Determination of the α-Tocopherol Inhibition Rate Constant for

Development of a tocopherol-mediated peroxidation (TMP) model and ... the inhibition rate constant of R-TOH in a lipoprotein of kinh ) 5.9 ( 0.5 × 10...
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Chem. Res. Toxicol. 2002, 15, 870-876

Determination of the r-Tocopherol Inhibition Rate Constant for Peroxidation in Low-Density Lipoprotein Sean M. Culbertson,† Fernando Antunes,‡ Christine M. Havrilla,† Ginger L. Milne,§ and Ned A. Porter§,* Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, Grupo de Bioquı´mica e Biologia Teo´ ricas and Centro de Estudos de Bioquimica e Fisiologia, Lisbon University, P-1749-016 Lisbon, Portugal, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California 90033, and Department of Chemistry, Duke University, Durham, North Carolina 27708 Received February 7, 2002

This work reports an estimate of the inhibition rate constant (kinh) for R-tocopherol (R-TOH) in low-density lipoproteins (LDL) based on cholesteryl linoleate hydroperoxide products formed during autoxidation of intact lipoproteins. The ratio of cis,trans/trans,trans product hydroperoxides was determined during the consumption of the antioxidant. For a reasonable determination of kinh in LDL, the pro-oxidant behavior of R-TOH was minimized by oxidizing LDL with an unsymmetrical amphiphilic azo initiator which significantly reduces phasetransfer mediated pro-oxidant effects of R-TOH. This initiator delivers a more constant flux of initiator radicals into LDL lipid regions and permits determination of R-TOH kinh in LDL. Development of a tocopherol-mediated peroxidation (TMP) model and analysis of cholesteryl linoleate hydroperoxide cis,trans/trans,trans product ratios provided an estimated value for the inhibition rate constant of R-TOH in a lipoprotein of kinh ) 5.9 ( 0.5 × 105 M-1 s-1

Introduction Lipid peroxidation of low-density lipoprotein (LDL)1 lipids is implicated in the initial events leading to atherosclerosis (1-3). Chain-breaking antioxidants are generally believed to protect LDL lipids from autoxidation by trapping the propagating peroxyl radical and “breaking” the chain reaction (4). The potent phenolic antioxidant R-tocopherol is one of the most effective lipidsoluble antioxidants (5, 6). The antioxidant activities (kinh) of R-TOH and other phenols have been the object of extensive investigations for autoxidations in homogeneous organic solvents (79). The number of chains terminated by each molecule of R-TOH is two and the overall kinetic rate law is given by eq 1

d[LOOH]/dt ) Rikp[LH]/(2kinh[R-TOH])

(1)

where Ri is the rate of generation of the radicals which initiate peroxidation chains. The kinh for R-TOH in * To whom correspondence should be addressed. Phone: (615) 3432693. Fax: (615) 343-5478. E-mail: [email protected]. † Department of Chemistry, Duke University. ‡ Grupo de Bioquı´mica e Biologia Teo ´ ricas and Centro de Estudos de Bioquimica e Fisiologia. § Department of Chemistry, Vanderbilt University. 1 Abbreviations: AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; AH, ascorbic acid; AMVN, 2,2′-azobis(2,4-dimethylvaleronitrile); R-TOH, R-tocopherol; R-TO‚, R-tocopheroxyl radical; C-0, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride; Ch18:2-OOH, cholesteryl linoleate hydroperoxide; DTBN, di-tert-butylhyponitrite; HBA, hydrogen bond acceptor; LDL, low-density lipoprotein; LH, lipid with bis-allylic hydrogen; LOOH, lipid hydroperoxide; LOO‚, lipid peroxyl radical; PBS, phosphate-buffered saline; PLPC, 1-palmitoyl-2-linoleoylsn-glycero-3-phosphatidylcholine; ROO‚, peroxyl radical; TMP, tocopherol-mediated peroxidation; UA, uric acid.

homogeneous aprotic solvents (styrene at 30 °C) is 3.2 × 106 M-1 s-1 (6). Polar solvents have been found to reduce the radical trapping activity of R-TOH (9). For example, R-TOH kinh is 50-fold lower in ethanol than in cyclohexane. Slower trapping of R-TOH in polar solvents was attributed to hydrogen bonding of the antioxidant phenolic hydrogen in hydrogen bond acceptor (HBA) solvents. Rate constants for peroxyl radical trapping by R-TOH appear to be substantially lower in micelles and lipid bilayers than those determined in isotropic media. Thus, the rate constants for inhibition in SDS micelles (kinh ) 3.7 × 104 M-1 s-1 at 37 °C) (10) and 1-palmitoyl-2linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) liposomes (kinh ) 4.7 × 104 M-1 s-1 at 37 °C) (11) are significantly lower than the values reported for any homogeneous system (tert-butyl alcohol, a good HBA solvent; kinh ) 5.1 × 105 M-1 s-1 at 37 °C) (12). Determinations of antioxidant activity are less reliable and more difficult to interpret in heterogeneous aqueous media such as micelles, phospholipid membranes, and especially lipoprotein than in isotropic solutions (11, 13). It should be noted, however, that the low rate constants for inhibition determined in micelles and liposomes cannot be explained by hydrogen bonding effects since water is not as good a HBA solvent as tert-butyl alcohol. In fact, it has been concluded that only a “...small fraction of the observed rate diminution in micelles and lipid bilayers can be attributed to hydrogen bonding of R-tocopherol to water...” (14). Other factors that may be responsible for reducing the antioxidant activity of phenols in heterogeneous media are the physical separation of the reactants and/or slow diffusion of the antioxidant and the chain-carrying peroxyl radicals (7, 15, 16). In a lipoprotein oxidation, an

10.1021/tx020012t CCC: $22.00 © 2002 American Chemical Society Published on Web 05/15/2002

Determination of R-TOH kinh in LDL

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Scheme 1. Oxidation of Cholesteryl Linoleate Forms Four Isomeric Hydroperoxidesa

a During oxidation of linoleate, a bis-allylic hydrogen at the 11-position is abstracted by an initiating radical or a lipid peroxyl. Addition of oxygen at the 9- or 13-position forms a peroxyl radical conjugated diene. Good hydrogen atom donors, such as R-TOH, can quickly trap the c,t peroxyl radicals to form c,t hydroperoxides (kinetic products).2 Poorer hydrogen atom donors, such as LH, allow conformational equilibration of the peroxyl radical and loss of oxygen leads to a new isomerized carbon radical. A second addition of oxygen forms t,t peroxyl radical conjugated dienes that can be trapped to t,t hydroperoxides (thermodynamic products).

R-TOH molecule in one particle cannot capture a LOO‚ in a second particle, unless LOO‚ can exchange between particles (17). The limited diffusion of antioxidant and radical species in heterogeneous systems creates complications because (1) lipid oxidation may arise in isolated pools of substrate locally depleted of antioxidant (2, 13, 18) and (2) the isolation of R-TO‚ from initiating or propagating radicals permits R-TOH to be involved in chain-transfer reactions (7, 17, 19). For PLPC liposomes, the most accurate value for R-TOH kinh was determined by measurement of lipid hydroperoxide products of linoleate, an approach which avoids complications due to chain-transfer reactions of antioxidant radicals (pro-oxidant effects) (11). This method makes use of the known direct relationship between the ratio of kinetic to thermodynamic peroxidation products formed and the hydrogen atom-donating ability of the medium (Scheme 1) (20, 21). In these liposome experiments, the cis/trans (kinetic products) to trans/trans (thermodynamic products) product ratios (c,t/t,t) of the 9- and 13-hydroperoxides in linoleate oxidation were determined in the presence of known amounts of R-TOH. The conditions of oxidation were such that the concentration of both the lipid and the antioxidant remained constant during the course of the experiment. This was accomplished by oxidizing only a small portion of the lipid (typically less than 5%) and regenerating R-TOH with the water-soluble antioxidants ascorbate and homocysteine3 (12, 22, 23, 24). Since there are two H-donors in the regions of lipid oxidation (linoleate and R-TOH), eq 2 controls the product ratio and 2 Recent findings have found that the linoleate bis-allylic 11hydroperoxide is one of the initial (kinetic) products of linoleate oxidation and can be trapped using high R-TOH concentrations [Brash, A. (2000) Lipids, 35, 947-952]. 3 Both ascorbate and homocysteine are partitioned into the aqueous phase and have been shown to be only weak H-donors to peroxyl radicals formed in the lipid phase of liposomes. Homocysteine regenerates ascorbate and ascorbate regenerates R-TOH.

kinh kp c,t ) [R-TOH] + [LH] + t,t kβII(1 - R) kβII(1 - R) RkβIII kβII(1 - R)

(2)

kinh can be determined because the values for kp and kβII(1 - R) have been determined for linoleate oxidation in PLPC bilayers (11, 21). A method that followed c,t/t,t product ratios during the consumption of antioxidant was also used for determinations of the kinh for R-TOH in PLPC bilayers (11). In this approach, the H-donating ability of the medium was permitted to change during oxidation. Therefore, the ratio of products at time t depends on the H-donating ability of the medium at that time and the accumulation of hydroperoxides preceding time t. Integration of eq 2 and assumption of a high kinetic chain-length (g10) gives eq 3,

(1 + Ratio0)(eG(t) - 1) )

kinh kβII(1 - R)

Ri(τ - eG(t)(τ - t)) (3)

where G(t) ) (Ratio(t) - Ratio0) ln(τ/τ - t)/(1 + Ratio(t)), Ratio0 ) (RkβIII + kp[LH])/(kβII(1 - R)), Ratio(t) is the cis,trans/trans,trans ratio at time t, τ is the length of the induction period, and Ri is the rate of initiation. The rate constant of inhibition for R-TOH determined by a linear fit of experimental data to eq 3 (kinh ) 2.1 × 104 M-1 s-1 at 37 °C) was less than half the value obtained when R-TOH was continuously regenerated (kinh ) 4.7 × 104 M-1 s-1 at 37 °C) (11). It was concluded, from experimental data and simulations, that consumption of antioxidant allowed pro-oxidant reactions to occur which influenced the rate of consumption of the antioxidant and consequently lowered the apparent kinh for R-TOH. Heterogeneous media, such as phospholipid bilayers, increase the pro-oxidant activity of an antioxidant com-

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pared to homogeneous systems. Thus, the best method for calculating kinh in heterogeneous media is to monitor c,t/t,t product ratios when the antioxidant is kept reduced and the pro-oxidant effects of the antioxidant are completely avoided. In vitro studies have shown that R-TOH in oxidizing lipoproteins can exhibit even more significant pro-oxidant behavior than is observed in liposomes (7, 17, 25). It nevertheless seems likely that R-TOH activity in vivo provides antioxidant protection for LDL because of the synergistic effects of the co-antioxidants ascorbate and ubiquinol-10 (19, 26). A range of values for kinh of R-TOH in LDL (6 × 103 to 3 × 106 M-1 s-1) has been assumed in the literature for this important reaction (13). These values were based on determinations of analogous reactions in micelles, liposomes, and organic solvents4 (6, 12, 27). The unusual behavior of R-tocopherol in lipoprotein particles indicates a need for an independent determination of the antioxidant activity of R-TOH in intact LDL. We report here an estimate of the inhibition rate constant for R-TOH in LDL based on c,t/t,t product ratios determined for oxidation of lipoproteins (11).

Materials and Methods Phosphate-buffered saline (PBS, pH 7.4, 50 mM) was placed over Chelex-100 resin for 24 h. The resin was removed and 100 µM ethylenediaminetetraacetic acid (EDTA) was added to remove trace metal contaminants (28). All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and used without further purification, unless otherwise noted. The azo initiator C-0 (VA-044) was generously donated by Wako Chemicals USA Inc. (Richmond, VA). Unsymmetrical azo initiators C-8, C-12, and C-16 were synthesized as described in detail previously (29). The antioxidants, R-tocopherol, and δ-tocopherol, were purified as described previously (30). Solvents were HPLC quality and purchased from commercial sources. Analytical HPLC was carried out on a single Beckman 5 µM Ultrasphere C-18 column (4.6 mm × 25 cm) used for reverse phase conditions, and two tandem Beckman 5 µM Ultrasphere columns (4.6 mm × 25 cm) used for normal phase conditions. The HPLC equipment used was as specified earlier (30). Lipoprotein Isolation. Briefly, whole blood from fasting, normolipidemic healthy subjects was collected and centrifuged for 10 min at 4200 rpm to obtain plasma. The LDL was isolated from plasma over 15 h by density gradient sequential ultracentrifugation at 14 °C using a Beckman Optima LE-80K centrifuge and a Ti 70 rotor. Each spin was performed at 504 000g for 5.5 h (31). Lipoproteins were desalted by passage through two consecutive PD-10 gel filtration columns and sterilized by passage through a Gelman 0.2 µm Supor membrane filter. Protein concentrations were determined by the method of Lowry as modified by Peterson (32). LDL isolation was confirmed with the use of SDS-PAGE separation of associated apoproteins (33) and Beckman Instruments Paragon LipoGel electrophoresis of intact lipoproteins (34). Oxidation of LDL. Lipoprotein concentrations were adjusted with PBS to give final concentrations of 0.75 mg of protein/mL and allowed to equilibrate to 37 °C for 5 min. To the stirred suspension were added water-soluble antioxidants as needed followed by addition of initiator dissolved in methanol (except for AAPH and C-0). Initiators were added to provide final initiator concentrations from 0.25 to 3.0 mM and e3% (v/v) methanol. Following addition of initiator (time zero) aliquots for R-TOH, cholesterol linoleate oxidation (Ch18:2-OOH), and uric acid (UA) were removed at various intervals and treated 4 Probably one of the more common estimates for R-TOH k inh in LDL has been ∼1 × 105 M-1 s-1.

Culbertson et al. as described below. Aliquots were taken for R-TOH analysis 200 µL, UA analysis 50 µL, and cholesteryl linoleate product analysis 200 µL and placed directly on ice. To the R-TOH aliquot was added butylated hydroxytoluene (BHT) and internal standard, δ-tocopherol. Extraction of all the aliquots was performed with ice-cold methanol and hexanes. The hexane phase was removed and concentrated under argon, and then stored at -78 °C until analysis. The UA aliquots were simply stored at -78 °C until analysis. Analysis for R-TOH and UA was by reversed-phase HPLC with electrochemical detection described previously (29). To the cholesteryl linoleate 200 µL aliquot was added BHT, the internal standard 13-hydroxyoctadecane,cis-9,trans-11-dienoate, and the hydroperoxides were reduced to alcohols with the addition of triphenylphosphine (∼1 mmol). These reduced cholesteryl linoleate oxidation (Ch18:2-OH) samples were then extracted the same as the R-TOH samples. However, analysis was by normal phase HPLC using 0.5% isopropyl alcohol in hexanes with ultraviolet detection (234 nm), which provided separation of the four isomeric Ch18:2-OH products as described previously (35). Local concentrations were calculated using the oxidation protein concentration, assuming apoprotein B100 ) 550 kDa and total lipid volume in a LDL particle ) 3.2 × 10-21 L (17). The local rate of radical initiation in the lipid (Ri) was calculated from the local rate of consumption of R-TOH (Ri ) 2‚d[R-TOH]/dt).

Results and Discussion Oxidation with Regeneration of R-TOH. To simplify the analysis of data, determination of R-TOH kinh in LDL should employ a method that eliminated prooxidant effects arising from chain-transfer reactions of R-TO‚ with LOOH and LH. For oxidation of PLPC liposomes, R-TOH pro-oxidant activity was avoided by others by regeneration of the R-TOH from the reduced R-TO‚ by addition of excess ascorbate and homocysteine (11). Both ascorbate and homocysteine are partitioned into the aqueous phase and they apparently do not affect, by themselves, lipid peroxidation in the lipid aggregates. Ascorbate is known to regenerate R-TOH across the lipid-aqueous interface in both liposome and lipoprotein systems (17, 22, 36) and homocysteine has been shown to regenerate ascorbate (24). Since the peroxyl radicals generated from watersoluble initiators were expected to be scavenged by ascorbate and homocysteine before they entered the lipid phase to initiate oxidation, lipid phase initiation was chosen for these studies. Unfortunately, any LDL oxidations initiated by di-tert-butylhyponitrite (DTBN) and 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) that included ascorbate and homocysteine gave no detectable cholesteryl linoleate hydroperoxides (see Supporting Information Figure SI-1). Eventual consumption of R-TOH was observed under these conditions, presumably due to termination reactions and adduct formation, but no oxidation of lipid occurred (37, 38). The amphiphilic unsymmetrical azo initiators C-8, C-12, and C-16 (30) were developed largely in hopes that

they would initiate LDL lipid oxidation in the presence of both R-TOH and ascorbate. Decomposition of the unsymmetrical azo initiator and escape from the geminate radical pair generates one free amphiphilic peroxyl

Determination of R-TOH kinh in LDL

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Figure 1. Analysis of R-TOH kinh using c,t/t,t ratios and the integrated rate eq 3. Conditions of oxidation: LDL 0.75 mg of protein/ mL was initiated at 37 °C in the presence of C-8 1.0 mM. C-8 was added as a concentrated solution in methanol (e3% v/v). (A) Product composition vs time. Cholesteryl linoleate oxidation products trans,cis-13-OH Ch18:2 (squares); trans,trans-13-OH Ch18:2 (diamonds); cis,trans-9-OH Ch18:2 (open circles); trans,trans-9-OH Ch18:2 (triangles) and R-TOH (crossed circles) were determined as indicated in the text. (B) Data fit. The c,t/t,t ratios were fit to eq 3 and the slope of this line gives Rif(kinh), where f(kinh) ) kinh/ [kβII(1 - R)]. Ri was determined by the induction method (Ri ) 2 [R-TOH]0/t, where t is the length of the induction period).

radical and one free hydrophilic peroxyl radical. The hydrophilic peroxyl was expected to partition to the aqueous phase where aqueous antioxidants, such as ascorbate, could scavenge the hydrophilic radical and thus decrease macromolecular cage and interparticle terminations from hydrophilic/lipophilic peroxyl radical recombination. Unsymmetrical azo initiators C-8 (0.53.0 mM), C-12 (0.5 mM), and C-16 (0.5 mM) were tested for initiation of LDL (0.75 mg protein/mL) lipid oxidation in the presence of ascorbate. Lipid peroxidation was prevented in all of these experiments, even when R-TOH was depleted from the LDL (11), and for this reason the regeneration method was abandoned. LDL Oxidation with Consumption of R-TOH. The failure of the regeneration method required that kinh be determined by an alternate strategy, one that involves monitoring the c,t/t,t hydroperoxide product ratios during consumption of R-TOH. In PLPC liposomes, this method resulted in a lower estimation of kinh than is obtained from the R-TOH regeneration method, presumably because pro-oxidant effects of R-TOH are not explicitly included in the analysis (11). For LDL experiments with this approach, several initiation conditions were investigated in order to minimize the pro-oxidant activity of R-TOH during the consumption of antioxidant. To avoid these effects, the rate of initiation needed to be rapid enough to terminate an R-TO‚radical before its reaction with LH could occur (15). On the other hand, oxidation of LDL should not be so rapid as to consume R-TOH without formation of any significant oxidation products. Indeed, very rapid oxidation of LDL with high concentrations of water-soluble 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) is a good method for depleting LDL of endogenous R-TOH with formation of only very minor amounts of lipid oxidation products (25). Measurable amounts of both cis,trans and trans,trans cholesteryl linoleate hydroperoxide products were formed when conditions were chosen such that the induction period was an hour or longer. Initiators tested were: water-soluble AAPH, lipid-soluble DTBN, and unsymmetrical amphiphilic initiators C-8, C-12, and C-16 (Figure 1 and Supporting Information Figure SI-2).

The four isomeric cholesteryl linoleate oxidation products were reduced with PPh3 and determined by normal phase HPLC with UV detection as described previously. The unsymmetrical amphiphilic azo initiator C-8, which significantly circumvents phase-transfer mediated pro-oxidant effects of R-TOH, was chosen as the optimal initiator for determination of R-TOH kinh in LDL using the antioxidant consumption method. Application of the integrated rate eq 3 to the c,t/t,t ratios obtained with a given initiator provides a linear fit where the slope is Ri[kinh/(kβII(1 - R))] (Figure 1) (11). (Examples of oxidation profiles using AAPH, DTBN, C-12, and C-16 can be found in the Supporting Information.) A value for kβII(1 - R) in lipoproteins has not been determined and would require substantial manipulation of LH levels in the absence of antioxidant. Considering that LDL contains several lipid H-donors and because manipulating the levels of a lipid, such as cholesteryl linoleate, in LDL would not be an easy task, if at all possible, the determination of kβII(1 - R) in lipoprotein was not pursued in this research. Thus, the determined ratio of kinh/[kβII(1 - R)] was reported in Table 1 as f(kinh). We believe the most appropriate estimate of kβII(1 R) for cholesteryl linoleate oxidation in lipoproteins is represented by the value reported for oxidation of methyl linoleate measured in benzene [kβII(1 - R) ) 142 M-1] (21). Therefore, we report estimated values of R-TOH kinh in LDL using this assumption for kβII(1 - R) (e.g., f(kinh)kβII(1 - R) ) 2478 M-1 × 142 s-1 ) 3.5 × 105 M-1 s-1). However, this assumption for kβII(1 - R) is made with the caveat that this parameter may be as low as the kβII(1 - R) measured for PLPC oxidation in multilamellar PLPC/DMPC liposomes [kβII(1 - R) ) 41 s-1] (11) (e.g., f(kinh)kβII(1 - R) ) 2478 M-1 × 41 s-1 ) 1.0 × 105 M-1 s-1). This assumption of kβII(1 - R) from benzene is supported by the notion that the fluidity of cholesteryl linoleate in the LDL lipid core is more comparable to that in benzene than the more limited motion expected in phospholipid regions (13, 39, 40). Best Estimate of kinh in Tocopherol-Mediated Peroxidation of LDL. The integrated rate eq 3 is derived from the conventional mechanism of lipid peroxidation and inhibition in which initiating radicals react

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Culbertson et al.

Table 1. Integrated Rate Equation Analysis Results Using a Variety of Azo Initiators initiatorc

concentration (mM)

tau (min)

Ratio0d

Ri (×10-6)e (M s-1)

slope

R2

f(kinh)a (M-1)

kinh (×105)b (M-1 s-1)

AAPH DTBN C-8 C-12 C-16

20 2.0 1.0 0.5 0.5

70 180 120 510 1200

0.50 0.50 0.50 0.41 0.41

2.4 0.70 1.2 0.29 0.12

0.0031 0.0015 0.0030 0.0005 0.0002

0.992 0.997 0.990 0.994 0.995

1800 2114 2478 1603 1915

2.6 3.0 3.5 2.3 2.7

a The integrated rate eq 3 was applied to determine values for f(k inh) [where f(kinh) ) kinh/(kβII(1 - R))] from LDL oxidation c,t/t,t product ratios initiated with the indicated azo initiator. b Estimated kinh assuming kβII(1 - R) ) 142 M-1 as reported for methyl linoleate oxidation in benzene. c The product profiles and experimental conditions for each initiator used are given in Figure 1A or Figure SI-2 (Supporting Information), as appropriate. An example of the linear analysis from Equation 3 is shown for C-8 initiated LDL oxidation in Figure 1B. d Ratio was determined from product profiles in R-TOH-depleted LDL oxidation when possible. e R was determined by the induction 0 i method (Ri ) 2[R-TOH]0/t, where t is the length of the induction period).

Scheme 2. Tocopherol-Mediated Model of Lipid Peroxidation in LDL

where

F(t) ) {kβII(1 - R) + kβIIRatio0(1 - R) + kinhRi/2τ}/ {kβII(1 - R) + kβIIRatio0(1 - R) + kinhRi/2(τ - t)}

directly with lipid before being scavenged by R-TOH (see Supporting Information Scheme SI-1) (7, 13). The evidence that LDL peroxidation may occur via a TMP mechanism suggests that pro-oxidant effects should not be ignored in the analysis. Therefore, reactions involving tocopherol-mediated peroxidation were included in the kinetic analysis to obtain the best estimate of R-TOH kinh in LDL (Scheme 2). The overall rate expression for this scheme, assuming a steady-state approximation for the production of LOOH, is given by eq 4,

Rp ) kiTOH([R-TOH]kinh + kp[LH])kTMP[LH]/(kt4kinh) (4) In this model, initiating peroxyl radicals react preferentially with R-TOH and lipid hydroperoxides are generated via reaction of L-H with R-TO‚. The TMP model better represents LDL oxidation in vitro with C-8 and a variety of other oxidants as well (15). Two different approaches were used to fit experimental c,t/t,t ratios to the kinetic TMP model presented in Scheme 2. This model was expanded to include β fragmentation of peroxyl radicals that leads to isomerization of the cholesteryl linoleate radicals (Scheme 1) (21). The new equation, which has terms for c,t/t,t hydroperoxide product formation (eq 5), is considerably more complex than the integrated rate expression (eq 3) and cannot be linearized.

Ratio ) {(kβII(R - 1)/kinht + Ri/2τt - Ri/4t2 + kβII2(R - 1)2)/(kinh2Ri/2)(1 + Ratio0)log[F(t)]}/ {-kβII(R - 1)/kinht - kβII2(R - 1)2/(kinh2Ri/2)(1 + Ratio0)log[F(t)]} (5)

Equation 5 was solved numerically by applying the Levenberg-Marquardt algorithm implemented in Mathematica (41) (Table 2). The Gepasi system for modeling biochemical systems (42) was also used to fit the experimental time course of the c,t/t,t ratio to the set of differential equations that describe the full TMP kinetic model (Table 2). This analysis was expanded to include two peroxidizable LH groups (one accounting for cholesteryl linoleate, Ch18:2, and the other for remaining LH groups in LDL)5 (17). The Gepasi fitting implemented a multistart Levenberg-Marquardt algorithm and used the rate constants that follow kp ) 30 M-1 s-1; kTMP ) 0.05 M-1 s-1; kperox ) 1 × 108 M-1 s-1; kt4 ) 1 × 108 M-1 s-1; kβI, kβIII ) 27 s-1 (21); kβII, kβIV ) 430 s-1 (21); a ) 0.67 (21). Since the fittings were done using the whole kinetic model, it was not necessary to assume the approximation of quasisteady state. The values for f(kinh) obtained by both methods were fairly independent of the values chosen for the other rate constants (kp, kTMP, kβII, kβIII, etc.). Because TMP lowers the apparent antioxidant “capacity” of R-TOH, it seems reasonable that the values determined for f(kinh) (and thus kinh) were higher than when the data was analyzed ignoring TMP. Both fittings, to eq 5 and to the set of differential equations, gave similar results, which gives us confidence that the analysis is appropriate. We believe that the Gepasi estimation of kinh, which considers TMP, is the best method to compensate for pro-oxidant effects of the antioxidant. With the assumption of the value of kβII(1 - R) in benzene6 (21), the estimated value for R-TOH kinh in LDL is 5.9 × 105 ( 0.5 × 105 M-1 s-1.7 Comparison of r-TOH Antioxidant Activity in LDL and PLPC Liposomes. Liposomes are oxidized with high kinetic chain lengths in the presence of ascorbate and R-TOH (11) while lipoproteins are completely protected against oxidation8 (43). Ascorbate ef5 The average molecules of Ch18:2 reported per LDL is 720 which represents a local concentration of 0.374 M Ch18:2. Likewise, the average number of total LH groups is reported as 1450 indicating a local concentration within the LDL of 0.752 M. The assumed concentration for remaining LH groups was 0.752-0.374 ) 0.378 M. 6 This value is reported with the caveat that k (1 - R) may be as βII low as the value reported in multilamellar PLPC/DMPC liposomes (11). For example, f(kinh)kβII(1 - R) ) 4169 M-1 × 41 s-1 ) 1.7 × 105 ( 0.14 × 105 M-1 s-1. 7 Where the upper limit is represented by R-TOH k inh reported in styrene (3.2 × 106 M-1 s-1) (6).

Determination of R-TOH kinh in LDL

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Table 2. r-TOH f(kinh) Analysis in LDL from C-8 Initiated Lipoprotein Oxidationsa

[C-8] (mM)

UA

0.5 1.0 1.0 2.0 0.5 2.0 2.0 2.0 1.0 1.0 mean ( SD

+ +

+

tau (min)

Rif (×10-6) (M s-1)

eq 3b f(kinh) (M-1)

Scheme 2 eq 5c f(kinh) (M-1)

Scheme 2 eq 5c kinh (×105)d M-1 s-1)

360 95 105 80 210 58 66 110 117 120

0.29 1.67 1.58 1.87 0.77 2.48 2.32 1.24 1.31 1.19

2610 2382 2370 2737 2204 2375 2815 2727 2549 2478 2525 ( 187

3817 [17] 3787 [2.9] 3843 [3.4] 4258 [9.4] 3932 [1.5] 4321 [2.1] 4450 [14] 4834 [0.9] 4390 [2.5] 4423 [1.0] 4206 ( 329

5.4 5.4 5.5 6.0 5.6 6.1 6.3 6.9 6.2 6.3 6.0 ( 0.5

Scheme 2 Gepasie f(kinh) (M-1) 3807 ( 151 3724 ( 50 3761 ( 51 4306 ( 102 3866 ( 41 4277 ( 64 4413 ( 165 4790 ( 37 4348 ( 36 4386 ( 63 4169 ( 337

Scheme 2 Gepasie kinh (×105)d (M-1 s-1) 5.4 5.3 5.3 6.1 5.5 6.1 6.3 6.8 6.2 6.3 5.9 ( 0.5

a LDL 0.75 mg of protein/mL was initiated at 37 °C in the presence of C-8 0.5-2.0 mM; some experiments also contained added uric acid (UA) ∼60 mM when indicated. b The integrated rate eq 3, derived from the conventional model of lipid peroxidation, was applied to determine values for f(kinh) as indicated in the caption of Figure 1 and Table 1. c Values of f(kinh) obtained from a numerical fitting of c,t/t,t ratios to eq 5. The chi-squared values for the numerical fitting of c,t/t,t data with one degree of freedom are given in the brackets ([]). d Estimated kinh assuming kβII(1 - R) ) 142 M-1 as reported for methyl linoleate oxidation in benzene (see text). e Values of f(kinh) obtained from a Gepasi biochemical kinetic model fitting of c,t/t,t ratios to TMP model (Scheme 2) implementing a multistart LevenbergMarquardt algorithm. f Rate of initiation, Ri, determined as indicated in caption of Table 1.

ficiently inhibits TMP (via radical chain-transfer) in LDL by reacting with R-TO‚ to regenerate R-TOH (eq 6, kAH) 3 × 102 - 2 × 105 M-1 s-1)9 (13, 44, 45, 46). kAH-

AH-(aq) + R-TO‚(lipid)98A•-(aq) + R-TOH(lipid) (6) This work suggests that kinh for R-TOH in LDL is approximately 12-fold greater than that reported in PLPC liposomes [5.9 × 105 M-1 s-1 in LDL and 4.7 × 104 M-1 s-1 in liposomes (11)] and this rate difference may account for the difference in oxidizability observed between liposomes and LDL. In both systems the regeneration of R-TOH by ascorbate significantly eliminates the pro-oxidant activity due to the reaction of R-TO‚ with LH (7, 11, 15). The lack of measurable oxidation products in LDL containing ascorbate suggests that in lipoproteins the initiating radicals do not directly react with lipid to a significant extent. Indeed, the ratio of ([R-TOH]kinh)/ ([LH]kp) for even one molecule of R-TOH in a LDL particle [(0.000 52 M × 5.9 × 105 M-1 s-1)/(0.752 M × 50 M-1 s-1)] suggests that initiating peroxyls will be more than eight times as likely to react with R-TOH than with LH in the lipoprotein. A 12-fold reduction of kinh in liposomes would make the reaction of LOO‚ with L-H competitive with its reaction with R-TOH, and this is consistent with the experimental observations. Significant linoleate hydroperoxide products are formed from oxidation of liposomes in the presence of ascorbate while none are observed from lipoproteins (data not shown). This suggests that ascorbate has ready access to tocopheryl radicals in lipoproteins while this is not the case for liposome structures. Of course, liposome and lipoprotein structures are quite different. An important question of lipoprotein oxidation in vivo concerns whether initiating radicals that can access the lipid regions of a lipoprotein exist (2, 22, 47). We are unaware of any free radical intiation system in vitro that can promote LDL oxidation directly in the presence of 8 It should be noted that the UV method used in these experiments is not the most sensitive method available for determination of cholesteryl linoleate hydroperoxides. Chemiluminescence detection of hydroperoxides is a much more sensitive method and would have provided much lower detection limits, however this equipment was not available. 9 The estimated range given for k AH- represents values determined in a variety of media excluding LDL.

both R-TOH and ascorbate (lipoxygenase enzymes might perform this indirectly) (15, 48, 49, 50). A recent report suggests that ascorbate can actively decompose linoleic acid hydroperoxides via one electron reduction, giving in the process a variety of reactive aldehyde and ketone products (51). It may be possible that ascorbate could act in a similar way in LDL oxidations. That is, if small amounts of lipid hydroperoxides are formed during a lipoprotein oxidation, they could be converted by ascorbate to reactive aldehyde or ketone compounds which could modify apoprotein. We doubt that this is the case in our studies, however, since we expect that cholesterol linoleate alcohols would be among the products of reaction of ascorbate with hydroperoxides and we do not find these compounds in LDL reactions that include ascorbate.

Acknowledgment. Support of this research from an NSF Award (Grant 9996188), a NIH (HL17921) grant, and a PRAXIS XXI/FCT fellowship (BPD/11779/97) are gratefully acknowledged. Supporting Information Available: Additional information available as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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