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RESEARCH COMMUNICATION |
INSERM U498, Biochimie des Lipoprotéines et Interactions Vasculaires, Université de Bourgogne, Dijon, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: : oxidation • LDL oxidation • red blood cell hemolysis • atherosclerosis • diabetes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the variety of biological mechanisms that have been proposed to explain the beneficial effects of higher albumin concentrations, a direct protective effect of the albumin molecule has been suggested. There is now evidence for a significant antioxidant activity of serum albumin. In fact, this molecule may represent the major and predominant circulating antioxidant in plasma known to be exposed to continuous oxidative stress (8 , 9 ). Albumin may thus represent a quantitatively important component that plays a role in the efficient antioxidant defense organisms have developed to protect against oxidative attack (10) .
In addition, alteration of the structure of albumin may result in modifications of its biological properties. These modifications could occur in insulin-dependent diabetes mellitus, which is one of the pathological conditions associated with early occurrence of vascular complications, together with structural and functional alterations of albumin, which undergoes increased glycation (11) . The usual half-life of albumin is 20 days. The binding of glucose to albumin typically occurs in vivo and is known to involve the nonenzymatic covalent attachment of glucose to a lysine side chain. Approximately 6 to 10% of the albumin in normal human serum is modified by nonenzymatic glycosylation (12) . This proportion typically increases between two- to threefold in hyperglycemia (13) . Moreover, diabetic patients exhibit elevated levels of iron and copper ions that, in the presence of glycated proteins, have been shown in vitro to generate free radicals (14) . These highly reactive species are able to induce oxidative degradation of protein in vitro (15) .
Oxidant stress is increasingly thought to be a key element in atherogenesis. Atherosclerosis is a complex multifactorial disease caused by an excessive accumulation of lipids in the vascular wall. Lipids are essentially found in foam cells, which result from the transformation of smooth muscle cells and macrophages (16 , 17 ). By damaging lipids of low density lipoprotein (LDL),2 free radicals generate proatherogenic oxidized particles (18 , 19 ). These antioxidant-depleted, oxidized lipid-enriched lipoproteins accumulate polyunsaturated fatty acid breakdown substances that secondarily react with the apolipoprotein B of LDL. These modified LDL are no longer recognized by their classical LDL receptor (20) but by scavenger receptors that are not down-regulated by excess cholesterol (21 , 22 ). This oxidative stress hypothesis is also reinforced by data provided by various studies of the beneficial effects of lipid-soluble antioxidants 23-27) . Indeed, although a precise cause-and-effect relationship between the susceptibility of LDL to oxidation and the potential benefits of antioxidant therapy with respect to vascular lesion development and/or reduction has not yet been established, particularly in clinical trials, studies in animals and humans indicate that supplementation with antioxidants increases the resistance of LDL to oxidation.
In the present work, we first investigated whether in vitro glycation and oxidation of bovine serum albumin (BSA) might alter its structural parameters. Second, using copper-mediated LDL oxidation and free radical-initiated hemolysis, we examined whether these modifications might be associated with changes in the antioxidant properties of this protein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro glycation of BSA
BSA was dissolved in phosphate-buffered saline (PBS), pH 7.4, to
yield a stock solution of 40 mg/ml. This solution was subsequently
diluted with glucose stock solutions made in PBS to form triplicate
incubation mixtures of 10 mg/ml BSA with 0, 5, 25, 100, and 500 mmol/l
glucose. After being sterilized by filtration (0.22 µm filters,
Millipore), the solutions were incubated at 37°C for 60 days under
argon gas in capped vials. Reversible and unbound glucose was removed
from BSA by extensive dialysis against PBS, pH 7.4 and stored at 4°C
in the dark prior to analysis for oxidative modification. As no
variations in the extinction coefficient were observed between the
different samples (CV<6%, n=15), the measurement of BSA
concentration was performed by absorbance at 278 nm (12)
.
Oxidative modification of BSA
Dialyzed samples of BSA (previously incubated with 0, 5, 25,
100, and 500 mmol/l glucose) were incubated with a water-soluble free
radical generator, 2,2' azo-bis(2-aminodinopropane) HCl (AAPH, >95%
pure, supplied by Lara-Spiral, Dijon, France). In a typical experiment,
2.5 mg/ml BSA was incubated for 1 h at 37°C with 12.5 mmol/l
AAPH. Reactions were stopped by cooling and extensive dialysis at 4°C
against PBS, pH 7.4. Thiol groups of native or modified BSA were
measured according to the Ellman's assay (28)
using
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Briefly, BSA samples (3.5
mg/ml) were mixed with 0.05 mol/l PBS pH 7.6 and incubated with 2.5
mmol/l DTNB for 15 min. The absorbance was measured at 410 nm. The free
thiol concentration of samples was calculated with the help of a
standard curve performed with various native BSA concentrations (0.8 to
4 mg/ml, corresponding to 19 to 96 nmol total thiols).
Fluorescence studies
As BSA contains two tryptophan residues (Trp213 and
Trp134), we could evaluate the effects of glycation and
free radical attack on its molecular conformation by assaying the
fluorescence intensity of BSA solutions. Fluorescence measurements of
BSA samples (0.01 mg/ml) were monitored using a spectrofluorometer
(LS50B Perkin-Elmer) at excitation and emission wavelengths of 280 and
340 nm, respectively, as previously described (29)
.
The production of fluorescent products or glycophore was monitored on samples with a concentration of 0.5 mg/ml BSA as previously described (30) at excitation and emission wavelengths of 350 and 425 nm, respectively.
Quenching of fluorescence
As acrylamide was previously shown not to alter the protein
conformation (31)
, it was thus used as a quenching agent
in our experiments to examine the effects of glycation and oxidation on
the fluorescence of Trp residues measured as above (
ex
280 and em 340 nm). Acrylamide was added to the various
BSA preparations in final concentrations of up to 0.32 mol/l. The
results of triplicate assays are expressed as mean values of
I0/I (Stern-Volmer constant), where I0 and I
were the albumin fluorescences before and after addition of the
quencher, respectively (31
, 32
).
Boronate affinity chromatography
The formation of Amadori products was assessed by the percentage
retention of modified materials on boronate affinity columns
(11
, 33
). Experiments were conducted as
follows: 1 ml separating gel (60 µmol m-aminophenylboronic
acid per ml, Sigma) was equilibrated with binding buffer (0.2 mol/l
ammonium acetate, pH 8.8) and 1 ml of 1 mg/ml BSA modified samples was
applied. Bound materials were eluted with 6 ml of elution buffer (0.15
mol/l NaCl, 0.01 mol/l MgCl2, 0.2 mol/l mannitol, pH 2.0).
The separating gels were regenerated with 4 ml of 0.02 mol/l NaOH, 5 ml
of 0.05 mol/l acetic acid, and 10 ml of distilled water.
LDL preparation and oxidation
LDL (1.019–1.055 g/ml) were isolated by sequential
ultracentrifugation of pooled plasma from normolipidemic subjects
(Beckman centrifuge). After dialysis against PBS, pH 7.4, LDL were
assayed for protein content by the bicinchoninic acid method
(34)
and stored at 4°C under argon in the dark for no
longer than 15 days.
Oxidation of LDL was conducted in a 500 µl sample volume using 100 µg/ml of LDL protein without or in the presence of the various BSA preparations (final concentration, 2 mg/ml). Accordingly, with the work of Schnitzer et al. (35) we confirmed that due to interferences at elevated BSA concentrations, the monitoring of the conjugated dienes for lipid oxidation usually measured at 234 nm (36) was improved at 245 nm. Consequently, oxidation of LDL was started by adding 40 µl of CuSO4 (40 µmol/l) and lipid oxidation was measured by spectrophotometry at 245 nm in a thermostated cuvette holder maintained at 37°C (Beckman DU 640).
The effects of the various BSA preparations were also studied on the Cu2+-induced production of thiobarbituric acid reactive substances (TBARS). After temperature equilibration (10 min) at 37°C, experiments were performed as above except that Cu-driven oxidations were stopped at 10 min by adding 10 µl of a solution containing 100 µmol/l BHT and 200 µmol/l EDTA in PBS. LDL TBARS are expressed in malondialdehyde equivalents as described (37 , 38 ). Agarose gel electrophoresis (0.5%) was performed to follow the LDL mobility with a Beckman's Paragon lipoprotein electrophoresis kit.
Free radical-induced hemolysis test
The antioxidant properties of the native and modified BSA
preparations were examined using a test based on in vitro
free radical-induced blood hemolysis 39-44)
. After an
overnight fast, blood samples were taken from rats using 10% (v/v)
buffered sodium citrate as anticoagulant. Results were expressed as
50% of maximal hemolysis time (HT50 in min), which refers
to the susceptibility of whole blood to free radicals. The measurement
of HT50 was very reproducible: intra- and interassay
coefficients of variation, 1.32% and 3.85%, respectively. In humans
and animal models where oxidative stress has been well documented,
HT50 was shown to be representative of the total defense
against free radicals (40
, 42
,
43
). Hemolysis was started by adding 52.4 mol/l AAPH and
assayed by measuring the decay of the optical density at 450 nm (MRX
Dynatech) of rat blood diluted to 1/50 in NaCl 0.15 mol/l without or in
the presence of the various BSA samples (2 mg/ml final concentration).
Statistical analysis
Data are expressed as the means ± standard deviation
(SD) from at least three experiments performed in
triplicate. The main effects of glycation and oxidation or their
interactions were evaluated using two-way analysis of variance (ANOVA)
performed with Prism (GraphPad Software Inc., San Diego, Calif.). When
the interactions were found to be statistically significant, the
individual significances of glycation and oxidation main effects were
not taken into consideration. After ANOVA, the significance of
differences (P<0.05) among means was analyzed by multiple
comparisons, using the Honestly Significant Difference test of Tukey or
the Newman-Keuls' test. Univariate correlation coefficients were
calculated according to Pearson's method.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of preliminary dose-response studies as well as time course studies have shown that AAPH can be used to produce minimally oxidized BSA under strictly controlled conditions. Consequently, free radical-mediated oxidation of glucose-modified BSA was also studied by exposure to the products of thermo-decomposition of AAPH. When compared with native BSA (Fig. 2 ), data indicate a drastic decrease in tryptophan fluorescence, which remained invariable (-70.1%, CV=2.8, n=4) whatever the glycation degree of BSA. No difference was observed in the glycophore fluorescence between native or glycated BSA with the same sample after AAPH-mediated oxidation (data not shown). This means that important changes in tryptophan fluorescence associated with glycated and oxidized BSA were not due to protein destruction. This was also confirmed by results of analyses by polyacrylamide gel electrophoresis (data not shown).
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As albumin is thought to represent the quantitatively most important source of thiols in plasma, we examined whether these groups were altered by our incubation conditions. We found that oxidation of BSA by free radicals strongly decreased the thiols (by more than 77%, P <0.01) whereas glucose-induced modifications did not appear to significantly affect them (Fig. 3 ). Results obtained by using two-way ANOVA indicate that interactions between glycation and oxidation were not significant and the oxidation process accounts for 77% of the total variance (P <0.0001). These effects were opposed by adding an antioxidant such as ascorbic acid (Fig. 3) .
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Glucose-induced modifications were assessed by the formation of Amadori products using boronate column chromatography. An elevation of the retained material boronate column was observed when BSA was incubated with increased concentrations of glucose (+362% for BSAG500 vs. BSAnative). The same results in boronate binding were observed whether glycated or native BSA was oxidized or not by AAPH (data not shown). Boronate affinity chromatography studies showed that, as already observed by Coussons et al. (45) , approximately 20% of nonglycated starting material (native BSA) was retained on boronate affinity columns (Fig. 4 ). This may be due to nonspecific interactions with the boronate gel.
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As illustrated in Fig. 5 , the fluorescence of the different modified BSA preparations could be quenched using increasing concentrations of acrylamide (31 , 32 ). the quasi-linearity of the Stern-Volmer plots indicated that the quenching process followed a collision-type mechanism. The calculated Stern-Volmer constants (Table 1 ), representing the slopes of the plots and expressing tryptophan accessibility to the quencher, were different for BSAnative, BSAG100, and oxidized BSA. These results indicate that the environment of the tryptophan residues is different with regard to the effect of glycation and/or oxidation.
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Antioxidant effect of modified BSA
To examine whether the glycation of BSA or its oxidation status
had any effect on its antioxidant properties, incubations were
performed in two different systems that involved different mechanisms:
the copper-induced oxidation of LDL and the free radical-mediated
hemolysis.
Copper-mediated LDL oxidation
The effects of the native and modified BSA on Cu-induced oxidation
kinetics of LDL are illustrated in Fig. 6
, which represents monitoring of the conjugated dienes adapted for
BSA at 245 nm (35)
. The usual three phases of the
oxidation kinetics were obtained (46)
. First, there is a
lag phase when endogenous antioxidants are progressively consumed.
Next, there is the propagation phase with a rapid increase in
absorbance. The last phase is the decomposition of hydroperoxides
during which no further increase in absorbance occurs. In the presence
of native BSA, a strong increase in the lag time was observed
indicating a marked antioxidant activity of native BSA. The shift of
the progress curves toward longer times was correlated with increasing
concentrations of BSA (not shown). Oxidation kinetics of LDL incubated
in the presence of BSA with increasing concentrations of glucose showed
that this protective effect was progressively lost. In addition, higher
glucose concentrations (100 and 500 mmol/l) resulted in pro-oxidant
effects as shown by shifts of the progress curves toward shorter times
by comparison with control conditions. When results are expressed as
the rate vs. time by the first derivative dA/dt, a maximum was obtained
by computer analysis that corresponds to the time necessary to obtain
50% oxidation (T1/2) (36)
. A value of 25.0
±1.0 min was calculated for native BSA (Fig. 7
), which represents an increase of +67% in comparison with LDL
alone (15.1±0.4 min). These peak times decreased to 18.5 ±0.7, 16.0
±0.4, 9.7 ±0.3, and 6.6 ±0.3 min for BSA modified by 5, 25, 100, and
500 mmol/l glucose, respectively.
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We also studied the effects of in vitro oxidation and glycation on the antioxidant properties of BSA; the results, expressed as T1/2 of the Cu-initiated oxidation of LDL, are illustrated in Fig. 7 . Oxidation of native BSA resulted in complete loss of its antioxidant capacity since a T1/2 similar to that obtained in the absence of BSA was found (12.6±1.6 and 15.1±0.4 min, respectively). Also, oxidized BSAG further decreased T1/2 for the LDL oxidation as compared with BSAG alone. This was particularly evident for BSA glycated by physiological (5 mmol/l) and diabetic glucose (25 mmol/l) concentrations, where 31.5% and 18.7% significant decreases were found (oxidized BSAG vs. BSAG, respectively). As for BSA preincubated in the presence of high glucose concentrations (500 mmol/l) alone, oxidation of BSAG500 also resulted in pro-oxidant effects, as evidenced by shorter T1/2 in comparison with control conditions (5.4±0.6 vs. 15.1±0.4 min, P<0.001, respectively).
Copper-mediated LDL oxidation was also measured as TBARS and the results of the changes of the antioxidant properties of native and modified BSA are summarized in Table 2 . To optimize the differences between the samples, incubations were stopped at 10 min according to the initial kinetic data such as those illustrated in Fig. 6 . In the presence of BSAnative, the TBARS produced in oxidized LDL were significantly reduced by 33%. Table 2 also shows that the more glycated the BSA, the more TBARS were produced. For BSA incubated in 500 mmol/l glucose, the change was even more drastic since results indicating a pro-oxidant effect (by 145%) were in line with the data reported in Figs. 6 and 7 . Furthermore, the antioxidant activity of native BSA was no longer observed in the presence of the various AAPH-oxidized BSA preparations (Table 2) . Increased TBARS were generated significantly during Cu-mediated LDL oxidation when incubated with BSAG compared to BSAnative.
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As oxidative modifications of the apo B moiety of LDL is known to produce an increase in the electrophoretic mobility, LDL oxidation was also monitored by agarose gel electrophoresis. As expected, the LDL mobility relative to appropriate controls was significantly reduced for incubations conducted in the presence of BSAnative. When LDL were Cu-oxidized in the presence of BSA glycated with increasing concentrations of glucose, their mobility became more and more augmented. Thus, with BSAG100 and BSAG500, oxidized LDL migrated faster than in the absence of BSA (control), indicating the pro-oxidant effect of BSA glycated by preincubation with high concentrations of glucose. As already observed for dienes and TBARS, the LDL relative electrophoretic mobility was markedly more elevated in the presence of oxidized BSAG than in the presence of BSAG.
Free radical-induced hemolysis
The antioxidant properties of BSAnative,
BSAG, and oxidized BSAG were also investigated
by means of our hemolysis test, which measures the susceptibility of
whole blood to free radicals. Typical hemolysis curves are illustrated
in Fig. 8A
. As expected, the free radical scavenging property of
native BSA was evidenced by the significant increase in the presence of
native BSA (+41%) as compared with controls (185.5±1.5 and 131.4±1.2
min, P<0.001, respectively). The antioxidant activity
of free radical-treated BSA was strongly impaired (Fig. 8A
;
176.6±1.9 min, P ±0.001 vs. native BSA). In contrast,
glycation tended to increase the antioxidant activity of BSA either
alone or with oxidation (Fig. 8B
). However, a comparison
with the corresponding unoxidized BSA preparation indicated that
oxidation consistently induced a significant decrease in the
antioxidant effect of modified BSA.
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Correlations
Univariate correlations were calculated for the parameters
affected by glycation-modified BSA (Table 3
). The glucose concentration was marginally correlated with the
binding to boronate chromatography, negatively correlated with the
fluorescence of the tryptophans (P=0.011), and positively
correlated with glycophore appearance (P=0.001), the
production of TBARS, and the increase in the relative electrophoretic
mobility of copper-oxidized LDL (P=0.013 and 0.017,
respectively). The decrease in tryptophan fluorescence correlated
negatively with boronate binding and positively with the appearance of
the glycophore and with parameters of the LDL oxidation. The latter
were positively related to the level of the glycophore.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence strongly suggest that a reduced serum albumin concentration, although within the normal range, is associated with mortality risk. From studies performed with healthy subjects and patients, it has been reported that the estimated increase in the odds of death ranges from 24% to 56% for each 2.5 g/l decrement in serum albumin concentration (1) . The association may predict overall and cause specific mortality, including cardiovascular mortality (2) . The serum albumin level thus appears to be an independent predictor of mortality risk. A direct protective effect of the albumin molecule is suggested by the persistence of the association after adjustment for other known risk factors, particularly for cardiovascular diseases. This idea may also be supported by the large variety of biological properties of albumin.
In the present work, we focused on the antioxidant activity of albumin because oxidative stress is thought to play a significant role in the pathogenesis of many diseases, including atherosclerosis 47-49) . There is now ample evidence suggesting that albumin, as the main circulating protein, is a quantitatively important antioxidant in the blood and extracellular fluids (50 , 51 ). Our present data are in full agreement with this concept, since by using different approaches we consistently found that BSA markedly delayed the copper-initiated oxidation of LDL as monitored by the appearance of conjugated dienes, changes in the electrophoretic mobility, and formation of end products of fatty acid oxidation such as TBARS. Second, BSA offered a significant protection against free radical-mediated blood hemolysis. These antioxidant effects were found to be concentration dependent, which is consistent with the idea of a beneficial effect of high albumin levels in humans.
However, the main purpose of this work was to extend the above-mentioned idea by proposing that besides its concentration, the integrity of the albumin molecule might be a key determinant of its activity. We found that when BSA was preincubated in the presence of various glucose concentrations, it progressively lost its antioxidant properties with regard to the propensity of LDL to oxidize. We present data consistent with the idea that under our conditions only minor modifications of the BSA molecule and no large damage occurred after incubation with glucose and free radicals. These changes have been demonstrated by a combination of fluorometry, boronate binding, and thiol group assay. First, a progressive glycation of BSA has been evidenced by the progressive increase in boronate binding of BSA incubated in vitro with increasing glucose concentrations. This has been attributed to the attachment of glucose to the amino group of the exposed lysine side chain, resulting at first in the formation of reversible Schiff's bases and Amadori products, which then irreversibly rearrange into the so-called advanced glycated end products (11 , 52 ). We next found that the concentration of these fluorescent glycophores markedly increased in BSA after incubation with glucose under our conditions (Fig. 1) . Finally, our finding that the glycophore formation was concomitant with the decrease in the tryptophan fluorescence confirms the data of Coussons et al. (45) . In keeping with previous work conducted with human albumin (12) , the decrease in the fluorescence of the tryptophanyl residues has been attributed to glycation-dependent conformational changes within the protein that affected the local environment of the tryptophan. It is well known that, although human serum albumin contains only one tryptophan compared to two in BSA, these proteins have a similar structure and conformation (53) . From studies of fluorescence behavior in the presence of surfactants, it has been proposed that Trp-214, the tryptophan residue of HSA, behaves similarly to the Trp-213 of BSA. Both tryptophans have lysine in their close vicinity that can bind glucose. Fluorescence quenching assessed the resulting conformational changes. We used acrylamide as a quenching agent to probe the change in the degree of exposure of tryptophan in modified BSA. As glycated and oxidized BSA had different Stern-Volmer constants, these data indicate that modified BSA preparations are in different conformational status. A glucose-induced decrease (by 23%) in the constant probably indicated that glycation led to a reduced accessibility of the probe to the hydrophobic fold where Trp-213 is located. Oxidation of native BSA resulted in a more drastic effect, since a 65% decrease was observed. However, the oxidation process did not appear to induce further changes in the conformation of the protein, as similar constants were found for the glycated BSA and the free radical-treated glycated BSA (Table 1) . However, as evidenced by polyacrylamide gel electrophoresis (data not shown), all these changes did not result in large damage to the protein such as fragmentation or aggregation.
The precise antioxidant mechanism of BSA is not known. However, from the present data dealing with hemolysis and as suggested by several studies conducted with LDL, BSA can work as a radical trapping and/or a metal sequestering agent (54 , 55 ). Glycation and oxidation may affect these processes differently. Although various amino acids such as His, Trp, and Lys are thought to play important roles, the peculiar effects of thiol-containing amino acids have been the subject of specific studies. In particular, the thiol group of Cys residues may play a role in free radical damage to proteins, as it is known that the formation of disulfide bonds occurs in protein aggregation and results in the loss of enzymatic activity (56 , 57 ). This is in line with our observation that free radical-treated BSA lost more than 77% thiols. As suggested by Davies et al. (56) when using EPR spin trapping with BSA, thiols may work either as a radical sink, thus protecting the protein from entire denaturation, or as agents transferring the damage to other sites such as the peptide backbone. This may explain why we found that in some instances, BSA not only lost its antioxidant effect but also became pro-oxidant (Fig. 6) . This markedly occurred with glycated BSA after incubations in glucose concentrations as low as 5 mmol/l and submitted to metal-catalyzed oxidation. This is in keeping with reports indicating that glycated proteins can generate free radicals in the presence of copper ions (14 , 58 ). Two mechanisms, both metal-catalyzed oxidations, by which glucose may induce oxidative structural changes in protein have been proposed. The first results from `glucose auto-oxidation', which generates oxidants and protein-reactive aldehydes by glucose in solution. The second, named `glycoxydation', generates similar products by glucose once attached to the protein. Although the glycophore formation was measured in our study, a more detailed analysis of the carbohydrate chemistry in modified BSA should be conducted in future studies. It would help to better characterize and compare the different effects observed between glycation alone and glycation plus oxidation. However, from our data obtained in AAPH-induced hemolysis, we provided new evidence to explain the mechanism of the antioxidant effects of native BSA. The increase in the hemolysis time observed with native BSA was significantly reduced with the glycated and oxidized BSA. Conversely, a certain antioxidant effect was still preserved with only glycated BSA. Although specific binding studies should be performed to precisely characterize the glucose-induced change in copper binding of BSA (35) , our data confirm the key role of metals in the pro-oxidant process. In our experiments, the effect of glycation could be to increase the pro-oxidant properties of BSA rather than decrease its antioxidant activity. These conclusions as well as the conformational changes of the native and modified BSA are summarized in the schematic representation of Fig. 9 .
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In conclusion, the present work confirms and extends the idea that serum albumin is an important protein that presents direct protective effects. These effects are based on a variety of biological mechanisms. In this report, we point to the antioxidative properties of albumin. Using fluorescence measurements, we found that structural changes occurred when albumin was incubated in increasing glucose concentrations, under oxidizing conditions, or both. These structural changes resulted in impairments of the biological activities of albumin as assessed in vitro for its antioxidative properties. Our data bring further support to the proposal that, in addition to its plasma concentration, the quality of the albumin molecule may be related to its biological properties. In particular, advanced glycated end-products accumulate at an accelerated rate in diabetes. A receptor system for these products has been described in various cells, including monocytes and vascular cells, that can trigger the secretion of reactive oxygen species and cytokines. Recently, glycated albumin has been reported to induce the expression of tissue factor in monocytes with the involvement of oxidative stress. This further confirms the role of modified albumin as a key element in the onset of hypercoagulability complications. As beneficial effects of high albumin concentrations have been reported in platelet activity, work is in progress in our laboratory to document the modulation of platelet function by modified forms of albumin. Albumin represents the quantitatively most important source of thiol in plasma, and this circulating store may be altered in situations where antioxidants become limiting resulting in changes in the redox status. For instance, compounds such as homocysteine, which accumulates in folate deficiency, may have pro-oxidant effects (59) and may combine with the thiols of albumin. Cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells (57 , 60 ), and this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and pathologies such as diabetes, atherosclerosis, and neurodegenerative diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: AAPH, 2,2' azobis 2-amidinopropane HCl; AGE, advanced glycation end products; ANOVA, analysis of variance; BSA, bovine serum albumin; BSAG0, native BSA or 0 mmol/l glucose-incubated BSA; BSAGX, BSA glycated by incubation with x mmol/l of glucose; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); HT50; 50% hemolysis time; LDL, low density lipoproteins; PBS, phosphate-buffered saline;TBARS, thiobarbituric acid reactive substances.
Received for publication July 3, 1998.
Revision received October 14, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was assessed using excitation and emission
wavelengths of 280 and 340 nm, respectively. Glycophore formation (
)
was measured at excitation and emission wavelengths of 350 and 425 nm,
respectively. The significance of the results expressed as mean
±SD, n=3 was determined by comparison with
control values using one-way ANOVA, followed by Dunnett's test.
***P < 0.001. Note that SD bars are sometimes
smaller than symbols.
) BSA; BSA incubated with 100 mmol/l glucose (
) and oxidized
(
) as described in Materials and Methods. I and I0 are
the fluorescence intensities with and without quencher, respectively.
Note that SD bars are not represented for reasons of
clarity.
