Aerobic oxidation of aminoacetone, a threonine catabolite: Iron catalysis and coupled iron release from ferritin

Article abstract of DOI:10.1021/tx015526r

Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cridu-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH₄⁺, and H₂O₂ has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH₄⁺. Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-^·OH adduct as a decomposition product from the DMPO-O₂^·- adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O₂^·- radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-·OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA^· enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O₂, leading directly to H₂O₂ generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O₂^·- and AA^· enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H₂O₂ and O₂^·-, triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.

Full text of DOI:10.1021/tx015526r

Chem. Res. Toxicol. 2001, 14, 1323-1329
1323
Aer obic Oxid a tion of Am in oa ceton e, a Th r eon in e
Ca ta bolite: Ir on Ca ta lysis a n d Cou p led Ir on Relea se
fr om F er r itin
Fernando Dutra, Fernanda S. Knudsen, Denise Curi, and
Etelvino J . H. Bechara*
Departamento de Bioqu ı´ mica, Instituto de Qu ´ı mica, Universidade de S a˜ o Paulo, CP 26077,
0
5513-970, S a˜ o Paulo, SP, Brazil
Received May 14, 2001
Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cri-
du-chat and threoninemia syndromes and, more recently, implicated as a contributing source
+
of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH
4
, and H
2
O
2
has been
reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO)
as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence
and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM)
autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of
+
MG and NH
4
. Superoxide radical was shown to propagate this reaction as indicated by strong
inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or
semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-
•
1
-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO- OH adduct as a
•
-
decomposition product from the DMPO-O
2
adduct. Accordingly, oxygen uptake by AA is
accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O
•-
2
radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen
uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-
•
•
OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA enoyl
radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA
to O , leading directly to H generation followed by the Fenton reaction is thought to take
place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen
2
2 2
O
•-
•
ferritin, putatively mediated by both O
2
and AA enoyl radicals, and the co-oxidation of added
hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron
release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of
diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and
•
-
2 2 2
iron-catalyzed oxidation with production of reactive H O and O , triggering pathological
responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated
with iron overload and oxidative stress.
In tr od u ction
paradoxally produces the cytotoxic and genotoxic MG (8,
).
Aminoacetone bears an amino group vicinal to the
9
Aminoacetone is a threonine and glycine catabolite (1)
long known to accumulate in the cri-du-chat (2) and
threoninemia (3) syndromes and, more recently, pointed
out as an alternative source of methylglyoxal (MG) to
triose phosphate and acetone in diabetes mellitus (4).
Threonine dehydrogenase catalyzes the oxidation of
carbonyl function and therefore, like 5-aminolevulinic
acid (ALA), a porphyrin precursor implicated in porphyric
disorders (10), is expected to undergo phosphate-cata-
lyzed enolization (11) and iron-catalyzed oxidation to
•
+
yield reactive oxygen species (ROS), including OH radi-
threonine by NAD to glycine and acetyl-CoA (5), but
cals (12-16). In this regard, Kawanishi et al. (17) recently
reported that AA oxidation catalyzed by Cu(II) present
in nucleus of human cultured cells is able to generate
ROS and thereby promote DNA damage.
The present work aims to clarify the mechanisms by
which AA undergoes direct and metal-catalyzed aerobic
oxidation to yield deleterious ROS, with emphasis on the
when the ratio acetyl-CoA/CoA increases in nutritional
deprivation (e.g., in diabetes) the enzyme produces AA
(Scheme 1). In turn, AA as well as many other endog-
enous (e.g., methylamine) and xenobiotic amines (e.g.,
benzylamine) are oxidized by dioxygen in the presence
of semicarbazide sensitive amine oxidases (SSAOs), a
group of poorly understood plasma circulating and mem-
brane bound Cu-dependent enzymes, yielding an alde-
catalytic role of iron given its well-known implications
in diabetes (18). We demonstrate here that the O
2
+
•-
hyde, H
2
O
2
and NH
4
ions (6, 7). With AA, SSAO activity
radical propagates oxygen oxidation of AA to imino-
+
acetone, whose hydrolysis yields MG and NH
4
ions. We
*
To whom correspondence should be addressed. Phone: 55-11-3818-
3
869. E-mail: ebechara@iq.usp.br.
also demonstrate that simultaneous electron transfer
1
0.1021/tx015526r CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/03/2001

1
324 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Sch em e 1. Th r eon in e Ca ta bolism to Am in oa ceton e (AA) a n d Meth ylglyoxa l (MG)
Dutra et al.
from both Fe(II) and AA to oxygen directly yields H
2
O
2
mixture (23). The mixture was heated for 3 h at 60 °C and the
crude product analyzed by HPLC-diode array detection (at 315
nm). In turn, ammonia was assayed spectrophotometrically in
the same final reaction mixture using a commercial kit (Am-
monia Diagnostic Kit, Sigma) based on the NAD -dependent
enzymatic amination of R-oxoglutarate.
•
and hence HO radical (Fenton reaction), which acceler-
ates the AA aerobic oxidation. Aminoacetone was also
found to release iron from added horse spleen ferritin and
to induce bovine hemoglobin (Hb) and myoglobin (Mb)
one-electron oxidation to their ferri forms, pointing to a
potential role of AA in impairing iron metabolism and
triggering oxidative stress in diabetes and other maladies
associated with AA overload such as threoninemia and
cri-du-chat (1-3).
+
Meth od s. Oxygen uptake was followed in a Yellow Spring
Instruments Model 53 Oxygraph equipped with a Clark-type
electrode. The oxygen consumption by AA (1-7 mM) was studied
in 100 mM Chelex-treated phosphate buffer (pH 7.4), at 37 °C
in the absence or presence of added metals. Iron release from
ferritin was followed spectrophotometrically by the increase in
absorbance at 530 nm due to the chelation of Fe(II) by batho-
-1
-1
Ma ter ia ls a n d Meth od s
phenanthroline sulfonate (ꢀ530 22.14 mM cm ) (20). EPR spin-
trapping with DMPO was studied at room temperature using a
Brucker EMX spectrometer, 10 min after AA addition. The
composition of the reaction mixtures and the operating condi-
tions of the EPR spectrometer for each case are given in the
legends to the figures. Aminoacetone-induced co-oxidation of
oxyhemoglobin or oxymyoglobin (both 10 µM) in 10 mM Tris/
glycine buffer (pH 7, 8) was followed by scanning the 500-700
nm region at selected time intervals or by monitoring the decay
of absorbance at 577 and 582 nm, respectively, in a thermostati-
cally controlled Hitachi U2000 spectrophotometer.
Rea gen ts. All reagents, purchased from Sigma-Aldrich, were
of purest analytical grade. Bovine oxy- and methemoglobin and
oxymyoglobin were prepared and assayed as described else-
where (19); their concentrations are expressed on a heme basis
throughout this work. Horse spleen ferritin was purified on
Sephadex G-25 as described by Oteiza et al. (20) to remove
loosely bound iron. Ferritin concentration was determined by
2
1).
the Bradford method ( Stock solutions of complexes of Fe(II),
Fe(III), and Cu(II) with ATP, ethylenediaminetetraacetic acid
(EDTA), and citrate were simply obtained by dissolving the
correspondent salt in Milli-Q purified water containing ATP,
EDTA, or citrate in a molar ratio of 1:1.2, and using the solution
immediately after preparation. Stock solutions of AA (500 mM)
were prepared in deaerated Milli-Q purified water. All buffers
were pretreated with Chelex-100.
Resu lts
P r od u ct An a lysis. Like in the SSAO-catalyzed reac-
tion (25, 26), but differently in the Cu(II)-catalyzed
Am in oa ceton e P r ep a r a tion a n d P r od u ct An a lyses. Ami-
noacetone hydrochoride was prepared according to Hepworth
+
oxidation (17), MG and NH
4
ions were found here to be
formed from AA in stoichiometric amounts with the
consumed oxygen: 205 ( 13 µM (triplicates) and 212 (
5 µM (triplicates), respectively.
Oxygen Con su m p t ion . Oxygen depletion occurs
within minutes upon addition of AA in Chelex-treated
phosphate buffer following a typical autoxidation kinetics
(Figure 1, line A). Maximum rates of oxygen uptake (kobs,
) respond linearly to the increase in AA concentration
3-20 mM) and allow the calculation of an apparent
(
22) and recrystallyzed from ethanol:ether (8:2). Proton NMR
analysis of AA (180 mM) was performed in a Varian T-200
spectrometer using dimethylsufoxide-d (DMSO-d ) or phos-
phate buffer prepared in D O (δ ppm, DMSO-d ): 2.16 (3H,
); 8.23 (2H, CH ); δ (ppm, D O): 2.09 (3H,
). NaOH voltametric titration of the R-am-
of 6.8 (see
1
6
6
2
6
CH
CH
3
3
); 3.56 (2H, NH
); 3.93 (2H, CH
2
2
2
2
monium group of AA hydrochloride resulted in a pK
a
-
1
results). Aminoacetone semicarbazone was also prepared and
recrystallyzed from aqueous ethanol to give colorless crystals
s
(
(mpobs 213 °C; mplit. 212-214 °C) (22). Methylglyoxal was
-1 -1
second-order rate constant, k
Figure 2). Neither Desferal (up to 3 mM) nor diethyl-
2
, as 0.160 ( 0.007 M
s
derivatized with 1,2-benzenediamine and analyzed by HPLC-
diode array detection, a procedure adapted from Deng and Yu
(
enediaminepentaacetic acid (DTPA) (up to 3 mM) affect
the AA oxidation rate (not shown), reassuring absence
of contaminant metals in the buffer that are putatively
able to catalyze autoxidation reactions (27). Addition of
radical scavengers such as CuZn-superoxide dismu-
tase (SOD) and semicarbazide (0.5-5 mM) (28) to the
(
23), as follows. Methylglyoxal was formed from the oxidation
of 5 mM AA in air-equilibrated 100 mM phosphate (pH 7.4), at
7 °C, where dissolved oxygen is expected to be roughly 200
3
µM (24), in a flask with minimal headspace. After 2 h both
perchloric acid (1 M), to stop the reaction, and o-phenylenedi-
amine, to derivatize MG, were added to the spent reaction
•
-
O
2

Iron-Catalyzed Aminoacetone Oxidation
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1325
F igu r e 1. Effects of iron and SOD on oxygen consumption by
aminoacetone. The time course of oxygen consumption by AA
was monitored in 100 mM Chelex-treated phosphate buffer (pH
F igu r e 3. Cooxidation of aminoacetone with the xanthine
oxidation reaction. The time course of oxygen consumption was
monitored in 100 mM phosphate buffer (pH 7.4), at 37 °C,
containing 1 mM AA (control, line A), the xanthine (50 µM)/
xanthine oxidase (60 nM) system (control, line B) or the complete
system (line C).
7
.4), at 37 °C, containing: 5 mM AA alone, in the absence
control, line A) or presence of 50 units/mL SOD (line B), and
with 30 µM Fe(II)EDTA in the absence (line C) or presence of
0 units/mL SOD (line D).
(
5
Ta ble 2. Ra te of Oxygen Up ta k e by Am in oceton e in th e
P r esen ce of Meta l Com p lexesa
metal complexes (µM)
oxygen uptake rate (µM O2/min)b
none
3.9
12.5
3.9
15.5
5.0
3.8
5.3
6.1
Fe(II)EDTA (30)
Fe(III)EDTA (30)
Fe(III)EDTA (200)
Fe(II)ATP (150)
Fe(III)ATP (150)
Fe(II)citrate (300)
Fe(III)citrate (300)
a
Oxygen uptake by 5 mM AA in 100 mM phosphate buffer (pH
7
.4) at 37 °C. Ratio chelator/metal ) 1.2. b Observed rate values
are reproducible within 15% (triplicates).
•
-
µM)/xanthine oxidase (6 nM) system as a O
source (30) (Figure 3).
2
radical
The Fe(II)EDTA complex (30 µM) was shown to ac-
celerate the rate of oxygen consumption by AA (Figure
, line C; Tables 1 and 2). Fe(III)EDTA and the Fe(II)
F igu r e 2. Effect of aminoacetone concentration on the observed
autoxidation rate. Maximal rates of oxygen consumption by AA
at increasing concentrations were measured in 100 mM Chelex-
treated phosphate buffer (pH 7.4), at 37 °C and used to calculate
1
and Fe(III) complexes with ATP or citrate were ineffec-
tive as catalysts in identical experimental conditions,
although the former showed catalytic activity at a higher
concentration (200 µM). For comparison, 50 µM Cu(II)(aquo)
increased 20-fold the rate of oxygen uptake, whereas 100
µM Cu(II)EDTA has no effect. Interestingly, SOD (50
units/mL) had only a small inhibitory effect on the rate
of oxygen uptake by AA in the presence of Fe(II)EDTA
k
2 2
obs and k values, assuming [O ] ) 200 µM.
Ta ble 1. Effect of Rea ctive Oxygen Sp ecies Sca ven ger s
on th e Ra te of Oxygen Up ta k e by Am in oa ceton ea
scavengers
metal
inhibition (%)
catalase
catalase
SOD
SOD
semicarbazide
+Fe(II)
-Fe(II)
+Fe(II)
-Fe(II)
(Fe(II)
53
40
22
90
80
(
Figure 1, line D; Table 1). This was interpreted as
resulting from the prevalence of a metal-catalyzed two-
electron reduction of oxygen to H and/or enzyme
2
O
2
a
Oxygen uptake by 5 mM AA in 100 mM phosphate buffer (pH
.4) at 37 °C, in the absence and presence of 30 µM Fe(II)EDTA.
•
inactivation by a high flux of HO radical production (see
spin-trapping studies below). Catalase (4.5 µM) addition
to the reaction mixture decreased the observed rate of
oxygen uptake by ca. 50% attesting stoichiometric forma-
7
Catalase, 4.5 µM; SOD, 50 units/mL; semicarbazide, 5 mM.
Inhibition values obtained from triplicates were reproducible
within 15%.
tion of H
2 2
O (Table 1). One cannot exclude the possibility
•
reaction mixture inhibits oxygen uptake, thus evidencing
that H O contributes to HO radical generation by the
2 2
•
-
the intermediacy of O
Table 1 and Figure 1, line B). Furthermore, as in the
case of the dihydroxyacetone phosphate autoxidation
2
radical in the propagation step
Fenton reaction, which is expected to oxidize AA directly
and amplify the AA oxidation chain.
(
Oxidation of deprotonated AA similarly to ALA is
preceded by its phosphate-catalyzed enolization (12, 31),
as indicated by the catalytic effect of phosphate on the
rate of oxygen uptake by 5 mM AA [1.14 and 2.47 µM
•-
studied by Mashino and Fridovich (29), the ability of O
ions to abstract one electron from the substrate initiating
the reaction is demonstrated here with the xanthine (50
2

1
326 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Dutra et al.
assignable to the DMPO- OH adduct, formed by decom-
•
•
-
position of the DMPO-O
2
adduct as suggested by the
quenching effect of added SOD (50 units/mL) on the
signal intensity (spectrum C); here, the presence of a
6
-line signal attributable to a carbon-centered radical
•
adduct (AA enoyl radical?) is also revealed (compare with
spectrum D). As DTPA (100 µM) addition has no effect
on the spectra, confirming absence of adventitious iron
in the Chelex-treated buffer and therefore excluding the
occurrence of the Fenton reaction strictu senso. It is
•
tempting to attribute OH radical formation in the
2 2
absence of iron to reduction of H O by the hypothetical
•
enoyl radical intermediate (AA ), similar to the so-called
organic Fenton reaction with semiquinones (33).
Upon addition of Fe(II)EDTA, the spin-trapping ex-
periments revealed two less intense spin adduct signals
(
spectrum D): a 6-line spectrum(a
N
) 15.5 G; a ) 23.2
H
G) assignable to a DMPO adduct with a carbon-centered
radical (hypothetical AA intermediate), and the 4-line
signal from the DMPO- OH adduct. Consistent with the
•
F igu r e 4. pH profile of aminoacetone iron-catalyzed oxidation.
Oxygen consumption by 5 mM AA in the presence of 30 µM Fe-
•
observed weak inhibitory effect of added SOD in the
oxygen uptake curve (Figure 1), the spin trapping experi-
ment with SOD in the presence of Fe(II)EDTA did not
change the ratio of the 6-line and 4-line signals (spectrum
(
II)EDTA was monitored in 100 mM phosphate buffer, 37 °C,
at differents pHs. The inset shows the titration curve of 5 mM
AA with 100 mM NaOH in Milli-Q water.
•
E). The observed relative increase in OH (and possibly
•
AA ) formation in the presence of SOD may result from
increased formation of H
2 2
O by concomitant two-electron
transfer from AA and Fe(II) to O2, followed by the Fenton
•
-
reaction, relative to O
2
-propagated oxidation. In fact,
dimethyl sulfoxide (DMSO) addition to the reaction
•
mixture caused replacement of the DMPO- OH adduct
•
with a DMPO- CH
(
3
adduct (a
H
) 23.2 G; a
N
) 15.9 G)
spectrum F). All radical signals reported here were
absent in control experiments without addition of AA
e.g., spectrum A), that is, those with DMPO-Fe(II)-
(
EDTA alone and in the absence or presence of either SOD
or DMSO.
F er r itin Ir on Relea se. As in the case of ALA (20),
•
-
we show here that O
2
and putative AA-derived enoyl
radicals generated during AA oxidation are able to
liberate Fe(II) from horse spleen ferritin (Figure 6).
Control experiments show minor release of persistent
loosely bound iron from the protein as previously reported
when studying ALA (20). Superoxide dismutase addition
partially inhibited Fe(II) release by 0.5 mM AA by 25%
at 100 units/mL (Figure 6, line C), whereas nitrogen
F igu r e 5. EPR spin-trapping studies of iron and SOD effects
on the aerobic oxidation of AA. EPR spectra of DMPO-radical
adducts were obtained after a 10-min incubation of 5 mM AA
at 25 °C in 100 mM phosphate buffer (pH 7.4) with 100 mM
DMPO: control without AA (A); plus 100 µM DTPA (B); plus
purging blocked the release (Figure 6, line D). These
•-
studies confirm the intermediacy of AA-generated O
2
•
and suggest the involvement of AA radical in the iron
liberation from ferritin like previously demonstrated with
ALA (34). In turn, the results in the absence of oxygen
show that AA itself cannot reduce ferritin ferric to ferrous
ions.
5
0 units/mL SOD and 100 µM DTPA (C); plus 30 µM Fe(II)-
EDTA (D); plus 30 µM Fe(II)EDTA and 50 units/mL SOD (E);
and plus 10% v/v DMSO and 30 µM Fe(II)EDTA (F). Instru-
mental conditions: microwave power, 20.2 mW; modulation
amplitude, 1.0; time constant, 1.63 s; scan rate 0.1 G/s; receiver
6
gain, 1.12 × 10 .
Hem oglobin a n d Myoglobin Co-oxid a tion . As pre-
viously reported with ALA (19), addition of oxyHb or
oxyMb (both at 10 µM) notably increased (10- and 8-fold,
respectively) the rate of oxygen consumption by 5 mM
AA, while undergoing co-oxidation to their ferri forms
(Figure 7). MetHb formation is attested to by the decay
of absorbance at 540 and 577 nm showing the well-
defined isosbestic points at 522 and 587 nm reported
elsewhere (35); metMb formation was monitored by the
decay of absorbance at 543 and 582 nm. In both cases,
the rate of oxyhemeprotein co-oxidation responds roughly
linearly the AA concentration (0.5-5.0 mM; not shown)
and the oxyHb oxidation could be inhibited by addition
2
O /min, at 50 and 100 mM phosphate (pH 7.4) respec-
tively; not shown] and by the pH profile of the iron-
catalyzed reaction showing an inflection at pH 7.2 (Figure
4
4
a
), therefore slightly above the AA pK (6.8; inset Figure
).
Ep r Sp in Tr a p p in g Stu d ies. Figure 5 summarizes
spin trapping studies conducted with AA (5 mM) in the
presence of DMPO (100 mM) and both in the presence
and absence of Fe(II). Hyperfine couplings were mea-
sured and radical adducts assigned from tables of known
values (32). The observed 4-line spectrum B (a
N
) a )
H
1
4.6 G) of the AA/DMPO-containing reaction mixture is

Iron-Catalyzed Aminoacetone Oxidation
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1327
HO• radical from H
O
plus Fe(II) and/or AA could
•
2
2
amplify the chain reaction.
Aminoacetone is demonstrated here to induce Fe(II)
release from isolated ferritin, an important biological iron
storage site (36), promoted by reduction of Fe(III)-ferritin
•
-
•
by AA-generated O
2
and AA radicals (Scheme 4). Our
experiments run in the presence of SOD (10-400 units/
•-
mL) indicate that O
2
accounts for maximally 25% of the
total iron mobilization (Figure 6), differently therefore
to that observed with ALA (50% of released iron) (20).
•
Enoyl AA radical like that formed by ALA oxidation is
possibly responsible for the release of remaining iron.
Indeed, during electrochemical one-electron oxidation of
ALA in a nitrogen atmosphere (34), we were able to
demonstrate the formation of a spin adduct with 3,5-
dibromo-4-nitrosobenzene sulfonic acid (DBNBS), char-
acterized by a 6-line signal, attributable to a resonant
•
ALA enoyl radical. Iron release from ferritin added to
F igu r e 6. Time course of aminoacetone-induced iron release
from ferritin. Iron release from ferritin (2.5 mg/mL) alone (line
A) and in the presence of 0.5 mM AA (line B) was followed by
measuring the increase in absorbance at 530 nm due to the
chelation of Fe(II) by bathophenanthroline sulfonate. Experi-
ments represented by lines C and D were performed in the
presence of 100 units/mL SOD and upon nitrogen purging,
respectively. Inset: Dependence of iron mobilization on AA
concentration (0.05-5 mM) was measured after 30 min incuba-
tion in the standard buffer, at 37 °C.
this anaerobic system could be coupled to the electro-
chemical ALA oxidation, suggesting that ALA enoyl
•
radical behaves like a semiquinone as an iron liberator
from ferritin (33). In turn, hemoglobin and myoglobin
catabolism are known to converge to hemin liberation and
dioxygenation with further iron release, which according
to Ryter and Tyrrell (37) could be involved in deleterious
reactions that compete with iron reutilization and se-
questration pathways.
The hypothesis involving AA oxidation by SSAO in
diabetes-associated angiopathy (responsible for the typi-
cal nephropathy, neuropathy, and retinopathy) is con-
sistent with the enzyme’s predominance in the plasma
membrane of vascular smooth muscle cells (7). However,
the SSAO-catalyzed oxidation of AA as an exclusive route
of either 1.0 µM catalase (95%) or 60 units/mL SOD
(
13%), indicating a principal role for H
2
O
2
via highly
•
oxidizing OH radical and Hb-H
and AA co-oxidation.
2 2
O complex in the oxyHb
Discu ssion
2 2
leading to MG and H O fails to take into consideration
The present study demonstrates that AA undergoes
autoxidation and iron-catalyzed oxidation propagated by
that AA is not an ordinary amine, but an easily autoxi-
dizable R-aminoketone, and that semicarbazide breaks
•
-
•
•-
O
2
, which is accompanied by HO formation which, in
O
2
-dependent chain reactions. In addition, the low
turn, can amplify the chain reaction (Schemes 2 and 3).
substrate specificity (several endogenous and synthetic
mono- and polyamines) and high species-specificity of
SSAOs, together with the production of MG, a highly
cytotoxic and cross-linking agent, intrigues a physiologi-
cal function for SSAOs (7).
+
Methylglyoxal, NH
4
2 2
ions and H O are the main oxida-
tion products. The AA oxygen-consuming reaction is
envisaged as preceded by phosphate-catalyzed AA eno-
lization, like with ALA (12), followed by unielectronic
reduction of oxygen by enol AA yielding a resonant enoyl
To our knowledge, the physiological and pathological
concentrations of AA in plasma and tissues have not yet
been reported, perhaps due to its instability toward
oxygen, especially in the presence of transition metal
ions, and to the expected rapid Schiff conjugation of its
oxidation product, MG, with proteins. Accordingly, (i)
preparation of AA hydrochloride (22) requires strict
dryness, glassware previously treated with metal chela-
tors, and a nitrogen atmosphere, and (ii) the presence of
AA in plasma has been inferred from the detection of a
MG adduct with o-diaminobenzene. The only available
in vivo AA estimation was long ago made by Urata and
Granick (38), who found “about 0.4 mg of a compound
with chromatographic properties of aminoacetone” in the
urine collected daily from normal human adults, i.e., 2
µM AA assuming excretion of 1.5 L of urine/day. There-
fore, the biological significance of our chemical studies
with millimolar concentrations of AA should be viewed
with perspective, although we have already demonstrated
that swelling of isolated rat liver mitochondria can be
attained with AA concentrations as low as 100 µM (work
in progress).
•
•-
radical (AA ) and O
2
, which propagate the reaction.
Hydrolysis of the oxidized AA, i.e., iminoacetone, results
+
•-
in MG and NH
4
ions, and O
2
2 2
dismutation yields H O ,
•
a source of HO radical by Fenton reactions with Fe(II)
•
and AA radical. Accordingly, our data obtained by
oxymetry and EPR spin-trapping experiments show that
the reaction is inhibited by added SOD and by semicar-
•-
bazide, both efficient O
2
scavengers. The same reaction
mechanism has been shown to be operative in the case
of ALA, a heme precursor which accumulates in some
inherited (e.g., AIP) and acquired (e.g., lead poisoning)
porphyric disorders and is able to provoke oxidative
injury to several biomolecules and cell structures (12-
1
4, 20).
The small effect of SOD in the iron- and Hb-catalyzed
AA oxidation, monitored by oxymetry and, in ther former
case, also by EPR spin trapping, suggests prevalence of
•
-
an O
complexation, followed by simultaneous two-electron
2 2
transfer from AA and Fe(II) to O with direct H O
2 2
-independent pathway (Scheme 3): AA-Fe(II)-O
2
formation, a reaction analogous to an oxidation reaction
studied by Caughey and collegues (35) with oxyhemo-
globin, oxygen and nucleophiles. Further formation of
Like ALA (also an R-aminoketone) (12) and simple
sugars (R-hydroxycarbonyls) (39), AA undergoes fast

1
328 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Dutra et al.
F igu r e 7. Cooxidation of aminoacetone and oxyhemeproteins. Temporal behavior of the spectra of oxyhemoglobin (10 µM, panel A)
and oxymyoglobin (10 µM, panel B) in the presence of AA (5 mM) in 50 mM Tris-Gly buffer (pH 7,8) at 36 °C. OxyHb (A): 0, 10, 15,
2
0 and 25 min, and 60 min (broken line). OxyMb (B): 0, 5, 10, 15, 20 and 25 min, and 60 min (broken line). In the absence of AA,
the absorption spectra underwent only very slight changes.
Sch em e 2. P r op osed Mech a n ism for th e Aer obic
Oxid a tion of Am in oa ceton e
Sch em e 4. F er r itin Ir on Liber a tion by O •- a n d
2
AA• Ra d ica ls
are also long known to accumulate in certain maladies
•
-
and hence might act as efficient chemical sources of O
2
by autoxidation or metal-catalyzed oxidation (40, 41). In
this regard, noteworthy are (i) the reported “metal-free”
oxidations of Amadori compounds prepared from fructose
•
-
and Arg and Gly, found to be effective sources of O
2
by
autoxidation of their enol forms (42) and (ii) the suggested
implication of metal-catalyzed oxidation of glucose and
glycated proteins yielding R-oxoaldehydes in protein
damage in diabetes (39).
Sch em e 3. Ca ta lytic Role of F e(II) in th e Aer obic
Oxid a tion of Am in oa ceton e
Several studies have suggested the role of iron in
triggering oxidative stress and tissue damage in human
and experimental animal diabetes (18, 43, 44). In ad-
vanced iron overload, known to be more effective than
chronic liver disease and cirrhosis in generating diabetes,
iron accumulation in pancreatic â-cells seems to impair
insulin secretion, leading to insulin-dependent diabetes
mellitus not reversible by iron removal. Accordingly,
Okunade et al. (45) found significant high levels of non-
heme iron and decreased reactive thiols in erythrocyte
membranes of noninsulin-dependent diabetic patients, as
compared to normal individuals, thus pointing to possible
association of diabetes complications with iron-induced
oxidative stress.
In conclusion, our studies point to AA as a potential
source of MG and ROS by nonenzymatic aerobic oxidation
and as an iron-releasing agent. The possibility raised
here is that the chemical pathway of AA oxidation and
iron liberation, in parallel to the reaction catalyzed by
enolization at physiological pHs to give highly oxidizable
enols, chemically similar to some extent to diphenols,
aminophenols and diaminoaromatics. These compounds

Iron-Catalyzed Aminoacetone Oxidation
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1329
SSAOs, may contribute significantly to oxidative stress
triggered by diabetes.
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Ack n ow led gm en t. This work was supported by
grants from the Funda c¸ a˜ o de Amparo a` Pesquisa do
Estado de S a˜ o Paulo (FAPESP), the Programa de Apoio
a N u´ cleos de Excel eˆ ncia (PRONEX), the Conselho Na-
cional de Desenvolvimento Cient ´ı fico e Tecnol o´ gico (CNPq),
and the von Humboldt Foundation. We thank Dr. Brian
Bandy and Dr. Al ´ı cia Kowaltowski for reading the
manuscript. Samples of human CuZnSOD were kindly
provided by Dr. Francisco Laurindo from the Instituto
do Cora c¸ a˜ o (INCOR).
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