(Received for publication, December 18, 1995; and in revised form, February 13, 1996)
From the
N-(Carboxymethyl)lysine (CML) is an
advanced glycation end product formed on protein by combined
nonenzymatic glycation and oxidation (glycoxidation) reactions. We now
report that CML is also formed during metal-catalyzed oxidation of
polyunsaturated fatty acids in the presence of protein. During
copper-catalyzed oxidation in vitro, the CML content of low
density lipoprotein increased in concert with conjugated dienes but was
independent of the presence of the Amadori compound, fructoselysine, on
the protein. CML was also formed in a time-dependent manner in RNase
incubated under aerobic conditions in phosphate buffer containing
arachidonate or linoleate; only trace amounts of CML were formed from
oleate. After 6 days of incubation the yield of CML in RNase from
arachidonate was
0.7 mmol/mol lysine compared with only 0.03
mmol/mol lysine for protein incubated under the same conditions with
glucose. Glyoxal, a known precursor of CML, was also formed during
incubation of RNase with arachidonate. These results suggest that lipid
peroxidation, as well as glycoxidation, may be an important source of
CML in tissue proteins in vivo and that CML may be a general
marker of oxidative stress and long term damage to protein in aging,
atherosclerosis, and diabetes.
Oxidative stress, an imbalance resulting from increased
pro-oxidant generation and/or decreased antioxidant defenses, is
implicated in the pathogenesis of numerous diseases, including
atherosclerosis, diabetes, hemochromatosis, ischemic reperfusion
injury, and rheumatoid arthritis, and in the normal aging
process(1) . In atherosclerosis, the oxidation of circulating
low density lipoproteins (LDLs) ()and their increased uptake
by the scavenger receptor is thought to promote the deposition of
lipid-laden macrophages in the vascular wall, leading to fatty streaks
that precede the development of plaque(2) . In diabetes,
oxidative stress, resulting from the metabolic sequelae of
hyperglycemia and/or nonenzymatic glycation of protein, leads to
increased formation of advanced glycation end products (AGEs) in tissue
proteins(3, 4) . Among these AGEs are the
glycoxidation products, N
-(carboxymethyl)lysine (CML) and pentosidine,
which are the only chemically characterized AGEs known to accumulate in
protein with age and at an accelerated rate in diabetes (5) .
Glycation and glycoxidation products and AGEs affect the structure and
function of proteins and cross-linking of extracellular proteins,
processes that are thought to contribute to the pathogenesis of
vascular disease in diabetes and
atherosclerosis(3, 4, 5) .
The observations that the extent of glycation of plasma proteins, including LDL, is increased in diabetes (6) and that glycated proteins are redox-active and may thus catalyze lipid peroxidation reactions have suggested that increased glycation of LDL in diabetes might accelerate its oxidation, providing a mechanism for the excess risk for atherosclerosis associated with diabetes(7) . Other studies have shown that plasma antioxidant defenses, including ascorbic acid and vitamin E concentrations, and total radical trapping activity are also compromised in diabetes(8) . Although the combination of increased glycation of LDL and decreased antioxidant defenses in diabetic plasma provides the potential for catalysis of lipid peroxidation in plasma lipoproteins, there is no direct evidence of a correlation between glycation and enhanced oxidation of LDL in diabetes.
In an effort to understand the potential interplay between glycation and lipid peroxidation reactions, we initiated studies on how changes in levels of glycation affect lipid peroxidation and formation of glycoxidation products during metal-catalyzed oxidation of LDL in vitro. We report here the unexpected observation that CML is formed during copper-mediated oxidation of LDL, independent of the presence of the putative carbohydrate precursor, the Amadori compound fructoselysine (FL). We also report that CML, heretofore described as a glycoxidation product, is formed during peroxidation of polyunsaturated fatty acids (PUFA) in the presence of ribonuclease A (RNase), a protein that contains neither enzymatically nor nonenzymatically attached carbohydrate.
To remove salts,
EDTA, and water-soluble antioxidants prior to copper oxidation
experiments, LDL was chromatographed on PD-10 columns (Pharmacia
Biotech Inc.) equilibrated in phosphate-buffered saline (PBS; (12) ) that had been gassed with nitrogen. The LDL was
sterilized by ultrafiltration (0.22-µ filters, CoStar, Cambridge,
MA) and then diluted to 100 µg of protein/ml using PBS that
had been bubbled with oxygen for at least 10 min. The diluted samples
were adjusted to 5 µM CuCl
, placed in loosely
capped plastic bottles, and incubated at 32 °C; copper was omitted
in control samples. The progress of the oxidation reaction was
monitored by measuring absorbance at 234 nm in aliquots removed at
various times. For analysis of CML, aliquots (
1 mg of protein)
were adjusted to 1 mM in diethylenetriaminepentaacetic acid
(0.1 M) in sodium borate, pH 9.2, and reduced overnight at 4
°C with a final concentration of 25 mM NaBH
.
The samples were then dialyzed against water, dried by centrifugal
evaporation (Savant Speed-Vac, Farmingdale, NY), and delipidated using
methanol:ether (3:10)(13) . For analyses of FL, samples were
treated with diethylenetriaminepentaacetic acid, dialyzed immediately,
dried, and delipidated.
Fig. 1describes the kinetics of metal-catalyzed
oxidation of normal LDL and concurrent changes in the protein's
content of CML and FL. Comparison of the kinetics of conjugated diene (panel A) and CML (panel B) formation indicates that
the rate of CML formation parallels the rate of fatty acid
peroxidation. Neither conjugated dienes nor CML increased in the
absence of copper. Because we assumed initially that the CML was
derived from oxidation of the Amadori adduct on LDL, the FL content of
the protein was also measured at various stages in the oxidation
reaction. The data in panel C demonstrate unexpectedly that,
unlike CML, the FL content of LDL remained constant during the course
of the copper oxidation. We concluded tentatively that the CML was
being formed from a product of oxidation of the lipid component of the
LDL. To verify that CML could be formed independent of the presence of
FL, the LDL was reduced with NaBH to convert FL to the
inert, redox-inactive hexitollysine adduct. Table 1shows that
5% of the original FL remained on the reduced LDL, yet following
oxidation of the reduced lipoprotein, the yield of CML was similar to
that formed during oxidation of the native protein. The absorbance
traces during metal-catalyzed oxidation of NaBH
-reduced LDL
were similar to those of the native protein (data not shown). These
results indicate that CML can be formed on lipoprotein from product(s)
of PUFA oxidation.
Figure 1: Copper-catalyzed oxidation of LDL results in an increase in CML without a change in the FL content of the protein. LDL was incubated with (open symbols) or without (closed symbols) 5 µM copper at 32 °C. The progress of the oxidation reaction was monitored by following conjugated diene formation at 234 nm (A). The amounts of CML (B) and FL (C) were measured by GC/MS as described under ``Materials and Methods.'' Data are shown for two different pools of LDL.
To further characterize the formation of CML during lipoxidation reactions, model experiments were carried out in which PUFAs were oxidized in the presence of RNase A, a protein devoid of carbohydrate. As shown in Fig. 2, oxidation of linoleate and arachidonate in the presence of RNase yielded a time-dependent increase in CML residues in the protein. In these experiments the fatty acids were progressively solubilized in buffer as they autoxidized, and the experiments were arbitrarily terminated at 6 days when the arachidonate reactions became a single phase. The overall amount of CML formed was dependent on the degree of unsaturation and oxidizability of the fatty acid, with the greatest yield of CML formed from arachidonate, an intermediate yield from linoleate, and only trace amounts formed from oleate. The difference in yields of CML from the various fatty acids probably reflects differences in their extent of oxidation at the end of the experiment.
Figure 2:
CML is formed during incubation of RNase
with PUFA. RNase (1 mM) was incubated with 100 mM arachidonate (), linoleate (
), or oleate (
) in
PBS at 37 °C, and aliquots were removed at indicated times. CML was
measured by GC/MS as described under ``Materials and
Methods.'' There was no CML detected in RNase incubated in the
absence of fatty acid. Data (mean ± S.D.) are for single
measurements of individual aliquots from three separate incubations;
absence of error bars indicates error was within size of symbol.
Because CML was originally identified as a
glycoxidation product(5, 17) , i.e. the
result of combined glycation and oxidation reactions, we also compared
the relative yield of CML from glucose and arachidonate. Fig. 3shows that at the end of 6 days' incubation the
yield of CML from autoxidizing arachidonic acid, 0.74 ± 0.03
mmol/mol RNase, was significantly greater than that from glucose, 0.03
± 0.003 mmol/mol RNase. These results indicate that both
carbohydrates and lipids may contribute to formation of CML during
autoxidation reactions in physiological buffer. However, oxidation of
fatty acid is clearly a more efficient source of CML, despite the fact
that the glucose is in solution throughout the course of the
experiment, while the PUFA are only progressively solubilized. Further,
after 6 days of incubation, a large fraction of the arachidonate was
oxidized based on its solubilization in the aqueous phase, while 2%
of the glucose is oxidized during this same time period(16) .
Finally, pentosidine, a second glycoxidation product known to form
during glycoxidation reactions in vitro and in vivo,
was detected in incubations of RNase and glucose, but no pentosidine
was detected either in incubations of RNase with PUFA or in
copper-oxidized LDL.
Figure 3:
Comparison of CML formation in RNase from
arachidonate or glucose. RNase (1 mM) was incubated with 100
mM arachidonate (, replotted from Fig. 2), or 100
mM glucose (
) in PBS. CML was measured by GC/MS as
described under ``Materials and Methods.'' Data are
expressed as described in the legend to Fig. 2.
We have previously shown that glyoxal is both a product of glucose autoxidation and a source of CML in protein(16) . Glyoxal formation has also been reported during UV irradiation of PUFA (18) and during oxidation of linolenic acid in an iron ascorbate model system(19, 20) , although in the latter case formation of glyoxal from iron ascorbate itself was not excluded. In the present experiments the formation of glyoxal from PUFA was monitored by trapping it as the Girard T adduct (16) . The data in Fig. 4show that there was a progressive increase in the amount of glyoxal formed in autoxidizing arachidonic acid incubations, which was not significantly affected by the presence of protein. The data also indicate that the amount of glyoxal formed during arachidonate oxidation was more than sufficient to account for the amount of CML formed on protein, if glyoxal were the only source of all the CML formed.
Figure 4:
Glyoxal formation from arachidonic acid.
Aliquots of reaction mixtures containing either arachidonate alone
() or ararchidonate and RNase (
) were removed at indicated
times, and glyoxal was measured by HPLC as described under
``Materials and Methods.'' Data are the average ±
range for two separate experiments. For comparison the data from Fig. 2for CML formation from arachidonate (
), expressed as
µM, are also shown; absence of error bars indicates error
was within size of symbol.
The observations described above indicate that CML, previously described as a glycoxidation product or AGE, may, in fact, be derived from PUFA during lipid peroxidation reactions. These observations require a reassessment of previous work on (a) the biochemical origin of AGEs, (b) the significance of carbohydrate oxidation, autoxidative glycosylation, and glycoxidation in the chemical modification of proteins in diabetes, and, in general, (c) mechanisms of oxidative stress and pathways of oxidative damage to protein and other biomolecules in aging, atherosclerosis, and diabetes.
The mechanism of formation of
CML and other AGEs during carbohydrate oxidation reactions is still
uncertain. The pathway may involve oxidation of free glucose or
protein-bound intermediates, including carbinolamine, Schiff base, and
Amadori adducts(17, 21, 22) . Wells-Knecht et al. (16) identified glyoxal as one intermediate in
the formation of CML from glucose. Since glyoxal is also formed during
peroxidation of PUFA ( (18, 19, 20) and Fig. 4), it may be a common intermediate in the formation of CML
during oxidation of both carbohydrates and lipids. However, other
common intermediates may also be involved, such as
glycolaldehyde(21) , -hydroxyaldehydes, or
,
-unsaturated
-hydroxyaldehydes or dicarbonyls, which
may be formed from both carbohydrate and PUFA oxidation. The
intersection of carbohydrate and lipid autoxidation reactions in the
formation of CML emphasizes the relationship between the fundamental
chemistry and biochemistry of these molecules.
To distinguish carbohydrate- from lipid-mediated damage and assess the relative importance of oxidation of these two substrates in the development of diabetic complications, it will be necessary to identify unique products derived from each of these precursors. In the meantime, since CML has been identified as a major AGE antigen in tissue proteins and a product of both carbohydrate and lipid peroxidation reactions, our results suggest that CML may be more useful as a general biomarker of oxidative stress and damage in tissue proteins.