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Biochemical and Biophysical Research Communications 311 (2003) 728734

Identication of carbonyl sulde and sulfur dioxide in porcine

coronary artery by gas chromatography/mass spectrometry,
possible relevance to EDHF
Michael Balazy,a,* Imad A. Abu-Yousef,b David N. Harpp,b and Joonsoo Parka

Department of Pharmacology, New York Medical College, Valhalla, NY 10595-1612, USA

Department of Chemistry, McGill University, Montreal, Canada
Received 23 September 2003

Incubation of porcine coronary artery rings and cardiac muscle tissue in Krebs buer followed by GC/MS analysis of the headspace gas revealed two gases, carbonyl sulde (COS) and sulfur dioxide (SO2 ). The gases were identied by characteristic ions obtained by electron ionization, and by comparison of the retention time on a chromatographic column (GS GasPro) with standards of
these gases. Stimulation of the arterial rings with acetylcholine and calcium ionophore A23187 increased the levels of SO2 and COS in
the vascular tissue. We also provide evidence that SO2 could originate from disproportionation of a very unstable gas, sulfur monoxide (S@O). We suggest potential origins of these gases and discuss their relevance to endothelium-derived hyperpolarizing factor.
2003 Elsevier Inc. All rights reserved.
Keywords: Carbonyl sulde; Sulfur dioxide; Sulfur monoxide; Coronary artery; Cardiac muscle; Acetylcholine; EDHF; Gas chromatography/mass

The vascular endothelium plays a pivotal role in the

control of the circulation. It secretes the potent vasodilators prostacyclin and nitric oxide and the vasoconstrictor peptide, endothelin-1, converts angiotensin I to
angiotensin II and metabolizes various vasoactive substances. The balance between these mediators determines the responses of the cardiovascular system in
diseases such as atherosclerosis, hypertension, and myocardial infarction [1]. Endothelium also produces a diffusible factor named EDHF of unknown chemical
structure, which can be released by acetylcholine and
other compounds from porcine coronary artery and
other arteries [2]. This factor presumably induces hyperpolarization of the smooth muscle cells and thereby
causes vasorelaxation. EDHF might play a compensatory role in cardiovascular system in pathologies which
reduce formation of NO and/or prostacyclin, both of
which are also known to hyperpolarize vascular smooth
muscle cells [3,4]. We wondered whether gases other than

Corresponding author. Fax: 1-914-594-3621.

E-mail address: (M. Balazy).
0006-291X/$ - see front matter 2003 Elsevier Inc. All rights reserved.

NO are released from vascular tissues and we have developed a method to analyze gases evolving from vascular
tissues by using GC/MS. According to this method, a
small piece of tissue is incubated in the biological buer in
a closed vial and the gases evolved during incubation
period accumulate in the space above the liquid (headspace). The headspace gas is sampled with a gas-tight
syringe and injected into a GC/MS containing a special
chromatographic column that separates tissue-derived
gases from air and water and concentrates them allowing
identication by mass spectrometry. This method allows
for structural identication of gases with good sensitivity
by specic detection of characteristic ions. Our work
shows that porcine coronary artery and cardiac muscle
produce two gases, carbonyl sulde (COS) and sulfur
dioxide (SO2 ).
Materials and methods
Materials. Standard gases, COS, SO2 , and hydrogen sulde (H2 S),
were obtained from SigmaAldrich in small tanks. The gases were
bubbled through distilled water to obtain saturated solutions at room

M. Balazy et al. / Biochemical and Biophysical Research Communications 311 (2003) 728734
temperature. The concentrations of these solutions were estimated
from available tables of gases solubility. The diluted solutions of these
gases (usually 103 104 -fold) were prepared by a sequential dilution in
water. Aqueous solutions of the gases in water or Krebs buer (1 ml)
were placed in 2-ml amber glass vials (Fisher) capped with a Teon
cap. After 30 min equilibration, the headspace gas was analyzed by
GC/MS (see below). Control experiments involved analysis of the
headspace gas from capped vials with and without the buer. Sulfur
monoxide (S@O) was prepared by thermal decomposition of
adamantylideneadamantanethiirane 1-oxide (AASO) prepared by the
reaction of triphenylmethanesulfenyl chloride with adamantylideneadamantane as described [5,6]. AASO (3.8 mg) was placed at the
bottom of a tightly capped vial, which was inserted in a heating block,
the temperature of which was raised to 150 C at 15 C/min. A ow of
high purity xenon (4 ml/min) was used to carry gases evolving from the
heating of AASO to the mass spectrometer (Hewlett-Packard model
5989A) via a short piece of capillary chromatographic column (DB-1,
0.5 m) maintained at room temperature. This column was directly inserted into the mass spectrometer operating in the electron ionization
mode (ionization energy was 70 eV).
Tissue preparation. Fresh porcine hearts were purchased from a
local slaughterhouse and the coronary arteries (PCA) were excised and
placed in oxygenated Krebs buer (Sigma). The arteries were cut into
rings (5 mm), placed in the buer (1 ml) in a 2-ml amber glass vials,
and capped. The arteries were prepared carefully so that the endothelial cells were not overly damaged. The PCA rings were then incubated at 37 C for a period of 3090 min. In another set of
experiments, the PCA rings were stimulated with calcium ionophore
A23187 (1 lM in DMSO or ethanol) or acetylcholine (1 lM in ethanol) followed by incubation for 1020 min and analysis of the headspace gas by GC/MS. The same experiments were also done with
porcine cardiac muscle (PCM) tissue (50 mg). Data are representative
of 37 measurements.
Analysis of tissue gases by GC/MS. The headspace gas (50 ll) was
sampled with a gas-tight syringe (Hamilton) and analyzed by GC/MS
(HP 5980 gas chromatograph connected to HP 5970 mass selective
detector). The instrument was under control of ChemStation software.
The tissue gases were separated on a gas capillary column (GS GasPro,
60 m  0.32 mm i.d.) from J&W Scientic. The initial column temperature of 70 C was maintained for 3 min after injection and then increased to 110 C at a rate of 10 C/min. The injector temperature was
220 C. Helium was used as a carrier gas at pressure 100 kPa and a ow
rate of 3.2 ml/min. The mass spectrometer operated in the electron
ionization mode (70 eV) at a source temperature of 220 C. The acquisition of ions was divided into three groups to specically detect
ions corresponding to COS (m/z 60, 4.55.4 min), H2 S (m/z 34, 5.4
9.5 min), and SO2 (m/z 64, 9.511 min). In preliminary experiments the
retention time of standards of COS, H2 S, and SO2 was established by
scanning and selected ion monitoring to be 5.05, 5.81, and 9.85 min,
respectively (Fig. 1). The change of ion acquisition over the period of
11 min revealed a higher background for ion at m/z 34, which resulted
in a sharp increase of the background in the middle portion of the
chromatograms. Nitrogen, oxygen, and water eluted within the rst
3 min after injection of the headspace gas.

Incubation of PCA and PCM followed by GC/MS
analysis of headspace gases resulted in detectable
amounts of COS and SO2 , whereas H2 S was not detected (Figs. 25). Control incubations of vials with
buer as well as analysis of the surrounding air did not
show these gases (Figs. 1C and D). COS and SO2 were
identied by comparison of retention times with au-


Fig. 1. Analysis of a mixture of standard gases by GC/MS on a GS

GasPro column (60 m  0.32 mm) using a temperature gradient from
70 C (isothermal for 3 min) to 110 C at a rate of 10 C/min. Carbonyl
sulde (COS), hydrogen sulde (H2 S), and sulfur dioxide (SO2 ) were
detected at indicated masses (m/z values), at retention times of 5.05,
5.61, and 9.85 min, respectively.

thentic synthetic standards of these gases and comparison of the mass spectra. COS produced ion at m/z 60 at
a retention time of 5.05 min and SO2 produced ion at
m/z 64 at a retention time of 9.85 min. COS appeared to
be continuously generated by PCA and PCM. Incubation of these tissues for 3090 min resulted in the
increased generation of COS. Formation of SO2 in PCA
was observed following stimulation with calcium ionophore (Fig. 2A) and acetylcholine (Fig. 3A). A control
experiment with ethanol (a solvent for acetylcholine)
showed a minor peak of SO2 (Fig. 3B), which increased
4-fold following addition of acetylcholine (Fig. 3A).
Calcium ionophore (A23187) stimulated formation of
COS in PCA 1.5-fold and activated formation of SO2
1.5-fold (Figs. 2A and B). In contrast to PCA, the incubations of PCM in the buer showed basal levels of
both COS and SO2 (Fig. 4). It was dicult to demonstrate stimulated release of COS and SO2 from PCM
because both these gases were generated by addition of
vehicles dimethyl sulfoxide (DMSO) and ethanol (not
shown) as well as calcium ionophore in DMSO and
ethanol (Figs. 4B and C). Incubation of PCM but not
PCA with DMSO showed increased levels of both COS
and SO2 (Fig. 5) as compared to incubations without
DMSO (Fig. 4A), suggesting that a sulfoxide could be a
precursor of these gases in the cardiac muscle. Thus,
whereas formation of SO2 in PCA was induced by acetylcholine and calcium ionophore, SO2 was continuously released from PCM. Both tissues also showed
basal release of COS, which in PCA was stimulated with
acetylcholine and calcium ionophore (Figs. 2 and 3). We
consistently observed formation of COS and SO2 in
PCA and PCM, and the GC/MS technique provided
condence as to their structure. In another experiment
we tested whether SO2 could originate from S@O. Fig. 6
shows the mass spectrum obtained during analysis of


M. Balazy et al. / Biochemical and Biophysical Research Communications 311 (2003) 728734

Fig. 2. Detection of COS and SO2 in coronary artery and controls by analysis of headspace gas by GC/MS. (A) Stimulation with calcium ionophore
A23187 (1 lM nal concentration in 1 ll DMSO); (B) DMSO (1 ll) control; (C) Krebs buer without tissue; and (D) air from a caped vial.

gases evolved from heating AASO, a S@O donor [5].

This mass spectrum shows ions at m/z 48 and 64 that
corresponded to S@O and SO2 . Since this analysis was
carried in the atmosphere of xenon, this experiment
shows that SO2 could originate from disproportionation
of S@O in the absence of other reactive molecules
according to the equation
4S@O ! 2SO2 1=4S8

where S8 denotes elemental sulfur. S@O also reacts with

oxygen to produce SO2 but its disproportionation
appears to be faster than oxidation [7].

Endothelium releases potent vasodilators, prostacyclin and nitric oxide, and a vasoconstrictor, endothelin1, which control the tone of vasculature. Because of its
sensitivity and specicity, mass spectrometry helped in
the past to structurally characterize these substances.
For example, a factor known as EDRF was denitively
identied as nitric oxide by mass spectrometry [8]. Our
method was specically designed for the detection of

sulfur containing gases released from vascular tissue.

Because gases display a wide range of polarities, the
capillary column liquid phase needs to be suitable for a
successful analysis of a particular gas. For example,
another kind of column allowed us to detect carbon
monoxide in the rat vascular tissue [9]. A GS GasPro
column, composed of bonded porous silica, was eective
for analysis of sulfur gases evolving from tissues. The
identication of two new gases generated by PCA, COS,
and SO2 , was based on comparison of their chromatographic and mass spectrometric properties with those of
chemical standards. The PCA tissue-derived COS and
SO2 had identical properties as the standard samples of
these gases. Moreover, stimulation of PCA with acetylcholine increased the release of these gases, suggesting
that muscarinic receptors are involved in formation of
COS and SO2 .
These gases have not been previously detected in
vascular tissue and at this stage of research we are
constructing a conceptual background for future
studies of potential origins of these two gases. COS
could originate from metabolism of thiocyanate
(SCN ) by thiocyanate hydrolase [10], and because the
occurrence of this enzyme has not been reported in

M. Balazy et al. / Biochemical and Biophysical Research Communications 311 (2003) 728734

Fig. 3. (A) Detection of COS and SO2 in coronary artery following

stimulation with acetylcholine (1 lM nal concentration in 10 ll ethanol); (B) control PCA incubation with 10 ll ethanol.

mammals, we cannot exclude the possibility that COS

detected in PCA could be of bacterial origin; however,
this type of thiocyanate metabolizing bacterium does
not grow heterotrophically [11]. Thiocyanate is found
in blood, milk, and saliva of mammals in concentrations of 30830 lM [12,13], and has been thought to
have antimicrobial function, because lactoperoxidase
and myeloperoxidase in the presence of H2 O2 oxidize
thiocyanate to cyanide and other compounds. Thiocyanate is biologically active [14] and one study reported
that it causes vasoconstriction of the rat aorta [15].
Cysteine, cystine, lanthionine, djenkolic acid, and
isothiocyanates have been also reported as biogenic
precursors of COS [16], but little is known about the
enzymology of the processes leading to COS in biological systems.


Fig. 4. Detection of COS and SO2 in cardiac muscle by GC/MS. (A)

Chromatogram showing analysis of the headspace gas from the incubation of the cardiac muscle for 30 min; (B) same as (A) with calcium
ionophore (1 lM) in ethanol (10 ll); and (C) same as (A) with calcium
ionophore (1 lM) in DMSO (1 ll).

A possible precursor of SO2 could be an intracellular

thiol such as cysteine that can be oxidized to cysteinesulnic acid by cysteine dioxygenase [17]. Cysteinesulnate is a substrate of glutamate-oxaloacetate
transaminase, and the product of this reaction, b-sulnylpyruvate, decomposes spontaneously to pyruvate
and SO2 [18]. In vivo, some of SO2 is hydrated to SO2
3 ,
which is oxidized by sulte oxidase to SO2
SO2 can escape this reaction and thus is found in the
headspace gas. Interestingly, all these elements appear to
be in place in endothelial cells, which produce sulte
from cysteine [19]. However, to our knowledge, formation of SO2 in vascular tissue has not been described
before and its vasoactivity on isolated porcine vessels is


M. Balazy et al. / Biochemical and Biophysical Research Communications 311 (2003) 728734

Fig. 5. Detection of COS and SO2 in cardiac muscle following incubation with dimethyl sulfoxide (DMSO, 10 ll).

not known. One study described that the acute exposure

of rats to SO2 has caused a drop in blood pressure [20],
and the other, that SO2 induces bronchial hyperreactivity to acetylcholine [21]. Physiologically active sulfur
compounds found in plants of the Allium family (such as
garlic) have been suggested to exert their eects through
metabolism of sulfur. For example, allicin causes vasodilation of rat pulmonary aorta that is independent of
the synthesis of nitric oxide, ATP-sensitive potassium
channels, and activation of cyclooxygenase [22], whereas
ajoene is antithrombotic [23]. While the origin of SO2 in
PCA remains to be established, we would like to suggest
another possibility, namely that SO2 could originate
from the disproportionation or oxidation of a very unstable molecule, sulfur monoxide (S@O), which has a
t1=2 of 1 s [7] but can be longer under certain conditions
[24]. Our work shows that S@O can be obtained in gas

phase by a moderate heating of adamantylideneadamantanethiirane oxide (AASO). The mass spectra of

AASO vapors in xenon showed detectable amounts of
S@O and SO2 (Fig. 6). Thus, because S@O is the sole
gaseous product of AASO decomposition [5], observation of SO2 in the mass spectrum of AASO vapors
(Fig. 6) suggests that SO2 can only originate from S@O
disproportionation via reaction (1). Additionally, according to reaction (1), elemental sulfur is another
product of S@O disproportionation [7]. Interestingly,
porcine endothelial cells grown on microcarrier beads
and examined by scanning electron microscopy X-ray
microanalysis at various times after initiation of culture
show the presence of elemental sulfur [25]. The sulfur
levels in porcine endothelial cells have doubled within
24 h of incubation and slightly increased following
stimulation with calcium ionophore A23187 [25]. One
possible explanation for the origin of elemental sulfur
[25] and SO2 (this study) in porcine endothelial cells is
that these cells produce S@O. A likely biological source
of S@O could be a sulfoxide or sulfenic acid [26,27].
Because S@O is unstable in aqueous solutions, SO2 as its
disproportionation/oxidation product should be detected in the headspace gas. In our experiments, addition
of dimethyl sulfoxide (DMSO) to PCM but not to PCA
increased the levels of SO2 , suggesting that DMSO could
be metabolized by cardiac muscle to SO2 . It is also
chemically feasible for S@O to originate from oxidation
of COS [7]. Also, we have not excluded the possibility
that the incubations of tissues in a closely capped vial
having a headspace volume of 1 ml might have caused
tissues to become hypoxic, a condition that might have
stimulated formation of these gases. However, shortening of the incubation time can decrease the amount of
COS and SO2 in the headspace below detection limits.

Fig. 6. GC/MS analysis of gases obtained during thermal decomposition of adamantylideneadamantanethiirane 1-oxide (AASO) in xenon as
described in Materials and methods. The mass spectrum shows sulfur monoxide (S@O) and sulfur dioxide (SO2 ). The intensity of ion at m/z 48
increased with temperature of AASO.

M. Balazy et al. / Biochemical and Biophysical Research Communications 311 (2003) 728734

Other studies have concluded that endothelial cells

secrete a factor of unidentied structure (EDHF) that
causes relaxation of vascular smooth muscle presumably
by opening of potassium channels. PCA is one of the
tissues that has been known to generate EDHF [28,29].
Endothelium-dependent hyperpolarizations are stimulated in PCA by acetylcholine [30], bradykinin [31], and
other substances [32]. Despite substantial amount of
work in this area, there is lack of experiments that would
help to establish the structure of EDHF. High instability
of EDHF appears to be a major impediment to its
structural characterization [33,34]. Moreover, a precursor and a stable metabolite of EDHF are unknown as
well as spectroscopic methods of its detection. One
bioassay study estimated that the half-life (t1=2 ) of
EDHF is in the range of 1.52 s [33]. A sandwich type
of bioassay suggested that the t1=2 of EDHF is about 1 s
[34]. The knowledge of the t1=2 of EDHF is important
because it could be used for comparison with the t1=2 of
chemical candidates in bioassay experiments. Measurements of t1=2 have helped in the past to establish chemical structures of endothelium derived prostacyclin and
nitric oxide [8,35,36]. While many candidates have been
suggested for the EDHF (the list includes H2 O2 ,
epoxyeicosatrienoic acids, K ions, cAMP, c-type natriuretic peptide, and also gap junctions), a comparison
of the t1=2 of these substances with the t1=2 of the endogenous endothelium-derived EDHF is lacking. The
t1=2 of EDHF, if it can be measured accurately, would be
very useful in selecting a chemical candidate for electrophysiological studies. Assuming that the t1=2 of
EDHF is in the range of seconds as indicated by available bioassay data [33,34], the likely candidates for
EDHF should be looked for among short-lived molecules, and S@O, COS, and SO2 deserve consideration.
Electrophysiological properties of these gases are not
known and clearly required to unravel their function in
vascular tissues.

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