 |
INTRODUCTION |
Inflammation associated with articular cartilage, bone, and dentin
surfaces is characterized by accumulation, adhesion, and activation of
neutrophils and monocytes, which results in the destruction of
cartilage and the loss of bone at these sites (1-4). These effects are
thought to be mediated in part by the production of reactive oxygen
species (ROS),1 a group of
reactants that includes superoxide (O
2), hydrogen peroxide
(H2O2), and the highly reactive species
hypochlorous acid (HOCl) (5-8). ROS are produced by neutrophils and
monocytes after they are recruited from the circulation to
extravascular spaces. Once outside of the circulation, they adhere to
extracellular matrix proteins (ECM) and undergo activation, which
results in the production of ROS and release of proteolytic enzymes
directly onto the matrix surface (4, 9-13). In inflammation associated with arthritic joints, the accumulation and activation of neutrophils and monocytes and increased synovial cell formation result in the loss
of synovial membrane integrity and eventually to irreversible damage
and destruction of articular cartilage in the afflicted joints (1, 4).
Similarly, in periodontitis or inflammation associated with the
tooth-supporting periodontal ligament, the recruitment of neutrophils
and monocytes from the circulation and the subsequent activation and
release of ROS and proteolytic enzymes can eventually result in a
significant and irreversible loss of underlying bone matrix at these
sites (2, 3). Despite the known participation of ROS in the
inflammatory-mediated loss of underlying ECM, the mechanism(s) by which
this occurs is not completely understood. This is particularly true for
collagen, which is the major ECM protein in cartilage and bone.
Neutrophils and monocytes contain two enzymes that are responsible for
producing ROS. The first is NADPH oxidase, which catalyzes the
formation of O2 by the transfer of electrons from NADPH to oxygen via cytochrome b558 (14-16). Superoxide
is rapidly dismutated to H2O2 either
spontaneously or via the enzyme superoxide dismutase (17). Neither
O2 nor H2O2 exhibit significant
reactivity with biologic compounds (18). The second enzyme is
myeloperoxidase (MPO), which catalyzes the formation of HOCl from
H2O2 and Cl
(19). MPO has been
localized to the primary granules of resting neutrophils and the
extracellular space and phagolysosomes of phagocytically stimulated
neutrophils (20, 21). MPO is also released from cytoplasmic granules of
monocytes and some macrophages (22). The cationic nature of MPO allows
it to adhere to cell and matrix surfaces and localize to sites of
inflammation (23). It is at these sites that MPO produces HOCl, a
highly reactive oxidant that readily reacts with primary amines to
generate long lived N-chloramines (24-26). Although
N-chloramines exhibit a lower oxidizing potential than HOCl,
their much longer effective lifetime (~18 h) would enable them to
cause damage at more distant sites than HOCl (26). Under acidic
conditions, similar to the environment found in phagolysosomes, MPO
generates Cl2 (27). MPO is also one of the pathways by
which neutrophils generate O2(1
g) (28).
Determining the ability of HOCl to contribute to the pathogenesis of
inflammatory processes associated with rheumatoid arthritis and
periodontitis is highly dependent on determining the relevant target(s)
at these sites. The most likely protein target for neutrophil oxidants,
including N-chloramines, is the ECM. ECM components found in
or associated with articular cartilage or bone include hyaluronate,
proteoglycans, fibronectin, several tissue-specific and nonspecific
protein components, and collagen, the major component of these tissues.
It has been reported that neutrophil-generated ROS mediate the
degradation of hyaluronate (29), modify proteoglycan structure
and/or synthesis, and alter the structure of fibronectin (29-31). Vissers and Winterbourn (32) reported an increase in proteolytic degradation of glomerular basement membrane collagen by
elastase in response to
myeloperoxidase/H2O2/Cl. Davies
et al. (33) reported that >1.0 mM HOCl was
required to cause extensive fragmentation of collagen type II isolated from bovine articular cartilage or collagen type I isolated from bovine
tendon. Davies et al. (33) also reported that
N-chloramines did not cause direct fragmentation but greatly
increased the degradation of collagen by collagenase and elastase. In
general, there are very few studies available as to the susceptibility
of collagen to oxidation by HOCl.
An important determinant for stability of the ECM is the degree of
cross-linking. One important type of cross-link is pyridinoline (Pyd),
which was first described by Fujimoto et al. (34) and later
confirmed to be a non-reducible intermolecular cross-link of mature
fibrillar collagen type I of bone (35). These cross-links were also
found to be especially abundant in mature fibrillar collagen type II of
articular cartilage (36), where they covalently link collagen type II
to other type II helical regions, collagen type IX to the surface of
type II, and bind collagen type IX to other molecules of collagen type
IX. Their function is to stabilize the collagen fibrillar
superstructure, or arrays, and make them more resistant to
collagenolysis or proteolytic degradation (37).
The present study focuses on the susceptibility of Pyd cross-links of
collagen to reaction with HOCl. Pyd cross-links were chosen as
potential oxidation targets because of their importance in maintaining
the collagen superstructure and because their chemical structure
suggested they would be targets of oxidative modification by
OCl/Cl2. Our findings indicate that these
cross-links react with OCl, Cl2, and
N-chloramines and suggest that Pyd would be a site for ROS
modification of collagen type I and II in bone and cartilage, respectively.
| |
EXPERIMENTAL PROCEDURES |
Pepsin-solubilized collagen types I and II isolated from human
bone and articular cartilage, respectively, were kindly provided by Dr.
David Eyre (University of Washington, Seattle). Pyridoxamine dihydrochloride, pyridoxine hydrochloride, 30% hydrogen peroxide, chloramine-T, 1,4-dimethylnaphthalene, methylene blue, and all other
salts and buffer components were purchased from Sigma. Glacial acetic
acid and dichloromethane were purchased from Fisher. Potassium iodide
was purchased from Acros Organics (Fairlawn, NJ). Disodium anthracene-9,10-dipropionic acid was purchased from Molecular Probes
(Eugene, OR). Sodium hypochlorite (NaOCl) (4-5% available chlorine)
was purchased from Sigma, Acros Organics, Fisher, Alfa (Ward Hill, MA),
and Cole-Palmer (Vernon Hills, IL); see comments about NaOCl below.
Preparation of Pyridoxamine and Vitamin B6--
10
mM stock solutions of pyridoxamine dihydrochloride
(pyridoxamine) and pyridoxine hydrochloride (vitamin B6)
were made fresh weekly and stored at 4 °C with protection from
light. Stock solutions were made in one of the following buffers: 0.5 M glacial acetic acid buffer, pH 3, or 0.5 M
sodium phosphate buffer, pH 5.5 ± 0.2, pH 7.2 ± 0.2, or pH
8.0 ± 0.2. Where appropriate, 0.1 M NaCl was added to
the buffers as a source of chloride ions.
Control and Reactive Oxygen Species Reaction with Pyridoxamine
and Vitamin B6--
On the day of use, stock solutions of
pyridoxamine and vitamin B6 were diluted in appropriate
buffer to 20 µM. 7.5 ml of the 20 µM
solutions of pyridoxamine or vitamin B6 were then added to
control and treated sample tubes so the final concentration of
pyridoxamine or vitamin B6 in all samples was 15 µM in a total volume of 10 ml. Samples were brought to a
total volume of 10 ml by the addition of 2.5 ml of appropriate buffer
to control samples or 1.25 ml to hydrogen peroxide
(H2O2) and sodium hypochlorite (NaOCl) samples.
No buffer was added to samples treated with the combination of
H2O2 and NaOCl
(H2O2/NaOCl). H2O2 and
NaOCl treatment solutions were prepared by diluting the stock solution
of H2O2 to 1, 2, or 4 mM and
diluting the stock solution of NaOCl to 0.1, 0.2, or 0.4 mM
in appropriate buffer immediately before use. 1.25 ml of a diluted
solution of H2O2 or NaOCl or 1.25 ml each of
diluted solutions of H2O2 and NaOCl were added
to appropriate tubes. The final concentrations of
H2O2 were 125, 250, or 500 µM,
and the final concentrations of NaOCl were 12.5, 25, or 50 µM. H2O2 was always added first
to either the H2O2 or
H2O2/NaOCl samples, and NaOCl was added
immediately after, where appropriate. Samples were mixed after each
addition. At pH 12, the NaOCl stock solution exists predominantly as
the conjugate base, hypochlorite (OCl).
H2O2 (30% or 12.92 M) and NaOCl
(0.65 M) stock solutions as supplied by the manufacturer
were stored tightly sealed at 4 °C with protection from light.
Despite these precautions, both stock solutions decomposed over a
period of 3-4 months after being opened. In general, decomposition of
NaOCl could be identified by a yellowing of the solution. Yellowing was
always accompanied by a distinctively greater reactivity of the NaOCl
solutions, which we attributed to the breakdown of NaOCl to
Cl and eventually reactive chlorate
(ClO3) (38-40). Therefore, caution should be
exercised when using NaOCl, since we found that even new solutions made
by different companies showed signs of decomposition.
UV Absorbance and Fluorescence--
After the addition of
buffer, H2O2, NaOCl, or
H2O2/NaOCl, control and treated samples were
incubated for 15 min at RT (approximately 25 °C) in a Precision
Scientific Low Temperature Incubator 815 (Chicago, IL). At the end of
each incubation, the UV absorbance of a 50-µl aliquot of each sample
was scanned from 400 to 200 nm in a Beckman DU-640 Spectrophotometer
(Fullerton, CA), and the fluorescence intensity of a 3-ml aliquot of
each sample was read in a PerkinElmer Life Sciences fluorescence
spectrophotometer. Optimal fluorescence excitation and wavelengths were
determined by referring to UV absorbance peaks and by prescanning
samples for maximal excitation and peaks. The known excitation and peak wavelengths for both pyridoxamine and vitamin B6 are 324 nm
excitation and 400 nm emission. A 200-µl aliquot of each control and
treated sample was also stored at 4 °C after incubation for later
N-chloramine analysis.
N-Chloramine Assay--
The presence of N-chloramines
was determined by the method of Witko et al. (41). This
method is based on the colorimetric measurement of triiodide
ions formed by the oxidation of potassium iodide (KI) in solution.
Chloramine-T (N-chloro-p-toluene-sulfonamide sodium salt), a commercially available source of
N-chloramine, was used to calibrate the assay. A 100 mM stock solution of chloramine-T was made fresh weekly in
distilled H2O and stored at 4 °C with protection from
light. The 100 mM chloramine-T solution was then diluted in
appropriate buffer to final concentrations of 25, 50, 75, or 100 µM immediately before use. We extended the RT incubation from 2 (41) to 5 min and found no significant difference in results.
The direct oxidation of KI by H2O2, NaOCl, or
H2O2/NaOCl was also determined, and these
values were subtracted as background from the correspondingly treated
samples. The resulting difference represented the amount of
N-chloramine present in each sample.
Chloramine-T Reactivity--
On the day of use, 10 mM stock solutions of pyridoxamine or vitamin
B6 were diluted to a concentration of 1 mM, and
a 100 mM stock solution of chloramine-T was diluted to
final concentrations of 25, 50, 75, or 100 µM in
appropriate buffer. Control and chloramine-T-treated samples contained
15 µl of the diluted 1 mM pyridoxamine or vitamin B6 solution plus 1 ml of appropriate buffer (control) or 1 ml of 25, 50, 75, or 100 µM chloramine-T solution. The
final concentration of pyridoxamine or vitamin B6 for all
control and chloramine-T-treated samples was 15 µM.
Following preparation, samples were incubated for 15 min at RT. At the
end of each incubation, the UV absorbance of a each sample was scanned
from 400 to 200 nm to look for any changes in the absorbance of
pyridoxamine or vitamin B6 due to a reaction with
chloramine-T and to identify the absorbance peaks for
N-chloramines and pyridoxamine-chloramine or vitamin
B6-chloramine reaction products. In addition, the
N-chloramine assay of Witko et al. (41) was
performed to determine if a reaction of chloramine-T with pyridoxamine
or vitamin B6 had occurred as indicated by a decrease in
the amount of chloramine-T available to oxidize KI.
1,4-Dimethyl-1,4-naphthalene Endoperoxide (DNE) Synthesis and
Release of O2(1g)--
DNE, a pure chemical
source of O2(1g) that thermally releases
O2(1g) at 37 °C, was synthesized by the
method of Wasserman and Larsen (42, 43). A duplicate set of coverslips
was coated with either dichloromethane or a solution of DNE in
dichloromethane (3.6 mg/100 µl) by surface evaporation at 4 °C.
200 µl of either a 15 µM pyridoxamine or vitamin
B6 solution in appropriate buffer was added to a duplicate set of dichloromethane- (control) and DNE-coated coverslips. One set of
control and DNE-coated coverslips was incubated overnight at 4 °C
and another at 37 °C. The release of
O2(1g) from DNE was confirmed after
overnight incubation at 37 °C by following the decrease in
absorbance at 400 nm of anthracene-9,10-diproprionic acid (AAP) (1 × 104 M) in 0.5 M
sodium phosphate buffer, pH 7.2, according to the method of Deby-Dupont
et al. (44).
Gas Chromatography-Mass Spectrometry--
Gas
chromatography-mass spectrometries (GC-MS) using electron
ionization-mass spectrometry were performed by M-Scan, Inc., West
Chester, PA. In brief, reacted samples containing vitamin B6 alone or vitamin B6 in a 1:1 ratio with
NaOCl were lyophilized, dissolved in 40 µl in dimethyl formamide,
and sialylated derivatives prepared by the addition of 100 µl
of N,O-bis(trimethylsilyltrifluoro-acetamide with
trimethylchlorosilane (Supelco, PA 16823) followed by heating to
35 °C for 5 min. Derivatized products were concentrated to ~50
µl under anhydrous N2 and analyzed on capillary column
(PerkinElmer Life Sciences PE-5MS, 30 m × 0.25 mm × 25 µm) by GC-MS (PerkinElmer Life Sciences Auto System XL gas
chromatograph with Turbomass Quadrupole mass spectrometer) in the
positive electron ionization mode. Electron ionization-mass
spectrometry was used to identify the structure of individual compounds
in each GC peak. The source and interface temperatures were both
200 °C. The injector temperature was maintained at 280 °C, and
the initial GC oven temperature was 70 °C for 2 min followed by an
increase to 140 °C/min to 300 °C.
Preparation of Pepsin-solubilized Collagen--
The lyophilized
collagen samples were dissolved in 0.5 M acetic acid at a
concentration of 1.2 mg/ml overnight at 4 °C with gentle stirring
and protection from light. Samples were then dialyzed against 0.02 M dibasic sodium phosphate buffer, pH 9.0, for 48 h at
4 °C using a dialysis cassette made by Pierce Slide-A-lyzer (Pierce)
according to manufacturer's instructions. The samples were split into
2 equal volumes and dialyzed for an additional 48 h at 4 °C
with protection from light against 0.5 M sodium phosphate buffer to bring the pH to approximately 5.0 ± 0.3 or 7.2 ± 0.3. After dialysis, samples were removed from cassettes and stored at
4 °C until use. The collagen suspensions were turbid and contained fibrils and/or a variety of polymorphic forms of collagen in
equilibrium with monomers (45, 46). These preparations represent a
mixture of fibrils, cross-linked trimers, dimers, and 
-subunits of
collagen (
-, 
-, and - bands, respectively) as visualized by
PAGE. At pH 7.2, collagen type II preparations also contained a small
amount of aggregated/particulate material, also in equilibrium with
monomers. The collagen preparations did not require sonication for
suspension (47) and could be quantitatively and reproducibly loaded
onto nitrocellulose or into wells for SDS-PAGE fractionation.
Reactive Oxygen Species Treatment of Collagen--
On the day of
use, 50 µg of collagen type I or II were added to 1.5-ml Eppendorf
tubes. Control samples received appropriate buffer only, and
ROS-treated samples received appropriate buffer containing
H2O2, HOCl, or the combination of
H2O2 and HOCl
(H2O2/HOCl). Final concentrations of
H2O2 were 125, 250, or 500 µM;
final concentrations of HOCl were 12.5, 25, or 50 µM in a
total volume of 200 µl. Samples were mixed after each addition and
then incubated for 1 h at 37 °C.
Oxyblot for the Detection of Carbonyl (Aldehyde and Ketones)
Formation--
Carbonyl groups are formed as a consequence of protein
oxidation and in the reaction of HOCl with pyridinium compounds. The 2,4-dinitrophenylhydrazine assay for carbonyls (48) was performed according to kit instructions in the Oxyblot-oxidized protein detection
kit (Oncor, Gaithersburg, MD) without SDS-PAGE separation. In brief,
aliquots of each collagen sample were reacted with
2,4-dinitrophenylhydrazine to derivatize carbonyl groups to the product
2,4-dinitrophenylhydrazone. After derivatization, aliquots of each
collagen sample were diluted in SDS-PAGE sample buffer and spotted onto
dry nitrocellulose, and the derivatized product was detected by
chemiluminescence using a horseradish peroxidase-conjugated antibody
that specifically recognized 2,4-dinitrophenylhydrazone. The spot
intensities were quantified by scanning densitometry (Arcus II flatbed
scanner) using NIH Image version 1.57 software (Wayne Rasband, National Institutes of Health, Bethesda).
Acetone Precipitation--
Cold precipitation of collagen
samples was performed according to the Pierce BCA Applications Note 13 (Pierce). 200 µl of 
20 °C acetone were added to 50 µl of each
sample, vortexed, and placed at 20 °C for 30 min. The samples were
then centrifuged at 12,000 × g for 10 min in a
microcentrifuge at 4 °C; supernatants were removed, and the
remaining acetone was evaporated by leaving samples uncovered for 30 min at RT.
Fluorescamine and o-Phthalaldehyde Primary Amine and Imino Acid
Measurements--
Fluorescamine reacts directly with primary amines or
imino acids to yield highly fluorescent derivatives that emit
fluorescence at 475 nm when excited at 390 nm (49) and was used
according to the method of Bohlen et al. (50).
o-Phthalaldehyde also reacts with primary amines and imino
acids and was used according to manufacturer's instructions (Pierce).
Both assays were performed on aliquots of each collagen sample, acetone
precipitates of each sample, or sample supernatants of acetone
precipitates after ROS and/or protease treatments. Triplicates of each
sample (200 µl) were placed in a 96-well cytoplate (CFCPN9610,
Millipore Corp.; Bedford, MA), and the fluorescence was read at an
excitation wavelength of 340 ± 20 nm and an emission wavelength
of 400 ± 20 nm (Cytofluor 2350, Perspective Biosystems, Inc.;
Cambridge, MA).
SDS-PAGE--
4-µg aliquots of acetone-precipitated collagen
or non-precipitated collagen samples were resuspended in SDS-PAGE
sample buffer and subjected to electrophoresis using a 5% stacking gel
and a 10% separating gel prepared according to a modified Laemmli
procedure previously described in detail (51) or using precast linear gradient gels (4-15% acrylamide) purchased from Bio-Rad. The
gels were stained with silver using the Bio-Rad silver stain kit
(Bio-Rad). The collagen gels were quantified by scanning densitometry
(Arcus II flatbed scanner). Band intensities were analyzed using NIH Image version 1.57 software (Wayne Rasband, National Institutes of
Health, Bethesda). To determine the reactivity of silver with protein after reaction with HOCl, [14C]bovine serum
albumin was reacted with increasing concentrations of HOCl under the
conditions described above for collagen. After reaction, the
[14C]bovine serum albumin was subjected to gel
electrophoresis, stained with silver, dried, and exposed to x-ray film.
Silver staining intensity of each band was then compared with
autoradiogram band intensity of the same gel.
Data Analysis--
Data are expressed as the mean ± S.E.
To evaluate the treatment effects, the data were grouped by experiment
and time point for statistical analysis. Statistical significance of
differences between the vehicle only (control) and treatment values for
an individual experiment and time point was determined by a pairwise comparison of correlated groups using Student's t test from
the GB-STAT statistics software version 5.4.1.
| |
RESULTS |
UV Absorbance and Fluorescence Emission of Pyridoxamine and Vitamin
B6--
Pyridoxamine dihydrochloride (Fig.
1B) and vitamin B6
(Fig. 1C) share the pyridinium ring structure and spectral
properties of the pyridinoline (Pyd) trifunctional cross-links (Fig.
1A) of collagens, including collagen types I, II, III, IX,
and XI. The spectral properties of pyridoxamine, vitamin
B6, and Pyd include their characteristic UV absorbance and
excitation maximum at 325 nm, pH 7.2 (Fig. 1B). The
absorbance characteristics of pyridoxamine and vitamin B6
include three peaks at 217-219, 251-252, and 321-325 nm. At pH 5.5, a hydrogen ion binds to the nitrogen group (52) of the pyridinium ring
of vitamin B6, resulting in an additional peak at 292 nm
(Fig. 1C).
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Chemical structures of pyridinoline
(A), a cross-link involved in maintaining the
interaction and structural integrity of collagen types I, II, III, IX,
and XI, pyridoxamine dihydrochloride (B), and vitamin
B6 (C), two
chemical substitutes for pyridinoline. B, UV absorbance
peaks of 15 µM pyridoxamine or vitamin B6 in
0.5 M sodium phosphate buffer, pH 7.2, are 217-219,
251-252, and 321-325 nm. C, at pH 5.5, vitamin
B6 has an additional absorbance peak at 292 nm.
|
|
Oxidative Modification of Pyridoxamine and Vitamin B6
after Exposure to the ROS, H2O2,
HOCl/OCl, or O2(1g)--
To
evaluate the oxidation of pyridoxamine or vitamin B6 by
H2O2, HOCl/OCl (which will be
referred to as HOCl), or O2(1g), a 15 µM solution of pyridoxamine or vitamin B6
dissolved in 0.5 M sodium phosphate buffer, pH 7.2 ± 0.2, was exposed for 15 min at RT to 125, 250, or 500 µM
of H2O2, 12.5, 25, or 50 µM of
HOCl, or a combination of H2O2/HOCl in a ratio
of 10:1. The above concentrations of HOCl and
H2O2 are within the predicted range generated
by activated neutrophils or monocytes at sites of inflammation (6).
Concentrations of H2O2 and HOCl in this range
are possible within specialized microenvironments, such as phagocytic
vacuoles or the neutrophil or macrophage attachment sites, because the
aqueous volumes in these microenvironments are thought to be nanoliters
or less, resulting in µM to mM concentrations of ROS (53). A ratio of 10:1 was used in the present study because the
amounts of HOCl generated by activated neutrophils are 5-20 times less
than relative amounts of H2O2 generated by the
same cells stimulated under the same conditions (6).
O2(1g) is generated when both HOCl and
H2O2 are added together (Reaction 1) (54) or
when HOCl is added in buffer containing Cl pH 7.2 (Reaction 2) (55).
The amount of O2(1g) generated in the
reaction of H2O2 with HOCl is
pH-dependent (56), with the greatest amount of
O2(1g) being produced at alkaline pH,
intermediate amounts at neutral pH, and essentially non-measurable
amounts at acidic pH due to assay limitations and interference by
chlorine (Cl2). No O2(1g) is
produced by HOCl in the absence of Cl or at pH < 4.2 or = 8.0 (55).
At pH 7.2, HOCl would exist in almost equal concentrations with
OCl (pKa = 7.4) (56), and
acidification of HOCl (pH below 6.0, peaking at pH 5.25) in the
presence of Cl results in the evolution of chlorine
(Cl2), Reaction 3 (38, 54, 56).
The UV absorbance data for a typical experiment using 0.5 M sodium phosphate buffer containing 0.1 M
NaCl, pH 7.2, is presented in Fig. 2. The
UV absorbance scans of pyridoxamine treated with HOCl showed an
immediate concentration-dependent shift in maximum absorbance at 217 and 325 nm (Fig. 2A). Accompanying the
shift in UV absorbance in response to increasing concentrations of HOCl was the appearance of two new absorbance peaks at 220-225 and 307-320
nm. In parallel, pyridoxamine excitation at 325 and fluorescence at 400 nm was dramatically decreased in response to increasing concentrations
of HOCl. The fluorescence data for HOCl-treated samples are presented
in Fig. 3A as the mean ± S.E. (n = 3, **p < 0.01 and
*p < 0.05). No other changes in fluorescence
excitation or emission were observed. These findings follow the
predicted increase in reactivity rate of HOCl in the presence of
Cl (38-40, 57).
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
The UV absorbance spectra of 15 µM pyridoxamine in 0.5 M
sodium phosphate buffer ± 0.1 M NaCl, pH 7.2, reacted
for 15 min at RT with 0 µM ( ),
12.5 µM ( ), 25 µM (), or 50 µM HOCl ( ) in the presence of 0.1 M NaCl (A) and without NaCl present
(B). The UV absorbance of pyridoxamine reacted
for 15 min at RT with H2O2 or a 10:1 mix of
H2O2 and HOCl;  , 500 µM
H2O2; , 0 µM
H2O2/HOCl; , 125 and 12.5 µM
H2O2/HOCl;  , 250 and 25 µM
H2O2/HOCl, or , 500 and 50 µM
H2O2/HOCl in the presence of 0.1 M
NaCl (C) or without NaCl present (D).
Graphs are a representative set of data from experiments
that were repeated three times.
|
|
View larger version (53K):
[in this window]
[in a new window]
|
Fig. 3.
Fluorescence of 15 µM pyridoxamine in 0.5 M
sodium phosphate buffer ± 0.1 M NaCl, reacted for 15 min at RT with () CT, ( ) HOCl, ( ) H2O2
(1 × 101) or ( ) H2O2
(1 × 101)/HOCl, at pH 7.2 in the presence of 0.1 M NaCl (A) or without NaCl present
(B). Pyridoxamine reacted at pH 5.5 in the
presence of 0.1 M NaCl (C) or vitamin
B6 in the presence of 0.1 M NaCl at pH 5.5 (D). After reaction, 3 ml of each sample were excited at 325 nm, and the emission intensity at 400 nm was determined. The
fluorescence data are presented as the mean ± S.E. of a set of
experiments repeated three times. Statistical significance of
differences between control and treatment values was determined by a
pairwise comparison of correlated groups using Student's t
test, and statistical significance is defined as **p < 0.01 or *p < 0.05.
|
|
The combination treatment of H2O2/HOCl also
shifted the absorbance (Fig. 2C) and decreased the
fluorescence (Fig. 3A) of pyridoxamine. The absorbance and
fluorescence changes in response to H2O2/HOCl were similar to the changes observed when pyridoxamine was treated with
the corresponding concentrations of HOCl alone. When 0.1 M
NaCl was omitted from the buffer system, only minor effects on the
absorbance and fluorescence were observed in response to either HOCl
(Fig. 2B and Fig. 3B) or
H2O2/HOCl (Fig. 2D and Fig. 3B). H2O2 alone had no effect on the
UV absorbance or fluorescence of pyridoxamine (Fig. 2C and
Fig. 3A) in either buffer system, although the absorbance of
H2O2 around 219 nm contributed to the slight
increase in absorbance at this wavelength in samples treated with this
ROS.
As shown in Fig. 1, vitamin B6 has essentially the same
chemical structure as pyridoxamine, except for a -CH2OH
group in the para position of the pyridinium ring instead of the
-CH2NH2 group. This difference is important
because a reaction of HOCl with the primary amine,
-CH2NH2, of pyridoxamine is favored at pH 7.2 (58) over a reaction with the nitrogen of the pyridinium ring. The reaction of HOCl with the primary amine of pyridoxamine would produce
an N-chloramine/-CH2NHCl
group/N-chloropyridoxamine. In contrast to HOCl, the
reactivity of O2(1g) with the nitrogen of
the pyridinium ring has been reported (59). Despite the potential of
O2(1g), and possibly HOCl, to react with the
ring nitrogen, there was essentially no reaction of
H2O2 or H2O2/HOCl and
little reaction of HOCl with vitamin B6 in the presence or
absence of NaCl, although the degree to which HOCl reacted with vitamin
B6 varied (data not shown).
Oxidation of Pyridoxamine and Vitamin B6 at pH 5.5 ± 0.2--
The pH of the phagolysosomes of neutrophils during the
first 15 min following the ingestion of opsonized particles is 7.4-7.8 (60, 61), but after 15 min, the pH within the phagolysosomes, and
presumably the underlying extracellular attachment site, decreases to
pH 5.5-6.0. pH 5.5 is also the optimal pH for the generation of
Cl2 and O2(1g) by HOCl in
solutions containing 0.1 M NaCl (53-54). To evaluate the
reactivity of HOCl, H2O2, or
H2O2/HOCl with pyridoxamine and vitamin
B6 at pH 5.5 ± 0.2, ROS were added to 15 µM pyridoxamine in 0.5 M sodium phosphate
buffer ± 0.1 M NaCl. Pyridoxamine or vitamin
B6 control and treated samples were then incubated at RT for 15 min. The UV absorbance data for
a typical pyridoxamine experiment is presented in Fig. 4. At pH
5.5 the absorbance of pyridoxamine at 217 and 325 nm, and to a lesser
extent at 252 nm, decreased after treatment with HOCl (Fig. 4,
A and B), and a new absorbance peak at 228-229
nm, which is within the range of known N-chloramine
absorbance peaks, was formed (27).
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
The UV absorbance spectra of 15 µM pyridoxamine in 0.5M
sodium phosphate buffer ± 0.1 M NaCl, pH 5.5, reacted
for 15 min at RT with 0 µM (),
12.5 µM (), 25 µM (), or 50 µM () HOCl in the presence or absence
of 0.1 m NACL (A) OR 15 µM pyridoxamine reacted with 500 µM H2O2 (),
0 µM
H2O2/HOCl (), 125 µM, 12.5 µM H2O2/HOCl
(), 250 µM, 25 µM H2O2/HOCl
(), or 500 µM, 50 µM H2O2/HOCl
() in the presence or absence of 0.1 M NaCl
(B). The UV absorbance spectra of 15 µM vitamin B6 in 0.5 M sodium
phosphate buffer ± 0.1 M NaCl, pH 5.5, reacted for 15 min at RT with 0 µM (), 12.5 µM (),
25 µM (), or 50 µM HOCl () in the
presence of 0.1 M NaCl (C) or without NaCl
present (D). Graphs present a representative set
of data from experiments that were repeated three times.
|
|
At pH 5.5, HOCl also reacted with vitamin B6, which
resulted in a decrease in the absorbance at 292 and 325 nm (Fig.
4C). In the absence of NaCl, vitamin B6 samples
treated with HOCl showed only a slight decrease in absorbance in
response to 50 µM HOCl (Fig. 4D), suggesting
that either Cl2 or O2(1g) are
involved in this reaction.
The fluorescence data for all samples are presented in Fig. 3,
C and D, as the mean ± S.E.
(n = 3, **p < 0.01, *p < 0.05). The fluorescence of pyridoxamine (Fig. 3C) and
vitamin B6 (Fig. 3D) at 400 nm was decreased in
response to either HOCl or H2O2/HOCl treatments. The decrease in absorbance (Fig. 4) and fluorescence (Fig.
3) of both pyridoxamine and vitamin B6 suggests that the ring structure is disrupted by this reaction.
N-Chloramine Formation in HOCl-treated Samples--
To determine
whether the reaction of HOCl with pyridoxamine or vitamin
B6 leads to the formation of N-chloramines, 15 µM pyridoxamine or vitamin B6 in 0.5 M sodium phosphate ± 0.1 M NaCl, pH 7.2, was reacted with increasing concentrations of HOCl. The oxidation of
potassium iodide (KI) to triiodide by HOCl or by
N-chloramines formed in the reaction of HOCl with
pyridoxamine was then determined after incubation for 15 min at RT.
In the absence of NaCl, HOCl in solution at pH 7.2 was slow to
decompose, and the direct oxidation of KI by HOCl remained high even
after 24 h incubation at 37 °C (Table
I). Due to the high background, the
formation of N-chloramines by HOCl in the absence of NaCl
could not be determined, although an immediate reaction of HOCl with
pyridoxamine could be observed as a decrease in KI oxidation in these
samples (data not shown).
View this table:
[in this window]
[in a new window]
|
Table I
N-Chloramine formation determined by the oxidation of potassium iodide
after the reaction of pyridoxamine with HOCl (0.5 M
sodium phosphate, pH 7.2)
The data are presented as the mean ± S.E. of a set of experiments
repeated three times. Statistical significance of differences between
control and treatment values was determined by a pairwise comparison of
correlated groups using Student's t test.
|
|
In the presence of NaCl, there was an immediate decrease in background
oxidation of KI by unreacted HOCl and the production of
N-chloramines could be detected, after 15 min, in
pyridoxamine samples reacted with 50 µM HOCl (Table I).
As would be expected from the limited effect of HOCl on the absorbance
and fluorescence of vitamin B6, there was no formation of
N-chloramines in the reaction of HOCl with vitamin
B6 at pH 7.2.
Despite the more rapid decomposition of HOCl or the rapid reaction of
HOCl with NaCl at pH 5.5, the formation of N-chloramines could not be determined at this pH. The increased oxidation of KI under
these conditions may be due to the production of
O2(1g).
Reaction of Chloramine-T with Pyridoxamine and Vitamin
B6--
To evaluate further the formation of
N-chloropyridoxamine in the reaction of HOCl with
pyridoxamine, the absorbance of chloramine-T in 0.5 M
sodium phosphate buffer, pH 7.2 and 5.5, was determined (Fig.
5A). The absorbance peaks for
chloramine-T were 219-224 nm and corresponded with the initial
absorbance peaks of HOCl-treated pyridoxamine samples (Fig. 2),
indicating the reaction of HOCl with pyridoxamine generates
N-chloropyridoxamine. The absorbance of unreacted
chloramine-T remained constant at both pH values. To evaluate the
reaction of N-chloramines with pyridoxamine or vitamin
B6, increasing concentrations of chloramine-T were added to
15 µM pyridoxamine or vitamin B6 in 0.5 M sodium phosphate buffer, pH 7.2 or 5.5, and the samples
were incubated for 15 min RT, 2 h at 37 °C, or overnight at
37 °C. Chloramine-T reacted with pyridoxamine at pH 7.2 as indicated
by the concentration-dependent shifts in the major
absorbance peak of pyridoxamine from 325 to 312-318 nm (Fig. 5,
B and C). There was essentially no reaction of
chloramine-T with vitamin B6.
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
The UV absorbance peaks for 0 µM (), 25 µM (), 50 µM (), 75 µM (), and 100 µM () chloramine-T in 0.5 M sodium phosphate buffer ± 0.1 M NaCl at
pH 7.2 (A) or pH 5.5 (C). One major peak at 219-224 nm
characterized the absorbance for chloramine-T. The UV absorbance for 15 µM pyridoxamine reacted with increasing concentrations of
chloramine-T ± 0.1 M NaCl for 15 min RT at pH 7.2 (B) or pH 5.5 (C). The UV absorbance scans for 15 µM vitamin B6 reacted with increasing
concentrations of chloramine-T in 0.5 M sodium phosphate
buffer pH 5.5 ± 0.1 M NaCl for 15 min at RT
(D), and then incubated overnight (16-18 h) at
37 °C.
|
|
At pH 7.2, the reaction of chloramine-T with pyridoxamine was verified
using the N-chloramine assay as described above. There was a
decrease over time in the amount of chloramine-T available to oxidize
KI after reaction with pyridoxamine, but not vitamin B6 at
pH 7.2 (data not shown). The reaction of chloramine-T with pyridoxamine
was immediate and continued to increase over time.
At pH 5.5, chloramine-T reacted with both pyridoxamine (Fig.
5C) and vitamin B6 (Fig. 5D),
although the vitamin B6 reaction was not immediate. The
reactivity of chloramine-T with vitamin B6 suggests that
the nitrogen in the pyridinium ring is reactive at this pH, although to
a lesser degree compared with the primary amine group of pyridoxamine.
Thermal Release of O2(1g) from
DNE--
DNE, a pure chemical source of
O2(1g), can provide a reaction system that
does not rely on the presence of other ROS that may interfere or react
with the target of interest to evaluate more definitively a reaction of
O2(1g) with pyridoxamine or vitamin
B6. We incubated 200 µl of 15 µM
pyridoxamine or vitamin B6 in 0.5 M sodium
phosphate containing 0.1 M NaCl, pH 7.2 or 5.5, on a
DNE-coated coverslip. Duplicate samples were incubated for 24 h at
4 or 37 °C, and the absorbance readings were compared. Control
coverslips were coated with the dichloromethane solvent. There were no
changes in the absorbance of either pyridoxamine or vitamin
B6 in response to O2(1g) (data
not shown). The release of O2(1g) was
verified using AAP (44). After 24 h at 4 °C, the absorbance of
AAP at 400 nm was 0.715 ± 0.003, and AAP incubated on a
DNE-coated coverslip had an absorbance of 0.702 ± 0.005. For
duplicate samples incubated at 37 °C, the absorbance of AAP after
24 h on control coverslips was 0.718 ± 0.003, and the
absorbance of AAP incubated on DNE-coated coverslips was 0.588 ± 0.008. The decrease in absorbance of AAP on DNE-coated coverslips after
incubation at 37 °C confirmed that O2(1g)
was released and reacted with AAP in the aqueous buffer systems used in
our study. Taken together these findings eliminate
O2(1g) in the reaction of HOCl with either
pyridoxamine or vitamin B6 at pH 5.5, leaving
Cl2 and HOCl as the remaining reactants.
Gas Chromatography and Mass Spectrometry--
Samples of vitamin
B6 and vitamin B6 reacted with 50 µM HOCl for 15 min were analyzed by GC-MS as described
under "Experimental Procedures." Fig.
6 presents the results of these analyses.
Electron ionization-mass spectrometry of the vitamin B6-GC
peak showed two peaks at 12.73 and 13.42 (Fig. 6A). After
reaction with 50 µM HOCl at pH 5.5 (Fig. 6B),
four chlorinated products were identified at 11.03, 12.29, 13.17, and
14.19 min as
4-chloro-2-hydroxymethyl-2,4-hexadiene-3-carboxaldehyde, 5-chloromethyl-3-hydroxy-4-hydroxymethyl-1,3,5-hexatriene,
N-chloro-3-chloromethyl-4-hydroxy-2-hydroxymethyl-1-imino-2,4-pentadiene, and 3-chloro-4,5-dihydroxymethyl-2-methylpyridine
(3-chloropyridinium), respectively. The product at 13.57 min was
not chlorinated, and no further characterization was carried out.
View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Gas chromatography and electron
ionization-mass spectrometry (GC-MS) of vitamin B6
(A) and vitamin B6 reacted with 50 µM HOCl for 15 min at pH 5.5 (B). Two peaks at 12.73 and 13.42 min
characterized the GC-MS for vitamin B6 (A), and
four chlorinated products were identified at 11.03, 12.29, 13.17, and
14.19 min for vitamin B6 (B) after reaction with
HOCl and were identified as
4-chloro-2-hydroxymethyl-2,4-hexadiene-3-carboxaldehyde,
5-chloromethyl-3-hydroxy-4-hydroxymethyl-1,3,5-hexatriene,
N-chloro-3-chloromethyl-4-hydroxy-2-hydroxymethyl-1-imino-2,4-pentadiene,
and 3-chloro-4,5-dihydroxymethyl-2-methylpyridine (3-chloropyridinium),
respectively. The product at 13.57 min was not chlorinated, and no
further characterization was carried out.
|
|
Carbonyl (Aldehyde and Ketone) Formation after Exposure of Collagen
to H2O2, HOCl, or
H2O2/HOCl--
To evaluate the oxidation of
human collagen types I (bone) and II (articular cartilage) by
H2O2, HOCl, or
H2O2/HOCl, 50 µg of collagen in 0.5 M sodium phosphate buffer containing 0.1 M NaCl, pH 7.2 or pH 5.0, were exposed for 1 h at 37 °C to 125, 250, or 500 µM H2O2, 12.5, 25, or
50 µM HOCl, or a combination of
H2O2/HOCl in a ratio of 10:1. The above
concentrations of HOCl and H2O2 are within the
predicted range generated by activated neutrophils or monocytes at
sites of inflammation (6). A ratio of 10:1 was used in the present
study because the amounts of HOCl generated by activated neutrophils is
5-20 times less than relative amounts of H2O2
generated by the same cells stimulated under the same conditions (6).
The pH values were chosen based on the neutral pH of the neutrophil
phagolysosomes during the first 15 min following the ingestion of
opsonized particles (60, 61) and the acidic pH after 15 min (62).
To determine the oxidation of primary amines or pyridinoline
cross-links by H2O2 or HOCl,
immuno-chemiluminescence analysis (Oxyblot kit) was used to assess the
presence of carbonyl groups within the protein of interest. A
concentration-dependent increase in the number of reactive
carbonyl groups in both collagen types was found after exposure to
HOCl, with the amount varying depending on collagen type. The results
for collagen reacted with 500 µM H2O2, 50 µM HOCl, or both are
presented in Fig. 7. Lesser amounts of
carbonyls were generated in the reaction of 25 and 12.5 µM HOCl with both collagen types (data not shown). In
general, collagen type I was less reactive at both pH 5.0 and pH 7.2 as
compared with collagen type II, as indicated by relative mean density
(Fig. 7).
View larger version (48K):
[in this window]
[in a new window]
|
Fig. 7.
50 µg of human
collagen type I or type II were incubated at pH 5.0 or pH 7.2 for
1 h at 37 °C in 0.5 M sodium phosphate buffer
(control) or buffer containing H2O2 and/or HOCl
as indicated. After incubation, 0.1 µg of each sample was
analyzed for carbonyl content. Only samples reacted with HOCl showed
increased carbonyl formation. The immuno-chemiluminescence (Oxyblot
kit) data is presented for a typical experiment performed three times.
The bar graph shows the relative mean density ± S.E.
for all three experiments.
|
|
No change in carbonyl content was observed relative to untreated
collagen control samples after treatment of either collagen type with
H2O2 or a combination of
H2O2/HOCl (Fig. 7). Adding H2O2 and HOCl together decreased the effect of
HOCl, suggesting that H2O2 is reacting with
HOCl and decreasing the availability of HOCl to react with collagen.
Pyridinoline Cross-link and Aromatic Amino Acid Fluorescence of
Collagen before and after Exposure to HOCl--
To assess oxidation of
the pyridinoline cross-links and aromatic amino acids of collagen types
I and II by HOCl, the fluorescence emission of HOCl-treated collagen
was measured and compared with untreated control samples subjected to
the same conditions. The fluorescence of collagen type I samples was
below measurable levels, and no further fluorescence studies were done
on this collagen type. Exposure of 0.125 mg/ml collagen type II to HOCl
at pH 5.0 and 7.2 for 15 min at 37 °C resulted in a
concentration-dependent decrease in fluorescence emission
at 400 nm (excitation 325 nm) (Table II).
No other changes in fluorescence emission or excitation were
observed.
View this table:
[in this window]
[in a new window]
|
Table II
Exposure of human articular collagen type II to HOCl resulted in a
decrease in fluorescence emission at 400 nm (excitation 325 nm) after a
15-min incubation at 37 °C
Data are presented as a percentage decrease in fluorescence emission
relative to unreacted collagen incubated under the same conditions.
Values represent the mean ± S.E. (n = 2).
|
|
SDS-PAGE Results of Collagen Types I or II Reacted with
H2O2, HOCl, or a Combination of
H2O2/HOCl--
After exposure to ROS as
described above, 4-µg aliquots of each collagen sample were subjected
to SDS-PAGE analysis (4-15% gradient) and stained with silver.
Results of a representative gel of an experiment performed three times
are presented in Fig. 8. Exposing
collagen types I (Fig. 8A, lanes 3 and 7) or II
(Fig. 8B, lane 3) to 50 µM HOCl resulted in a
60-80% decrease in the intensity of collagen electrophoretic band
staining by silver (arrows indicating form top to bottom,
, , and ). Except at pH 7.2, the decrease in silver staining
for collagen type II reacted with HOCl was much less (Fig. 8B,
lane 7). Concentration-dependent decreases in silver
staining are shown in Fig. 8, C and D, for collagen reacted with HOCl at pH 5.0 and 7.2. No smearing
(fragmentation) or low molecular weight bands were observed after any
treatment. A decrease in reactivity of silver with the HOCl-reacted
collagen monomers is consistent with the formation of
N-chloroamines (33) in the reaction of HOCl with collagen
and ultimately the spontaneous deamination and decarboxylation of
N-chloramines (-amino groups) to form aldehydes (26, 63),
thus suggesting that the collagen monomers are still intact but have
been oxidatively modified and no longer react with silver. The
oxidative modification of amine groups by HOCl would no longer make
them available for reaction with silver.
View larger version (52K):
[in this window]
[in a new window]
|
Fig. 8.
50-µg samples of
collagen type I (A )and collagen type II
(B) were reacted at pH 5.0 and pH 7.2 or collagen
types I or II were reacted at pH 5.0 (C) or pH 7.2 (D) for 1 h at 37 °C in 0.5 M
sodium phosphate buffer alone or buffer containing
H2O2 and/or HOCl as indicated. After ROS
exposure, 4 µg of human collagen type I, type II, or
[14C]bovine serum albumin
(14C-BSA) were subjected to SDS-PAGE
analysis and stained with silver. E, 14C-BSA was
reacted at pH 7.2 in buffer alone or buffer containing HOCl at 12.5 (lane 2), 25 (lane 3), or 50 µM
(lane 4). Exposing collagens type I, type II, or
[14C]BSA to 12.5-50 µM HOCl resulted in a
concentration-dependent decrease in the intensity of the
electrophoretic band staining by silver (arrows) without
causing a loss of protein as indicated by the single and equally
intense (F) autoradiograph band for [14C]BSA
in lanes 1-4. Lanes marked M contain molecular
mass markers (kDa). The presented data represent typical results
of an experiment performed three times.
|
|
To determine if the decrease in silver reactivity with collagens
after oxidation by HOCl is due to oxidative modification of primary
amine groups and not protein fragmentation or both, we reacted
14C-labeled bovine serum albumin (BSA) with increasing
concentrations of HOCl (12.5-50 µM). After exposure to
HOCl as described above, 4-µg aliquots of [14C]BSA were
subjected to SDS-PAGE analysis (10% acrylamide), stained with silver,
then dried and exposed to autoradiographic film. Silver staining and
autoradiogram results are presented in Fig. 8, C and
D, respectively. A concentration-dependent
decrease in silver staining intensity was observed after treatment of
[14C]BSA with 12.5-50 µM HOCl, without
affecting the autoradiogram band intensity of these same samples. This
provided us with some supportive evidence that the decrease in collagen
staining after reaction with HOCl is due to oxidative modification
rather than protein fragmentation or a combination of both processes.
Fluorescamine and o-Phthalaldehyde Primary Amine and Imino Acid
Measurements--
To determine if the decrease in detectable
electrophoretic band staining was the result of complete collagen
fragmentation by HOCl, aliquots of each collagen sample, acetone
precipitates of each sample, and sample supernatants of acetone
precipitates were analyzed by fluorescamine and
o-phthalaldehyde assays. Both assays detect primary amines
and imino acids, which would increase if collagen were fragmented.
However, neither assay detected an increase in free amine or imino acid
groups in any of the collagen samples.
| |
DISCUSSION |
ROS are produced and released into extracellular spaces and
contribute to the development and progression of inflammatory diseases.
However, little is known about direct ROS-induced molecular modifications of individual matrix proteins and the consequence of
these modifications on the structure, physical properties, or function
of the matrix. The present study tested the hypothesis that HOCl
participates in the inflammatory-mediated loss of connective tissue
collagen by oxidizing the Pyd cross-links found in abundance in adult
articular cartilage. HOCl, produced by the enzyme MPO, is the major
highly reactive oxidant produced by activated neutrophils and to a
lesser extent by monocytes and some macrophages. Based on its
reactivity, HOCl has the potential to cause the bulk of the tissue
damage at sites of acute inflammation (64). Pyd cross-links are of
particular importance because their function is to help maintain the
structure of the collagen fibrils and make them more resistant to
collagenolysis or proteolytic degradation (37, 65). Our current
findings indicate that OCl/Cl2, and to a
lesser extent N-chloramines, chlorinate pyridinium compounds
with structures similar to Pyd and that these HOCl species react with
both collagen types I and II, resulting in the oxidation of amine
groups and Pyd cross-links.
Our findings indicate that HOCl rapidly reacts with and chlorinates
pyridinium compounds, but the chlorination reaction and the
N-chlorination sites are pH-dependent. At pH
7.2 ± 0.2, HOCl/OCl preferentially reacted with the
para-CH2NH2 group of pyridoxamine to
form N-chloropyridoxamine, which was detected using the KI oxidation assay for N-chloramine and by the appearance of
the characteristic N-chloramine absorbance peak at 220-225
nm. In pyridoxamine samples treated with HOCl, a second absorbance peak at 307-320 nm was observed. This peak corresponds to a peak observed for the product that results from the reaction of pyridoxamine with
chloramine-T, a commercial N-chloramine standard. Based on the presence of these two peaks, we suggest that as soon as
N-chloropyridoxamines are formed in the reaction of
pyridoxamine and HOCl, they in turn react with other pyridoxamine
molecules and initiate the formation of
N-chloropyridoxamine-pyridoxamine dimers. The reaction of
HOCl/OCl with the
para-CH2NH2 of pyridoxamine is in
agreement with the report of Davies et al. (33) stating that
the preferred reaction of HOCl/OCl with collagen at
neutral pH is with the primary amine groups.
At pH 5.5 ± 0.2 and in the presence of Cl, HOCl,
and N-chloramines reacted with both pyridoxamine and vitamin
B6. The reactivity of HOCl at this pH with the pyridinium
compounds is consistent with the reported increase in reactivity of
OCl with compounds that possess extensive p
electrons (ring nitrogen) (66). The significant loss in absorbance and
fluorescence of either compound after reaction with HOCl at pH 5.5 suggests that a percentage of the pyridinium ring structure was
disrupted in these reactions. Disruption of the ring structure was
confirmed by GC-MS analysis and is consistent with the formation of an
aldehyde as a result of the spontaneous deamination and decarboxylation of an N-chloramine (in this case the ring nitrogen) to form
an aldehyde (58, 63).
The reactivity of HOCl with vitamin B6 at pH 5.5 in the
presence of Cl is also consistent with the evolution of
Cl2 (55). GC-MS analysis also identified 3-chloropyridinium
as one of the products of this reaction, similar to a previous report
of 3-chlorotyrosine formation in the reaction of Cl2 with
the aromatic amino acid, L-tyrosine (27). The reactivity of
Cl2 is also of interest because it has been demonstrated
that neutrophils generate Cl2 via the
MPO-H2O2-Cl system (66).
In contrast to HOCl, H2O2 alone was without
effect under any condition, which is in keeping with its low reactivity
with biological molecules. The third of the nonradical species tested
in this study, O2(1g), is a relatively long
lived (2 µs) and highly reactive oxidant produced by HOCl at pH 5.5 and to a lesser extent at pH 7.2 in the presence of Cl
(55). Because O2(1g) would be produced by
both of these ROS systems, it would seem likely that
O2(1g) would react with the pyridinium ring
nitrogen (59). However, O2(1g) did not
significantly contribute to the derivatization of pyridoxamine, and at
pH 7.2 the production of O2(1g) by the
reaction of H2O2 with HOCl actually interfered
with the reaction of HOCl and pyridoxamine. Similarly, at pH 5.5 HOCl should generate maximal amounts of O2(1g) in
the presence of NaCl (55); however, even at this pH our findings
indicate that HOCl and not O2(1g) is the
major reactant with either pyridoxamine or vitamin B6.
Finally, the thermal release of O2(1g) by
DNE, a pure chemical source of O2(1g), did
not result in an absorbance or fluorescence change in the pyridoxamine
or vitamin B6 spectra. It has been reported that O2(1g) can react with pyridinium compounds,
resulting in cleavage of the nitrogen-carbon bond between the
nitrogen-containing pyridinium ring and the terminal carbon of a
substituted group at this site (59). This reaction leaves the
pyridinium ring structure intact and unchanged. Although we did not
detect a reaction of O2(1g) with an
unsubstituted nitrogen group in the pyridinium rings of pyridoxamine
and vitamin B6, it is still possible that a reaction between O2(1g) and the Pyd cross-links could
take place in vivo when the nitrogen is covalently linked to
the triple helical region of a collagen molecule. This type of reaction
would also result in the disruption of the intermolecular bond between
two molecules of collagen.
In a previous study by Davies et al. (33) the oxidation of
bovine collagen type I isolated from tendon and collagen type II
isolated from articular cartilage was assessed as the amount of
collagen fragmentation taking place in response to HOCl or N-chloramines. Only at superphysiological concentrations of
1-5 mM did HOCl cause extensive fragmentation (smearing)
of collagen. In contrast, the addition of N-chloramines
(5-50 µM) did not cause fragmentation but, instead,
greatly increased the degradation of collagen by collagenase and
elastase. The mechanism by which N-chloramines increased the
proteolytic susceptibility of collagen was not specifically determined,
although it was assumed that N-chloramines were reacting
with amine groups and disr
, and
TOP
ABSTRACT
INTRODUCTION
