Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid
2003; Elsevier BV; Volume: 44; Issue: 3 Linguagem: Inglês
10.1194/jlr.m200407-jlr200
ISSN1539-7262
AutoresRebecca A. Patterson, Elizabeth Horsley, David S. Leake,
Tópico(s)Biochemical effects in animals
ResumoOxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of Mr below 500 was prepared as a model for the low Mr components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low Mr serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL.These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants. Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of Mr below 500 was prepared as a model for the low Mr components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low Mr serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL. These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants. The oxidation of LDL may be an important event in the development of atherosclerosis (1Steinberg D. Low density lipoprotein oxidation and its pathobiological significance.J. Biol. Chem. 1997; 272: 20963-20966Google Scholar). The mechanisms of LDL oxidation in atherosclerotic lesions are uncertain, but there is evidence that catalytically active transition metal ions are present in human lesions (2Smith C. Mitchinson M.J. Aruoma O.I. Halliwell B. Stimulation of lipid peroxidation and hydroxy radical generation by the contents of human atherosclerotic lesions.Biochem. J. 1992; 286: 901-905Google Scholar, 3Lamb D.J. Mitchinson M.J. Leake D.S. Transition metal ions within human atherosclerotic lesions can catalyse the oxidation of low density lipoprotein by macrophages.FEBS Lett. 1995; 374: 12-16Google Scholar, 4Swain J. Gutteridge J.M.C. Prooxidant iron and copper, with ferroxidase and xanthine oxidase activities in human atherosclerotic material.FEBS Lett. 1995; 368: 513-515Google Scholar). There is also a suggestion that oxidation may be catalysed by metal ions in advanced atherosclerotic lesions (which cause clinical problems), but not early ones, based on the levels of protein-bound o-tyrosine in human atherosclerotic lesions (5Leeuwenburgh C. Rasmussen J.E. Hsu F.F. Mueller D.M. Pennathur S. Heinecke J.W. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques.J. Biol. Chem. 1997; 272: 3520-3526Google Scholar).In vitro, low concentrations of serum (6Leake D.S. Rankin S.M. The oxidative modification of low-density lipoproteins by macrophages.Biochem. J. 1990; 270: 741-748Google Scholar, 7Kalant N. McCormick S. Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein.Biochim. Biophys. Acta. 1992; 1128: 211-219Google Scholar) or interstitial fluid (8Dabbagh A.J. Frei B. Human suction blister interstitial fluid prevents metal ion-dependent oxidation of low density lipoprotein by macrophages and in cell-free systems.J. Clin. Invest. 1995; 96: 1958-1966Google Scholar) can protect LDL against oxidation. Under normal circumstances, the antioxidant protection offered by interstitial fluid should be sufficient to protect LDL against oxidation in the arterial wall. Indeed, antibodies against oxidized LDL (oxLDL) do not detect the presence of oxLDL in the normal arterial wall (9Palinski W. Rosenfeld M.E. Ylä-Herttuala S. Gurtner G.C. Socher S.S. Butler S.W. Parthasarathy S. Carew T.E. Steinberg D. Witztum J.L. Low density lipoprotein undergoes oxidative modification in vivo.Proc. Natl. Acad. Sci. USA. 1989; 86: 1372-1376Google Scholar, 10Javed Q. Leake D.S. Weinberg P.D. Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall.Exp. Mol. Pathol. 1999; 65: 121-140Google Scholar), but oxLDL has been detected in atherosclerotic lesions (9Palinski W. Rosenfeld M.E. Ylä-Herttuala S. Gurtner G.C. Socher S.S. Butler S.W. Parthasarathy S. Carew T.E. Steinberg D. Witztum J.L. Low density lipoprotein undergoes oxidative modification in vivo.Proc. Natl. Acad. Sci. USA. 1989; 86: 1372-1376Google Scholar, 10Javed Q. Leake D.S. Weinberg P.D. Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall.Exp. Mol. Pathol. 1999; 65: 121-140Google Scholar), implying that LDL oxidation occurs in diseased arteries.We reported that ascorbate switched from being an antioxidant for native (nonoxidized) LDL to become a prooxidant toward partially oxidized LDL (11Stait S.E. Leake D.S. Ascorbic acid can either increase or decrease low density lipoprotein modification.FEBS Lett. 1994; 341: 263-267Google Scholar). Antioxidant to prooxidant switches have since been reported for a number of different compounds, including dehydroascorbate (12Stait S.E. Leake D.S. The effects of ascorbate and dehydroascorbate on the oxidation of low-density lipoprotein.Biochem. J. 1996; 320: 373-381Google Scholar), flavonoids (13Yamanaka N. Oda O. Nagao S. Green tea catechins such as (-)-epicatechin and (-)-epigallocatechin accelerate Cu2+-induced low density lipoprotein oxidation in propagation phase.FEBS Lett. 1997; 401: 230-234Google Scholar), caffeic and chlorogenic acid (14Yamanaka N. Oda O. Nagao S. Prooxidant activity of caffeic acid, dietary non-flavonoid phenolic acid, on Cu2+-induced low density lipoprotein oxidation.FEBS Lett. 1997; 405: 186-190Google Scholar), catecholestrogens (15Markides C.S.A. Roy D. Liehr J.G. Concentration dependence of prooxidant and antioxidant properties of catecholestrogens.Arch. Biochem. Biophys. 1998; 360: 105-112Google Scholar), ferulic acid (16Bourne L.C. Rice-Evans C.A. The effect of the phenolic antioxidant ferulic acid on the oxidation of low density lipoprotein depends on the pro-oxidant used.Free Radic. Res. 1997; 27: 337-344Google Scholar), Trolox C (a water-soluble analog of vitamin E) (17Albertini R. Abuja P.M. Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein.Free Radic. Res. 1999; 30: 181-188Google Scholar), aminoguanidine (18Philis-Tsimikas A. Parthasarathy S. Picard S. Palinski W. Witztum J.L. Aminoguanidine has both pro-oxidant and antioxidant activity toward LDL.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 367-376Google Scholar), and uric acid (19Bagnati M. Perugini C. Cau C. Bordone R. Albano E. Bellomo G. When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.Biochem. J. 1999; 340: 143-152Google Scholar, 20Abuja P.M. Ascorbate prevents prooxidant effects of urate in oxidation of human low density lipoprotein.FEBS Lett. 1999; 446: 305-308Google Scholar).Here we report that a human serum ultrafiltrate of Mr below 500, prepared as a model for the low Mr components of interstitial fluid, is antioxidant toward native LDL, but prooxidant toward mildly oxidized LDL. We have identified the component of the ultrafiltrate responsible for both its antioxidant and prooxidant activity as uric acid and discuss the possible mechanisms involved.EXPERIMENTAL PROCEDURESIsolation of LDLLDL was isolated by a modification of the method of Vieira et al. (21Vieira O.V. Laranjinha J.A.N. Madeira V.M.C. Almeida L.M. Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants.J. Lipid Res. 1996; 37: 2715-2721Google Scholar). Normal human plasma containing 3 mM EDTA was adjusted to a density of 1.21 g/ml by the addition of solid KBr, and overlayed with a KBr solution of density 1.006 g/ml containing 300 μM EDTA. Following centrifugation in a near vertical rotor at 365,000 g for 50 min at 4°C, the LDL band was removed and its density was adjusted to 1.15 g/ml. The LDL was overlayed with a KBr solution (containing 300 μM EDTA) of 1.063 g/ml and centrifuged as above for 3 h. KBr and EDTA were removed by rapid filtration through two disposable desalting columns (PD 10, Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) using PBS pretreated with washed Chelex-100 (Sigma-Aldrich, Poole, Dorset, UK) containing 10 μM EDTA. LDL was sterilized by membrane filtration (Minisart-plus, Sartorius, Goettingen, Germany) and was stored aseptically under argon in the dark for up to 4 days.LDL oxidationLDL was incubated at 37°C in modified HBSS containing CuSO4 (usually 5 μM net above the concentration of EDTA carried over from the storage buffer, which was below 1 μM), with or without the addition of the ultrafiltrates (0.3%, v/v), as indicated in the figure legends. CuSO4 was the last component to be added (except when the ultrafiltrates or uric acid were added after the oxidation was already underway). The HBSS was pretreated with Chelex-100 (washed prior to use with distilled H2O to remove any contaminating antioxidant activity) (22van Reyk D.M. Brown A.J. Jessup W. Dean R.T. Batch-to-batch variation of Chelex-100 confounds metal-catalyzed oxidation, leaching of inhibitory compounds from a batch of Chelex-100 and their removal by a pre-washing procedure.Free Radic. Res. 1995; 23: 533-535Google Scholar), and consisted of 113 mM NaCl, 5.36 mM KCl, 5 mM H3PO4, 1.26 mM CaCl2, and 0.81 mM MgSO4, and adjusted to pH 7.4 by the addition of NaOH. LDL oxidation was monitored continuously by following the formation of conjugated dienes (23Esterbauer H. Striegl G. Puhl H. Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein.Free Radic. Res. Commun. 1989; 6: 67-75Google Scholar), and the lag phase determined by extrapolating the tangent to the most rapid part of the propagation phase to the x axis. The oxidation of LDL was also measured noncontinuously after halting oxidation with EDTA (1 mM) and butylated hydroxytolulene (BHT; 20 μM from a stock solution of 2 mM in ethanol) using a tri-iodide lipid hydroperoxide assay (24El-Saadani M. Esterbauer H. El-Sayed M. Goher M. Nassar A.Y. Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent.J. Lipid Res. 1989; 30: 627-630Google Scholar).LDL was oxidized by 1 mM 2,2′-azo-bis(2-amidinopropane) dihydrochloride (AAPH; Polysciences, Warrington, PA) at 37°C in the above buffer and conjugated dienes measured. AAPH was also added to the reference cuvettes and its absorbance subtracted automatically from that of the test cuvettes.Preparation of serum ultrafiltratesNormal human serum was centrifuged at 9,774 g for 1 h at 4°C through a filtration unit with a membrane selecting for components of Mr below 100,000 (Whatman International Ltd, Maidstone, Kent, UK) to remove large serum proteins. The ultrafiltrate of Mr below 100,000 was then refiltered by centrifugation at 2,000 g overnight at 4°C through a filtration unit fitted with a membrane to select for components of Mr below 500 (Micropartition kit, Amicon, Beverly, MA).Uric acid (Sigma) was dissolved in 1 M or 5 M NaOH and the pH value adjusted to about pH 7.4 using HCl.Uricase and catalase treatment and uric acid analysis of the serum ultrafiltrateThe serum ultrafiltrate was incubated for 15 min at room temperature with uricase (Type V from porcine liver; Sigma) at a concentration of 2 mg/ml (equivalent to 0.066 units of uricase/ml) and/or catalase (450 ng/ml, equivalent to 5 U/ml; thymol-free, derived from bovine liver; Sigma). To separate the ultrafiltrate from the uricase and/or catalase, the samples were then loaded into filtration units fitted with a membrane selecting for components of Mr below 5,000 (Whatman) and centrifuged at 9,774 g for 30 min at 4°C. The uric acid content of the ultrafiltrates was analyzed by UV absorbance at 292 nm, and compared with a series of standards prepared from uric acid in 700 mM glycine of pH 9.Copper reduction by serum ultrafiltratesThe reduction of Cu2+ to Cu+ was measured using bathocuproine disulphonic acid, which binds Cu+ to give a complex with an absorbance at 480 nm (25Lynch S.M. Frei B. Reduction of copper, but not iron, by human low density lipoprotein (LDL) - implications for metal ion-dependent oxidative modification of LDL.J. Biol. Chem. 1995; 270: 5158-5163Google Scholar). A serum ultrafiltrate (0.3%, v/v), uric acid (1 μM), or LDL (50 μg protein/ml) was incubated in modified HBSS at 37°C with bathocuproine disulphonic acid (360 μM) in the presence of CuSO4 (5 μM). The absorbance at 480 nm was recorded prior to the addition of CuSO4 and was subtracted from subsequent readings. The A480 was recorded every 30 s and the concentration of Cu+ was calculated using a molar absorption coefficient for the Cu+-bathocuproine disulphonic complex of 12,200 M−1 cm− 1, which was calculated from a standard plot prepared from CuSO4 reduced by excess ascorbic acid (1 mM; Sigma) to form Cu+. To investigate copper reduction following uricase treatment of the ultrafiltrate or uric acid, the ultrafiltrate (0.3%, v/v) or uric acid (1 μM) was added to CuSO4 (5 μM) in modified HBSS, and bathocuproine disulphonic acid (360 μM) was added immediately and the absorbance was measured at 480 nm. The absorbance of the appropriate mixture in the absence of CuSO4 was subtracted and the concentration of Cu+ was calculated from its molar absorption coefficient. The ultrafiltrate did not contain any copper detectable by this method either with or without the addition of ascorbic acid.StatisticsA Student's t-test was used to detect differences between conditions. A difference was deemed significant if the P value was below 0.05.RESULTSAntioxidant activity of the serum ultrafiltrate toward native LDL oxidized by copperLDL (50 μg protein/ml) was oxidized by 5 μM copper ions and the accumulation of conjugated dienes was measured. There was a lag phase prior to the rapid propagation phase, as expected (23Esterbauer H. Striegl G. Puhl H. Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein.Free Radic. Res. Commun. 1989; 6: 67-75Google Scholar) (Fig. 1). An ultrafiltrate containing components of Mr below 500 was prepared from normal human serum and tested for antioxidant activity toward LDL oxidation by copper. The ultrafiltrate potently inhibited the oxidation of native LDL, with substantial inhibition being obtained even with 0.3% (v/v) of the ultrafiltrate (P < 0.005; n = 10 experiments). In the example shown in Fig. 1, the lag phase was increased from 32 min to 94 min without significantly changing the rate of oxidation during the propagation phase. A similar effect was seen when lipid hydroperoxides were measured (a LDL concentration of 100 μg protein/ml, rather than 50 μg protein/ml, was used for this experiment because of the lower sensitivity of the lipid hydroperoxide assay than the conjugated diene assay) (Fig. 2).Fig. 2The effect of serum components of Mr below 500 on the oxidation of native or mildly oxidized LDL. Native LDL (100 μg protein/ml) was incubated in triplicate at 37°C with CuSO4 (10 μM net) in modified HBSS (line 1; diamond). Additions of ultrafiltrate were made at zero time (line 2; square) and after 45 min (line 3; triangle). Oxidation was halted at each time point by the addition of EDTA (1 mM) and BHT (20 μM), and the lipid hydroperoxide content was measured. The mean ± SEM for the triplicate samples is shown except where the error bar is smaller than the symbol. The results shown were confirmed by two other experiments. The ultrafiltrate (0.3%, v/v) did not have significant absorbance at 234 nm and did not interfere with the lipid hydroperoxide assay.View Large Image Figure ViewerDownload (PPT)An antioxidant effect of the ultrafiltrate was also seen when LDL was oxidized by 10 μM copper and conjugated dienes were measured (Fig. 3). No antioxidant effect was observed, however, when 1 μM copper was used to oxidize the LDL (Fig. 3).Fig. 3The effect of serum components of Mr below 500 on the oxidation of native or mildly oxidized LDL by different concentrations of copper. Native LDL (50 μg protein/ml) was incubated at 37°C with CuSO4 at a net concentration of 1 μM (open symbols) or 10 μM (closed symbols) in modified HBSS (line 1, open diamond or line 2, closed diamond). A serum ultrafiltrate of Mr below 500 (0.3%, v/v) was added at zero time (line 3, open square or line 4, closed square) or after 68 min (line 5; open triangle) or 48 min (line 6, closed triangle) of oxidation by copper. (The conjugated dienes had increased to about the same levels at 68 min and 48 min with 1 μM and 10 μM copper, respectively.) To monitor conjugated diene formation, the absorbance at 234nm was monitored against appropriate reference cuvettes without LDL. The results are expressed as the change in A234 and were confirmed by another experiment.View Large Image Figure ViewerDownload (PPT)Prooxidant activity of the serum ultrafiltrate toward mildly oxidized LDL in the presence of copperThe ultrafiltrate, at the same concentration (0.3%, v/v) that had previously resulted in antioxidant activity toward native LDL with a copper concentration of 5 μM or above, was also added to LDL in an early stage of oxidation by 5 μM copper (which contained 4% ± 0.5 (mean ± SEM for 10 independent experiments) of the maximum levels of conjugated dienes) (Fig. 1). The ultrafiltrate immediately increased the rate of oxidation of the mildly oxidized LDL (Fig. 1). A similar result was obtained when lipid hydroperoxide formation was measured (Fig. 2). Similar antioxidant and prooxidant effects toward native and mildly oxidized LDL, respectively, were seen with ultrafiltrates of Mr below 100,000, 30,000, 20,000, 12,000, or 5,000, and with serum components of Mr below 12,000–14,000 prepared by dialysis of normal human serum (data not shown).A similar rapid prooxidant effect was also seen when the ultrafiltrate was added to partially oxidized LDL in the presence of 10 μM copper and conjugated dienes were measured, but the prooxidant effect was less apparent when 1 μM copper was used (Fig. 3).Identification of the component responsible for the prooxidant and antioxidant activities as uric acidTo identify the active component in the ultrafiltrate responsible for these observations, the effects of cysteine, ascorbate, and uric acid toward LDL oxidation by copper were investigated. At the concentration predicted to be present in the ultrafiltrate (0.3%, v/v), neither cysteine nor ascorbate (0.1 μM; ∼0.3% of their serum concentrations (26Brigham M.P. Stein W.H. Moore S. The concentration of cysteine and cystine in human blood plasma.J. Clin. Invest. 1960; 39: 1633-1638Google Scholar, 27Riemersma R.A. Wood D.A. Macintyre C.C.A. Elton R.A. Gey K.F. Oliver M.F. Risk of angina pectoris and plasma concentrations of vitamin A, vitamin C, and vitamin E and carotene.Lancet. 1991; 337: 1-5Google Scholar) were able to cause the antioxidant-prooxidant effects shown by the ultrafiltrate (data not shown). Uric acid is present in serum at a concentration of around 300 μM (28Nyyssönen K. Porkkala-Sarataho E. Kaikkonen J. Salonen J.T. Ascorbate and urate are the strongest determinants of plasma antioxidative capacity and serum lipid resistance to oxidation in Finnish men.Atherosclerosis. 1997; 130: 223-233Google Scholar), and would be present at approximately 1 μM in the ultrafiltrate at 0.3% (v/v). Uric acid (1 μM) had antioxidant and prooxidant activities toward the oxidation of native and mildly oxidized LDL, respectively (Fig. 4). These observations agree with the findings of Abuja (20Abuja P.M. Ascorbate prevents prooxidant effects of urate in oxidation of human low density lipoprotein.FEBS Lett. 1999; 446: 305-308Google Scholar) and Bagnati et al. (19Bagnati M. Perugini C. Cau C. Bordone R. Albano E. Bellomo G. When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.Biochem. J. 1999; 340: 143-152Google Scholar).Fig. 4The effect of uric acid on the oxidation of native or mildly oxidized LDL. Native LDL (50 μg protein/ml) was incubated at 37°C with CuSO4 (5 μM net) in modified HBSS (line 1; diamond). Additions of uric acid (1 μM) were made at zero time (line 2; square) or after 33 min (line 3; triangle). The absorbance at 234 nm was monitored every 2 min against appropriate reference cuvettes without LDL. The results shown were confirmed by five other experiments.View Large Image Figure ViewerDownload (PPT)Pretreatment of the ultrafiltrate with uricase [an enzyme that catalyses the conversion of urate into allantoin (Reaction 1)] completely abolished both the antioxidant effect of the ultrafiltrate toward native LDL and the prooxidant effect toward mildly oxidized LDL (Fig. 5).Fig. 5Identification of the component responsible for the antioxidant and prooxidant activity of serum ultrafiltrates toward native or mildly oxidized LDL. Native LDL (50 μg protein/ml) was incubated at 37°C with CuSO4 (5 μM net) in modified HBSS (line 1; open diamond). Addition of 0.3% (v/v) ultrafiltrate of Mr below 500 (T = 0, line 2, square; T = 45, line 5, triangle), uricase-treated ultrafiltrate (T = 0, line 3, cross; T = 45, line 6, X), or uricase-treated ultrafiltrate plus 1 μM uric acid (T = 0, line 4, circle; T = 45, line 7, closed triangle) were made at zero time or after 45 min. The absorbance at 234 nm was monitored every 2 min against appropriate reference cuvettes without LDL. The results are expressed as the change in A234 and were confirmed by three other experiments.View Large Image Figure ViewerDownload (PPT)uricaseuricacid+2H2O+O2→allantoin+CO2+H2O2(Reaction 1)The ultrafiltrate was analyzed for uric acid content before and after uricase treatment. The uric acid concentration of the ultrafiltrates varied between donors over a range of 235 μM to 300 μM and was decreased to 4.5 ± 2 μM (mean ± SEM of nine independent experiments) following uricase treatment. The addition of uric acid [1 μM; the concentration predicted to be present in the ultrafiltrate (0.3%, v/v)] to the uricase-treated ultrafiltrate confirmed that uric acid was responsible for the antioxidant and prooxidant effects, as this restored the antioxidant and prooxidant effects of the uricase-treated ultrafiltrate (Fig. 5).The degradation of uric acid by uricase produces H2O2 (Reaction 1), which may possibly have been prooxidant toward LDL. To ensure that any prooxidant activity of H2O2 did not mask any antioxidant activity of other molecules within the uricase-treated ultrafiltrate, the ultrafiltrate was treated with uricase plus catalase to decompose H2O2. The ultrafiltrate treated with both uricase and catalase was not significantly different in its antioxidant and prooxidant effects toward LDL compared with the ultrafiltrate treated with uricase alone (unpublished observations).Copper reduction by the serum ultrafiltrateWe investigated the ability of the serum ultrafiltrates of Mr below 500 to reduce Cu2+ to Cu+. In the absence of any addition, the reduction of Cu2+ to Cu+ was slow (Fig. 6A). Addition of the ultrafiltrate, however, resulted in a rapid reduction of Cu2+ to Cu+, and within the first minute of addition, the ultrafiltrate reduced 2.4 μM of the available 5 μM Cu2+ to Cu+. Similarly, uric acid (1 μM) reduced 2.3 μM Cu2+ to Cu+ within the first minute of addition. The concentration of Cu+ increased only very slowly after 1 min. Taking into account the reagent blank, the stoichiometry of Cu2+ reduction was two copper ions reduced per molecule of uric acid. LDL (50 μg protein/ml), at the same concentration as used in the oxidation experiments, reduced Cu2+ to Cu+ at a slower rate than the ultrafiltrate or uric acid, taking about 4 min until the rate of Cu2+ reduction became very low.Fig. 6The reduction of Cu2+ to Cu+ by serum ultrafiltrates. A: CuSO4 (5 μM) was incubated alone (line 1; diamond) with LDL (50 μg protein/ml; line 2; triangle), a serum ultrafiltrate (0.3%, v/v; line 3; square), or uric acid (1 μM; line 4; circle) at 37°C in the presence of bathocuproine disulphonic acid (360 μM). The absorbance at 480 nm was monitored against time, and the Cu+ concentration was calculated from the molar absorption coefficient of Cu+-bathocuproine disulphonic acid. Similar results were obtained in two other experiments. B: Cu2+ (5 μM) was added to serum ultrafiltrates (0.3%, v/v) or uric acid (1 μM) or to ultrafiltrates or uric acid treated with uricase. Bathocuproine disulphonic acid was added immediately to quantify the amount of copper present as Cu+. The mean ± SEM for triplicate samples from three individual experiments using serum ultrafiltrates prepared from three different individuals are shown. P < 0.05 for the control versus ultrafiltrate, control versus uric acid, ultrafiltrate versus uricase-treated ultrafiltrate, and for uric acid versus uricase-treated uric acid.View Large Image Figure ViewerDownload (PPT)To demonstrate the importance of uric acid within the ultrafiltrate toward copper reduction, the uricase-treated ultrafiltrates were also tested for their copper reducing activity (Fig. 6B). Treatment of the ultrafiltrates with uricase greatly diminished their ability to reduce Cu2+ to Cu+, implying that uric acid was the main reductant of Cu2+ within the ultrafiltrate.Antioxidant to prooxidant switch and lipid hydroperoxide availabilityThe switch from antioxidant to prooxidant activity may have been dependent on the availability of lipid hydroperoxides. To test this, we added a lipid hydroperoxide in the form of 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid [HPODE; 30 nmol/mg LDL protein (about 4% of the maximum content of lipid hydroperoxides in oxLDL (see Fig. 2)] to native LDL in the presence or absence of the ultrafiltrate (0.3%, v/v) (Fig. 7). The addition of HPODE to native LDL shortened the lag phase compared with control LDL, as expected (29O'Leary V.J. Darley-Usmar V.M. Russell L.J. Stone D. Prooxidant effects of lipoxygenase-derived peroxides on the copper-initiated oxidation of low-density lipoprotein.Biochem. J. 1992; 282: 631-634Google Scholar). The lag phase in the presence of added lipid hydroperoxides was further decreased upon addition of the ultrafiltrate. In the absence of added lipid hydroperoxides, however, adding the ultrafiltrate to native LDL resulted in a much longer lag phase.Fig. 7Induction of the antioxidant to prooxidant switch of a serum ultrafiltrate by the addition of lipid hydroperoxides to native LDL. Native LDL was incubated as described in Fig. 1 (A) in the absence (line 1, diamond) or presence (line 2, square) of a serum ultrafiltrate of Mr below 500 (0.3%, v/v). HPODE (Affinity, Exeter, Devon, UK) was added to give a final concentration of 30 nmol/mg LDL protein from a stock solution of 160 μM in ethanol in the absence (line 3, circle) or presence (line 4, triangle) of the ultrafiltrate (0.3%, v/v). Oxidation was monitored by following the formation of conjugated dienes, and values are expressed as the change in A234. Ethanol at the final concentration of 0.975% (v/v) did not affect the oxidation of LDL. Similar results were obtained in two other experiments.View Large Image Figure ViewerDownload (PPT)Effects of the presence of serumThe effects of adding human whole serum to the system was investigated because interstitial fluid contains proteins, as well as low Mr components (8Dabbagh A.J. Frei B. Human suction blister interstitial fluid prevents metal ion-dependent oxidation of low density lipoprotein by macrophages and in cell-free systems.J. C
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