Airway peroxidases catalyze nitration of the β2-agonist salbutamol and decrease its pharmacological activity

Article abstract of DOI:10.1124/jpet.110.170027

β₂-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β₂-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H₂O₂) and nitrite (NO₂ ^-), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pK_a of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β₂-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H₂O₂/NO₂^- being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β₂-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β₂-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.

Full text of DOI:10.1124/jpet.110.170027

0022-3565/11/3362-440–449
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 336:440–449, 2011
Vol. 336, No. 2
170027/3655827
Printed in U.S.A.
Airway Peroxidases Catalyze Nitration of the ␤2-Agonist
Salbutamol and Decrease Its Pharmacological Activity
Krzysztof J. Reszka, Larry Sallans, Stephen Macha, Kari Brown, Dennis W. McGraw,
Melinda Butsch Kovacic, and Bradley E. Britigan
Research Services (K.J.R., B.E.B.) and Department of Internal Medicine (D.W.M., B.E.B.), Veterans Affairs Medical Center,
Cincinnati, Ohio; Departments of Internal Medicine (K.J.R., K.B., D.W.M., B.E.B.), Chemistry (L.S., S.M.), and Pediatrics
(M.B.-K.), Cincinnati Children Hospital and University of Cincinnati College of Medicine, Cincinnati, Ohio
Received May 5, 2010; accepted October 19, 2010
ABSTRACT
␤2-Agonists are the most effective bronchodilators for the rapid (m/z 255.13) by mass spectrometry. It is noteworthy that the
relief of asthma symptoms, but for unclear reasons, their effec- latter metabolite was detected in exhaled breath condensates
tiveness may be decreased during severe exacerbations. Be- of asthma patients receiving salbutamol but not in unexposed
cause peroxidase activity and nitrogen oxides are increased in control subjects, indicating the potential for ␤2-agonist nitration
the asthmatic airway, we examined whether salbutamol, a clin- to occur in the inflamed airway in vivo. Salbutamol nitration was
ically important ␤2-agonist, is subject to potentially inactivating inhibited in vitro by ascorbate, thiocyanate, and the pharmaco-
nitration. When salbutamol was exposed to myeloperoxidase, logical agents methimazole and dapsone. The efficacy of inhi-
eosinophil peroxidase or lactoperoxidase in the presence of bition depended on the nitrating system, with the lactoperoxi-
hydrogen peroxide (H2O2) and nitrite (NOϪ2 ), both absorption dase/H2O2/NOϪ2 being the most affected. Functionally, nitrated
spectroscopy and mass spectrometry indicated formation of a salbutamol showed decreased affinity for ␤2-adrenergic recep-
new metabolite with features expected for the nitrated drug. tors and impaired cAMP synthesis in airway smooth muscle
The new metabolites showed an absorption maximum at 410 cells compared with the native drug. These results suggest that
nm and pKa of 6.6 of the phenolic hydroxyl group. In addition to under inflammatory conditions associated with asthma, phe-
nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, nolic ␤2-agonists may be subject to peroxidase-catalyzed ni-
formed by elimination of the formaldehyde group, was detected tration that could potentially diminish their therapeutic efficacy.
exchange. Treatment of acute asthma exacerbations typically
includes inhalation of ␤2-adrenergic agonists, which activate
Introduction
Asthma is a chronic inflammatory disease characterized by
␤2-adrenergic receptors (␤2ARs) on bronchial smooth muscle
bronchial smooth muscle contraction and episodic narrowing
cells, triggering an increase in cAMP that leads to smooth
of the airway. This, along with edema of the bronchial wall
muscle relaxation. ␤2-Agonists are currently the most potent
and accumulation of airway mucus, limits airflow and gas
and effective bronchodilators in clinical use. However, for
unknown reasons, their ability to relieve bronchoconstriction
This study was partially supported by a Veteran Administration Merit
during severe asthma exacerbations (i.e., status asthmati-
Review Grant and a Research Initiative Project, the Cystic Fibrosis Founda-
cus) may be reduced and even harmful (Suissa et al., 1994;
tion, and the National Institutes of Health National Heart, Lung, and Blood
Abramson et al., 2003).
Institute [Grant HL092121].
The contents of this article do not represent the views of the Department of
Veterans Affairs or the United States Government.
Portions of this work were presented previously: Reszka KJ, McGraw D,
and Britigan B (2009) Airway peroxidases can catalyze inactivating oxidation/
nitration of ␤-agonists, at the American Thoracic Society International Con-
ference; 2009 May 15–20; San Diego, CA. American Thoracic Society, New
York, NY.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.170027.
Elevated levels of inflammatory cells, particularly neutro-
phils (PMN) and eosinophils (EOS), as well as their secretory
products, have been detected in asthmatic airways, and their
amounts are further increased during clinical exacerbations
of the disease (Bousquet et al., 2000; Wu et al., 2000; Al-
dridge et al., 2002; Andreadis et al., 2003). Upon activation,
EOS and PMN generate superoxide (O•2Ϫ) and hydrogen per-
ABBREVIATIONS: ␤2-AR, ␤2-adrenergic receptor; AA, ascorbic acid; EBC, exhaled breath condensate; EPO, eosinophil peroxidase; LPO,
lactoperoxidase; MPO, myeloperoxidase; NaSCN, sodium thiocyanate; EOS, eosinophil; PMN, neutrophil; DTPA, diethylenetriamine pentaacetic
acid; MS/MS, tandem mass spectrometry; FT, Fourier transform; ICR, ion cyclotron resonance; CYP, cyanopindolol; HPLC, high-performance
liquid chromatography; ppb, parts per billion.
440

Airway Peroxidases Catalyze Nitration of Salbutamol
441
oxide (H2O2) and secrete unique peroxidases, eosinophil per- a mechanism that underlies impaired ␤2-agonist efficacy in
oxidase (EPO) by EOS and myeloperoxidase (MPO) by PMN. certain clinical settings.
These enzymes functionally resemble lactoperoxidase (LPO)
that is normally present in lung lining fluid (Conner et al.,
Materials and Methods
2002; El-Chemaly et al., 2003).
Materials. Salbutamol hemisulfate (1) [4-[2-(tert-butylamino)-1-
hydroxyethyl]-2-(hydroxymethyl)phenol sulfate (2:1), also known as
albuterol] was from MP Biochemicals, Inc. (Solon, OH). LPO (from
bovine milk; EC 1.11.1.7) and all other chemicals (hydrogen perox-
Nitric oxide (NO•) is generated by constitutive and induc-
ible forms of nitric-oxide synthases that are present in many
cell types within the respiratory tract, including airway and
alveolar epithelial cells, macrophages, neutrophils, mast
cells, and vascular endothelial and smooth muscle cells. In
asthmatic airways there is increased synthesis of NO• as
evidenced by increased levels of NO• in exhaled breath, ac-
cumulation of nitrite (NO2Ϫ) and nitrate (NOϪ3 ), and the pres-
ence of nitrated tyrosine residues in proteins (Gaston et al.,
1994; MacPherson et al., 2001; Ramesh et al., 2001; Andrea-
dis et al., 2003). Previous work indicated that nitrogen oxides
may reduce the bronchodilatory effect of salbutamol in asth-
matics (Kanazawa et al., 2002), presumably by inactivation
of adenylate cyclase. Impairment of ␤2AR function has also
been attributed to peroxynitrite oxidation of cysteine resi-
ide, 30%), ascorbic acid (AA), methimazole (1-methyl-3H-imidazole-
2-thione), dapsone (diamino-diphenyl sulfone), and diethylenetri-
amine pentaacetic acid (DTPA) were obtained from Sigma-Aldrich
(St. Louis, MO). LPO concentration was determined using ε412 of
1.12 ϫ 105 MϪ1 ⅐ cmϪ1 (Jenzer et al., 1986). MPO from human
leukocytes [lyophilized powder, 25 U, RZ (reinheit zahl purity value)
(A429/A280) of 0.61] was obtained from Axxora, LLC (San Diego, CA)
and reconstituted with 0.25 ml of distilled water before use. Human
EPO (460 U/mg protein) (Calbiochem, San Diego, CA) was reconsti-
tuted with 0.240 ml of water. The concentration of H2O2 was deter-
mined using ε240 of 39.4 MϪ1 ⅐ cmϪ1 (Nelson and Kiesow, 1972).
Intact salbutamol shows absorption maximum at 277 nm, and its
molar absorptivity (ε277) has been determined to be 1.8 ϫ 103 MϪ1
⅐
dues and/or nitration of tyrosine residues present in the cmϪ1 in pH 7.0 phosphate buffer. All chemicals were used as re-
ceived, and stock solutions of reagents were prepared in glass-dis-
tilled water.
extracellular domains in the ␤1- and ␤2-receptors (Lewis et
al., 2005).
Exhaled Breath Condensate Samples. EBC samples were col-
lected from childhood asthmatics hospitalized after an asthma exac-
erbation during 15 min of quiet breathing through a single-use
disposable RTube collector (Respiratory Research, Inc., Charlottes-
ville, VA) while subjects were wearing nose clips. Two aluminum
sleeves were used to cool the device at an initial temperature of
Ϫ80°C before collection. All EBC samples were taken within 3 h of
the last albuterol dose. After collection, the plunger was used to pool
the condensed material within the tube into a single sample (approx-
imately 1.0 ml). Samples were stored in the reaction tube at a
temperature of Ϫ80°C until thawed and processed for analysis.
Spectrophotometric Measurements. Spectra were measured
by using an Agilent diode array spectrophotometer model 8453 (Agi-
lent Technologies, Santa Clara, CA). Nitration of salbutamol was
studied by measuring absorption spectra at designated time points
after the start of the reaction. Samples were prepared in 100 mM
phosphate buffers, pH 7.0. All buffers contained DTPA (200 ␮M) or
were pretreated with Chelex-100 before use, and measurements
were performed at an ambient temperature of 20°C. Typically, the
reaction was started by the addition of a small aliquot of H2O2 or the
desired peroxidase. Time course measurements were carried out
after changes in absorbance at 410 nm in 30-s intervals versus
absorbance at 800 nm, where none of the compounds absorb.
To determine molar absorptivity of the mixture of salbutamol-
derived metabolites, the absorbance at 410 nm was measured after
multiple additions of peroxidases, H2O2, and NOϪ2 until A410 stabi-
lized, indicating that all salbutamol was consumed. These experi-
ments were conducted at various initial concentrations of the drug.
Based on these measurements ε410 for the mixture of metabolites
was estimated to be 4550 Ϯ 227 MϪ1 ⅐ cmϪ1 (n ϭ 14) at pH 7.0. The
effect of pH on ionization of the compound’s phenolic moiety was
determined by measuring absorption spectra of samples prepared by
adding equal volumes of nitrated salbutamol (25 ␮l) to buffers (0.1
M, 475 ␮l) of pH ranging from 4 to 9.
Because of their phenolic character, ␤-agonists such as
salbutamol (also referred to as albuterol) (Fig. 1) are poten-
tially subject to peroxidative metabolism. Consistent with
this notion, we have shown that the airway peroxidases LPO
and MPO can catalyze oxidation of ␤2-agonists in vitro
(Reszka et al., 2009). Given that inflamed airways are also
the source of NOϪ2 , we were interested to examine the effect
of NOϪ2 on the peroxidative metabolism of the agonist. Nitrite
is an excellent substrate for peroxidases, which oxidize it to
the nitrogen dioxide radical (NO•2), a species involved in
oxidation and nitration of biological targets (Reszka et al.,
1997; van der Vliet et al., 1997; Burner et al., 2000; Bru¨ck et
al., 2001; Brennan et al., 2002; Van Dalen et al., 2006).
Results of our study show that in the presence of NOϪ2 airway
peroxidases can convert salbutamol to nitrated species with
reduced capacity to bind ␤2-ARs and stimulate cAMP syn-
thesis. Putative metabolites of salbutamol nitration were
also detected in exhaled breath condensates (EBCs) from
asthmatics, thereby suggesting that metabolism of salbuta-
mol by peroxidases and NO2Ϫ in the inflamed airway may be
Mass Spectrometry. Experiments were performed on a Thermo
Fisher Scientific (Waltham, MA) LTQ-FT, a hybrid instrument con-
sisting of a linear ion trap and a Fourier transform ion cyclotron
resonance (ICR) mass spectrometer (Micromass, Manchester, UK).
Liquid chromatography separation of the sample components used a
Waters (Milford, MA) XBridge C18 3.5-␮m, 2.1 ϫ 100-mm column,
Finnigan Surveyor MS pump (Thermo Fisher Scientific), and Finni-
gan Micro AS autosampler (Thermo Fisher Scientific). A 200 ␮l/min
gradient elution of water and acetonitrile, both containing 0.1%
Fig. 1. Structures of salbutamol (1), 6-nitrosalbutamol (2), and salbuta-
mol-derived nitrophenol (3).

442
Reszka et al.
formic acid, took place as follows: 5 to 32% acetonitrile in the first 8 confluence in 96-well plates. After washing with phosphate-buffered
min followed by a rapid rise to 95% acetonitrile within the next
minute and held at 95% for an additional 7 min. The entire eluant
saline, cells were treated for 15 min with either phosphate-buffered
saline vehicle or the indicated concentration of salbutamol. The
was introduced into the LTQ-FT using the standard electrospray media were then removed, and the reaction was halted by using the
ionization source for the instrument with a spray voltage of 5 kV and supplied stop buffer, after which the resultant sample was processed
a capillary temperature of 275°C. Autogain control was used and set for fluorescent detection per the manufacturer’s instructions. The
at 500,000 with a maximum injection time of 1250 ms for FT-ICR full cAMP content for each sample was determined by extrapolating
values to a standard curve prepared with known concentrations of
cAMP.
scans. Collision-induced dissociation, MS/MS, was executed in the
linear trap with an autogain control setting of 10,000 and a maxi-
mum injection time of 500 ms. FT-ICR full scans were acquired in the
positive ion mode at 100,000 resolving power at m/z 400. Mass
accuracy errors for salbutamol and its nitrated analogs were below
250 ppb. The positive-ion MS/MS experiments were performed si-
multaneously in the linear trap portion of the instrument using
helium as a collision gas, isolation widths of 2 atomic mass units,
normalized collision energies of 35 to 40, a q value of 0.250, and an
excitation time of 30 ms.
Samples for MS experiments were prepared using ϳ50 ␮M salbu-
tamol, 5.9 mM H2O2, 5.8 mM NaNO2, and 0.25 ␮M LPO (or 0.19
U/ml MPO). The reaction was allowed to proceed until A410 ceased to
increase. Before the MS analysis, proteins were removed by using a
Centricon centrifugal filter device with a 10-kDa cut-off filter (Mil-
lipore Corporation, Billerica, MA). Experiments were also performed
on samples prepared using lower concentrations of reactants, 20 ␮M
salbutamol, 200 ␮M NaNO2, and 200 ␮M H2O2, MPO, or EPO (0.1
U/ml). All of these systems produced similar results. The newly de-
tected salbutamol nitration products are 6-nitrosalbutamol (2) 4-[2-(tert-
butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)-6-nitrophenol and
salbutamol-derived nitrophenol (3) 4-[2-(tert-butylamino)-1-hy-
droxyethyl]-2-nitrophenol.
Efficacy of Salbutamol Conversion. Salbutamol (10 ␮M) was
exposed to MPO (0.1 U/ml), NaNO2 (100 ␮M), and H2O2 (100 ␮M) in
phosphate buffer (0.1 M, pH 7.0). After 3-h reaction, when no further
changes in the absorbance at 410 nm were observed, the sample was
analyzed by a Q-TOF2 mass spectrometer (Micromass, Manchester,
UK). Before the analysis, equal volumes of the sample were mixed
with either 50/50 acetonitrile/H2O, 0.1% formic acid, or methanol,
then each sample was directly infused into the mass spectrometer.
The presence and relative amount of unreacted salbutamol was
monitored by measuring its mass at m/z 240.16 (MϩHϩ), 260.14
(MϩNaϩ), and their relative amounts based on their ion counts. Five
runs were performed for each solvent. Similar analysis was per-
formed on intact salbutamol (10 ␮M) without performing nitration. A
comparison of the salbutamol ion counts in the sample before nitra-
tion to the salbutamol ion counts in the same sample after nitration
indicated that approximately 98% of the salbutamol was converted
in the process.
Extraction of Products from EBC. Salbutamol and derived
products were extracted from the exhaled breath condensate using
an Oasis MCX 1cc extraction cartridge (Waters). The cartridge was
first washed with 1 ml of methanol followed by 1 ml of water. The
sample was acidified with formic acid to pH ϳ 3, loaded, and washed
with 1 ml of formic acid (2% in water) and vacuum-dried. Neutral
substances were eluted with 0.25 ml of methanol and then bases
(salbutamol and related products) were extracted with 0.25 ml of
ammonia (5% in methanol) then acidified and diluted with 2% formic
acid.
Results
Reaction of Salbutamol with Peroxidase/H2O2/NO؊2 .
We have reported that in the presence of LPO(MPO)/H2O2
salbutamol undergoes oxidation to a new species, most likely
a o,oЈ-dimer, absorbing in the UV range, approximately 315
nm (Reszka et al., 2009). The reaction was, however, slow,
presumably because of the inability of the drug to react
efficiently with compound II of the peroxidases. In prelimi-
nary studies, we found that NOϪ2 substantially facilitates the
peroxidative reaction and affords a different metabolite,
clearly distinguishable by its absorption spectrum. Figure 2A
shows spectra observed during interaction of salbutamol
with LPO and H2O2 in the presence of NaNO2. The concen-
tration of LPO used is in the range found in the lavage fluid
of human subjects, 3.2 nM (0.25 ng/␮l) (El-Chemaly et al.,
2003). During the reaction, the color of the sample changed to
yellow, and the new species formed exhibited absorption
maxima at 410 and 314 nm. The inset in Fig. 2A shows the
time course of the reaction measured at these two wave-
lengths. The yellow color and the absorption maximum at
410 nm suggest formation of a nitrated phenol with the nitro
group in position ortho with respect to the phenolic –OH
group. These features are similar to those described for 3-ni-
trotyrosine, in which the phenolic -OH and -NO2 groups are
ortho to each other (Sokolovsky et al., 1966; Monzani et al.,
2004). Because there is only one unoccupied ortho position in
salbutamol, the new product absorbing at 410 nm was ten-
tatively attributed to 6-nitrosalbutamol (Fig. 1, compound 2),
although related salbutamol-derived nitrated phenol and
other oxidation products may also be formed. No character-
istic absorption at 410 nm was observed when either LPO or
NOϪ2 or H2O2 was omitted, confirming that nitration of sal-
butamol is an enzymatic process totally dependent on the
simultaneous presence of the drug, NOϪ2 , LPO, and H2O2.
The molar absorptivity of the reaction mixture at 410 nm
derived from salbutamol was estimated to be 4550 Ϯ 227
MϪ1 ⅐ cmϪ1 (n ϭ 14) at pH 7.0. This value is not far from the
molar absorptivity reported for 3-nitrotyrosine of 4000 MϪ1
⅐
cmϪ1 at 422 nm at pH 7.5 (Monzani et al., 2004) and 4100
MϪ1 ⅐ cmϪ1 at 428 nm at pH 8 (Sokolovsky et al., 1966;
Riordan et al., 1967). It is also close to molar absorptivities
determined for a number of ortho-nitrophenols in alkaline
solutions (Rapoport et al., 1961).
␤2-AR Receptor Binding and cAMP Generation. Receptor
affinity was assessed by measuring the ability of native and nitrated
salbutamol to displace binding of the ␤2AR antagonist 125I-CYP as
described previously (McGraw and Liggett, 1997). Binding assays
were carried out with crude membrane preparations from airway
smooth muscle cells that transgenically overexpress the human
␤2-AR (McGraw et al., 1999).
The ability of native and nitrated salbutamol to stimulate cAMP
production in airway smooth muscle cells was measured by a fluo-
rescent detection assay using a commercially available kit (Mediom-
The dependence of salbutamol nitration on [H2O2] was
investigated measuring ⌬A410 at constant [NaNO2] of 250
␮M. When the concentration of H2O2 was varied from 0 to
1000 ␮M, ⌬A410 initially increased reaching maximum at
near 250 ␮M H2O2, and then decreased (Fig. 2B). This de-
crease in efficacy at higher concentrations of the peroxide
was probably caused by inactivation of the enzyme by excess
H2O2 because A410 increased to near the maximal value
ics, St. Louis, MO). For these studies, cells were grown to near- when additional doses of LPO were applied (data not shown).

Airway Peroxidases Catalyze Nitration of Salbutamol
443
Fig. 2. Interaction of salbutamol with
LPO/H2O2/NaNO2 (A and B) and MPO/
H2O2/NaNO2 (C and D) systems. A and
C, absorption spectra recorded during in-
teraction of salbutamol (20 ␮M), with
H2O2 (310 ␮M), NaNO2 (300 ␮M), and
LPO (4 nM) (A) or MPO (0.2 U/ml) (C).
Spectra were recorded every 30 s. Arrows
indicate direction of changes. Absorption
peaks at 350 and 276 nm in spectrum a
(before H2O2 addition) in A and C are
from NOϪ2 and salbutamol, respectively.
Insets show time course of absorbance
changes at 410 and 314 nm after bolus
addition of H2O2. B, nitration of salbuta-
mol (100 ␮M) measured at 410 nm versus
[H2O2] (F) in the presence of NaNO2 (250
␮M) and LPO (6.5 nM), and versus
[NaNO2] (f) in the presence of H2O2 (104
␮M) and LPO (10 nM). D, nitration of
salbutamol (10 ␮M) measured at 410 nm
versus [H2O2] (F) in the presence of
NaNO2 (1000 ␮M) and versus [NaNO2]
(f) in the presence of H2O2 (220 ␮M) and
MPO (0.1 U/ml). All experiments were
carried out in phosphate buffer (0.1 M,
pH 7.0) containing DTPA (0.2 mM).
When the concentration of NaNO2 was varied from 0 to 250
␮M at constant H2O2 concentration (250 ␮M), ⌬A410 monot-
onously increased, reaching a maximum value approximately
250 ␮M NaNO2 (Fig. 2B). At higher concentrations of
NaNO2, ⌬A410 started to plateau, suggesting that nearly all
of the H2O2 was consumed under these conditions.
Because either MPO or EPO may be present in the airways
of patients with asthma (Andreadis et al., 2003), we next
examined whether these peroxidases also support nitration
of salbutamol. Figure 2C shows spectra observed during in-
teraction of salbutamol with MPO/H2O2/NOϪ2 . It may be ap-
preciated that these spectra are similar to those generated by
the LPO/H2O2/NOϪ2 system (Fig. 2A), suggesting formation
of the same products. The inset in Fig. 2C shows kinetic
traces recorded at 410 and 314 nm. As with LPO, the efficacy
of salbutamol nitration by MPO depends on [H2O2] and
[NaNO2] as shown in Fig. 2D. Similar observations were
made when MPO was replaced with EPO (not shown), con-
firming that all three peroxidases can support nitration and
oxidation of salbutamol.
Fig. 3. Absorption spectra of nitrosalbutamol measured at pH 7.9, 7.0,
6.7, 6.3, 5.2, and 4.2 (spectra a-f, respectively). Inset, A410 plotted versus
pH. The apparent pKa of 6.6 was determined for the phenolic –OH group
in nitrated salbutamol-derived products.
We further found that the absorption spectrum of nitrated
products derived from salbutamol depended on pH (Fig. 3), ysis of the sample by Q-TOF2 mass spectrometer. Salbuta-
most likely caused by changes in protonation of the com- mol (10 ␮M) was exposed to MPO (0.1 U/ml), NaNO2 (100
pounds’ phenolic –OH group. The intensity of the peak at 410 ␮M), and H2O2 (100 ␮M) in phosphate buffer (0.1 M, pH 7.0),
nm increased when the pH was raised from 4 to 8 and and the reaction was continued until A410 reached maximum.
remained essentially unchanged above pH 8. Based on the A comparison of the salbutamol ion counts in the sample
dependence of A410 on pH (Fig. 3, inset), the apparent pKa of before nitration to the salbutamol ion counts in the same
the phenolic –OH in the nitrated compound was determined sample after nitration indicated that approximately 98% of
to be 6.6. This value is in agreement with pKa of 6.8 reported the salbutamol was converted in the process.
for 3-nitrotyrosine (Riordan et al., 1967) and is close to pKa
To relate these observations to more physiological condi-
values of other ortho-nitrophenols (Rapoport et al., 1961). All tions we compared the efficacy of the reaction (measured as a
of these observations further support the conclusion that the change in ⌬A410) carried out at 20°C with that performed at
product absorbing at 410 nm can be linked to 6-nitrosalbu- 37°C using MPO of low (0.2 U/ml) and high (1 U/ml) activity.
tamol (2) and/or a related ortho-nitrophenol.
The higher MPO activity is to mimic the severe inflammation
Efficacy of Salbutamol Conversion. The efficacy of the during acute asthma attack. Concentrations of other reac-
enzymatic conversion of salbutamol was determined by anal- tants were kept low, salbutamol 5 ␮M, H2O2 45 ␮M, NaNO2

444
Reszka et al.
50 ␮M, in pH 7.0 phosphate buffer. When the experiment
was performed at 37°C and with MPO of 1 U/ml the maxi-
mum level of nitro-salbutamol was attained in 5 min (⌬A410 ϭ
0.026; n ϭ 2). In contrast, at 20°C and with MPO of 0.2 U/ml
even 60 min was not enough to reach the same level (n ϭ 2).
Thus, under more physiologic conditions the reaction is
rapid, which suggests that it may play a role in rapid inac-
tivation of the agonist during a severe asthma attack.
Mass Spectrometry. Samples of native salbutamol and
salbutamol treated with LPO(MPO, EPO)/H2O2/NOϪ2 in pH
7.0 phosphate buffer containing DTPA were analyzed by
HPLC/mass spectrometry. Figure 4A shows HPLC elution
profile of intact salbutamol (retention time 3.15 min), and
Fig. 4C shows its mass spectrum (m/z 240.15947; error 207
ppb). Two mono-nitrated products were detected in samples
of salbutamol treated with LPO(MPO)/H2O2/NOϪ2 with re-
tention times of 6.19 and 6.75 min (Fig. 4B). These species
were identified as nitrated salbutamol, 2 (m/z 285.14456,
error: 215 ppb; Fig. 4D) and a salbutamol-derived nitrophe-
nol 3 (m/z 255.13397, error: 142 ppb; Fig. 4E), consistent
with the elemental composition of their protonated ions
C
13H21N2Oϩ5 and C12H19N2Oϩ4 , respectively. Structures of
these metabolites are shown in Fig. 1. To further confirm
identity of these ions, the MS/MS spectra of ions 2 and 3 were
acquired (Fig. 4, F and G). The fragmentation pattern of the
parent ion 2 indicates elimination of a water molecule (m/z
267.08), the isobutene fragment (C4H8) (m/z 229.00), loss of
both C4H8 and H2O (m/z 211.08), and another water mole-
cule (m/z 193.08) (Fig. 4F). The fragmentation pattern of the
parent ion 3 indicates elimination of water molecule (m/z
237.08), the isobutene fragment (C4H8) m/z 199.00, and the
loss of both C4H8 and H2O (m/z 181.00) (Fig. 4G). These data
further support the assignment of the parent ions to nitrated
phenolics 2 and 3, respectively. MS spectra of salbutamol
treated with MPO and EPO nitrating systems showed for-
mation of the same nitrated species (not shown).
We also considered formation of salbutamol dimers during
enzymatic nitration of the drug. However, mass spectrometry
investigations did not indicate the presence of these products
in our samples, possibly because in the presence of high
concentrations of nitrite nitration of phenolics is preferred
over their dimerization.
Mass Spectrometry of Exhaled Breath Condensate.
The preceding in vitro experiments offered strong evidence
that salbutamol and other ␤2-agonists are subject to peroxi-
dase-catalyzed nitration. Because both peroxidase activity
and nitrite content are elevated in the inflamed airway, we
next wanted to determine whether drug nitration could occur
in vivo. To examine this possibility, we performed mass spec-
trometric analysis of exhaled breath condensates from asth-
matic patients who were being treated with salbutamol for
an asthma exacerbation. Preliminary mass spectroscopy
analyses were suggestive but not definitive for the presence
of nitrated species similar to those observed in the in vitro
reactions caused by high signal-to-noise ratio. Therefore to
increase the concentration of potential nitrated drug metab-
Fig. 4. A and B, HPLC elution profiles of salbutamol (1, retention time
3.15 min) (A) and two mono-nitrated metabolites of salbutamol (2, reten-
tion time 6.19 and 3, retention time 6.75 min) (B). Nitration of salbutamol
was carried out using a LPO/H2O2/NOϪ2 nitration system in phosphate
buffer, pH 7.0, containing DTPA (0.1 mM). C-E, positive ion electrospray
ionization mass spectra of salbutamol 1 (m/z 240.15947, error: 207 ppb,
eluted at 3.15 min) (C), nitrosalbutamol 2 (m/z 285.14456, error: 215 ppb,
eluted at 6.19 min) (D), and salbutamol-derived nitrophenol 3 (m/z
255.13397, error: 142 ppb, eluted at 6.75 min) (E). F and G, MS/MS
spectra and fragmentation patterns of the molecular ions 2 (m/z 285.14)
(F) and 3 (m/z 255.13) (G).

Airway Peroxidases Catalyze Nitration of Salbutamol
445
olites, we pooled condensates from three different subjects ural constituents of airway lining fluids with concentrations
and analyzed it as a single concentrated sample. Figure 5A near 100 ␮M and 20 to 120 ␮M, respectively (Tenovuo and
shows the HPLC elution profile of the EBC sample with a Ma¨kinen, 1976; Cross et al., 1994). Figure 6A shows that AA
peak at 6.80 min, which coincides with the HPLC peak (6.75 in a concentration-dependent manner delayed the appear-
min) from salbutamol-derived nitrophenol 3, m/z 255.13 (Fig. ance of the peak at 410 nm, suggesting that nitration of
5B). The MS/MS spectrum of the EBC fraction eluting at 6.8 salbutamol by LPO/H2O2/NOϪ2 is also delayed. However, the
min (Fig. 5C) shows product ions of m/z of 237.17, 199.00, resumption of the reaction after a lag period suggests that
181.00, and 130.25, which are the same as those from nitro- the inhibitory action of AA is not caused by inhibition/inac-
phenol 3 eluting at 6.75 min prepared in vitro (Figs. 4G and tivation of the enzyme, but rather by its interaction with the
5D), which indicates a similar pattern of fragmentation and drug-derived phenoxyl radicals (Reszka et al., 2009) and/or
the same parent ion. The m/z 181 product ion arising from with NO•2 radicals (Reszka et al., 1999). Thus, although AA at
the loss of isobutene and water was isolated in the linear trap physiological concentrations only transiently inhibits nitra-
and further fragmented, resulting in a MS3 experiment. MS3 tion of the drug, supplementation of the system with AA at
product ions from the parent matched the standard; both higher than physiological concentrations, e.g., ϳ 1 mM, com-
gave m/z 136 as the most abundant ion along with strong pletely prevented nitration of salbutamol (not shown).
abundances for m/z 117, 133, 135, and 136 (not shown).
Figure 6B shows the effects of NaSCN on nitration of the
Together, these preliminary data (the same retention times, agonist by LPO/H2O2/NaNO2. On increasing the concentra-
the same parent ion of m/z 255.13, and the same fragments tion of NaSCN from 0 to 50 ␮M there is a gradual inhibition
observed in MSn from both samples) strongly suggest that of the reaction. However, NaSCN at 50 ␮M completely pre-
oxidation and nitration of salbutamol can occur in the air- vented nitration of the drug (Fig. 6, trace d). This inhibitory
ways of asthmatics.
action is most likely caused by preferential oxidation of
Effect of Ascorbate and Thiocyanate on Nitration of SCNϪ by the LPO/H2O2 system and rapid depletion of H2O2.
Salbutamol. AA and sodium thiocyanate (NaSCN) are nat-
Effect of Methimazole and Dapsone on Nitration of
Salbutamol. Methimazole, an antithyroid drug, and dap-
sone, an anti-inflammatory and antileprotic agent, are po-
tent inhibitors of LPO and MPO (McGirr et al., 1990; Kettle
and Winterbourn, 1991; Bozeman et al., 1992). In agreement
with this, both compounds in a concentration-dependent
Fig. 6. Nitration of salbutamol by LPO/H2O2/NOϪ2 : effect of ascorbate (A)
and thiocyanate (B). A, ascorbate in a concentration-dependent manner
causes a lag in net nitration of the drug. Shown are A410 versus time
traces recorded during interaction of salbutamol (100 ␮M), with H2O2 (4
mM), NaNO2 (3.5 mM), and LPO (67 nM) in the absence and presence of
55, 88, and 109 ␮M ascorbate (traces a-d, respectively). B, thiocyanate
inhibits nitration of salbutamol (20 ␮M) by LPO (10 nM), H2O2 (312 ␮M),
and NaNO2 (300 ␮M). Shown are A410 versus time traces recorded in the
absence (trace a) and presence of 10, 25, and 50 ␮M NaSCN (traces b-d,
respectively). n ϭ 2.
Fig. 5. A and B, HPLC elution profile of molecular ions of m/z 255.13 in
breath condensate of asthma patients treated with salbutamol (A; reten-
tion time 6.80 min) and from a salbutamol nitrated in vitro (with
MPO(LPO)/H2O2/NOϪ2 ) (B; retention time 6.75 min). C and D, MS/MS
spectra of the parent ion of m/z 255.13 in breath condensate (C) and from
compound 3 produced by nitration of salbutamol by LPO/H2O2/NOϪ2 in
vitro (D). Peaks at m/z 237.17, 199.00, 181.08, and 130.25, originating
from the parent ion of m/z 255.13 in D, coincide with peaks in C.

446
Reszka et al.
Fig. 9. Inhibition by dapsone of salbutamol nitration by peroxidase/H2O2/
NOϪ2 systems. The extent of nitration is expressed as a change in ⌬A410
versus control (dapsone omitted) taken as 100% after 10, 30, and 60 min
reaction for LPO, MPO, and EPO nitrating systems, respectively. LPO system:
[Salbutamol] ϭ 100 ␮M, [NaNO2] ϭ 3.4 mM, [H2O2] ϭ 4 mM, [LPO] ϭ 65
nM. MPO system: [Salbutamol] ϭ 50 ␮M, [NaNO2] ϭ 5 mM, [H2O2] ϭ 5
mM, [MPO] ϭ 0.2 U/ml. EPO system: [Salbutamol] ϭ 50 ␮M, [NaNO2] ϭ
5 mM, [H2O2] ϭ 2 mM, [EPO] ϭ 0.075 U/ml. Reactions were carried out
in phosphate buffer, pH 7.0, containing dimethyl sulfoxide (5% v/v) (n ϭ 2).
ever, in contrast to AA or NaSCN, the methimazole- and
dapsone-dependent inhibition was permanent, presumably
caused by inactivation of the enzyme, because the reaction
resumed when LPO was readded (Fig. 7A, trace c, and Fig. 8,
traces d and e).
The effects of methimazole on nitration catalyzed by MPO
and EPO were somewhat different. In contrast to its appar-
ent irreversible inhibition of LPO, the inhibitory action of
methimazole on MPO was transitory. The A410 versus time
traces recorded at various concentrations of methimazole are
shown in Fig. 7B. The S shape of these traces indicates that
methimazole affects the process only at its initial stage, pre-
sumably because it is a good MPO substrate and competes
with other reactants for the active site of the enzyme and/or
the nitrite-derived oxidants. At low concentrations, however,
methimazole is rapidly consumed, after which the nitration
process is resumed as suggested by the increased rate of the
reaction. A similar effect of methimazole was observed when
MPO was replaced by EPO (not shown).
Fig. 7. Inhibition by methimazole of salbutamol nitration by LPO/H2O2/
NOϪ2 (A) and MPO/H2O2/NOϪ2 (B) systems measured as a change in
absorbance at 410 nm. A, salbutamol (100 ␮M) reacted with LPO (73 nM),
NaNO2 (3.5 mM), and H2O2 (4 mM) in pH 7.0 buffer in the absence (trace
a) and presence of 27, 55, and 109 ␮M methimazole (traces b-d, respec-
tively). In traces c and d additional doses of LPO were added at the time
indicated by arrows. n ϭ 2. B, salbutamol (50 ␮M) reacted with MPO (0.2
U/ml), NaNO2 (5 mM), and H2O2 (2.1 mM) in pH 7.0 buffer in the absence
(trace a) and presence of 25, 50, and 100 ␮M methimazole (traces b-d,).
Shown are typical kinetic traces.
Figure 9 illustrates the effects of dapsone on nitration
carried out by LPO, MPO, and EPO systems. Dapsone seems
to be a potent blocker of nitration catalyzed by LPO even at
high concentrations of NaNO2 and H2O2 used in these exper-
iments. Its inhibitory effect on nitration catalyzed by EPO
was stronger than on nitration supported by MPO (Fig. 9), in
agreement with a prior report that EPO is more sensitive to
dapsone than is MPO (Bozeman et al., 1992; Thomas et al.,
1994).
Effect of Salbutamol Transformation on ␤2AR Bind-
ing and cAMP Generation. The ability of ␤2-agonists such
as salbutamol to stimulate receptor signaling depends on
their ability to bind to the receptor and induce conforma-
tional changes in ␤2AR structure that facilitate activation of
its associated G proteins. Addition of a nitrite moiety to a
␤2-agonist such as salbutamol could potentially impair ago-
Fig. 8. Inhibition by dapsone of salbutamol nitration by LPO/H2O2/NO2Ϫ
measured as a change in absorbance at 410 nm. Salbutamol (97 ␮M)
reacted with LPO (65 nM), NaNO2 (3.4 mM), and H2O2 (4 mM) in phos-
phate buffer, pH 7.0, in the absence (trace a) and presence of 2.5, 5, 10, 50,
and 100 ␮M dapsone (traces b-f, respectively). In traces d and e additional
doses of LPO were added at the time indicated by arrows. n ϭ 2.
manner inhibited nitration of salbutamol by LPO/H2O2/NO2Ϫ nist-mediated signal transduction by altering the agonist’s
(Figs. 7A and 8). Methimazole at a concentration close to 100 ability to bind to the ␤2AR, or alternatively, the nitrated
␮M completely stopped the reaction (Fig. 7A, trace d). Dap- agonist might bind the receptor but fail to induce the confor-
sone at the concentration of 50 ␮M inhibited LPO/H2O2/NO2Ϫ mational changes in receptor structure necessary for signal
nitration of salbutamol by approximately 86% (Fig. 8). How- transduction. Therefore, to determine whether the affinity of

Airway Peroxidases Catalyze Nitration of Salbutamol
447
nitrated salbutamol for the ␤2AR was different from that of airway inflammation characterized by an influx of cells with
the native drug, we carried out competition binding assays significant peroxidase content and activity (i.e., EOS and
using primary airway smooth muscle cells that were derived PMN). The in vitro studies presented here show that, in the
from transgenic mice overexpressing the human ␤2AR presence of NOϪ2 , salbutamol can undergo oxidation and ni-
(McGraw et al., 1999). As shown in Fig. 10A, salbutamol tration in reactions catalyzed by airway peroxidases. These
transformed by LPO/H2O2/NOϪ2 displaced binding of the non- findings raise the possibility that a similar process may occur
selective ␤2AR antagonist 125I-CYP to airway smooth muscle in severe asthma exacerbations, which are characterized by
cell membranes by nearly 1.7 orders of magnitude less than increased airway peroxidase activity and elevated content of
that of native salbutamol, indicating that nitration of the nitrogen oxides. Preliminary results indicating the presence
parent compound markedly reduced its capacity to bind the of the oxidatively modified drug (compound 3) in breath
␤2AR.
To establish whether the reduction in receptor affinity was butamol therapy strongly support this hypothesis.
associated with decreased signal transduction, we compared In vitro-generated nitrated salbutamol shows both sub-
condensate samples from asthmatic patients undergoing sal-
the ability of native salbutamol and the nitrated reaction stantially diminished affinity for the ␤2-AR and decreased
products to stimulate cAMP synthesis in murine airway ability to stimulate cAMP production in airway smooth mus-
smooth muscle cells. Consistent with the reduction in bind- cle cells. Because the therapeutic activity of ␤2-agonists de-
ing affinity, we found that the ability of the nitrated salbu- pends on molecular interactions between the agonist and
tamol product to stimulate cAMP was more than two orders amino acid residues that induce or stabilize an “active” re-
of magnitude less than that of the parent compound (Fig. ceptor conformation, the resultant change in agonist struc-
10B), with EC50 1.02 Ϯ 0.05 ϫ 10Ϫ7 M [confidence interval ture caused by the introduction of the highly electrophilic
(0.588–1.77) ϫ 10Ϫ7 M] versus 5.74 Ϯ 1.20 ϫ 10Ϫ9 M [con- nitro group to the aromatic ring could play a role in the
fidence interval (3.86–8.52) ϫ 10Ϫ9 M], respectively (p Ͻ agonist’s pharmacologic activity. Given the resultant reduc-
0.01, n ϭ 3).
tion in ␤2-AR activation observed in airway smooth muscle
cells, the occurrence of salbutamol nitration in vivo could
have important clinical implications for therapeutic efficacy
of ␤2-agonists in severe asthma exacerbations. However, our
study also demonstrates that it may be possible to prevent
the peroxidase- and nitrite-dependent inactivation of the
drug by using antioxidants or peroxidase inhibitors. Should
nitration of the ␤2-agonist be responsible for its diminished
physiological responsiveness, these approaches might be use-
ful in maintaining the therapeutic activity of the drug in
some clinical settings.
Discussion
Salbutamol is a short-acting ␤2-adrenergic agonist fre-
quently used to relieve acute airway bronchospasm. How-
ever, its therapeutic effectiveness may be decreased in severe
exacerbations of the disease. The molecular mechanisms re-
sponsible for this decreased effectiveness are poorly under-
stood. One feature of severe asthma exacerbations is marked
Mechanistic Considerations. Salbutamol is a phenolic
compound (Fig. 1) and, therefore, similar to tyrosine and
other phenolics, may be subject to the oxidative metabolism
and nitration when exposed to peroxidases and NOϪ2 . The
process probably involves formation of free radical interme-
diates, the corresponding phenoxyl radical (PhO•) from sal-
butamol and the NO•2 radical from NOϪ2 . Both species can be
formed enzymatically by LPO, MPO, and EPO systems
(Burner et al., 2000; Bru¨ck et al., 2001; Brennan et al., 2002;
Monzani et al., 2004; Van Dalen et al., 2006; Reszka et al.,
2009). At concentrations of NOϪ2 markedly higher than the
concentration of salbutamol, oxidation of NOϪ2 to NO2• may
prevail, and oxidation of salbutamol may be accomplished by
its reaction with NO•2. In either case, formation of a nitrated
phenol could then be accomplished via radical-radical recom-
bination: PhO• ϩ NO2• 3 PhOH-NO2.
The scheme in Fig. 11 illustrates the proposed pathway for
the oxidation and nitration of salbutamol using MPO as a
typical peroxidase. It is suggested that MPO/H2O2 concomi-
tantly oxidizes 1 to the corresponding phenoxyl radical (1a)
and oxidizes NOϪ2 to NO2•. 1a rearranges to 1b (only one of
several resonance forms is shown), which reacts with NO2•
according to eq. 1, yielding 2.
The species, which eluted at 6.75 min and produced a
molecular ion of m/z 255.13 (Fig. 4E), was assigned to a
mono-nitrated salbutamol derivative 3 (Fig. 1). This assign-
ϩ
ment is consistent with elemental analysis (C12H19N2O4
,
Fig. 10. A, 125I-CYP displacement from ␤2-receptors by intact (E) and
nitrated (F) salbutamol. B, cAMP production by smooth muscle cells
stimulated by intact (E) and nitrated (F) salbutamol.
exact mass 255.13397) and further supported by MS/MS
spectrum obtained from this molecular ion (Fig. 4G). Forma-

448
Reszka et al.
reduces it to native LPO, accelerates the enzyme turnover
and generates NO•2. Thus, all enzymes studied generate spe-
cies required for nitration of the drug.
The absorption spectrum of nitrated salbutamol depends
on pH with the pKa (phenol) of 6.6. This is significantly less
than the pKa (phenol) of 9.4 reported for salbutamol itself
(Croes et al., 1995). For comparison the corresponding pKa
values for nitrotyrosine and tyrosine are 6.8 and 10.2, respec-
tively (Riordan et al., 1967; Sokolovsky et al., 1967). Thus,
the presence of the electron-withdrawing nitro group renders
the phenolic –OH group in 2 and 3 almost 1000-fold more
acidic than in salbutamol. Thus, at physiological pH, nitro-
salbutamol will be present mostly with its phenolic –OH
group ionized. Therefore, if interaction of salbutamol with its
␤2-AR involves hydrogen bonding, the nitrated agonist will
be unable to participate in this type of interaction. This may
be another possible reason the affinity of the nitrated agonist
for the ␤2-ARs decreased (Fig. 10A) in addition to its struc-
tural incompatibility to the receptor.
Fig. 11. Proposed mechanisms of the enzymatic nitration of the aromatic
moiety of salbutamol. Salbutamol (1) is oxidized to the phenoxyl-type
radical (1a), which rearranges to a resonance structure 1b (only one of
several resonance forms is shown), in which the unpaired electron is
localized in the aromatic ring. The concomitantly generated NO•2 radicals
add to 1b to form nitrated salbutamol, which rearranges to 2 (m/z
285.14). In an alternative pathway, the radical 1a undergoes intramolec-
ular hydrogen atom transfer from the –CH2OH moiety at C3 to form a
methoxyl-type radical 1c, which after elimination formaldehyde (H2CO),
produces the aromatic radical 1d, which reacts with NO•2 to form the
nitrophenol 3 (m/z ϭ 255.13). In general, the mechanisms of nitration
catalyzed by EPO and LPO systems may be similar to that shown in the
scheme for MPO. R ϭ CH(OH)CH2NHC(CH3)3.
We found that AA and NaSCN at physiological concentra-
tions inhibit nitration of salbutamol. However, the effect of
AA was transient because of its rapid consumption. It is
expected that in inflamed airways concentrations of these
compounds will be significantly decreased, even in the ab-
sence of the drug, so their interference with nitration reac-
tion of this nitrophenol was unexpected as it required the tions should be minimal. However, one can speculate that
side chain at C3 of the aromatic ring, –CH2OH, to be elimi- administration of ascorbate could be a potential mean to
nated. We verified that the putative phenolic precursor of 3, control or even prevent the oxidative inactivation of the drug
a salbutamol analog, in which the –CH2OH moiety was re- in vivo.
placed with –H, (m/z 210), was not present as an impurity in
Pharmacological agents methimazole and dapsone were
salbutamol samples. This indicates that the corresponding also effective in inhibiting the reaction. Methimazole, a sui-
radical (1d in Fig. 11) had to be produced in situ, during cidal inhibitor of thyroid peroxidase and LPO (McGirr et al.,
enzymatic oxidation of salbutamol itself. In the proposed 1990), is used in the treatment of hyperthyroid conditions.
mechanism (Fig. 11), the initially generated phenoxyl radi- Salbutamol nitration was also inhibited by dapsone, an anti-
cal, 1a, undergoes intramolecular hydrogen atom transfer inflammatory and antileprotic drug that has been reported to
from the adjacent –CH2OH moiety to generate the aryl- have corticosteroid-sparing activity in severe asthma (Dewey
methoxyl-type radical 1c. After formaldehyde elimination, et al., 2002). Although both drugs displayed inhibitory activ-
the remaining aromatic radical 1d adds NO•2 to form 3. It is ity, there were differences between methimazole- and dap-
noteworthy that results of our analyses of a sample prepared sone-dependent inhibition of nitration that depended on the
from pooled asthmatic EBCs suggests that 3 could be gener- enzymatic systems used. Both of these agents efficiently and
ated in the asthmatic airway (Fig. 5). It is noteworthy that if permanently inhibited nitration catalyzed by LPO, presum-
this transformation does indeed occur, it should be a matter ably by poisoning the enzyme. However, in contrast to LPO,
of concern because of the cytotoxic and carcinogenic nature of the effect of dapsone on the reaction catalyzed by MPO and
formaldehyde. Further studies are needed to confirm un- EPO was less pronounced, even at high concentrations of the
equivocally the oxidative degradation of salbutamol and re- inhibitor (Fig. 9). Nitration by EPO systems seemed to be
lease of formaldehyde in vivo.
more sensitive to dapsone than nitration catalyzed by MPO.
Nitration supported by EPO most likely proceeds via a This is in agreement with prior reports showing that dapsone
similar mechanism. We note that nitration catalyzed by LPO affected EPO to a larger extent than MPO (Bozeman et al.,
may differ from that driven by MPO or EPO, because, in 1992).
contrast to these two peroxidases, the compound I of LPO
We emphasize that there are multiple targets in the air-
probably does not oxidize NOϪ2 to NO2• but converts NOϪ2 ways, which may undergo enzymatic modification during
directly to nitrate (NOϪ3 ) in one step (Bru¨ck et al., 2001). The severe exacerbation of the disease. This includes tyrosine
NO•2 radical presumably is generated by oxidation of NOϪ2 by residues in proteins (Gaston et al., 1994; MacPherson et al.,
LPO compound II (Bru¨ck et al., 2001), the latter could be 2001; Ramesh et al., 2001; Andreadis et al., 2003) and com-
produced by the reduction of LPO compound I by salbutamol. ponents of ␤2-AR itself, such as cysteine and tyrosine present
We have previously reported that salbutamol undergoes ox- in the extracellular domains of the receptor (Lewis et al.,
idation by LPO/H2O2 alone, but the process is not very effi- 2005). In addition, ascorbic acid and glutathione, the natural
cient, because only a small portion of the drug was oxidized antioxidants present in the airway lining fluid undergo rapid
(Reszka et al., 2009). It was concluded that this was caused depletion, which may facilitate oxidative injury. Although
by the slow interaction of the drug with LPO compound II, the present study focused on the enzymatic transformation of
which accumulates in the system. In this situation, addition salbutamol, the clinically most important bronchodilator, as
of NOϪ2 , which readily reacts with LPO compound II and an additional factor that potentially may play a role in the

Airway Peroxidases Catalyze Nitration of Salbutamol
449
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therapeutic control of asthma, the presented data do not
preclude the possibility that the diminished bronchorelax-
ation activity of the agonist may also be caused by oxidative
modification of these other targets.
In summary, we have demonstrated that airway peroxi-
dases catalyze nitration of the ␤2-agonist salbutamol. The
transformed salbutamol binds to ␤2-AR on smooth muscle
cells with diminished affinity and reduced capacity to stim-
ulate cAMP synthesis. If nitration of the agonist is responsi-
ble for its diminished bronchorelaxation effects during a se-
vere asthma attack, results of this study suggest that
antioxidants or peroxidase inhibitors may be useful in main-
taining the drug’s therapeutic activity. Results of our prelim-
inary studies show that related phenolic ␤2-agonists (fenot-
erol, formoterol, salmeterol, and terbutaline) undergo similar
oxidation and nitration reactions. Further studies are needed
to verify formation of nitrated salbutamol and related ␤2-
agonists in the airways of asthmatic patients and their role
in bronchodilator resistance and treatment failure.
Authorship Contributions
Monzani E, Roncone R, Galliano M, Koppenol WH, and Casella L (2004) Mechanistic
insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite
and hydrogen peroxide. Eur J Biochem 271:895–906.
Participated in research design: Reszka, Sallans, Macha, McGraw,
Butsch Kovacic, and Britigan.
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catalase at 25°C (with molar extinction coefficients of H2O2 solutions in the UV).
Anal Biochem 49:474–478.
Ramesh G, Jindal SK, Ganguly NK, and Dhawan V (2001) Increased nitric oxide
production by neutrophils in bronchial asthma. Eur Respir J 17:868–871.
Rapoport M, Hancock CK, and Meyers EA (1961) The correlation of the electronic
spectra and acidity of 4-substituted 2-nitrophenols with substituent constants.
J Am Chem Soc 83:3489–3494.
Conducted experiments: Reszka, Sallans, Macha, and Brown.
Contributed new reagents or analytic tools: Butsch Kovacic.
Performed data analysis: Reszka, Sallans, Macha, McGraw, But-
sch Kovacic, and Britigan.
Wrote or contributed to the writing of the manuscript: Reszka,
Sallans, Macha, McGraw, Butsch Kovacic, and Britigan.
Other: Reszka, McGraw, and Britigan acquired funding for the
research.
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Address correspondence to: Dr. Krzysztof J. Reszka, Department of Internal
Medicine, University of Cincinnati College of Medicine, P.O. Box 670557, 231
Albert Sabin Way, Cincinnati, OH 45267-0557. E-mail: reszkakj@ucmail.uc.edu