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J. Biol. Chem., Vol. 275, Issue 28, 21177-21184, July 14, 2000
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From the
Received for publication, April 17, 2000, and in revised form, May 8, 2000
Maillard or browning reactions lead to formation
of advanced glycation end products (AGEs) on protein and contribute to
the increase in chemical modification of proteins during aging and in
diabetes. AGE inhibitors such as aminoguanidine and pyridoxamine (PM)
have proven effective in animal model and clinical studies as
inhibitors of AGE formation and development of diabetic complications. We report here that PM also inhibits the chemical modification of
proteins during lipid peroxidation (lipoxidation) reactions in
vitro, and we show that it traps reactive intermediates formed during lipid peroxidation. In reactions of arachidonate with the model
protein RNase, PM prevented modification of lysine residues and
formation of the advanced lipoxidation end products (ALEs) N Aging and age-related diseases, such as diabetes, atherosclerosis,
and neurodegenerative diseases, are characterized by increases in
oxidative chemical modification of tissue proteins (1, 2). Some
oxidation products, e.g. methionine sulfoxide and
o-tyrosine, are formed by direct oxidation of amino acids in
proteins. Glycoxidation products or advanced glycoxidation end products
(AGEs),1 such as the
cross-links pentosidine and vesperlysines (3, 4), are produced by
secondary modification of proteins by products of carbohydrate
oxidation. Advanced lipoxidation end products (ALEs), including
malondialdehyde (MDA) and hydroxynonenal (HNE) adducts to lysine
(MDA-Lys, HNE-Lys) (5) and pyrroles (6), are formed during lipid
peroxidation reactions, whereas other compounds, such as
N The AGE inhibitor PM was introduced by Hudson and colleagues (12, 13)
as an inhibitor of AGE formation from Amadori compounds, the first
stable sugar-protein adducts formed during glycation of protein. In
animal model studies, PM also inhibited AGE formation and
retarded the development of nephropathy
in streptozotocin-induced diabetic
rats.2,3
However, the renal protection was accompanied by a significant reduction in hypertriglyceridemia, and there were strong correlations between plasma triglyceride concentration and the levels of CML and
CEL, fluorescence, and cross-links in skin collagen. These observations
suggested that, in addition to its AGE-inhibitory activity, PM might
also affect lipid homeostasis and lipid-dependent chemical
modification of protein in vivo. In this paper we describe experiments in vitro on the effects of PM on the chemical
modification of proteins during lipid peroxidation reactions, and we
show that PM is a potent inhibitor of modification of lysine by
peroxidizing lipids, both in a model protein-lipid system and during
copper-catalyzed oxidation of LDL. We also identify the major products
formed on reaction of linoleic acid (LA) with PM under autoxidative
conditions, the hexanoic acid amide and nonanedioic acid monoamide
derivatives of PM, and we propose a mechanism for PM inhibition of
chemical modification of proteins during lipoxidation reactions. Our
results provide insight into the mechanism of action of PM as an AGE
and ALE inhibitor, and we suggest that PM may be useful for inhibiting the increased chemical modification of tissue proteins, whether derived
from carbohydrate or lipids, in diabetes, atherosclerosis, and other
chronic diseases.
Chemicals--
Arachidonic acid (AA), LA, oleic acid, palmitic
acid, PM·(HCl)2, pyridoxal·(HCl), pyridoxine·(HCl),
ribonuclease A (type II-A from bovine pancreas; RNase),
diethylenetriaminepentaacetic acid (DTPA), phytic acid, butylated
hydroxytoluene, NaBH4, hexanoyl (caproyl) chloride, methyl
linoleate, and nonanedioic (azelaic) acid monomethyl ester were
purchased from Sigma. Boron trichloride-methanol was
purchased from Pierce and heptafluorobutyric acid from Acros Chemicals (Atlanta, GA). [2H4]- and
[2H8]lysine were from Isotec
(Miamisburg, OH). Deuterated and natural abundance internal
standards (CML and [2H4]CML, CEL and
[2H4]CEL, MDA-Lys and
[2H8]MDA-Lys, and HNE-Lys and
[2H4]HNE-Lys) were prepared as described
previously (5, 18, 19).
Reaction of PM with Fatty Acids--
PM (1 mM) was
incubated with AA, LA, or oleic acid (5 mM) in 5 ml of
filter-sterilized 200 mM sodium phosphate buffer, pH 7.4, in 20-ml scintillation vials for 6 days at 37 °C in a shaking water
bath. Reactions were initially biphasic but, in the case of the
polyunsaturated fatty acids (PUFA) AA and LA, became monophasic by day
3. Aliquots were removed from the aqueous phase and stored at
Effect of PM on Oxidation of LA--
LA (5 mM) was
oxidized alone or in the presence of PM (1 mM), as
described above. Individual reaction vessels were removed at various
times (0, 1, 3, and 6 days); DTPA was added (final concentration = 1 mM from 100 mM stock), and the samples were then stored at Reaction of RNase with AA--
RNase (1 mM, 13.7 mg/ml; equivalent to 10 mM lysine concentration) was
reacted with AA (100 mM) in the absence or presence of PM
(1 mM) in 5 ml of 0.2 M sodium phosphate
buffer, pH 7.4. The reactions were prepared under sterile conditions,
using ultrafiltered solutions. Aliquots were removed at 0, 1, 3, and 6 days, DTPA was added (final concentration = 1 mM), and
the samples were frozen at Oxidation of LDL--
LDL was isolated from plasma of healthy
donors by single-vertical spin centrifugation and used within 24-48 h
of preparation, as described previously (18). LDL (50 µg of
protein/ml) was incubated at 37 °C in phosphate-buffered saline
alone or in the presence of 5 µM CuCl2 or 5 µM CuCl2 plus PM (100-250 µM).
Formation of conjugated dienes, an index of lipid peroxidation, was
monitored at 234 nm. Aliquots corresponding to ~1 mg of LDL protein
were removed at various times, reduced with NaBH4, and
hydrolyzed for amino acid analysis and measurement of ALEs, as
described previously (18).
Other Methods--
N-Hexanoyl-pyridoxamine was
synthesized from PM·(HCl)2 and hexanoyl chloride. PM (20 mg) was dissolved in 50 ml of 2 M NaOH in a 250-ml round
bottom flask. Hexanoyl chloride (22 mg in 30 ml of dry ether) was added
dropwise over an hour while the reaction was kept on ice and stirred
vigorously. The reaction was stirred for an additional 2 h and
then the ether layer was removed and dried under nitrogen, yielding a
white powder. N-Hexanoyl-PM was identified and isolated by
RP-HPLC with detection by absorbance and electrospray ionization/mass
spectrometry (ESI-LC/MS), as described below.
N-Nonanedioyl-pyridoxamine was synthesized according to
D'Alelio and Reid (23). Briefly, 0.3 g of PM in 0.3 ml of
deionized water was added to 0.3 g of azelaic acid monomethyl
ester and heated at 140 °C for 10 h. The brown reaction mixture
was dried in vacuo and reconstituted in deionized
water:acetonitrile (1:1, v/v). N-Nonanedioyl-PM was
identified and isolated by RP-HPLC with detection by absorbance and
ESI-LC/MS, as described below.
N-Acyl-PM derivatives were hydrolyzed in 2 M HCl
for 4 h at 95 °C, releasing free PM and carboxylic acids. The
hydrolysate was dried in vacuo, and the resulting free
carboxylic acids (hexanoic and nonanedioic acids) were analyzed by
GC/MS as their propyl and dimethyl esters, respectively. Esterification
was performed in 1 ml of 1 M HCl in n-propyl
alcohol or methanol at 65 °C for 1 h. After drying in
vacuo, the esters were extracted into 2 ml of hexane:water (2:1,
v/v). Following vortexing and centrifugation, the hexane phase was
analyzed by GC/MS, as described below. The aqueous phase, containing
PM, was dried in vacuo, and the PM was acetylated by
reaction with acetic anhydride:pyridine (1:1, v/v) for 2 h at room
temperature. Following evaporation in vacuo, the product was
reconstituted in methylene chloride for GC/MS analysis.
Mass Spectrometry--
ESI-LC-MS was carried out in the positive
ion mode on a VG (Manchester, UK) Trio-3 triple quadrupole mass
spectrometer equipped with a Hewlett-Packard (Palo Alto, CA) series
1100 HPLC system and a Keystone (Bellefonte, PA) Aquasil C-18 microbore
column. The solvent system consisted of 0.1% heptafluorobutyric acid
(solvent A) and acetonitrile (solvent B), flow rate 0.05 ml/min. The
gradient used was as follows: 100% A, hold for 2 min; 6 min ramp to
60% B, hold for 4 min; 3 min ramp to 75% B, hold for 10 min. GC/MS was carried out on a Hewlett-Packard model 6890 gas chromatograph/5970 mass selective detector, using a 30-meter HP-5MS (5% phenyl methyl siloxane) capillary column (Restek, Bellefonte, PA). The temperature program for analyses of PM and carboxylic acid esters was as follows: initial temperature 75 °C, 6 °C/min ramp to 110 °C,
10 °C/min ramp to 180 °C hold 5 min, 12 °C/min ramp to
270 °C/min, hold 5 min. Fast atom bombardment-MS was performed on a
VG (Manchester, UK) 70 SQ high resolution, magnetic sector mass spectrometer.
Reaction of PM with PUFAs--
To study the interaction of PM with
products of lipid peroxidation, PM was incubated with oleate or PUFA in
phosphate buffer at physiological pH, relying on endogenous metal ions
in the phosphate buffer to catalyze PUFA oxidation. As shown in Fig.
1A, PM incubated alone or in
the presence of oleate was recovered unchanged over the 6-day
incubation period. However, in the presence of LA and AA, PM was
gradually consumed, with ~60% PM loss at 6 days, based on HPLC
analysis (see inset to Fig. 4). When the same samples were
analyzed by the TNBS assay (Fig. 1B), ~60% of the free
amino groups of PM was also modified during peroxidation of PUFA. The close agreement between the loss of PM by HPLC and the loss of amino
groups by the TNBS reaction suggested that PM was reacting with
products of lipid peroxidation via its benzylic amino group. PUFA were
also solubilized at similar rates in the presence and absence of PM,
and as shown in Fig. 2, the kinetics
of formation and yield of TBARs and the rate of loss of LA, the primary
PUFA in LDL (see below), were only slightly affected by the presence of
PM in reaction mixtures at 1 mM concentration. These
experiments indicate that PM, at the concentration used (1 mM) in the above experiment, did not prevent lipid
peroxidation by chelation or antioxidant activity.
PM Inhibition of Lipoxidative Modification of RNase by AA--
The
activity of PM as an inhibitor of lipoxidative modification of proteins
was studied in reactions of AA with RNase. As shown in Fig.
3, CML, CEL, MDA-Lys, and HNE-Lys were
formed continuously on RNase during the incubation with AA. These
studies confirm previous reports on the formation of CML during lipid
peroxidation reactions (18) and also identify CEL as a product of
arachidonate oxidation. The decline in MDA-Lys recovered after 1 day
resembles that observed during copper oxidation of LDL and is thought
to result from further reaction of MDA-Lys to form other products, such
as Lys-MDA-Lys (5). Addition of 1 mM PM to the AA + RNase mixture resulted in almost complete inhibition of formation of all the
ALEs. Notably, PM provided potent protection against lipoxidative modification of RNase, although it was present at only 1% the concentration of AA, and 10% the concentration of lysine residues in
RNase. The small increase in formation of lipoxidation products by day
6 likely reflects the fact that all of the PM had been consumed by that
time, as shown in Fig. 4. Amino acid
analysis indicated that there was a progressive loss of lysine during
the reaction, with about 58% of lysine residues lost at the end of 6 days in the absence of PM (Fig. 5). In
contrast, inclusion of PM in the incubation resulted in loss of only
about 5% of lysine residues by day 6.
Inhibition of LDL Oxidation by PM--
Because PM inhibited
lipoxidation product formation in the AA + RNase system, we also
evaluated its effectiveness as an inhibitor of lipoxidation during
copper-catalyzed oxidation of LDL, a common in vitro model
for studies on lipoxidative modification of proteins. PM was used at
concentrations of 100 and 250 µM concentrations, comparable to the ~120 µM concentration achieved in
rats treated with 1 g/liter in drinking water.2,3 At these
concentrations, PM caused a 2-3-fold increase in the lag phase of LDL
oxidation and an ~20% decrease in absorbance at 234 nm during the
later plateau phase of the reaction (Fig. 6A). Confirming the results of
experiments with AA and RNase, PM was an effective inhibitor of
formation of CML, CEL, MDA-Lys, and HNE-Lys (Fig. 6B.)
during metal-catalyzed oxidation of LDL. The relative yields of the
ALEs during oxidation of LDL differed significantly from yields
obtained in reactions of AA with RNase, probably reflecting the
heterogeneous fatty acid composition of LDL. PM was more effective in
limiting formation of CML and CEL, compared with MDA-Lys and
HNE-Lys (Fig. 6B), but was a potent inhibitor of the
overall modification of lysine residues in oxidized LDL (Fig.
6C), with only 6% loss in the presence of PM compared with
29% in its absence.
Identification of PM Adducts--
To identify products trapped by
PM during PUFA oxidation, we studied the reaction of PM (1 mM) with LA, the primary PUFA in LDL. HPLC analyses of the
LA + PM reaction, described in Fig. 7,
indicated formation of products with fluorescence (Fig. 7, B
versus A) and absorbance (Fig. 7C)
maxima characteristic of PM. The consumption of PM over a period of 6 days of reaction is evident, as well as the time-dependent
formation of new products eluting later than PM, indicating that these
adducts were more hydrophobic than PM itself. Two major fluorescent
products were consistently observed, eluting at ~24 min. Based on
their fluorescence intensity, these two products accounted for
Semi-preparative RP-HPLC analysis (Fig. 7C) showed that a
number of additional products with absorbance maxima at 294 nm were also detected after 6 days of reaction. Those eluting after about 23 min (indicated by an asterisk) had absorbance maxima below 260 nm and were also formed from LA, in the absence of PM. However, products eluting between 19 and 22 min had extracted absorbance maxima
at 294 nm (photodiode array detector), consistent with the presence of
the intact pyridine ring. The peaks eluting in this region of the
chromatogram were pooled and subjected to direct injection ESI-MS.
Major products identified had m/z values of 267, 305, 339, 377, and 479 (Fig. 8A); none
of these were present in incubations of LA or PM alone. Assignment of
the various species to particular chromatographic peaks was
accomplished by collecting individual peaks by semi-preparative RP-HPLC
(Fig. 8B) and analysis by direct injection ESI-MS. Once peak
assignments were made, the kinetics of formation of the three most
abundant products (267, 305, and 339) were determined by RP-HPLC (Fig.
8C). The yields of all three compounds increased with time,
with products 267 and 339 consistently forming in the highest yields.
These products were chosen for further characterization.
Product Characterization--
Products 267 and 339 were identified
as the hexanoic acid amide and nonanedioic (azelaic) acid monoamide
derivatives of PM (24). The hexanoic acid amide derivative of lysine
has been identified previously by Kato et al. (25, 26) in
reactions of linoleic acid hydroperoxides with
N
The hexanoic acid amide and nonanedioic acid monoamide derivative of PM
were prepared synthetically, as described under "Materials and
Methods." Analysis of the synthetic products by RP-HPLC-ESI-MS confirmed that they co-eluted with products 267 and 339 obtained from
the reaction mixture and yielded identical molecular ions by ESI-MS
analysis. Acid hydrolysis (2 M HCl, 2 h, 95 °C) of
products 267 and 339 (both synthetic and from reaction mixtures) also
yielded PM by HPLC and either hexanoic or nonanedioic acid by GC/MS as the propyl or dimethyl ester, respectively (shown for reaction product
339 in Fig. 9, C and D). Both PM and the
respective carboxylic acid were formed in approximately equal
proportions (>90% yield), confirming the identity of products 267 and 339.
Product 305 has not yet been isolated in sufficient amounts for
complete characterization. Based on its molecular formula, determined
by high resolution fast atom bombardment-MS, product 305 is a 9-carbon
mono-oxygenated adduct to PM, possibly an epoxy or hydroxy acid
derivative of PM. It was not formed by reaction of HNE with PM in
phosphate buffer (data not shown). Product 479 was a minor reaction
product. Its molecular weight indicates that it is formed by adduction
of PM to an intact, dioxo-derivative of LA. Further characterization of
these and other reaction products is in progress.
PM Inhibition of Advanced Lipoxidation Reactions--
PM was
originally described as an AGE inhibitor and, like other AGE
inhibitors, is thought to act as a nucleophilic trap for reactive
carbonyl intermediates in AGE formation. Reactive carbonyl compounds
are also involved in the formation of ALEs, so it seemed likely that
this compound would also inhibit chemical modification of proteins
during lipid peroxidation reactions. Indeed, PM was a potent inhibitor
of chemical modification of both RNase and LDL during lipid
peroxidation reactions, inhibiting both the modification of lysine
residues and the formation of specific lipoxidation products, CML, CEL,
MDA-Lys, and HNE-Lys. Some weak antioxidant activity, a general
characteristic of phenolic compounds, was also observed, as judged by a
marginal effect of PM on the kinetics of oxidation of linoleate and
LDL. However, the gradual solubilization of the PUFA and chemical
modification of PM in reactions in either the absence or presence of
protein, the formation of TBARS and decomposition of LA (Fig. 2), as
well as the formation of conjugated dienes during oxidation of LDL
(Fig. 6) document that lipid peroxidation occurred, even in the
presence of PM. The overall observations were consistent with the
hypothesis that PM acts as a sacrificial nucleophile, trapping reactive
intermediates in lipoxidation (and glycoxidation) reactions. The
involvement of the amino group of PM in inhibition of ALE formation was
confirmed by the loss of TNBS reactivity in reactions of PM with
oxidizing PUFA (Fig. 1B).
The fact that PM protects against modification of lysine residues and
formation of lipoxidation products in RNase, despite the 100-fold
excess of PUFA and the 10-fold higher concentration of lysine residues
on RNase (Figs. 3 and 4), illustrates its efficiency in trapping
reactive carbonyl intermediates. The efficiency of PM in protecting
lysine amino groups suggests either that only a fraction of lipid
peroxidation products participate in chemical modification of protein
or that PM intercepts critical, early lipid peroxidation products,
preventing their decomposition to a broader range of shorter -chain
reactive carbonyl compounds. Consistent with the latter hypothesis,
none of the anticipated ALE derivatives of PM, e.g.
carboxymethyl-PM, carboxyethyl-PM, MDA-PM, or HNE-PM, were identifiable
in the PM + AA reaction mixture, based on scanning for their molecular
weight by LC-ESI-MS. Thus, it appears that, by trapping early
intermediates in glycoxidation and lipoxidation reactions, PM protects
against the formation of a range of later precursors of ALEs on proteins.
The inhibition of lipoxidation in the RNase model system suggested that
PM would exert similar effects during copper-catalyzed oxidation of
LDL. As shown in Fig. 6, despite the high rate of lipoprotein oxidation
catalyzed by 5 µM Cu2+ in this reaction
system, PM, at a pharmacologically relevant concentration, was an
effective inhibitor of chemical modification of LDL, including both the
formation of specific ALEs and the modification of lysine residues.
These inhibitory effects, coupled with the lipid lowering activity of
PM in diabetic rats,3 suggest that PM may be effective in
protecting against lipid-dependent, oxidative chemical
modification of proteins in vivo and may, therefore, have
anti-atherogenic activity. Experiments to assess the effects of PM on
formation of lipoxidation products in vivo and development of diabetic vascular disease are currently under way.
Mechanism of Action of PM--
PM is unique among current
inhibitors of advanced glycation reactions because of its
characteristic fluorescence spectrum. Thus, the carbonyl intermediates
trapped by PM are not only amenable to RP-HPLC analysis but are also
readily identifiable by fluorescence detection (Fig. 7). These features
of PM, combined with previous work of Kato et al. (25, 26),
facilitated the identification of the hexanoic acid amide and
nonanedioic acid monoamide derivatives of PM. These compounds are
clearly formed by adduction of PM to the 9- and 13-carbons of LA,
followed by oxidative cleavage of the 9-10 or 12-13 carbon-carbon
bonds (Scheme I). We propose that carbonyl species, such as 9- and 13-oxo-octadecadienoic acids (keto-octadecadienoic acids; 9- and 13-KODE) (27), formed from the
lipid peroxides (hydroperoxyoctadecadienoic acids) are involved as
intermediates in the reaction mechanism. The reaction pathway proposed
in Scheme I involves the formation of a carbinolamine adduct of PM to
the KODE, followed by metal-catalyzed, oxidative cleavage of the
carbon-carbon bond adjacent to a carbonyl group, yielding the hexanoic
amide from 13-KODE and nonanedioic acid monoamide from 9-KODE.
Theoretically, bond cleavage could occur on either side of the C-9- or
C-13 carbonyl group, yielding the alternative 10-carbon and 13-carbon
amide derivatives of PM; however, products with these molecular weights
were not detectable by RP-HPLC-ESI-MS. Thus, the mechanism of formation
of the hexanoic and nonanedioic acid adducts must account for selective
cleavage of the PM-LA intermediate on the conjugated, unsaturated side
of the alkane chain. We propose that this preferred cleavage is driven
by stabilization of an intermediate radical released from the
conjugated side of the hydrocarbon system during the cleavage
reaction.
In support of this mechanism, KODEs have been identified as products of
both non-enzymatic (27, 28) and enzymatic (29, 30) peroxidation of LA
and have also been detected by chemical methods in atherosclerotic
plaque (31). Thiol compounds are known to react directly with KODEs,
whereas their reaction with hydroperoxyoctadecadienoic acids requires
autoxidizing conditions (Fe3+ and O2) (16, 17).
PM also fails to react with lipid peroxides (prepared from LA using
soybean lipoxygenase) under anti-oxidative conditions (anaerobic
incubation in the presence of DTPA; data not shown.). Thus, PM may
function as a surrogate, non-thiol nucleophile, trapping and
inactivating KODE intermediates and thereby inhibiting further chemical
modification of proteins by peroxidizing lipids.
Concluding Remarks--
Based on the proposed mechanism of
formation of PM adducts, the hexamide derivative of lysine is likely to
be formed from any We acknowledge the helpful support of Drs.
William E. Cotham and Michael D. Walla, Mass Spectrometry
Center, Department of Chemistry and Biochemistry, University of South
Carolina. We also thank Dr. Gerhard Spiteller, University of Bayreuth,
Germany, for helpful discussions and suggestions regarding reaction mechanisms.
*
This work was supported by NIDDKD Research Grant DK-19971
from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom all correspondence should be addressed: Dept. of
Chemistry and Biochemistry, University of South Carolina, Columbia SC
29208. Tel.: 803-777-7272; Fax: 803-777-7272; E-mail:
baynes@mail.chem.sc.edu.
Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M003263200
2
T. P. Degenhardt, N. L. Alderson,
D. D. Arrington, R. J. Beattie, S. R. Thorpe, and
J. W. Baynes, submitted for publication.
3
T. P. Degenhardt, N. L. Alderson,
R. J. Beattie, J. M. Basgen, M. W. Steffes, S. R. Thorpe, and J. W. Baynes, submitted for publication.
The abbreviations used are:
AGE, advanced
glycation end product;
AA, arachidonic acid;
CEL, N
Pyridoxamine, an Inhibitor of Advanced Glycation Reactions, Also
Inhibits Advanced Lipoxidation Reactions
MECHANISM OF ACTION OF PYRIDOXAMINE*
,, and¶
Department of Chemistry and
Biochemistry, University of South Carolina,
Columbia, South Carolina 29208 and the § Department of
Medicine, Medical University of South Carolina,
Charleston, South Carolina 29425
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-(carboxymethyl)lysine,
N-(carboxyethyl)lysine,
malondialdehyde-lysine, and 4-hydroxynonenal-lysine. PM also inhibited
lysine modification and formation of ALEs during copper-catalyzed
oxidation of low density lipoprotein. Hexanoic acid amide and
nonanedioic acid monoamide derivatives of PM were identified as major
products formed during oxidation of linoleic acid in the presence of
PM. We propose a mechanism for formation of these products from the 9- and 13-oxo-decadienoic acid intermediates formed during peroxidation of
linoleic acid. PM, as a potent inhibitor of both AGE and ALE formation,
may prove useful for limiting the increased chemical modification of
tissue proteins and associated pathology in aging and chronic diseases,
including both diabetes and atherosclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-(carboxymethyl)lysine (CML) and
N-(carboxyethyl)lysine (CEL), are formed
during both glycoxidation and lipoxidation reactions (7). Age-adjusted
levels of pentosidine and CML are increased during chronic
hyperglycemia and correlate with the severity of renal, retinal, and
vascular complications in diabetes (8). Inhibitors of AGE formation,
such as aminoguanidine (9), tenilsetam (10), OPB-9195 (11), and
pyridoxamine (PM) (12, 13), also retard the development of diabetic
complications in animal models, and aminoguanidine and PM are being
evaluated in clinical trials for treatment of diabetic nephropathy.
Although AGE inhibitors vary widely in structure, they are all
nucleophilic compounds and are thought to function by trapping reactive
carbonyl intermediates in AGE formation. Because they are relatively
nonspecific nucleophiles, AGE inhibitors also affect other oxidative
chemistry, e.g. the formation of ALEs. Aminoguanidine, for
example, inhibits chemical modification of proteins during lipid
peroxidation reactions (14) and inhibits metal-catalyzed oxidation of
low density lipoprotein (LDL) and uptake of oxidized LDL into
macrophages via the scavenger receptor (15).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C until analyzed. Modification of the free amino group of PM
was measured by the trinitrobenzenesulfonic acid (TNBS) assay (20),
using PM as standard. Reversed-phase high performance liquid
chromatographic analysis of the samples was carried out on a Waters
(Milford, MA) HPLC system, using a Supelco (Bellefonte, PA) C-18 column
(4.6 mm × 25 cm). The gradient (flow rate = 1 ml/min;
solvent A = 0.1% aqueous heptafluorobutyric acid and solvent B = acetonitrile) was as follows: 0-2 min, 100% A; 2-20 min,
linear gradient to 60% B; hold 10 min; 30-31 min, linear gradient to 75% B; hold 10 min; 41-42 min, linear gradient to 100% A, hold 13 min to re-equilibrate column. PM and derivatives were detected by
fluorescence at Ex = 328 nm and
Em = 393 nm and were quantified by absorbance
detection at 294 nm.
20 °C until analyzed. Aliquots of well mixed
reaction mixtures were removed for measurement of thiobarbituric acid
reacting substances (TBARs), as described by Sawicki et al.
(21), using MDA as external standard. For measurement of residual LA,
butylated hydroxytoluene (antioxidant, 0.01%) and palmitate (internal
standard, 5 mM) were added, and then the lipids were
extracted with CHCl3:CH3OH (2:1), as described
by Folch et al. (22), and the organic layer was dried
in vacuo. Linoleate was converted to methyl linoleate using
0.5 ml of boron trichloride:methanol heated at 65 °C for 1 h.
The derivatizing agent was evaporated under nitrogen, and the methyl
esters were extracted with hexane:water (3:1, v/v). The organic layer
was dried under nitrogen, reconstituted in 0.15 ml of
CH2Cl2, and analyzed by selected ion
monitoring-gas chromatography/mass spectrometry (SIM-GC/MS) (see below)
using ions 294 and 220 for methyl linoleate and methyl palmitate, respectively.
20 °C. Aliquots (~1 mg of protein)
were extracted with CHCl3:CH3OH, according to
Folch (22), and the lower organic phase was removed. The upper aqueous
phase and interfacial protein were reduced by addition of a 5-fold
excess of NaBH4 over lipid in 0.1 M borate buffer, pH 9, for 4 h at room temperature. The samples were then transferred to dialysis tubing (6,000-8,000 molecular weight cut-off) and dialyzed against 100 volumes of deionized water for 24 h at 4 °C with several water changes. An aliquot of the dialysate, equivalent to ~100 µg of protein, was removed, diluted with an equal volume of concentrated HCl, and hydrolyzed (110 °C for 24 h) for amino acid analysis. Modification of lysine residues was measured by amino acid analysis, conducted by cation-exchange chromatography, using post-column derivatization with
o-phthalaldehyde and fluorescence detection. For analysis of
lipoxidation products, the remainder of the sample was dried in
vacuo, deuterated internal standards
([2H8]Lys,
[2H4]CML, [2H4]CEL,
[2H8]MDA-Lys, and
[2H4]HNE-Lys) were added, and the sample was
hydrolyzed in 6 N HCl, as above. The hydrolysate was dried
in vacuo, reconstituted in 1 ml of 0.1% trifluoroacetic
acid, and applied to a 1-ml C-18 Sep-Pak (Waters Associates, Milford,
MA) equilibrated with 0.1% trifluoroacetic acid. The flow-through
fraction from the column was collected, and the column was washed with
3 ml of 0.1% trifluoroacetic acid containing 20% methanol. The eluate
was pooled with the flow-through fraction, dried in vacuo,
and the amino acids then derivatized as their
N,O-trifluoroacetyl methyl esters and analyzed by selected ion monitoring (SIM)-GC/MS, as described previously (5, 18, 19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pyridoxamine is consumed during peroxidation
of fatty acids. PM (1 mM) was incubated alone ( 
) or
with oleate (
), linoleate (
), or arachidonate (
) (5 mM) in 200 mM sodium phosphate buffer, pH 7.4, for 6 days at 37 °C. A, loss of PM was quantified by HPLC
(see inset to Fig. 4). B, loss of primary amino
groups, measured by the TNBS assay. Data are means ± S.D. of
three independent experiments.
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Fig. 2.
Pyridoxamine has minimal effect on the
kinetics of oxidation of linoleate. Linoleate (5 mM)
was incubated alone ( 
) or in the presence of PM (1 mM)
() in 200 mM sodium phosphate buffer, pH 7.4, for 6 days
at 37 °C. Aliquots removed at various times were analyzed for
reactivity in the TBAR assay expressed as nmol MDA equivalents
(A) and residual linoleate by GC/MS (B). Data are
means ± S.D. of three independent experiments.
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Fig. 3.
Pyridoxamine inhibits formation of ALEs
during peroxidation of arachidonate in the presence of RNase.
RNase (1 mM) was incubated with arachidonate alone ( 
)
(100 mM) or in the presence of PM () (1 mM)
in 200 mM phosphate buffer, pH 7.4, for 6 days at 37 °C.
By 3 days of incubation, all of the arachidonate had been solubilized,
yielding a monophasic system. At various times, aliquots of the
reaction were reduced with NaBH4, then dialyzed and
prepared for GC/MS as described under "Materials and Methods."
Approximately 1 mg of protein was analyzed by selected ion monitoring
(SIM)-GC/MS for CML (A), CEL (B), MDA-Lys
(C), or HNE-Lys (D). Data are means ± S.D.
of three independent experiments.
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Fig. 4.
Pyridoxamine is consumed during inhibition of
chemical modification of RNase by arachidonate. RNase was
incubated with arachidonate and PM (1 mM) as described in
the legend to Fig. 3. Aliquots were removed at various times and
assayed for residual PM by HPLC (inset). Pyridoxal
(PL) was added as an internal standard. Data are means ± S.D. of three independent experiments.
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Fig. 5.
Pyridoxamine inhibits lysine modification
during oxidation of arachidonate in the presence of RNase.
Aliquots of the reaction mixture described in the legend to Fig. 3 were
analyzed by amino acid analysis for their lysine content; data are
expressed relative to lysine content of native RNase. Data are
means ± S.D. of three independent experiments.
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Fig. 6.
Pyridoxamine does not prevent
copper-catalyzed oxidation of LDL but does inhibit ALE formation and
lysine loss. LDL (50 µg/ml) was incubated at 37 °C in
phosphate-buffered saline alone ( ) or in the presence of 5 µM Cu2+ (
) or with 5 µM
Cu2+ and 100 µM () or 250 µM
() PM. A, conjugated diene formation was monitored at 234 nm. At the end of the incubation ~1 mg of protein was removed,
reduced with NaBH4, dialyzed, delipidated, and then
analyzed for CML, CEL, MDA-Lys, and HNE-Lys content by selected ion
monitoring (SIM)-GC/MS (B) and lysine loss (C) by
amino acid analysis. Results are mean and range for analysis of two
independent pools of LDL and are representative of two separate
experiments.
10%
of the PM in the starting reaction, whereas ~65% of the starting PM
was consumed by 6 days (Fig. 1). The fluorescence of the total reaction
mixture was also measured at 6 days, to establish that fluorescent
reaction products were not binding irreversibly to the C-18 column.
These measurements confirmed a significant ~50% decrease in
fluorescence during the reaction, consistent with the overall loss of
fluorescence observed in the HPLC analyses in Fig. 7C. This
observation suggested that the PM derivatives were less fluorescent
than PM itself.
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Fig. 7.
Pyridoxamine is consumed, and fluorescent and
absorbant products are formed during oxidation of linoleic acid.
PM (1 mM) was incubated with LA (5 mM) in 0.2 M sodium phosphate buffer, pH 7.4, for 6 days at 37 °C.
A and B, aliquots of the aqueous phase were
analyzed by analytical RP-HPLC at various times using fluorescence
detection with pyridoxal added as an internal standard, just prior to
chromatography. Products indicated by brackets were
consistently formed in reaction mixtures in the presence of PM.
C, semi-preparative chromatographic analysis of a sample at
6 days, monitoring absorbance at 294 nm. The brackets in
C indicate the region pooled from several semi-preparative
column runs and were used for mass spectrometry analysis. The *
indicates products produced during LA oxidation in the absence of
PM.
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Fig. 8.
Identification and kinetics of formation of
pyridoxamine adducts in reactions of pyridoxamine with linoleic
acid. A, products formed after 6 days of reaction and
eluting between 19 and 22 min (Fig. 7D) were analyzed by
direct injection ESI-MS. Five major products were identified, with
m/z = 267, 305, 323, 339, and 479. B,
semi-preparative RP-HPLC (absorbance detection), identifying major
products with m/z = 339, 267, and 305, in order of
elution. C, kinetics of formation of 267 ( ), 339 (),
and 305 (), based on their area ratios to pyridoxal, added as
internal standard. The data shown are the mean and S.D. from three
independent experiments.
-blocked lysine, was shown to be formed
during copper-catalyzed oxidation of LDL, and was identified in the
vascular wall by immunohistochemical methods. Data on product 339 are
therefore presented in more detail here. Products 267 and 339 were
isolated by semi-preparative RP-HPLC. Based on absorbance area units on
HPLC, both products yielded PM (>90%) on acid hydrolysis (2 N HCl, 2 h at 95 °C) (shown for product 339 in Fig.
9, A versus
B). The fluorescence response of these products on HPLC
analysis was ~30% that of PM, consistent with the loss of PM
fluorescence during reaction of LA with PM (Fig. 7, B
versus A). Neither product contained a free amino
group, based on lack of reactivity with TNBS, consistent with the loss of TNBS reactivity during lipid peroxidation reactions (Fig.
1B).
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Fig. 9.
Characterization of reaction product 339, the
nonanedioic acid monoamide derivative of PM. A, RP-HPLC
of product 339, isolated by semi-preparative RP-HPLC. B,
RP-HPLC of product 339, following acid hydrolysis, yielding PM in
>90% yield. C, GC/MS analysis of the organic phase
following extraction and derivatization as the methyl ester.
NDA, nonanedioic acid dimethyl ester; PA,
palmitic acid methyl ester (derived from the internal standard,
palmitic acid). D, mass spectrum of the product identified
as NDA in C. Mass assignments: m/z
216 = molecular ion (not detected); m/z 185 = M CH3O; base peak, m/z 152 = M 2(CH3OH); m/z 143 = M CH2COOCH3; m/z 125 = M (COCH3 + CH3OH); m/z 111 = M (CH2COOCH3 + CH3OH);
m/z 97 = M (CH2CH2COOCH3 + CH3OH);
m/z 83 = M (CH2CH2CH2COOCH3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Scheme I.
HPODE, hydroperoxyoctadecadienoic
acid.
-6 PUFA. A number of other mono- and dicarboxylic
acid amide derivatives should also be formed on oxidation of other
families of PUFA. Analysis of the amide content of tissue proteins
should therefore provide insight into both the origin of lipid-derived chemical modifications of proteins and their contribution to the overall chemical modification of proteins in vivo. These
non-enzymatic lipid adducts to protein promote protein-membrane
interactions, affecting the endocytosis, subcellular transport,
compartmentation, and catabolism of lipoxidized proteins. These and
other lipid adducts may also contribute to the accumulation of
lipofuscin in postmitotic tissues during aging and to the accumulation
of lipids and lipoproteins in foam cells in the vascular wall in atherosclerosis. We are encouraged by the possibility that inhibition of lipoxidative modification of proteins, using compounds such as PM,
may provide a route for limiting age-dependent and
disease-related damage to proteins by both glycoxidation and
lipoxidation reactions and may be useful for treatment of diseases
involving hyperlipidemia and/or oxidative stress.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-(carboxyethyl)lysine;
CML, N-(carboxymethyl)lysine;
DTPA, diethylenetriaminepentaacetic acid;
HNE-Lys, Michael addition adduct of
lysine to 4-hydroxynonenal;
KODE, keto-octadecadienoic acid
(9-oxo-11,13-octadecadienoic acid or 13-oxo-9,11-octadecadienoic acid);
LA, linoleic acid;
LC-ESI-MS, liquid chromatography-electrospray
ionization-mass spectrometry;
LDL, low density lipoprotein;
MDA, malondialdehyde;
MDA-Lys, malondialdehyde-lysine, Schiff base adduct;
PM, pyridoxamine;
PUFA, polyunsaturated fatty acid;
RNase, bovine
pancreatic ribonuclease A;
RP-HPLC, reverse-phase high performance
liquid chromatography;
TBARs, thiobarbituric acid-reactive substances;
TNBS, trinitrobenzenesulfonic acid;
MS, mass spectrometry;
GC/MS, gas
chromatography/mass spectrometry;
ALEs, advanced lipoxidation end
products.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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