and CO (4, 5). Signaling may
be mediated by H2S, HS?, or, less likely, S2?, species related by
2 (7), resulting in molar fractions of H2S, HS?, and S2?at 0.26,
0.74, and 1.7 ? 10?12, respectively, at a physiological pH of 7.4.
Herein, the term H2S refers to totality of free sulfide in solution.
In vivo and in vitro cardiovascular effects of H2S include de-
creased blood pressure (8), cardioprotection against ischemic
reperfusion damage (9), and O2-dependent vasorelaxation (10).
In the vascular system, H2S is produced in vascular smooth
muscle cells via cysteine metabolism by the pyridoxal 5?-
phosphate-dependent enzyme cystathionine-?-lyase (8). Nonen-
zymatic H2S production may also occur in the vasculature, as
ietary garlic (Allium sativum) has been recognized for its
beneficial health effects for centuries. In particular, garlic
Searcy and Lee (11), corroborated by ourselves (data not
shown), have demonstrated that human RBCs produce H2S
when provided with elemental sulfur (S8) or inorganic polysul-
fides (S32?and S52?). However, because inorganic polysulfides
are not likely dietary, we predicted that the organic polysulfide
compounds found in dietary garlic may react with RBCs in a
similar way, perhaps representing a substantial potential source
of vascular H2S.
Here we demonstrate that garlic-derived organic polysulfides
are H2S donors via glucose-supported and thiol-dependent
cellular as well as glutathione (GSH)-dependent acellular reac-
tions, and we propose the chemical mechanism by which H2S is
produced leading to vasorelaxation. Taking these observations
together, we propose that the major beneficial effects of garlic-
rich diets, specifically on cardiovascular disease and more
broadly on overall health, are mediated by the biological pro-
duction of H2S from garlic-derived organic polysulfides.
To define the real-time kinetics of H2S production by RBCs, we
used a polarographic H2S sensor (PHSS) in a temperature-
controlled closed-chamber respirometer (12). All experiments
were performed at pH 7.35 and 37°C. Freshly collected and
prepared human RBCs suspended in anoxic PBS containing 50
?M diethylenetriaminepentaacetic acid (DTPA) initiated sub-
stantial H2S production upon the addition of 1 mg/ml garlic (Fig.
1A), a concentration equivalent to two garlic cloves (5–6 g)
dissolved in the blood volume of a typical adult human (?5
liters). Because abundant cellular thiols may reduce polysulfides
to liberate H2S, GSH, near 2 mM concentration in freshly
collected RBCs (13), represents a likely candidate for such a
reaction. This led us to predict that, if H2S production is
mediated by GSH, then sustained H2S production by RBCs
would depend on the presence of glucose to maintain GSH
levels. Freshly collected RBCs (20% vol/vol) placed in two
identical respirometer chambers, one with glucose and one
without glucose, exhibited rapid H2S production at a rate near
12 ?M/min and yielded a concentration of ?8 ?M H2S upon first
injection of aliquots from a single garlic stock solution (to
eliminate interclove variations in organic polysulfide concentra-
Author contributions: G.A.B., G.L.S., V.M.D.-U., J.E.D., and D.W.K. designed research;
G.A.B., R.W.M., H.D.P., T.S.I., and D.W.K. performed research; G.L.S., T.S.I., R.P.P., and
D.W.K. contributed new reagents/analytic tools; G.A.B., G.L.S., J.E.D., and D.W.K. analyzed
data; and G.A.B., G.L.S., T.S.I., R.P.P., V.M.D.-U., J.E.D., and D.W.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: H2S, hydrogen sulfide; GSH, glutathione; GSSG, GSH disulfide; DATS, diallyl
trisulfide; DADS, diallyl disulfide; DAS, diallyl sulfide; DPDS, dipropyl disulfide; AMS, allyl
methyl sulfide; DTPA, diethylenetriaminepentaacetic acid; L-NAME, N?-nitro-L-arginine-
See Commentary on page 17907.
¶To whom correspondence should be addressed at: Departments of Biology and Environ-
Boulevard, Birmingham, AL 35294-0022. E-mail: firstname.lastname@example.org.
© 2007 by The National Academy of Sciences of the USA
November 13, 2007 ?
vol. 104 ?
no. 46 ?
tions) (Fig. 1A). The slow decline in H2S after the initial rapid
rise suggested ongoing H2S removal from solution, indicating
that all H2S production rates should be considered net produc-
tion rates. After the second and third garlic injections, RBCs
with glucose showed sustained H2S production whereas RBCs
without glucose gradually exhausted their H2S production ca-
pacity (Fig. 1A). PBS and glucose alone did not exhibit garlic-
induced H2S production.
We further predicted that, in RBCs without glucose, the
cytosolic GSH pool would be depleted after exposure to garlic,
compared with RBCs with glucose. Fig. 1B shows that RBCs
incubated for 20 min with or without glucose or with glucose and
garlic all had statistically similar GSH and glutathione disulfide
(GSSG) concentrations. The GSH/GSSG ratios in these RBCs
indicated that GSH was largely in its reduced form, consistent
with the literature (14, 15). However, RBCs without glucose,
after a single bout of garlic-induced H2S production, exhibited
decreased GSH and increased GSSG concentrations, leading to
a large decrease in the GSH/GSSG ratio compared with control,
although total GSH equivalents were conserved (Fig. 1B). In
cysteine) reacted directly, in the absence of RBCs, with garlic to
produce H2S (Fig. 1C), with rates and yields that indicate a
reaction preference for GSH in the production of H2S.
Other redox active components that may participate in this
reaction include membrane protein thiols, perhaps on both
membrane faces. To test this, RBCs were pretreated with
thiol-blocking reagents, either membrane-impermeable
iodoacetic acid (IAA) or membrane-permeant iodoacetamide
production and GSH and GSSG concentrations were measured.
IAA blockade of exofacial thiols did not statistically alter total
intracellular GSH equivalents or the GSH/GSSG ratio (Fig. 1B)
but inhibited garlic-induced H2S production by 75% compared
with control (Fig. 1D). In contrast, IAM blockade of both
exofacial and intracellular thiols resulted in elimination of both
the entire GSH pool (Fig. 1B) and garlic-induced H2S produc-
tion (Fig. 1D).
To explore the chemical efficacy of specific garlic-derived
organic polysulfides in GSH-mediated H2S production, 2 mM
GSH was combined with 100 ?M each of DATS, DADS, DAS,
allyl methyl sulfide (AMS), and dipropyl disulfide (DPDS) in
PBS (Fig. 2A). Total H2S yield (Fig. 2B) was highest with DATS
followed by DADS and was lowest with DPDS.
Increased garlic consumption in some populations is associ-
ated with lower incidence of hypertension (1). To test the
vasorelaxant effect of garlic-induced H2S production, phenyl-
ephrine (PE)-precontracted aorta rings were suspended in a
37°C organ bath containing 1 mM GSH under physiological O2
conditions and provided with 50, 200, and 500 ?g/ml garlic.
These additions resulted in concentration-dependent simulta-
neous vasorelaxation and H2S production (Fig. 3 A and C). To
establish the chemical efficacy of H2S production on vasoactiv-
ity, specific garlic-derived organic polysulfides were added to
aorta ring preparations, again under physiological O2conditions.
PE-precontracted aorta rings showed maximum relaxation with
DATS and DADS and minimum relaxation with DPDS and
AMS (Fig. 3 B and D), effects that paralleled their H2S yields
(Fig. 2), suggesting a link between bioactivity and production of
this signal molecule. In the presence of authentic H2S donors
such as the lipophilic garlic polysulfides, H2S is formed either on
the cell surface or in the cytoplasm, most likely leading to a
40 % RBCs
sentative polarographic traces of garlic-induced H2S production in 20% (vol/
(blue line) 10 mM glucose (Glc), with sequential 1 mg/ml garlic additions at
arrows, compared with the same garlic additions to 10 mM PBS and 50 ?M
of GSH (black bars) and GSSG (expressed in GSH equivalents; white bars) in
40% (vol/vol) RBCs subject to no treatment, 10 mM glucose (Glc), 10 mM
each bar represents the mean ? SD of three to five experiments.*, GSH and
GSSG levels are statistically different compared with levels in untreated RBCs
H2S production in anoxic 10 mM PBS with 50 ?M DTPA in the absence of RBCs,
with 2 mM each GSH, cysteine (Cys), homocysteine (Hcys), or N-acetylcysteine
(NAC) (pH 7.35) at 37°C upon addition of 1 mg/ml garlic at arrow (C) and
garlic-induced H2S production in 20% (vol/vol) RBCs with 10 mM glucose
previously treated with 10 mM IAA (blue line) or 10 mM IAM (red line)
compared with untreated RBCs (black line) with 1 mg/ml garlic additions at
Garlic-induced H2S production and GSH and GSSG levels. (A) Repre-
H2S ( M)
*, Yields are statistically different compared with yield from DPDS by Student’s t test (P ? 0.005).
Single garlic-derived organic polysulfide-induced (nonenzymatic) H2S production by GSH. (A) Representative polarographic traces of H2S production
www.pnas.org?cgi?doi?10.1073?pnas.0705710104Benavides et al.
substantially higher local and relatively sustained H2S dose that
is more potent in producing vascular relaxation than are direct
bolus additions of H2S.
Mechanically denuded aortic segments exhibiting KCl-mediated
contraction but no acetylcholine-mediated relaxation remained
responsive to the relaxation effects of DADS (Fig. 3D), demon-
strating that a functional endothelium was not required for this
experiments conducted with and without GSH do not show a
difference in vasorelaxation (Fig. 3E). Furthermore, H2S produc-
tion by reaction of garlic-derived organic polysulfides with GSH in
the absence of RBCs is most likely supplemented by H2S produc-
tion by aorta directly (Fig. 3F).
Garlic has been used for centuries in the treatment of diverse
ailments in many cultures (1). Here we demonstrate that garlic
and garlic-derived organic polysulfides induce H2S production in
a thiol-dependent manner and that this signal molecule mediates
the vasoactivity of garlic (Fig. 4). Organic polysulfides such as
DATS and DADS act as H2S donors when they react with
biological thiols including GSH, and glucose is necessary to
maintain the reduced GSH pool, likely via the pentose phos-
phate pathway-mediated NADPH production that supports
GSH reductase activity. We observed that exofacial membrane
protein thiols play an important role in H2S production from
garlic-derived organic polysulfides, suggesting the participation
of a transplasma membrane reductase system (16), perhaps
driven by NADPH and GSH, that acts to maintain exofacial
thiols in the reduced state, thereby sustaining H2S production. In
addition, we observed that these compounds can cross the
membrane, as previously reported for allicin (17), to react with
intracellular GSH because blocking the exofacial membrane
thiols did not completely abrogate H2S production. The data
presented here suggest that garlic-induced H2S production can
occur in any thiol-containing cell and that H2S thus produced
diffuses through plasma membranes causing vascular smooth
muscle cell relaxation, likely via activation of KATPchannels (8).
Other membrane channels may also be affected by garlic. For
example, garlic-derived compounds activate TRP channels in
sensory neurons and human embryonic kidney cells (18, 19).
Reports on garlic-mediated (20–22) and H2S-mediated (8)
vascular smooth muscle relaxation indicate that both are based
in part on NO signaling pathways, but the main action of both
garlic (20) and H2S (8) is likely the opening of vascular smooth
muscle cell membrane KATPchannels, leading to depolarization
and blood vessel dilation. Garlic-mediated vasorelaxation has
previously been studied under the traditional high 95% O2(900
?M) (20, 21) where spontaneous and biological H2S oxidation
would be accelerated. High O2 levels also likely increase the
activity of NO synthase (23), thereby increasing the observed
contribution of the NO signaling pathways. Interestingly, under
high O2 conditions, the effects of endothelium removal or
196 50 M O2
30 M O2
o i t a
a l e
50 200 500
Garlic ( g/ml)
o i t a
a l e
o i t a
a l e
tivity and H2S production of PE-precontracted rat aorta rings at physiological
O2levels. Representative simultaneous traces of vasoactivity (blue line) and
H2S production (red line) in a 15-ml organ bath of Krebs–Henseleit buffer (pH
at the dashed line and 500 ?g/ml garlic addition at the arrow (A) and
aorta rings supported by 50, 200, and 500 ?g/ml garlic (black bars) compared
with 20 ?M H2S (white bar) (C; each bar represents the mean ? SD of four to
six experiments) and 100 ?M each DPDS, AMS, DAS, DADS, or DATS, as well as
ethanol vehicle and 20 ?M H2S (D; each bar represents mean ? SD of three to
nine experiments). Endothelium-intact aortic segments (black bars) are com-
pared with endothelium-denuded segments (white bars). Levels are statisti-
cally different compared with DPDS (*, P ? 0.001; n ? 6–9) and AMS (**, P ?
with or without 1 mM GSH; each bar represents mean ? SD of four to six
experiments. (F) Representative polarographic traces of DADS-induced H2S
production by entire aorta (eight segments, ?30 mg fresh weight; red line)
with 10 mM glucose (Glc) and 100 ?M DADS additions at the arrows, in the
2-ml closed multisensor respirometer chamber.
Garlic- and single garlic-derived organic polysulfide-induced vasoac-
in the vascular system. Garlic-derived organic polysulfides with allyl moieties
and more than two sulfur atoms (see Fig. 5) react with exofacial membrane
thiols and cross the cell membrane to react with GSH to produce H2S. Glucose
is the main energy source of RBCs, supporting glycolysis and pentose phos-
phate pathway (PPP) reduction of NADP?to NADPH, a cofactor of GSH
reductase (GR), which maintains the intracellular GSH pool. GSH may also
H2S production then leads to vasorelaxation via vascular smooth muscle cell
KATP-linked hyperpolarization (8).
Proposed model of garlic-induced H2S production and H2S function
Benavides et al.PNAS ?
November 13, 2007 ?
vol. 104 ?
no. 46 ?
N?-nitro-L-arginine-methyl ester (L-NAME) addition to elim-
inate NO production are minor if the concentration of garlic
extract (or allicin) is increased (21). H2S-mediated vasorelax-
ation decreases at higher O2levels in part as a result of H2S
oxidation but also because sulfide oxidation products mediate
vasoconstriction (10). In contrast, at lower, more physiological
O2 levels, a decrease in NO synthase activity and a relative
increase in H2S persistence may alter the observed efficacy of
these signaling pathways. We have found that H2S, acting as a
reductant and nucleophile, reacts with S-nitrosothiol species to
release NO, providing another mechanism by which H2S could
augment vasorelaxation (24).
We observed that the chemical conversion of garlic-derived
organic polysulfides to H2S is facilitated by allyl substituents and
by increasing numbers of tethering sulfur atoms (see Fig. 2 for
chemical structures). A similar structure–activity correlation has
been observed for the cancer-preventative effects of garlic-
proceeds at least in part by a different mechanism from the more
common thiol/disulfide exchange because the exchange reaction
does not produce H2S (Fig. 5A, blue). We propose that, in a
reaction competing with thiol/disulfide exchange, DADS under-
goes nucleophilic substitution at the ? carbon, yielding S-allyl-
glutathione (27) and allyl perthiol, a key intermediate in the
formation of H2S (28–31) (Fig. 5A, red). The resulting allyl
perthiol then undergoes nucleophilic substitution at the S-atom
in a manner analogous to thiol/disulfide exchange, yielding
allyl-GSSG and H2S. The allyl-glutathione disulfide is an addi-
to further H2S production. The allyl perthiol may also undergo
nucleophilic substitution at the ? carbon yielding S-allyl-
glutathione and H2S2that in turn reacts with GSH to produce
H2S (Fig. 5, reactions B and C). Organic disulfides substituted
with only one allyl group may also produce H2S, albeit in lower
yields. In contrast, organic disulfides with propyl substituents
such as DPDS show only trace H2S production, confirming
Streitwieser (32), who estimates that the rate of nucleophilic
substitution at the ? carbon in an allylic residue is ?100 times
faster than with an N-propyl residue. Organic trisulfides and
probably higher polysulfides may undergo nucleophilic substi-
tution at an S-atom, yielding a perthiol or higher hydropolysul-
fides that then react further with GSH to form H2S (Fig. 5,
reactions D and E). Elemental sulfur also reacts with GSH to
yield H2S in a similar manner (data not shown). Protein thiols
and other biological thiols may compete with GSH for organo-
sulfur compounds in certain microenvironments such as plasma
membranes, resulting in H2S formation as well as protein
Few plants other than garlic contain allyl-substituted sulfur
compounds, and garlic is the only one of these with a dietary use.
We propose that H2S production from these garlic-derived
organic polysulfides provides the basis for the long-term bene-
ficial effects obtained from the habitual consumption of garlic.
Although numerous clinical studies have demonstrated the
beneficial effects of garlic on cardiovascular disease progression,
there exist a sufficient number of studies that show little or no
beneficial effects. Critical reviews attempting to address these
contradictory results often site differences in subject health
status, trial duration, and unknown active constituents in various
garlic preparations as nonuniform factors contributing to incon-
sistent outcomes (33). If boosting endogenous H2S production in
blood and tissues represents an essential benefit of ingesting
garlic, then differences in this important variable may well
influence the outcome and should be addressed in future clinical
H2S can be used to standardize garlic dietary supplements.
Materials and Methods
Materials. GSH, IAA, IAM, PBS, sodium sulfide, L-cysteine,
N-acetylcysteine, homocysteine, glucose, m-phosphoric acid,
L-NAME, DADS, DAS, DPDS, and AMS were obtained from
Sigma (St. Louis, MO). DATS was purchased from LTK Labs
(St. Paul, MN).
tubes from healthy volunteers after obtaining informed consent,
according to University of Alabama at Birmingham Institutional
Review Board-approved procedures. Freshly collected blood
was centrifuged at 1,000 ? g for 10 min, and plasma and upper
buffy coat were removed by aspiration. RBCs were washed by
centrifugation three times with 10 mM PBS containing 50 ?M
DTPA (used to chelate metals) (pH 7.35) and finally resus-
pended to a hematocrit of 20% or 40% (vol/vol) using 10 mM
PBS with 50 ?M DTPA. To limit spontaneous oxidation of any
H2S produced during respirometer experiments, RBCs in 10 mM
PBS and 50 ?M DTPA were first deoxygenated in a gently
swirling argon-perfused tonometer to avoid hemolysis that could
occur by bubbling. Initial experimental use of 40% (vol/vol)
(vol/vol) in subsequent experiments. Freshly collected RBCs
required a preparation time of ?4 h. Buffer sampled after
respirometry experiments indicated ?1% hemolysis.
Garlic. Garlic bulbs were obtained from local supermarkets.
Fresh garlic cloves were weighed (1–3 g), then pressed to obtain
the juice, which was kept on ice. Before use, the juice was diluted
1:10 with water to obtain a stock of 100 mg/ml, then used within
2 h of preparation.
and allows real-time measurement of dissolved H2S concentra-
tion in physiological solution (12). PHSS performance and
calibration were evaluated in a temperature-controlled multi-
sensor oxygraph respirometer (Oroboros, Innsbruck, Austria)
SSH + GSH
H2S2 + +
posed mechanism of H2S production by reaction of DADS and GSH via ? carbon
nucleophilic attack (red); H2S is not produced by thiol/disulfide exchange (blue).
(B) Nucleophilic substitution of allyl perthiol at the ? carbon followed by (C)
and higher polysulfides. See text below for description.
H2S production from organic polysulfides by thiol reactions. (A) Pro-
www.pnas.org?cgi?doi?10.1073?pnas.0705710104Benavides et al.
chamber containing 2 ml of stirred (500 rpm) 10 mM PBS with
50 ?M DTPA (pH 7.35) at 37°C. The PHSS polarizing voltage
Sarasota, FL), which also recorded the PHSS signal. The PHSS
was calibrated before each experiment with freshly prepared
anoxic sodium sulfide stock solution (0–200 ?M), as described
previously (see figure 5 in ref. 12), using the same buffer and
conditions as the experiment. The respirometer polarographic
oxygen sensor was calibrated in air-equilibrated (196 ?M O2) 10
mM PBS with 50 ?M DTPA; argon was used to deoxygenate the
buffer in the respirometer before RBC addition.
H2S Production Measurement by Multisensor Respirometry. H2S pro-
duction by RBCs and thiols was demonstrated by injecting garlic
juice or garlic-derived organic polysulfides (DAS, DADS, PDS,
polarographic oxygen sensor containing 2 ml of 10 mM PBS with
50 ?M DTPA for controls and thiols, or 20% (vol/vol) RBCs
previously deoxygenated to 0–2 ?M O2. For aorta H2S produc-
tion, clean isolated rat aorta segments (?30 mg wet weight) were
placed in the respirometer chamber with 10 mM PBS, 50 ?M
DTPA, and 10 mM glucose (pH 7.35) at 37°C. H2S production
was initiated by injection of 100 ?M DADS at physiological O2
concentrations (50 and 25 ?M O2). Data were analyzed with
respirometer DatLab software (Oroboros) coupled with Apollo
4000 software (WPI).
GSH/GSSG Determination by HPLC-ECD. GSH and GSSG were
analyzed by using an HPLC procedure similar to the method of
Melnyk et al. (34) for aminothiols but adapted to coulometric
electrode array detection and optimized for GSH and GSSG.
Briefly, freshly collected and prepared RBCs in anoxic 10 mM
PBS with 50 ?M DTPA (0.5 ml, 40% vol/vol) were placed in
microtubes, treated with substrates, and incubated at room
temperature for 20–30 min under an argon blanket, then cen-
trifuged at 1,000 ? g for 5 min at 4°C. RBCs were washed three
times with 2 volumes of 10 mM PBS with 50 ?M DTPA and
resuspended again to 40% (vol/vol). RBCs (0.5 ml, 40% vol/vol)
were mixed with equal volume cold 10% m-phosphoric acid with
2 mM EDTA and vigorously shaken for 1 min, then centrifuged
at 16,000 ? g for 2 min at 4°C. The supernatant was stored at
?80°C and processed within 1 month. For HPLC analysis,
samples were diluted 1:10 with 5% m-phosphoric acid and
filtered. The sample (20 ?l) was then separated on a Phenome-
nex Luna reversed-phase column 5? C18(2) 250 ? 4.60 mm
provided with a Phenomenex guard column (ODS, 4 mm
length ? 3.0 mm inner diameter) using a mobile phase flow of
0.6 ml/min. An isocratic mixture of 50 mM phosphate buffer (pH
3.1) containing 100 ?M sodium 1-octanesulfonate and acetoni-
trile (98.25:1.75) was used on a completely integrated HPLC
system (Shimadzu LC-2010CHT). An ESA CoulArray Model
5600A was connected in tandem. Data were analyzed by using
the ESA CoulArray for Windows 32 software. GSH and GSSG
concentrations in control and treated RBCs, determined ?4–6
h after blood was collected, were measured simultaneously for
comparative purposes. Because RBC preparation and treatment
were carried out with argon-sparged buffers, the RBCs would
have experienced periodic hypoxic conditions, perhaps resulting
in GSSG export and a lowered intracellular GSH pool (35).
RBCs in vivo would likely have higher total GSH as well
as GSH/GSSG ratios (15), which should enhance RBC H2S
300 g) were housed in University of Alabama at Birmingham
animal care facilities according to Institutional Animal Care and
Use Committee procedures on a 12-h light/dark cycle with food
and water available ad libitum. Isolated aorta was cleaned of
adventitious tissue, and aorta rings were cut to 3 mm long. Some
aorta rings were denuded of endothelium by gently rolling each
ring on a small dowel, the efficacy of which was tested with KCl,
PE, and acetylcholine. Aorta rings were mounted in an organ
bath containing 15 ml of Krebs–Henseleit buffer (pH 7.35), with
one end connected to a force transducer (Isolated Tissue Bath
System; Radnoti, Monrovia, CA). The vessel bath chamber
housing a PHSS and polarographic oxygen sensor was bubbled
with a mass flow-controlled (Series 100; Sierra Instruments,
Monterey, CA) mixture of N2and air, each containing 5% CO2,
to maintain a specific O2concentration from 30 to 50 ?M (36).
precontracted with 100 nM PE. Data were analyzed with Acq-
Knowledge software (BIOPAC Systems, Goleta, CA). Prelimi-
nary experiments with 10% (vol/vol) RBCs in the vessel bath
showed garlic-induced vasorelaxation and H2S production (data
not shown); however, bubbling the bath solution caused exces-
sive foaming and eventual hemolysis, which precluded the use of
RBCs. Accordingly, subsequent experiments were designed by
using garlic or garlic-derived organic polysulfides with GSH at a
concentration comparable to a 20% (vol/vol) RBC solution.
We acknowledge Michael Fallon, Junlan Zhang, Jo Morrison, and Asaf
Stein for greatly appreciated help with the pilot in vivo blood pressure
experiments. This work was supported by American Heart Association
Grants 0625292B (to G.A.B.), 0455296B (to D.W.K.), and 0655312B (to
R.P.P.) and National Institutes of Health (NIH) Grants HL58031 (to
V.M.D.-U) and 08RGM073049A (to D.W.K.). T.S.I. is supported by NIH
Cardiovascular Training Fellowship T32 HL007918 (to V.M.D.-U.).
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Benavides et al.PNAS ?
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no. 46 ?