Nitrating reactive nitric oxygen species transform acetaminophen 3-nitroacetaminophen

Article abstract of DOI:10.1021/tx000115g

Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [¹⁴C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO₂^-, NO₂Cl, and ONOO^- or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO^-, spermine NONO, Sin-1, or H₂O₂. HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO₂^-. ONOO^--derived 3NAP was identified by ¹H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [¹⁴C]APAP and stimulated with β-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO₂^-. Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO₂^-. Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO₂^-. Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.

Full text of DOI:10.1021/tx000115g

Chem. Res. Toxicol. 2000, 13, 891-899
891
Nitr a tin g Rea ctive Nitr ic Oxygen Sp ecies Tr a n sfor m
Aceta m in op h en to 3-Nitr oa ceta m in op h en
Vijaya M. Lakshmi,† Fong Fu Hsu,‡ Bernard B. Davis,† and Terry V. Zenser*,†,§
VA Medical Center, Division of Geriatric Medicine, and Department of Biochemistry,
St. Louis University School of Medicine, St. Louis, Missouri 63125-4199, and Department of
Medicine, Washington University, St. Louis, Missouri 63130
Received May 24, 2000
Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the
inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult.
Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated
by HPLC. When [14C]APAP was incubated at pH 7.4, the same new product (3NAP) was
produced by at least three separate pathways represented by the following conditions:
myeloperoxidase oxidation of NO2-, NO2Cl, and ONOO- or Sin-1. Diethylamine NONO and
spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at
pH 7.4 with ONOO-, spermine NONO, Sin-1, or H2O2. HOCl transformed 3NAP, which was
prevented by APAP, ascorbic acid, taurine, or NO2-. ONOO--derived 3NAP was identified by
1H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass
was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated
with [14C]APAP and stimulated with â-phorbol 12-myristate 13-acetate produced 3NAP in the
presence of NO2-. Neutrophil 3NAP formation was verified by mass spectrometry and was
consistent with myeloperoxidase oxidation of NO2-. Spermine NONO supported 3NAP
formation by stimulated cells in the absence of NO2-. Results demonstrate that 3NAP is a
product of nitrating RNOS generated by at least three separate pathways and may be a
biomarker for nitrating mediators of inflammation.
Effects of RNOS can include nitrosation, nitration, and
oxidation (7). Nitrosation of primary amines results in
the reaction of diazonium ions with water and subsequent
deamination. Deamination of DNA by RNOS can lead to
mispairing, depurination, and strand breakage, contrib-
uting to mutagenesis (5, 10). ONOO- nitrates certain
phenolic compounds such as tyrosine to yield 3-nitro-
tyrosine. A considerable amount of evidence has ac-
cumulated suggesting that nitration of LDL, formation
of 3-nitrotyrosine, is an important event in atherogenesis
(11, 12). Nitration of certain tyrosine residues may exert
dramatic physiologic effects by altering kinase reactions
and subsequent signaling pathways (7).
In tr od u ction
Nitrating reactive nitric oxygen species (RNOS)1 are
thought to contribute to the deleterious effects of inflam-
mation in a variety of disease states (1-5). Upregulation
of the inducible isoform of nitric oxide synthase (iNOS)
during inflammation can produce high levels of nitric
oxide (NO) (6). RNOS can be derived from a series of
complex reactions involving NO with either oxygen or
superoxide (7). RNOS include NO, peroxynitrite anion
(ONOO-), dinitrogen trioxide (N2O3), and nitrogen diox-
ide radical (NO2 ). Nitrite (NO2-) is a relatively stable
•
NO product and a major end product and is used in
combination with nitrate (NO3-) as a measure of the
At least three pathways generate RNOS capable of
nitrating tyrosine. As mentioned above, intermediates
derived from ONOO- produce 3-nitrotyrosine (13). The
same product can be derived from human polymorpho-
nuclear neutrophils by a myeloperoxidase-catalyzed H2O2-
-
extent of iNOS upregulation. Levels of NO2 in healthy
humans range from 0.5-3.6 µM in plasma (8) to 30-210
µM in saliva (9). Concentrations of NO2 as high as 0.1
mM are found in respiratory fluid and gastric juice as
well as plasma during inflammation (9).
-
-
•
dependent oxidation of NO2 to NO2 -like radicals (14).
Nitryl chloride (NO2Cl) also reacts with tyrosine to form
3-nitrotyrosine. NO2Cl is formed by the reaction of
hypochlorous acid (HOCl) with NO2- (15). HOCl is formed
by myeloperoxidase-mediated oxidation of chloride anion.
3-Nitrotyrosine is used as a marker for RNOS.
* To whom correspondence should be addressed: VA Medical Center
(GRECC/11G-J B), St. Louis, MO 63125-4199. Phone: (314) 894-6510.
Fax: (314) 894-6614. E-mail: zensertv@slu.edu.
† VA Medical Center and Division of Geriatric Medicine, St. Louis
University School of Medicine.
‡ Washington University.
§ Department of Biochemistry, St. Louis University School of
Medicine.
Acetaminophen (APAP), a phenolic compound, is a
popular analgesic antipyretic drug tolerated well at the
recommended oral adult dose of 300-1000 mg for adults
and children 12 years of age and older (16). Following a
therapeutic dose, peak plasma concentrations occur at
0.5-2 h and range up to 0.2 mM APAP. APAP is rapidly
and completely absorbed from the gastrointestinal tract
1 Abbreviations: RNOS, reactive nitric oxygen species; iNOS, induc-
ible nitric oxide synthase; NO, nitric oxide; ONOO-, peroxynitrite
anion; NO2•, nitrogen dioxide radical; APAP, acetaminophen; DETA-
PAC, diethylenetriaminepentaacetic acid; PMA, â-phorbol 12-myristate
13-acetate; DEA-NONO, diethylamine NONOate; Sin-1, 3-morpho-
linosydnonimine; ESI, electrospray ionization; CAD, collisionally
activated dissociation; EI, electron impact.
10.1021/tx000115g CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/10/2000

892 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Lakshmi et al.
ONOO-, and 30 min for other conditions. Blank values were
obtained in the absence of either RNOS generating agent or
H2O2. The reaction was stopped by adding 0.1 mL of 10 mM
ascorbic acid and/or 10 µg/mL catalase, and the mixture was
placed on ice. Formation of 3NAP was assessed by HPLC as
described below.
with 90-100% of it recovered in urine within 24 h. A
small amount of APAP is excreted unchanged in urine,
and about 90% is excreted after conjugation in liver as
an O-glucuronide or sulfate. Bioactivation by cytochrome
P450 contributes to hepatotoxicity. APAP is also a
substrate for peroxidases, which catalyze one- and/or two-
electron oxidation (17-19).
Determination of the role of RNOS in disease processes
depends on their identification and measurement in
lesions of tissues or in biological fluids from individuals
affected by or at risk for a specific disease. The high
reactivity and short half-life of RNOS make their analysis
difficult. An indirect measure of RNOS can be achieved
P r ep a r a tion of Hu m a n P olym or p h on u clea r Neu tr o-
p h ils. Human blood was mixed with EDTA (0.2% final concen-
tration) and immediately layered over an equal volume of
neutrophil isolation media from Robins Scientific Corp. (Sunny-
vale, CA). Neutrophils were isolated by centrifugation using the
manufacturer’s specifications. Red blood cell contamination was
eliminated by hypotonic lysis at 4 °C. Cells were resuspended
in Hank’s balanced salt solution at a density of 10 × 106 cells/
mL. The Human Studies Committee approved the use of human
polymorphonuclear neutrophils, and informed consent was
obtained from participants. To assess PMA responsiveness of
different preparations of cells, superoxide production was as-
sessed (20). Superoxide-specific reduction of cytochrome c was
assessed spectrophotometrically (ꢀ550 ) 21.1 mM-1 cm-1) and
was inhibited by superoxide dismutase (10 µg/mL). Values
observed with cells in the absence of PMA were considered as
blanks.
by assessing products formed by their reaction with a
-
particular target molecule. 3-Nitrotyrosine and NO2
/
NO3- have been used as biomarkers to assess increased
levels of RNOS. However, NO2-/NO3 determinations
-
reveal little about the RNOS and can be influenced by
diet (9). While NO has a very short half-life, 3-nitro-
tyrosine levels in tissue likely reflect an estimate of
-
steady-state values over time and protein turnover. NO2
/
-
NO3 determinations utilize body fluids, while 3-nitro-
tyrosine measurements employ protein hydrolysates from
tissue or serum. Biomarkers are useful in interpreting
the effects of treatment variables such as pharmacologi-
cal intervention, lifestyle habits, nutritional deficits, and
nutritional supplements. This study was designed to
assess the in vitro effects of different RNOS on APAP, a
popular over-the-counter drug, and apply these results
to intact cells with the ultimate goal of finding biomar-
kers for nitrating RNOS that can be utilized in humans.
The concentrations of NO2- and APAP that are used are
those expected during an inflammatory response or after
ingestion of the recommended dose of APAP (9, 16).
Meta bolism of AP AP by Neu tr op h ils. Neutrophils (2 ×
106 cells in 0.3 mL) were incubated in 12 mm × 75 mm
polypropylene tubes at 37 °C for 30 min. Cells were incubated
with 0.02 mM [14C]APAP in the presence or absence of 30 ng/
mL PMA. Blank values were obtained in the absence of cells.
The reaction was stopped by placing the mixture on ice, the
mixture sonicated three times for 15 s, and 0.3 mL of methanol
added. The supernatant was evaporated, and the residue was
dissolved in 0.1 mL of methanol and analyzed by HPLC.
HP LC An a lysis of Meta bolites. Metabolites were assessed
using a Beckman HPLC system with System Gold software,
which consisted of a 5 µm, 4.6 mm × 150 mm C-18 ultrasphere
column attached to a guard column. The mobile phase contained
20 mM ammonium acetate buffer (pH 7.0) in 5% acetonitrile
from 0 to 2 min, 5 to 10% from 2 to 10 min, 10 to 50% from 20
to 25 min, and 50 to 5% from 35 to 45 min, at a flow rate of 1
mL/min. Using these solvent conditions, the 3NAP standard
eluted at 20.1 min. The radioactivity in HPLC eluents was
measured using a FLO-ONE radioactive flow detector. Data are
expressed as a percentage of total radioactivity or picomoles
recovered by HPLC. The amount of APAP that was metabolized
was determined by subtracting the percentage of APAP recov-
ered (unmetabolized) from 98% (purity of APAP).
Exp er im en ta l P r oced u r es
Ma ter ia ls. [ring-U-14C]APAP (46 mCi/mmol) was purchased
from American Radiolabeled Chemicals, Inc. (St. Louis, MO).
APAP, NaNO2, NaOCl, H2O2, cytochrome c (horse heart),
superoxide dismutase (bovine erythrocytes), catalase (bovine
liver), ascorbic acid, diethylenetriaminepentaacetic acid (DE-
TAPAC), NaCN, and â-phorbol 12-myristate 13-acetate (PMA)
were purchased from Sigma Chemical Co. (St. Louis, MO).
Diethylamine NONOate (DEA-NONO), spermine NONOate,
3-morpholinosydnonimine (Sin-1), and myeloperoxidase from
human polymorphonuclear leukocytes (180-220 units/mg of
protein) were purchased from Calbiochem (San Diego, CA).
Ma ss Sp ectr a l An a lysis. A Finnigan SSQ-7000 GC/MS
system (interfaced with a Varian 3300 gas chromatograph),
controlled by Finnigan ICIS software operated on a DEC alpha
station, was used for the analysis. The machine was tuned by
auto-tune mode and further tuned manually with PFTFA as
the calibrant. The TMS derivatives of both standard and
biological samples of 3NAP were separated by a J & W DB-1
capillary column (15 m × 0.25 mm, 0.25 µm film thickness)
operated in a splitless injection mode. The injector, transfer line,
and source temperatures were set at 250, 250, and 200 °C,
respectively. The GC oven was programmed at 100 °C for 1 min,
increased to 220 °C at 12 °C/min, and kept at 220 °C for 1 min.
A standard electron energy at 70 eV (300 µA) was used. The
multiplier voltage was set at 2 kV (the detector is operated with
a conversion dynode at 15 kV).
-
Alkaline solutions of ONOO- were prepared from acidified NO2
and H2O2, and quantitated spectrophotometrically (ꢀ302 ) 1.67
mM-1 cm-1) as described previously (13). Stock solutions were
kept at -70 °C. The 3NAP standard was chemically synthesized
from p-aminophenol. The nitrated product was prepared by
mixing p-aminophenol with concentrated HNO3 and H2SO4 at
0-5 °C for 1 h. The purified product was added to acetic
anhydride and heated under reflux for 2 h. Silica gel column-
purified 3NAP was identified by NMR/MS analysis. Ultima-Flo
AP was purchased from Packard Instruments (Meriden, CT).
Rea ction of AP AP w ith RNOS. [14C]APAP (0.06 mM) was
incubated in 100 mM potassium phosphate buffer (pH 7.4),
containing 0.1 mM DETAPAC in a total volume of 0.1 mL at
37 °C. For incubation with myeloperoxidase, 1 µg/mL peroxidase
was added in the presence or absence of the indicated concen-
tration of NO2-, and the reaction was started by addition of 0.05
mM H2O2. Incubations with DEA-NONO, spermine-NONO,
ONOO-, Sin-1, or HOCl were started immediately following
their addition. The pH was checked at the conclusion of these
incubations and did not change by more than ( 0.1 pH unit.
Incubation times were 10 min for myeloperoxidase, 1 min for
Electrospray ionization (ESI) MS analyses were performed
on a Finnigan (San J ose, CA) TSQ-7000 triple-stage quadrupole
spectrometer equipped with a Finnigan ESI source and con-
trolled by Finnigan ICIS software operated on a DEC alpha
workstation. Samples were loop injected onto the ESI source
with a Harvard syringe pump at a flow rate of 5 µL/min. The
electrospray needle and the skimmer were at ground potential,
and the electrospray chamber and the entrance of the glass
capillary were at 4.4 kV. The heated capillary temperature was
200 °C. For collisionally activated dissociation (CAD) tandem
mass spectra, the collision gas was argon (2.2-2.5 mTorr), and

3-Nitroacetaminophen Formation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 893
hundred twenty-eight transients were signal averaged with a
recycle delay of 5 s and a 45° tip. The time domain data were
processed on a SUN workstation using VNMR software. A line-
broadening parameter of 0.5 Hz was used for the Fourier
transformation. Chemical shifts were referenced to TMS at 0.0
ppm.
Resu lts
Metabolism of 0.06 mM [14C]APAP by myeloperoxidase
was assessed in the presence of 0.05 mM H2O2. Under
the conditions of this incubation, approximately 63% of
APAP was metabolized (Figure 1, top panel). Previous
studies have identified polymers as the major products
of APAP peroxidatic metabolism and have characterized
these products in detail (21, 22). The products eluting at
15 (dimer) and 24.8 min (trimer) represented 14 and 19%
of the total radioactivity recovered by HPLC, respectively.
The masses of these myeloperoxidase products were
determined by ESI/MS and the products not character-
-
ized further. Addition of 0.1 mM NO2 resulted in 73%
metabolism of APAP and the formation of a new peak at
20.1 min (Figure 1, bottom panel). The new peak was
labeled 3NAP and represented 7% of the total radioactiv-
ity recovered by HPLC. Dimer and trimer products were
also observed in the presence of NO2-. The results
suggest formation of the new product may involve RNOS.
A variety of conditions, which generate RNOS, were
evaluated for conversion of APAP to 3NAP (Table 1).
When 0.06 mM APAP was incubated with myeloperoxi-
-
dase in the presence of 0.05 mM H2O2 and 0.1 mM NO2
,
245 ( 21 pmol of 3NAP was formed. This reaction is
•
thought to generate NO2 as a RNOS (14). Myeloperoxi-
dase-mediated metabolism was prevented by 1 mM
NaCN, a peroxidase inhibitor. In the presence of physi-
ologic concentrations of chloride (100 mM), a myeloper-
oxidase substrate, 166 ( 23 pmol of 3NAP was observed.
Taurine (0.5 mM), an HOCl scavenger, did not alter the
metabolism in the absence or presence of chloride. DEA-
NONO (0.2 mM) and spermine-NONO (1 mM) are both
NO-donor molecules and are thought to generate a
variety of RNOS, including N2O3 (23). Neither compound
was effective in producing detectable levels of 3NAP. NO2-
F igu r e 1. HPLC analysis of myeloperoxidase metabolism of
0.06 mM [14C]APAP in the absence and presence of 0.1 mM
NaNO2. Reactions were started by addition of 0.05 mM H2O2.
A2 and A3 refer to metabolites with masses corresponding to an
APAP dimer and trimer, respectively.
the collision energy was set at 22 eV. Product ion spectra were
acquired in the profile mode at a scan rate of one scan per 3 s.
NMR An a lysis. Samples were prepared in 100% DMSO-d6
(Aldrich gold label) under a dry nitrogen purge. About 30 µg of
the sample was dissolved in 1 mL of DMSO and the mixture
transferred into high-quality 5 mm tubes which were capped
under nitrogen. NMR spectra were acquired on a Varian Inova-
500 instrument with a proton resonance frequency of 499.97
MHz with an INVERSE probe (1H 90 pulse of 9 µs). One
-
Cl, generated by incubation of NO2 with HOCl (15),
elicited a small amount of 3NAP (43 ( 4 pmol). A bolus
addition of ONOO- was effective in eliciting formation
of 3NAP. The amount of product observed with 0.1 mM
ONOO- (467 ( 73 pmol) was more than that observed
with myeloperoxidase (245 ( 21 pmol). Preincubation of
ONOO- in buffer for 10 min prior to APAP addition failed
Ta ble 1. F or m a tion of 3NAP by Differ en t Rea ctive Nitr ic Oxygen Sp eciesa
conditions
3NAP formed (pmol)
RNOS
NO2
myeloperoxidase, 0.05 mM H2O2, and 0.1 mM NaNO2
with 1 mM NaCN
245 ( 21
0
with 0.5 mM taurine
with 100 mM NaCl
with 100 mM NaCl and 0.5 mM taurine
0.2 mM DEA-NONO
208 ( 22
166 ( 23
146 ( 19
0
NO, N2O3
NO, N2O3
NO2Cl
1 mM spermine-NONO
0
0.1 mM NaNO2 and 0.1 mM HOCl
43 ( 4
469 ( 73
0
0.1 mM ONOO-
ONOO-
with a 10 min preincubation before APAP addition
2 mM Sin-1
59 ( 7
ONOO-
a
Incubations contained 0.06 mM [14C]APAP, 100 mM potassium phosphate buffer (pH 7.4), and 0.1 mM DETAPAC in 0.1 mL and
were incubated at 37 °C. Incubation times were 10 min for myeloperoxidase, 1 min for ONOO-, and 30 min for other conditions. 3NAP
formation was analyzed by HPLC, and values represent means ( SEM. Some RNOS thought to be generated by each condition are
indicated.

894 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Lakshmi et al.
Ta ble 2. Sta bility of [14C]3NAP a
conditions
phosphate buffer
[
14C]3NAP remaining (nmol)
pH 5.0
pH 7.4
pH 9.0
10 ( 0
10 ( 0
10 ( 0
0.1 mM HOCl
with 0.5 mM APAP
with 0.5 mM ascorbic acid
with 0.25 mM taurine
6.3 ( 0.1
9.6 ( 0.1
10 ( 0
10 ( 0
-
with 0.1 mM NaNO2
9.6 ( 0
9.8 ( 0.1
9.7 ( 0.1
9 ( 0.1
10 ( 0
0.1 mM ONOO-
1 mM spermine-NONO
2 mM Sin-1
0.1 mM H2O2
a
[
14C]3NAP (0.1 mM, 10 nmol) was incubated in 100 mM
phosphate buffer at the indicated pHs for 18 h (0.1 mL). Additional
incubations were carried out in 100 mM phosphate buffer (pH 7.4)
containing 0.1 mM DETAPAC for 5 min at 37 °C. Reaction
mixtures containing HOCl were stopped by addition of 1 mM
methionine. Samples were analyzed by HPLC, and values repre-
sent means ( SEM.
Ta ble 3. 1H NMR Sp ectr a l P a r a m eter s for
Aceta m in op h en a n d 3-Nitr oa ceta m in op h en
F igu r e 2. EI mass spectra of the 3-nitroacetaminophen-TMS2
standard (A) and the biological product isolated from PMA-
stimulated human polymorphonuclear neutrophil incubations
(B). ESI tandem mass spectrum of the 3-nitroacetaminophen
standard (C). Scheme 1 illustrates the fragmentation pattern
of the corresponding EI or ESI mass spectra.
the new product are given in Table 3. The D2O-exchange-
able amide NH proton at position 1 of APAP (9.133 ppm)
was detected in the new product (10.065 ppm). The CH3
protons of APAP (2.000 ppm) at position 7 were also
detected in the new product (2.030 ppm). A single D2O-
exchangeable phenolic proton at position 4 was present
in both APAP (9.650 ppm) and the 20.1 min peak (10.656
ppm). While both protons meta to OH in position 4 are
present along with the ortho proton in position 5, the new
product is missing the ortho proton in position 3. Thus,
to demonstrate product formation. Sin-1 elicits a sus-
tained release of ONOO- and was also evaluated (24).
At 2 mM Sin-1, a small amount of 3NAP was produced
(59 ( 7 pmol).
The stability of 3NAP was assessed to determine if this
could explain its low yield under certain conditions used
above. An equal molar concentration of HOCl (0.1 mM)
resulted in an ∼40% loss of [14C]3NAP (Table 2). A new
product, which eluted in front of 3NAP, was observed,
but not characterized further. Loss of 3NAP was pre-
vented by 0.5 mM APAP, 0.5 mM ascorbic acid, or 0.25
mM taurine. Inclusion of an equal molar concentration
1
the H NMR spectrum of the new product is consistent
with that expected for 3-nitroacetaminophen or 3-nitro-
N-acetyl-p-aminophenol (3NAP).
The ONOO--derived product was analyzed further by
MS (Figure 2). Using electron impact (EI) MS, the
trimethylsilyl (TMS) derivative of the synthetic 3NAP
yields the M+• ion at m/z 340, corresponding to a di-TMS
molecule (3-nitroacetaminophen-TMS2) (Figure 2A). The
derivative gives a prominent ion at m/z 325 by loss of
CH3-. Further losses of TMSOH and TMSH from m/z 325
give rise to m/z 235 and 251 ions, respectively. The ion
-
of NO2 to generate NO2Cl also prevented HOCl loss of
3NAP. ONOO- (0.1 mM) did not cause depletion of [14C]-
3NAP. [14C]3NAP was also stable in the presence of 2
mM Sin-1, 1 mM spermine-NONO, and 0.1 mM H2O2.
To determine its pH stability in 100 mM potassium
phosphate buffer, 3NAP was incubated at pH 5, 7.4, and
9 for 18 h. No loss of 3NAP was observed at any pH.
Except for HOCl, 3NAP was stable under the conditions
that were tested. Thus, the stability of 3NAP did not
influence its synthesis reported in Table 1.
-
corresponding to an NO2 loss is observed at m/z 294.
The postulated fragmentation pathways for ion formation
are illustrated in Scheme 1. These results are consistent
with the synthetic product derived from the reaction of
ONOO- with APAP being 3-nitroacetaminophen. This
structural assignment is further supported by the ESI
mass spectrum (Figure 2C). In negative ion mode, the
synthetic 3NAP yields a prominent deprotonated molec-
ular ion (M - H)- at m/z 195, which gives abundant
1H NMR analyses were used to determine the structure
of the new product derived from the reaction of ONOO-
1
with APAP. H NMR spectral parameters for APAP and

3-Nitroacetaminophen Formation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 895
Sch em e 1. F r a gm en ta tion P a th w a ys Obser ved for (A) Nega tive Ion ESI Ta n d em Ma ss Sp ectr om etr y of
3-Nitr oa ceta m in op h en a n d (B) th e EI Ma ss Sp ectr u m of th e 3-Nitr oa ceta m in op h en -TMS2 Der iva tive
fragment ions upon CAD. The tandem mass spectrum
yields fragment ions at m/z 150 and 165 corresponding
to (M - NO2)- and (M - H - NO)-, respectively. Ions at
m/z 178 and 152 arise from loss of OH- and CH3CO-,
respectively. Further loss of NO from m/z 152 gives an
ion at m/z 122. The ion at m/z 46, corresponding to an
NO2 ion, was also observed. These analyses further
confirmed that the structure of the new product is
3-nitroacetaminophen (3NAP).
phil metabolism of [14C]APAP was assessed with 30 ng/
-
mL PMA in the presence and absence of NO2 (Figure
-
3). Metabolism of APAP in the absence of NO2 was
similar to that observed by myeloperoxidase in Figure
1A with metabolite peaks at 15.1, 24.6, and 25.8 min
-
(Figure 3, top panel). In the presence of 1 mM NO2
,
-
3NAP formation was observed (Figure 3, bottom panel).
This metabolite represented about 13% of the total
radioactivity recovered by HPLC. In separate experi-
ments, the material eluting at 19-21 min was collected,
a TMS derivative prepared, and analysis performed by
To assess the effect of RNOS generated by intact cells
on 3NAP formation, human polymorphonuclear neutro-

896 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Lakshmi et al.
Ta ble 4. F or m a tion of 3NAP by Hu m a n
P olym or p h on u clea r Neu tr op h ilsa
3NAP
formed
(pmol)
conditions
PMNs and APAP
with PMA
0
0
-
with 1 mM NO2
with 1 mM NO2- and PMA
520 ( 31
14 ( 14
89 ( 27
480 ( 23
with 1 mM NO2-, PMA, and NaCN
with 1 mM NO2-, PMA, and catalase
with 1 mM NO2-, PMA, and taurine
with 1 mM NO2-, PMA, and superoxide dismutase 558 ( 40
with 0.1 mM NO2- and PMA
89 ( 20
104 ( 6
63 ( 6
8 ( 5
77 ( 17
with 0.1 mM NO2-, PMA, and taurine
with PMA and spermine-NONO
with PMA, spermine-NONO, and catalase
with PMA, spermine-NONO, and taurine
a
Human polymorphonuclear neutrophils (PMNs; 2 × 106 cells)
were incubated with 0.02 mM [14C]APAP in 0.3 mL of HBSS at
37 °C for 30 min. The following test agents were present at the
indicated concentrations: 30 ng/mL PMA, 0.1 or 1 mM NO2-, 1
mM NaCN, 1 mM taurine, 330 munits/mL superoxide dismutase,
10 µg/mL catalase, and 0.25 mM spermine-NONO. When present,
PMA and spermine-NONO were added 5 and 6 min, respectively,
after the start of the 37 °C incubation. Formation of 3NAP was
assessed by HPLC, and values represent means ( SEM.
Sch em e 2. Th r ee P a th w a ys for 3NAP F or m a tion a
F igu r e 3. Illustrated is the HPLC elution profile of incubations
of human polymorphonuclear neutrophils with 0.02 mM [14C]-
APAP in the absence and presence of 1 mM NaNO2. PMA (30
ng/mL) was added 5 min after the incubations were started.
a
Mediators of inflammation (endotoxin and cytokines) induce
iNOS, increasing the extent of NO synthesis from L-arginine
(L-Arg). NO reacts with superoxide (O2•-) to form peroxynitrite
anion (ONOO-) which can form several RNOS intermediates (i.e.,
EI/MS. The mass spectrum of the metabolite from PMA-
stimulated neutrophils (Figure 2B) is identical to that
of ONOO--derived 3NAP (Figure 2A). Thus, the metabo-
lite isolated from neutrophils is 3-nitroacetaminophen.
In addition, the material eluting at 19-21 min was
collected and rechromatographed on the same gradient
with HPLC buffer at pH 5.5, instead of pH 7.0. The
rechromatographed sample eluted as a single peak at
26.2 min with a purity of >95%. This decrease in the pH
of the HPLC buffer increased the elution time of 3NAP
by 6 min, consistent with the lower pKa of phenols with
an ortho-substituted nitro group (25).
•
NO2 ), some of which can nitrate acetaminophen (APAP) to form
-
3NAP (pathway 1). NO2 is the major end product of NO and
ONOO- metabolism. NADPH oxidase reduces molecular oxygen
to O2•-, which dismutates to H2O2. Myeloperoxidase (MPO)
•
oxidizes NO2- to a RNOS, NO2 -like radicals, that nitrates APAP
(pathway 2). HOCl oxidizes NO2- to NO2-Cl (nitryl chloride) which
•
reacts with APAP in forming 3NAP (pathway 3). [NO2 ] denotes
known and unknown intermediates. Inhibitors are available to
prevent 3NAP formation by each pathway.
Discu ssion
This is the first paper to demonstrate nitration of
APAP to form 3NAP by RNOS associated with the
inflammatory response (Table 1). 3NAP was formed by
at least three separate pathways (Scheme 2) represented
by the following conditions: myeloperoxidase oxidation
of NO2-, NO2Cl, and ONOO-. With myeloperoxidase, a
chloride concentration (100 mM) 1000-fold higher than
that of NO2- only reduced the extent of 3NAP formation
by 30%. Previous studies have demonstrated that oxida-
Further assessment of the formation of 3NAP by
neutrophils indicated that it was not detected in the
-
absence of either PMA or NO2 (Table 4). 3NAP forma-
-
tion in the presence of both PMA and 1 mM NO2 (520
( 31 pmol) was completely inhibited by 1 mM NaCN and
reduced by more than 80% with catalase. However,
neither taurine (1 mM) nor superoxide dismutase altered
3NAP formation. Formation of 3NAP could also be
-
-
detected with 0.1 mM NO2 (89 ( 20 pmol) which was
tion of NO2 is preferred over chloride by myeloperoxi-
dase (14). ONOO- was much more effective than either
also not altered by taurine. Spermine-NONO (0.25 mM)
initiated 3NAP formation (63 ( 6 pmol) by PMA-
stimulated neutrophils in the absence of NO2- which was
prevented by catalase, but not taurine.
-
myeloperoxidase/NO2 or NO2Cl in producing 3NAP.
Sin-1 produces a sustained release of NO and superoxide,
causing continuous formation of ONOO- (24). The amount

3-Nitroacetaminophen Formation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 897
of ONOO- produced by 2 mM Sin-1 should exceed the
0.1 mM ONOO- bolus addition (Table 1). However, 3NAP
produced by Sin-1 was only about 13% of that observed
with 0.1 mM ONOO-. As demonstrated with Sin-1
formation of 3-nitrotyrosine (26), secondary effects of
Sin-1 byproducts (i.e., NO and superoxide) with APAP
may cause a decreased level of 3NAP formation. NO
donors, DEA-NONO and spermine-NONO, elicit con-
trolled release of NO and did not produce 3NAP. Using
very different reaction conditions (ambient temperature
for a 12 h incubation), 1 mM spermine-NONO reacted
with 1 mM tyrosine to produce a 0.3% yield of 3-nitro-
tyrosine (27). A proportional yield of 3NAP during our
30 min incubation would not be detectable using our
HPLC method.
lecular hydrogen bonding between these two functional
groups. Ionization of the hydroxyl serves to extend the
conjugation of the aromatic ring and shifts the secondary
UV maximum of 3NAP from 370 nm at pH 3.5 to 430
nm at pH 9.5. This characteristic is shared with 3-nitro-
tyrosine in which the pKa of the phenol for tyrosine is
about 10, while that of 3-nitrotyrosine is shifted to about
6.8. 3-Nitrotyrosine exhibits UV absorption maxima at
pH 3.5 and 10 of 357 and 430 nm, respectively (25).
3NAP, formed by reaction of APAP with NO2- at acidic
pH, is a clinical assay for diagnosing APAP overdose in
serum samples (31). 3NAP has been termed 2-nitro-4-
acetamidophenol and characterized by its absorption
spectra (32, 33). Its first reported synthesis may have
been in 1891 (34). We can find no prior evidence for
structural identification of 3NAP, using techniques simi-
lar to those in this report.
The formation of 3NAP may occur by the same mech-
anism as formation of 3-nitrotyrosine. Peroxynitrite
nitrates tyrosine solely through a radical mechanism,
which involves reaction of tyrosine phenoxyl radical with
The lack or slight formation of 3NAP observed with
some reagents is not due to their reaction with 3NAP
(Table 2). A variety of biological antioxidants, such as
ascorbic acid, taurine, and glutathione, are oxidized by
HOCl (35). Thus, the inhibition of 3NAP transformation
•
-
NO2 (28). Oxidation of NO2 by myeloperoxidase also
•
yields a NO2 -like radical (14), and a similar radical could
be derived from NO2Cl (15). Dityrosine, a product of
myeloperoxidase tyrosine metabolism (29), is indicative
of a free radical-coupling reaction of two tyrosine phe-
noxyl radicals. Peroxidatic metabolism of APAP to phe-
noxyl radicals has been demonstrated (18), and the
identification of polymeric products in the myeloperoxi-
dase reaction mixtures is consistent with this conclusion.
APAP reactions with ONOO- or NO2Cl also involve a
dimer and trimer of APAP (not shown). The NO donor-
controlled release of NO results in the generation of
-
by APAP, ascorbic acid, taurine, and NO2 is consistent
with these reagents competing with 3NAP for reaction
with HOCl. It is likely that APAP and biological anti-
oxidants would protect 3NAP from loss by HOCl in vivo,
and that 3NAP would be recovered relatively unaltered
in biological samples. Recent studies have demonstrated
a similar stability profile for 3-nitrotyrosine (36).
With the recognition that 3-nitrotyrosine represents an
in vivo biomarker of nitrating RNOS-associated inflam-
matory conditions (1-5), a number of methods were
developed for its measurement (37). Protein-associated
3-nitrotyrosine can be analyzed by either immunohis-
tochemical or enzyme-linked immunosorbent assays.
These measurements allow tissue localization and, while
only semiquantitative, have demonstrated a role of RNOS
in tissue injury associated with many diseases. HPLC
methods can be more sensitive and specific. However,
HPLC analysis provides no structural information about
the analyte and can be confounded by coeluting but
structurally different molecules. A quantitative, selective,
sensitive stable isotope dilution GC/MS method has been
developed to measure the level of 3-nitrotyrosine (38).
The primary source of artifactual nitration with these
•
nitrosation species such as N2O3, not NO2 (23). Thus,
APAP phenoxyl radicals would be expected to react with
•
NO2 , forming 3NAP by a radical mechanism.
Human polymorphonuclear neutrophils were used as
a representative component of the inflammatory re-
sponse. These cells exhibit an oxidant burst following
stimulation with PMA. Membrane-associated NADPH
oxidase reduces molecular oxygen to superoxide anion
which dismutates to form H2O2. Myeloperoxidase cata-
lyzes the metabolism of H2O2 in the presence of reducing
-
cosubstrates, such as NO2- (14). In the presence of NO2
,
PMA-stimulated neutrophils metabolized APAP to 3NAP.
This product was not observed in the absence of either
PMA or NO2-. The lack of inhibition by taurine indicates
that NO2Cl is not mediating 3NAP formation (Scheme
2, pathway 3). In addition, the lack of superoxide dis-
mutase inhibition suggests ONOO--mediated APAP me-
tabolism is also not contributing to 3NAP formation
(pathway 1). Inhibition of 3NAP formation by catalase
and NaCN is consistent with myeloperoxidase-mediated
formation by pathway 2. Spermine-NONO, which did not
directly elicit 3NAP formation (Table 1), supported 3NAP
formation by PMA-stimulated cells in the absence of
NO2-. The extent of 3NAP formation with spermine-
NONO was reduced by catalase, but not taurine. This is
consistent with one-electron oxidation of NO to NO+ with
-
methods is due to tissue NO2-/NO3 (the level of which
is expected to be elevated in inflammation). This is a
special problem with methods requiring acid hydrolysis
of proteins to amino acids, because of the high efficiency
-
of acid-catalyzed NO2-/NO3 nitration. Antibody detec-
tion techniques using peroxidases or acidic conditions
also generate artifactual increases in the level of 3-ni-
trotyrosine. While 3-nitrotyrosine measurements are
taken with serum proteins, the use of atherosclerotic and
other tissues requires invasive techniques. 3-Nitrotyro-
sine levels in protein are likely to reflect its average
accumulation over time and protein turnover rate.
-
immediate hydration to NO2 (30), which is oxidized by
The current method for assessing 3NAP has been
modified, using a stable isotope (3-nitro-N-acetyl-p-
aminophenol-d3) to increase precision. The pharmacoki-
netics of APAP specify a relatively narrow window during
which it is available to react with nitrating RNOS be-
fore being excreted in urine (16). 3NAP measurements
are thought to complement and extend current measure-
ments of RNOS, using 3-nitrotyrosine. 3-Nitrotyrosine
neutrophil myeloperoxidase (Scheme 2, pathway 2).
Results suggest 3NAP formation during an inflammatory
response in vivo.
Direct chemical synthesis of this biological product was
achieved by nitration and acetylation of p-aminophenol.
A characteristic property of 3NAP shared by other
phenols with an ortho-substituted nitro group is intramo-

898 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
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Ack n ow led gm en t. We thank Cindee Rettke and
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work was supported by the Department of Veterans
Affairs (T.V.Z.) and National Cancer Institute Grant
CA72613 (T.V.Z.). MS was performed at the Mass Spec-
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