Bilirubin sensitized photooxidation of human plasma low density lipoprotein

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Biochimica et Biophysica Acta 1304 (1996) 197-209

Bilirubin sensitized photooxidation of human plasma low density lipoprotein S t e f a n A. H u l e a

a

T e r r a n c e L. S m i t h

a,

Erwin Wasowicz

b,

Fred A. Kummerow

a,*

a University of Illinois, Department of Food Science, Burnsides Research Laboratory, 1208 W. Pennsylvania Avenue, Urbana, IL 61801, USA b Agricultural University, Poznan 60-624, Poland

Received 16 April 1996; accepted 15 July 1996

Abstract Previous investigations have shown that the bile pigment bilimbin can act as peroxyl radicals scavenger and transition metals trap, but also as a prooxidant, to erythrocyte ghost membranes through 102-driven photooxidation. In the present study we examined the changes occurring in the lipoprotein particle following bilirubin-sensitized photooxidation of isolated plasma LDL. The oxidative stress resulted in increased TBA reactivity, diene formation, free cholesterol oxidation, apo B fragmentation and enhanced uptake of the modified particle by the mouse macrophage scavenger receptors as well as the decrease binding to the native B, E-receptor on fihroblasts. The marked increase in TBARS production in D20-enriched medium and the inhibition of lipid peroxidation of azide is consistent with singlet oxygen involvement in the oxidation process. The apo B-bound Cu 2+ appears to become redox active during photooxidation since the presence of EDTA in the reaction mixture greatly reduced protein fragmentation. It was also found that BHT inhibited almost completely the lipid peroxidation, as determined by the TBA reaction but could not totally abolish the formation of 5a-hydroxycholesterol, which is the main product formed by the direct attack of l o 2 on cholesterol. The results of this work strongly suggest that, through photooxidation by light-activated bilirubin, the lipoprotein particle may be modified in the blood stream as well, besides being modified in the well known oxidation site within the arterial wall. Our findings provide the rationale for extending these studies to clinical investigations, which aim at developing strategies for minimizing damage to arterial tissue following phototherapy of hyperbilirubinemic newborns or cancer patients after systemic administration of photosensitizers. Keywords." LDL; Photooxidation; Bilirubin

1. Introduction The involvement of oxidatively modified lipoproteins, particularly low density lipoproteins (LDL) in the initiation of the atherosclerotic lesion is now well

* Corresponding author. Fax: + 1 (217) 3337370.

documented (for a review see [1-3]). L D L is shown to be oxidized by a variety of agents such as molecular oxygen [4], oxygen reactive species, e.g. O 2- and O H [5,6], metal ions [7,8], hemin [9], hemoglobin [10], as well as monocytes [11], smooth muscle [5] and endothelial cells [12]. Protection against the oxidative stress is provided by ascorbate [13], vitamin E [14] and hemoglobin degradation products, particu-

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larly bilirubin (BR) as recent in vitro work has shown [15-17]. On the other hand, photo-oxidized BR has been found to promote lipid peroxidation and polypeptide association in the human erythrocyte membrane [18], as well as causing changes in membrane functions such as transport of metabolites across the lipid bilayer [19]. There is as yet no evidence that BR is associated with the photooxidation of plasma lipoproteins. However, it was reported earlier that newborns with hyperbilirubinemia exhibited an increased level of plasma lipid peroxides following phototherapy [20]. Whether lipid peroxidation occurred in plasma, as a result of photodynamic action of the bile pigment or in the liver itself, is not clear at present. It should also be noted that recent findings would suggest a possible involvement of BR in the oxidation of tissue polyunsaturated fatty acids since there appears to be a direct relation between the concentration of plasma BR and the level of lipid peroxides in several liver disorders [21,22]. The aim of this study was to investigate whether lipid peroxidation could occur in LDL subjected to illumination with visible light in the presence of the bile pigment. It was found that BR-induced photooxidation caused marked alterations of the physiochemical and biological properties of the LDL particle, as demonstrated by the release of thiobarbituric acid reactive substances, increased concentration of cholesterol oxidation products, shifts in electrophorteic mobility, apo B fragmentation as well as increased recognition by the receptors on the mouse macrophages. These findings may have important clinical implications, particularly when phototherapy is used to treat hyperbilirubinemia of the newborn as well as certain types of tumors, after systemic administration of photosenitizers to cancer patients.

were purchased from Sigma (St. Louis, MO). 7-Ketocholesterol (7-keto-C) and 7/3-hydroxycholesterol (7/3-OH-C) were obtained from Research Plus (Bayonne, NJ). Linoleic acid hydroperoxide (9(S)hydroperoxy-octadecadienoic acid) was obtained from Oxford Biomedical (Oxford, MI) and Na125I (carrier-free) in 0.1 M NaC1 (17 m C i / m g ) was purchased from NEN Research Products (Boston, MA). Thin-layer chromatography silica G plates were products of Analtech (Newark, DE). Fibroblast cells (Hs68) were obtained from American Type Culture Collection (Rockville, MD). All other reagents were of the highest purity commercially available. Nanopure water was used throughout. Fresh plasma was obtained from the local blood bank. 2.2. Isolation of L D L

The lipoprotein was isolated from fresh plasma supplemented with 1 m g / m l EDTA by sequential ultracentifugation as described by Goldstein et al. [23], sterilized by passage through a 0.45 /zm filter, stored under argon at 4°C and used within two weeks. The experiments described in this work were performed using different batches of LDL, which were carefully tested for TBA reactivity, cholesterol oxides concentration and electrophoretic mobility in agarose gel. Only those preparations exhibiting TBARS levels of less than 1 n m o l / m g protein were used. The LDL solution was dialyzed extensively against deoxygenated PBS immediately before use. Before labeling, LDL was dialyzed for 24 h against 0.14 M NaC1, 0.24 mM EDTA (pH 7.4). The iodination was carried out in 1 M glycine-NaOH buffer (pH 10.0), generally according to Fidge and Poulis [24] providing a specific activity of labeled preparations between 100 and 200 c p m / n g protein. 2.3. Oxidation o f L D L and the TBA assay

2. Materials and methods 2.1. Materials

Bilirubin, Rose Bengal, thiobarbituric acid (TBA), agarose, trinitrobenzenesulfonic acid (TNBS), deuterium oxide, 7a-hydroxycholesterol (7a-OH-C), 20a-hydroxycholesterol (20a-OH-C), chemicals for gel electrophoresis and a protein assay kit (P5656)

For the oxidation experiments the standard incubation of LDL was done in PBS (pH 7.4) at a concentration of 0.2 mg protein/ml at 37°C for 24 h in the presence of 5 /~M Cu SO 4 or 20-80 /zM BR. The BR stock solution was 0.1 mM in 0.01 M NaOH pH 8.5 and used within 1 h. The photo-oxidation experiments were performed by using laboratory fluorescent light and the test tubes containing the incubation

S.A. Hulea et al. / Biochimica et Biophysica Acta 1304 (1996) 197-209

mixture were positioned 10 cm from the light source. The tubes were placed in horizontal position and no stirring was performed during the incubation period. Incoming light was passed through a broad band blue filter (Coming CS-57) with maximum transmittance at about 430 nm. Light intensity was measured with a radiometer (Yellow Springs Instruments) and maintained at = 5 m W / c m 2. At the conclusion of the incubation time, EDTA and butyl hydroxy toluene (BHT) (final concentrations 0.1 mM and 0.2 mM, respectively) were added to the reaction mixture. In the photoxidation experiments, the cholesterol 5 a-hydroperoxide (5a-OOH) formed by the action of ~O2 on cholesterol, was reduced to the corresponding alcohol (5 c~-OH), by the method of Girotti et al. [25]. Aliquots of 0.5 ml from the incubation mixture containing LDL and the photosenitizer (BR or Rose Bengal) were mixed with 4.5 ml chloroform/methanol (2:1, v / v ) and 0.1 ml (200 ng) of 20ce-OH-C in ethanol. After centrifugation, the organic phase was removed and evaporated to dryness under nitrogen. One ml of chloroform/methanol (1:1, v / v ) was added, followed by 50 /.d of 50 mM NaBH 4 in methanol/10 mM NaOH. After 20 min incubation at room temperature the solvent was evaporated at 37°C under nitrogen and further processed for oxysterol analysis (see below). The TBA assay was performed according to the method of Schuh et al. [4]. Briefly, after the incubation period was completed, to 0.5 ml of the reaction mixture were added 0.2 ml of 25% TCA and 0.3 ml H20, followed by 1 ml of 1% TBA in 50% acetic acid. The mixture was vortex-mixed for 10 s. and heated in capped tubes at 95°C for 45 min. After cooling on ice, the tubes were centrifuged at 3000 rpm for 20 min and the absorbance of the supernatant read at 532 nm. Since BR and its oxidation products were found to form a chromogen with TBA (absorbing between 525-540 nm), upon heating at 95°C for 45 min, test tubes containing only BR in PBS were run concurrently in order to correct for the absorbance due to the TBA-BR adduct. It was found that neither freshly prepared BR nor the photooxidized one, exhibited a band with the same Rf as the standard TBA-MDA adduct, following TLC on silica gel plates (Table 1). In addition, we showed previously that either fresh or oxidized BR, when added before measuring TBARS, had no effect on the development of color [26]. The amount

199

Table 1 Thin-layer chromatography of TBA adducts formed between TBA and BR, oxidized BR and the BR-oxidized LDL TBA adduct formed from reaction Rf 0.56; 0.50 Freshly prepared BR (40/xM) BR (40/zM, 37°C, 24 h 0.53; 0.46 0.54; 0.46 BR (40/xM), light, 24 h 0.62; 0.63; 0.46 LDL + BR (40/zM) light, 24 h LDL+Cu 2+ (5/zM), 37°C, 24 h 0.635; 0.46 0.64 MDA (20 ~M) The standard incubation mixture contained 200 /xg LDL and the indicated concentrations of BR and CuSO4. After incubation for 24 h, the TBA reaction was performed and an aliquot was applied to a silica gel plate and chromatography carried out as described in Section 2. The MDA standard was prepared by the hydrolysis of l,l,3,3-tetramethoxypropane in 5 mM HCI at 20°C overnight. Values represent the means of four separate experiments and typically varied by less than 5%. of TBARS (TBA reactive substances) was determined by comparison to a standard curve of malonaldehyde (MDA) equivalents, prepared from acidcatalyzed hydrolysis of 1,1,3,3-tetramethoxypropane. The results were expressed in nmoles TBARS per mg protein. 2.4. Chromatographic procedures

For the detection of TBA-BR adducts the analysis included thin-layer chromatography (TLC) performed on heat activated silica G 60 plates (20 × 20 cm), in the solvent system chloroform:methanol:acetic acid (60:20:10, v / v ) . After completion of the run, the plates were allowed to dry in the air and the products of the chromatographic separation appeared as darkpurple spots on a white background. Lipid extracts from the incubation mixture containing LDL and the photosensitizer were analyzed by TLC using the solvent system, heptane:ethyl acetate (1:1, v / v ) . Cholesterol oxidation products were detected by spraying the plates with 50% sulfuric acid and briefly heating at 100°C [25]. The formation of linoleic acid hydroperoxides (18:2-OOH) following the incubation of BR with linoleic acid, was analyzed by a HPLC instrument equipped with an Adsorbsphere C 18 (5 /xm) column, 150 × 4.6 mm (Alltech). Methanol (1 ml/min) was used as an eluant and the absorbance of the effluent was monitored at 234 nm.

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Cholesterol oxidation products were analyzed by gas chromatography (GC). Before lipid extraction, to an aliquot of the reaction mixture (0.5 ml), 0.1 ml (200 ng) of 20a-OH-C in ethanol, as internal standard, was added. Lipids were extracted by adding 4.5 ml of chloroform/methanol (2:1, v / v ) , and after centrifugation, the organic phase was removed and dried under nitrogen. The oxysterol fraction was separated from phospholipids, glycolipids and their oxidation products by passage through two sets of SepPak cartridges; i.e., NH 2 and C-18 in that order. The prepurified sample was dissolved in 0.1 ml isopropanol/acetonitrile/water (45:45:10, v / v ) and injected into a 10 cm long column (Supelcosil LC-18) connected to a Waters pump (M-6000A) and a Waters absorbance detector (M-484) set to 210 nm. Elution was carried out with the above solvent at 1.8 m l / m i n . The oxysterol fraction, which eluted between 3 and 14 min, was silylated by treatment with dimethylformamide and bis(trimethylsilyl)trifluoroacetamide. GC analysis of isolated oxidation products was performed with a Hewlett-Packard 5890 Series II instrument linked to a HP Series II Integrator. The peaks on gas chromatography were identified by referring to the retention times of standard oxysterols injected into the column prior to running of the sample. The standard oxysterols were: 7ce-OH-C, 7/3OH-C, 7-keto-C, Triol, 5a-OH-C and 20o~-OH-C (internal standard). Quantification was done by comparison to peak areas relative to that of the internal standard. 2.5. Gel electrophoresis

Agarose gel electrophoresis was performed using 0.7% agarose in 0.05 M Tris-0.30 M glycine pH 8.5, diluted 1:1 in the gel and 1:4 in the running buffer. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [27], with a 6% separation gel and 4% stacking gel. The running buffer was 0.005 M Tris-0.038 M glycine (pH 8.3). The lipoproteins were detected by staining with 0.1% Sudan Black in 60% ethanol for 2 h. The gels were destained by washing with 60% ethanol, 5% acetic acid. Proteins were stained with 0.1% Coomassie brilliant blue R-250 in 30% methanol, 10% acetic acid.

2.6. Other assays

Free lysine amino groups in LDL were estimated with trinitrobenzene sulfonic acid (TNBS) as described by Aviram et al. [28]. Protein concentration was determined according to Peterson [29] using the Sigma kit P5656. 2.7. Interaction of oxidized macrophages in culture

lipoproteins

with

Mouse macrophages were isolated from the peritoneal cavity 2 days after thioglycollate stimulation and cultured in 12-well plastic culture plates (0.5 ml per well) at a density of 10 6 cells/ml in Ham's F-10 medium supplemented with 10% fetal calf serum for 24-48 h. The cells were washed with serum free Ham's medium then native and ~25I-oxidized LDL, at a concentration of 10 / x g / m l were added to the cells in Ham's medium supplemented with 2 m g / m l lipoprotein deficient serum and 1 m g / m l BSA to minimize cytotoxicity. After incubation at 37°C for 5 h, the medium was removed and assayed for trichloroacetic acid-soluble organic iodide radioactivity. Cells were washed 5-times with cold PBS, dissolved in 0.1 N NaOH and assayed for protein content. For the LDL receptor binding analysis, fibroblasts (Hs68) were grown in 60 m m culture dishes in Dulbecco's modified Eagle's medium with 4.5 g/1 glucose and 10% fetal calf serum for three days in a humidified atmosphere of 5% CO 2. The binding experiments were performed essentially as described by Goldstein et al. [23].

3. Results 3.1. Oxidative damage to LDL by the photodynamic action of bilirubin

Illumination of a mixture of LDL and BR with blue light (filtered fluorescent light) for 24 h caused changes in the lipoprotein particle, which are characteristic of oxidative stress. Following the TBA reaction, a spectrum exhibiting three peaks - - i.e., 420, 460 and 528-535 nm - - was recorded (Fig. 1). As the TLC experiments have demonstrated the TBA

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makes it a convenient model when investigating the

oxidative stress in plasma lipoproteins [30]. A lag period was recorded during the first 2 h of incubation

d

at low (20 /xM) and intermediate (40 /xM) BR concentrations, as indicated by the lack of TBARS production and conjugated diene formation. It was worth mentioning that the extent of oxidative stress

0.3 Z m

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500

WAVELENGTH

600

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Fig. 1. Absorption spectra of TBA reaction products with: (a) freshly prepared BR, (b) photooxidized BR, (c) LDL+ BR, (d) LDL + Cu2+. LDL (200/xg/ml) was incubated in 1 ml PBS with BR (40 /xM) or CuSO4 (5 ~M) at room temperature for 24 h. The TBA reaction was performed as described in Section 2.

30

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chromogen of the reaction mixture containing LDL and BR migrated on the silica gel plate very close to the T B A - M D A adduct suggesting that M D A was formed during photooxidation of LDL in the presence of bile pigment (see Table 1). In the absence of BR the extent of lipid peroxidation in the LDL particle incubated at room temperature for 24 h was rather low and the band corresponding to the T B A - M D A adduct could not be detected after thin-layer chromatography. The above qualitative results are supported by the data obtained after the quantification of the TBA reaction. The production of TBARS and the formation of conjugated diene with visible light are presented in Fig. 2. At the physiological BR concentration in plasma (20 /zM), there was no apparent TBA reactivity, but as the concentration of the bile pigment increased to 80 /xM, which is well in the range recorded for some liver disorders, the level of TBARS increased significantly reaching approximately 27% of that observed for the Cu2+-mediated LDL peroxidation. The metal-catalyzed LDL peroxidation was chosen as a reference system because the changes observed in the lipid and protein moieties of the lipoprotein particle are well characterized and this

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Fig. 2. Time-course of TBA reactivity (A) and diene formation (B) during photooxidation of LDL in the presence of bilirubin. The incubation mixture contained lml PBS with 200 /xg (A) and 100/zg (B) of LDL as well as BR at the indicated concentrations. For the purpose of comparison, LDL (200 /zg) was also incubated with CuSO4 (5 /xM) as a standard oxidation system (X). The reaction mixture was illuminated for 24 h with fluorescent light. 20 /xM BR: (zx); 40 /xM BR: (D); 80 tzM BR: ( • ) .

S.A. Hulea et al. / Biochimica et Biophysica Acta 1304 (1996) 197-209

202

was much less apparent if LDL was incubated with BR at 37°C in the dark. Thus, at the highest BR concentration tested (80 /xM), the TBARS production reached only 10% of that recorded in the light reaction (data not shown). Since BR could cause lipid peroxidation in LDL following illumination with fluorescent light, it was important to know whether BR can also induce the oxidation of one of the polyunsaturated fatty acids present in the lipoprotein particles, e.g. linoleic acid. As the results presented in Fig. 3 clearly demonstrate, linoleic acid hydroperoxides were formed, when a mixture of linoleic acid and BR in chloroform was irradiated with fluorescent light. There was a direct correlation between the amount of 18:2-OOH formed and the concentration of both BR and linoleic acid. In contrast to the light reactions, in which the oxidation of linoleic acid is mainly due to the action of IO 2, the limited LDL peroxidation occurring in the dark reactions appears to be initiated by free radical processes (data not shown).

3.2. Effect of singlet oxygen It is well known that the self-sensitized photooxidation of BR in vitro is mediated by singlet molecular oxygen [31]. As earlier work indicated that IO 2 plays a role in the BR-sensitized photoinactivation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase [32] as well as in the photodynamic action of BR on erythrocyte membranes [19], it was of interest to know whether this reactive oxygen species is implicated in the BR-sensitized oxidation of LDL. As seen in Table 2, in deuterated water, in which the lifetime of lO 2 is much greater than in H 2O, there was a higher production of TBARS than in H20 control. These findings clearly demonstrate that singlet oxygen is involved in the oxidation of LDL. In both types of water no significant increase in lipid peroxidation was noted in the first 2 h of irradiation. The fact that sodium azide, which is a known quencher of IO 2, inhibits by more than 50% the release of TBARS from LDL, further supports the

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Fig. 3. Production of linoleic acid hydroperoxide (18:2-OOH) in mixtures of linoleic acid and bilirubin. The reaction mixture (1 ml) contained linoleic acid and BR in chloroform. The reaction was started by placing the tubes under fluorescent light, 10 cm from the light source. Incubation was carried out at room temperature for 24 h. The solvent was then evaporated to dryness under a stream of N 2 and the residue taken up in 1 ml of methanol. 20 /xl of this solution was injected into the HPLC column and elution performed as described in Section 2. Four concentrations of linoleic acid in the mmolar range were used as follows: 5 mmoh (<~); 10 mmol: ( zx); 25 mmol: ([~) and 50 mmol (O).

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203

Table 2 Effect of deuterated water and azide on the BR-sensitized oxidation of LDL Incubation time

nmol TBARS/mg protein

(h)

H 20

D20

NaN 3

0 1 2 6 24

4.2±0.3 4.3±0.3 5.4±0.4 7.5±1.8 16.4±2.2

4.5±0.4 5.0±0.6 6.8±0.5 11.6±1.8 24.6±2.9

4.1±0.2 4.4±0.3 4.4±0.4 5.1±0.8 3.8±0.6

The incubation mixture contained in 1 ml: 0.05 ml LDL (2 mg/ml), 0.1 ml PBS tenfold concentrate, prepared in H20 (pH 7.6) and D20 (pD 7.6) and 80 /zl of 1 mM BR. A duplicate preparation of LDL and BR in normal water was irradiated in the presence of 10 mM NaN 3. The final concentration of D20 in the reaction mixture was approx. 85%. Incubation was performed under fluorescent light. Values represent mean ± S.D. of duplicate determination for three separate experiments. The P-values of the determination of TBARS for incubation times greater than 1 hour were highly significant ( P < 0.001).

role of this reactive oxygen species in the oxidation of lipoprotein. Our results are consistent with those of Girotti who showed that singlet oxygen is responsible, to a great extent, for the loss of erythrocyte cell membrane spectrin following the irradiation of cell ghosts in the presence of the bile pigment [18].

Fig. 4. Shifts in electrophoretic mobility of LDL illuminated with fluorescent light in the presence of bilirubin. The reaction mixture contained 1 ml PBS (pH 7.6) with 200 /xg LDL protein and where indicated, 5/zM CuSO 4, 10/xM RB and different concentrations of BR. incubation was for 24 h at room temperature. Electrophoresis was performed in 0.7% agarose gel in Tris-glycine buffer (pH 8.5) as described in Section 2. Lane 1, native LDL; 2, LDL + BR (20 /xM); 3, LDL + BR (40 /zM); 4, LDL + BR (60 /,tM); 5, L D L + B R (80 /xM); 6, LDL+RB (10 /zM); 7, LDL+ Cu2+; 8, native LDL.

extensive breaks in the apo B polypeptide chain, but more so in the case of RB (Fig. 5). Less apo B fragmentation, in the RB containing sample was ap-

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3.3. Effect of photooxidation on the electrophoretic mobility of LDL There is a large body of evidence to demonstrate that metal ion or cell-mediated oxidation of LDL results in modified particles with increased negative charge, which confers to the lipoprotein a higher anodic mobility in agarose gels. Incubation of freshly prepared LDL with BR under fluorescent light leads to shifts in electrophoretic mobility of the lipoprotein particle as compared to the native one (Fig. 4). At physiological BR concentrations (20 /xM) there was a very small shift in mobility which increased at higher BR concentration. Rose bengal (RB), a well known oxygen sensitizer, at 10/zM induced a mobility shift similar to that of BR at 60-80 /xM. For the purpose of comparison we also incubated LDL with copper ions. The analysis of apo B integrity, following the illumination of LDL in the presence of RB and BR, revealed that both oxygen sensitizers caused

Fig. 5. Apo B electrophoretic pattern following bilirubin-sensitized photooxidation of LDL. The reaction was performed in 1 ml PBS (pH 7.6), which contained, where indicated, LDL (200 /.tg protein), BR (40, 80 /~M), RB (10 /zM), EDTA (0.1 mM) and BHT (0.1 mM). The test tubes were illuminated with fluorescent light for 24 h at room temperature as described in Section 2. Lane a, native LDL; b, LDL with no additions; c, LDL+RB; d, LDL + RB + EDTA; e, LDL + BR (80) + BHT + EDTA; f, LDL +BR (40); g, L D L + B R (80); h, L D L + B R (80), reaction performed under argon; s, marker proteins (Sigma Cat. No. M-3788) containing 8 marker proteins with molecular weights ranging from 205 000 to 36000.

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parent when EDTA was present (lane d) suggesting that protein-bound Cu 2+, which is known to have binding sites on apo B [33], may be partly responsible for polypeptide chain degradation. The protein electrophoretic pattern did not change significantly when the BR concentration increased from 40 to 80 /zM (lanes f and g). This may be explained by the fact that at higher BR concentration ~O2 produced becomes also involved in the oxidation of the bile pigment due to increased substrate availability, so that less singlet oxygen is available to catalyze apo B breakdown. When BHT and EDTA were added to the reaction mixture lipid peroxidation was inhibited and hardly any degradation of apo B was noted (lane e). In addition, the absence of oxygen from the mixture containing LDL and BR resulted in complete inhibition of apo B degradation (lane h) indicating that oxygen is absolutely required for the reaction to proceed. Previous reports indicated that the shifts in electrophoretic mobility are due to the covalent modification of lysine residues in the LDL protein moiety [33,34]. Illumination of LDL in the presence of BR resulted in a significant decrease of free amino groups concentration (Table 3). The decrease was concentration-dependent and smaller than that recorded for the copper-mediated oxidation of LDL. When RB was used as oxygen sensitizer there was an even higher decrease in the concentration of free amino groups in

123

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Fig. 6. Thin-layer chromatography of lipid extracts from bilirubin-sensitized photooxidized LDL. Incubation (1 ml) in PBS (pH 7.6) contained, where indicated, LDL (200 g g protein), BR (20, 40 /xM), RB (10 /zM), BHT (1 raM). After illumination with fluorescent light for 24 h lipid extraction and reduction with sodium borohydride were carried out as described in Section 2. The 5a-OH cholesterol standard was purified by TLC from the mixture of cholesterol (500 /xg) and RB (20 /zM) in ethanol that had been illuminated with fluorescent light for 24 h. Lane 1, LDL+RB; 2, L D L + B R (40); 3, L D L + B R (20); 4, LDL+RB + BHT (after reduction); 5, LDL + BR (20) + BHT (after reduction); 6, L D L + B R (40)+BHT (after reduction); 7, 5a-OH cholesterol + 7a-OH cholesterol; 8, cholesterol + 5a-OH cholesterol; 9, 7-OH epimers (prepared by the reduction of 7-keto cholesterol).

LDL, which is consistent with the electrophoretic data. Table 3 TNBS reactivity following bilirubin-sensitized photooxidation of LDL Compound

TNBS reactivity % of native LDL

Native LDL LDL, 37°C LDL + BR (20) LDL + BR (40) LDL + BR (80) LDL + RB (20) LDL + Cu 2+, 37°C

100 95 86 74 61 55 48

The reaction mixture in PBS (1 ml) at pH 7.4 contained, where indicated, LDL (200/xg protein), CuSO4 (5 p,M), BR (20, 40, 80 /xM) and a well known oxygen sensitizer, Rose bengal (20 /xM). The test tubes were illuminated with fluorescent light at room temperature for 24 h. Values are means of duplicate determinations from three independent experiments.

3.4. Oxysterol products of LDL photooxidation In an effort to further define the changes occurring in the LDL particle during photooxidation we analyzed the cholesterol oxidation products [35] by TLC and GC. As seen in Fig. 6 several cholesterol oxides as well as the 5 a-OOH cholesterol could be detected after TLC of lipid extracts from LDL that had been illuminated with fluorescent light in the presence of RB and BR. After borohydride reduction the 5 ce-OOH component was no longer detected on the silica gel plate and the spot corresponding to the 5ce-OH cholesterol became more intense (Fig. 6, lane 4). When the reaction was performed in the presence of BHT and EDTA, the formation of 7-OH epimers was strongly inhibited as the spots corresponding to the

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Table 4 Cholesterol oxides concentration in LDL following photooxidation in the presence of oxygen sensitizers BR and RB Addition LDL, native LDL, 37°C no additions LDL + Cu2+ LDL + RB (10) LDL + RB (20) LDL + RB (20) + BHT (200/xM) LDL + BR (20) LDL + BR (40) LDL + BR (80) LDL + BR (80) + BHT (1 mM)

ng oxysterol/mg LDL protein 7c~-OH C

7/3-OH C

7-keto C

Triol

5 c~OHC

7.6 _+0.2 120 + 4.8 5202 +_68 854 _+35 1692 _ 78 32.4 + 2.6 236 _+ 10.2 625 + 18.5 960 + 31.6 25.4 _+ 1.7

5.8 _+0.2 225 +_9.6 6430 _+ 110 723 _+26 1427 _+ 195 27.9 + 2.1 208 _+9.5 552 _+ 14.2 834 _+25.0 21.8 _+ 1.6

2.2 _+0.1 54.2 _+2.7 214 _+8.5 72.8 _+4.9 99.1 +_5.6 7.4 _+0.5 15.8 + 0.8 24.6 _+ 1.1 41.2 +_ 1.9 6.1 + 0.3

3.0 _+0.1 78.2 +_3.5 473 + 15.2 195 _+7.8 245 4- 11.6 11.4 _+0.8 98.5 +_5.2 108 _+5.6 150 _+6.4 10.8 _+0.5

N.D. N.D. N.D. 1145 _+66 2856 _+ 124 980 _+35 451 +_ 12 839 + 22 1926 _ 28 524 _+9.5

Incubation (1 ml) in PBS (pH 7,6) contained, where indicated, LDL (400 p.g protein), BR (20, 40, 80 /xM), RB (10, 20 /xM), CHSO 4 (5 /xM) and BHT (200 and 1000 /xM). The incubation mixture was illuminated with fluorescent light a room temperature for 24 h. An alignot of 0.5 ml was withdrawn and the excitation and purification of oxysterols were performed as described in Section 2. After lipid extraction all samples except for the native LDL were treated with sodium borohydride to reduce the hydroperoxides to the corresponding alcohols. Values are means _+S,D. of duplicate determinations from three independent experiments (P < 0.001).

above oxysterols were not detected after the TLC run. These findings would suggest that the 7-OH epimers may have been produced through general decomposition a n d / o r allylic decay of 5c~-OOH to the more stable 7ol, 7/3-hydroperoxide (cf. Ref. [36]), but possibly also through copper-catalyzed reactions, as it is known that L D L possesses two binding sits for this metal ion [33]. Our results are consistent with those reported earlier by Girotti and his colleagues [25] on the photooxidation of membrane cholesterol in the erythrocyte ghosts. A quantitative analysis of the cholesterol oxidation products present in the lipid extracts from photooxidized L D L was afforded by GC. As seen in Table 4 both oxygen sensitizers induced considerable cholesterol oxidation, but RB more so. It is well known that the main component o f the reaction between cholesterol and ~O 2 is 3/3-hydroxy-5 a-cholest6-ene-5-hydroperoxide ( 5 a - O O H ) . Its concentration increased with the concentration of the pigment in a dose-dependent fashion. All samples were treated with sodium borohydride in order to reduce the hydroperoxides to their corresponding alcohols. It should be noted however, that 7-OH epimers may have also originated from 7-keto cholesterol, which upon reduction, yields the above oxysterols. When the reduction step is omitted the concentration of the 7-keto component is higher (data not shown). Besides

5 a - O H , significant amounts of 7-OH epimers and Triol were also detected. These latter oxysterols may have arisen through type I (radical) photochemistry. Indeed, when B H T was added to the incubation mixture, the production of 7-OH epimers was greatly inhibited suggesting that both pigments photooxidize partially via a type I mechanism. In contrast to the findings of Girotti et al. [25], who showed that B H T inhibited the formation of 7-OH epimers during the illumination of erythrocyte ghosts in the presence of protoporphyrin IX, while not affecting 5 a-OH, in our system the production of this oxysterol decreased significantly. The reason for this discrepancy is not clear, but B H T is known to react with or quench ~O 2 rather than acting as a free radical trap [37]. Therefore, it is possible that in our system BHT, which is highly lipophilic, could intercept the singlet oxygen entering the L D L particle, thus greatly diminishing the oxidation of cholesterol and unsaturated fatty acyl groups. 3.5. Binding macrophages

of

phootoxidized

LDL

to

mouse

There is ample information to show that modified L D L is avidly taken up by the macrophage scavenger receptor. As seen in Table 5, photooxidation of L D L in the presence of BR led to a modified L D L particle

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Table 5 LDL degradation by mouse macrophages, and fibroblast LDL receptor binding activity following bilirubin-sensitized LDL photooxidation Macrophage degradation ( #g LDL/mg cell protein)

B, E-receptor binding activity (ng L D L / m g cell protein)

LDL, native

0.38 ± 0.04

LDL, alone LDL + BR (20) LDL + BR (40) L D L + B R (80) LDL +RB (20) L D L + C u 2+

0.92 + 0.07 1.07 + 0.08 1.68 + 0.09 2.15+0.10 2.58-+-0.12 2.88±0.13

LDL, native

114 + 5

LDL, alone LDL + BR (40) LDL+ Cu 2+

101 ± 6 82 + 9 64__ 10

Incubations (0.5 ml) in PBS (pH 7.6) contained, where indicated, t25I-LDL (100/xg/protein), CuSO 4 (5/xM), bilirubin (20, 40, 80 /zM) and rose bengal (20 /zM). After illumination with fluorescent light at room temperature for 24 h, an aliquot was withdrawn and incubated with mouse macrophage or fibroblasts as described in Section 2. The cells were digested for 30 min in 0.1 N NaOH and aliquots taken for protein determination and counting of radioactivity. Values are means +__S.D. of duplicate determinations of three separate experiments ( P < 0.05).

that was recognized by the receptors on the mouse macrophages and the uptake of lipoprotein increased with BR concentration in the incubation mixture. Modification of the LDL particle following photooxidation is further demonstrated by the fibroblast receptor binding activity. Thus, it was found that BR-sensitized photooxidation of LDL resulted in decreased binding to the B, E-receptor as compared to native lipoprotein. At the same concentration, BR was found to be a much less potent oxygen sensitizer than RB, as judged by the LDL uptake by mouse macrophages. These findings clearly show that during light exposure in the presence of oxygen sensitizers, LDL undergoes structural changes that alter its biological properties making it no longer recognizable by the native receptor. On the other hand, it is worth mentioning that at the LDL concentration used in our photooxidation experiments (100 /xg protein/ml) some aggregation may have occurred, due to intermolecular cross-linking of apoB molecules by reactive aldehydes. When iodine-labeled LDL at the above

concentration was oxidized in the presence of either photosensitizers, about 20% of the label was recovered in the insoluble fraction after centrifugation at 10 000 × g for 10 min (not shown). Previous workers have demonstrated that aggregated Cu2+-oxidized LDL was not recognized by the macrophage scavenger receptor and suggested that the insoluble oxidized LDL may be taken up by an alternative mechanism [37].

4. Discussion

Despite a large number of studies on various aspects related to the biochemistry and metabolism of BR in normal and pathological states, there are still many unsolved problems concerning the structure and function of this hemoglobin degradation product [38]. As for the biological role of BR it is interesting to note that both pro-oxidant and ant±oxidant activities were ascribed to this molecule. Based on previous observations that BR, and to a lesser extent biliverdin, could scavenge peroxyl radicals [15,16], Stocker and his associates have recently shown that BR may function, together with ascorbate and ubiquinol-10, as a a-tocopheroxyl radical ( T O ) reductant, thus protecting LDL against lipid peroxidation [17]. In addition, recent findings from this laboratory have demonstrated that free and albumin-bound BR could inhibit transition metals catalyzed LDL peroxidation [26]. Finally, a recent clinical investigation on apparently healthy U.S. Air Force servicemen revealed that a 50% decrease in total serum BR was associated with a 47% increase in the odds of being in a more severe coronary artery disease category [39]. On the other hand, earlier reports indicated that singlet oxygen, generated through the transfer of energy from photoactivated BR to the ground state oxygen, could promote lipid peroxidation and protein cross-linking in erythrocyte ghosts irradiated with blue light [18], as well as impair membrane transport function and cause lysis of resealed red blood cell ghosts [19]. In addition, recent animal and clinical studies demonstrated a positive correlation between plasma and tissue lipid peroxides concentration and BR level, suggesting that this bile pigment may be involved in tissue lipid peroxidation [20-22]. The results presented in this work clearly show,

S.A. Hulea et al. / Biochimica et Biophysica Acta 1304 (1996) 197-209

for the first time, that the LDL particle undergoes profound changes, affecting both its physicochemical and biological properties, following photooxidation in the presence of oxygen-sensitizer bilirubin. These changes represent an intricate process involving both the lipid and protein moieties. The high lipophilicity of BR makes it a powerful initator of lipid peroxidation, when mixtures of plasma lipoproteins and the bile pigment are irradiated with visible light. Our results indicate that significant lipid oxidation occurred in the lipid moiety of LDL involving the unsaturated fatty acyl groups and free cholesterol, as judged by the increase in TBARS production, diene formation and the analysis of cholesterol oxides by gas chromatography. That unsaturated fatty acids are peroxidized under these conditions is further demonstrated by the formation of linoleic acid hydroperoxides following illumination of linoleic acid and BR in organic solvents (Fig. 3). The involvement of 102 in LDL peroxidation is well documented by the data presented in Tables 2 and 4. An increased reactivity in D20 enriched medium, the marked inhibition of lipid peroxidation by azide ion and the formation of 5-OH cholesterol argue in favor of ~O2 implications in LDL modification. Furthermore, it was found that mixtures of LDL and the bile pigment irradiated under argon showed no TBA reactivity (unpublished observations). These findings indicate that the primary reactions responsible for lipid peroxidation and apo B degradation (see Fig. 5, lane h) were oxygen-dependent. Photooxidation also caused extensive breaks in the apo B molecule as shown by SDS-gel electrophoresis. Indirect evidence based on the calculated distribution of ~O2 quenching in LDL suggested that apo B might be the main target for this reactive oxygen species [40]. However, it is not clear at present if ~O2 is directly involved in apo B modification or through by-products of lipid peroxidation. On the other hand, apo B was shown to possess two binding sites for Cu 2+, one of which allows the metal ion to be redox active [32]. The marked decrease in apo B fragmentation in the presence of EDTA (Fig. 5, lane d) supports the notion that apo B-bound Cu 2÷ is partly responsible for protein degradation in LDL. The loss of certain amino acid residues in apo B such as lysyl, histidyl, prolyl as well as fragmentation of the polypeptide

207

chain are features characteristics of metal-catalyzed oxidation of proteins [41]. It is also apparent from our study that BR-sensitized LDL photooxidation does not involve MDA-based polypeptide cross-linking, since in the presence of BHT no such effects were observed. There is ample evidence to show that an important implication of cholesterol oxide-induced cytotoxicity is its role in the development of atherosclerotic lesions (for a review see Ref. [42]). It is reasonable to assume that in pathological conditions such as hyperbilirnbinemia of the newborn, which is generally treated with phototherapy, a higher level of cholesterol oxides may arise in plasma following the attack of ~O2 on free cholesterol in lipoproteins. As the results presented in Table 5 clearly show, the BRsensitized photooxidation of LDL produced a large amount of 5 a-OH cholesterol after the reduction of the corresponding 5ot-hydroperoxides with borohydride. Other oxysterols present included cholestane3/3,5ol,6/3-triol (Triol), 7-OH epimers and 7-keto cholesterol. The latter was not completely reduced to the 7-OH epimers in our experimental conditions. Most of the 7-OH epimers originate in the 7-keto fraction, since in the absence of the reduction step the concentration of these oxysterols is much lower. Somewhat similar results regarding the production of cholesterol oxides were obtained when the photooxidation experiments were performed on whole plasma supplemented with BR to a final concentration of about 0.1 raM, a condition that matches hyperbilirubinemia in newborns and some liver disorders (unpublished observations). These findings lend additional support to the notion that oxidatively stressed LDL, with its load of oxidized cholesterol, is cytotoxic to endothelial as well as to other cells of the arterial tissue. It is widely believed that the initiation of lipoprotein oxidation leading to uptake by the macrophage scavenger receptor occurs mainly in the confinement of the arterial wall and hardly any attention was paid to exploring other possible sites, such as blood plasma. The prevailing view is that very little, if any, lipoprotein oxidation occurs in circulation because on the one hand, LDL is well endowed with antioxidant devices of its own and on the other hand, the transition metals, which are powerful prooxidants are rendered redox inactive by tight binding to several

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plasma proteins. Nevertheless, conditions such as photodynamic therapy of the newborn with hyperbilirubinemia or after systemic administration of photosensitizers to cancer patients could promote LDL peroxidation because of the action of 102 produced in those circumstances. The results of this study clearly demonstrated that BR-sensitized photooxidation of LDL led to extensive changes in the lipid and protein moieties of the lipoprotein and subsequent recognition by the scavenger receptor on the mouse macrophage. The data in Fig. 4 and Table 5 would suggest that the modified LDL particle is taken up by the mouse macrophage via an alternative mechanism, besides the scavenger receptor pathway. The increase in agarose gel electrophoretic mobility by only 2-fold and the aggregation of photo-oxidized LDL occurring at the concentration used in our study, as demonstrated by the precipitate recovered after centrifugation at 10000 × g for 10 rain, support the above assertion. Hoff et al. [37] showed recently that the soluble oxidized LDL was taken up by the scavenger receptor(s) and suggested that aggregated oxidized LDL is dealt with by the macrophage through a different mechanism, possibly one involving phagocytosis. Although we did not carry out any competitive binding assays, it is plausible that in our case too, the aggregated photooxidized LDL might be taken up a pathway similar to that proposed by Hoff et al. Although the LDL particle is well endowed with antioxidant devices conditions favoring the above mentioned alterations are likely to occur during photodynamic therapy of the newborn with hyperbilirubinemia or after systemic administration of photosensitizers to cancer patients, when the 102 produced would quickly exhaust the antioxidant molecules associated with the lipoprotein. The reaction pathway involved in LDL peroxidation could be as follows: BR + h t , ~ BR* (1) BR* + 0 2 ~ BR + I o 2

(2)

LH + lO 2 ~ LOOH

(3)

LOOH + C u 2 + ~ L O O ' + C u + + H + L O O + LH ~ LOOH + L L+ 0 2 ~ LOO

(4) (5) (6)

L O O + L O O + H + ~ L O + LOH + 10 2 (7) L O + LH ~ LOH + L (8) The above reaction pathway suggests that IO 2 is

not generated only as shown by reaction [2] but also by reaction [7]. There will be a competition for I o 2 between BR and the unsaturated fatty acyls in phospholipids and cholesterol esters. It should also be noted that during photooxidation, as earlier work suggested, BR could be degraded through a type I mechanism and this possibility increases when the bile pigment is albumin-bound [31]. During these reactions it is likely that a hydrogen atom could be abstracted from an unsaturated fatty acyls to yield a carbon-centered radical, which will react with molecular oxygen. It is conceivable that in vivo, the modified LDL particle, exhibiting sufficient alterations of specific apo B domains, may not be cleared from the circulation because it is not recognized by the native LDL receptor on the Kupffer cells. In this way, the altered LDL particle, with its load of oxidized free cholesterol may interact with and damage the endothelial layer in specific sensitive areas. Although serum albumin, which has BR binding sites, was not included in the photooxidation experiments described in this work, we have observed that when albumin, at the physiological plasma concentration (500 /zM) was mixed with BR (0.1 mM), the bile pigment was not so tightly bound to the protein as one might have expected at this high albumin to BR molar ratio. Thus, when the albumin-BR complex at the above molar ratio, was incubated for 30 min with LDL in the dark at room temperature, then the incubation mixture subjected to gel filtration on Sephacryl S-200 at 4°C in the dark, it was found that some 40% of the bile pigment was associated with the lipoprotein fraction (unpublished observations). These findings would suggest that in pathological conditions, such as the hyperbilirubinemia of the newborn or certain liver diseases, some of the 'excess' plasma BR may enter the LDL particle and trigger lipid peroxidation when activated by visible light. One should also bear in mind that BR occurs in plasma in free and conjugated forms and that all the oxidative stress-related studies on the role of BR in this process were performed using free BR. It is not clear what role, if any, has the conjugated BR in preventing or favoring the oxidative stress as well as the ability of plasma albumin to distinguish between the two forms, in relation to the suggested role of albumin-BR complex in the protection of plasma lipid against oxidation.

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Despite its limitations, the experimental system described in this paper provides the rationale for extending these studies to include also the albuminB R c o m p l e x and lipoprotein-free serum so that a better insight into the in vivo events m a y be achieved. Further clinical studies could determine to what extent phototherapy contributes to increased oxidative stress in blood p l a s m a and what kind of strategies can be devised to minimize the photooxidation of lipoproteins and hence, injury to the arterial wall, a condition that is associated with the initiation of the atherosclerotic process.

Acknowledgements This work was supported by a grant f r o m the Wallace Genetic Foundation, Tarrytown, NY. W e also thank Dr. Radu Olinescu for his helpful suggestions and Dottie Slavik for excellent editorial assistance.

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