Review

Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of the hydroperoxide-reducing proteins from human blood plasma

Correspondence to: Yorihiro Yamamoto.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS and DISCUSSION REFERENCES

Plasma glutathione peroxidase (GSHPx) has been suggested to reduce submicromolar levels of free fatty acid hydroperoxides and phosphatidylcholine hydroperoxides (PC-OOH), and therefore these hydroperoxides are undetectable in human blood plasma. The capacity for the reduction should be about 2.5 µM as the level of glutathione in human plasma is about 5 µM. However, 2 h of aerobic incubation of 58 µM PC-OOH in human plasma at 37°C resulted in the formation of 36 µM phosphatidylcholine hydroxide (PC-OH). The presence of PC-OOH-reducing protein other than plasma GSHPx was suggested by the results. a) The same rates of PC-OOH decay and PC-OH formation were observed in both sera from rats with selenium-deficient and selenium-supplemented diet; b) the PC-OOH-reducing activity was observed only in the high molecular weight fraction but not in the low molecular weight fraction; and c) albumin did not work as a reducing substrate of plasma GSHPx. We have isolated two hydroperoxide-reducing protein fractions from human plasma by a sequential purification scheme, comprising an ammonium sulfate precipitation followed by sequential chromatography on anion exchange, hydrophobic interaction, and heparin columns. One of the proteins was identified as apolipoprotein A-I by N-terminal amino acid sequence analysis. Moreover, the hydroperoxide-reducing activity of one of the fractions was inhibited almost completely by the addition of anti-apolipoprotein A-I antibody.

These findings demonstrate that apolipoprotein A-I in high density lipoprotein can reduce PC-OOH to PC-OH.—Mashima, R., Y. Yamamoto, and S. Yoshimura. Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of th hydroperoxide-reducing proteins from human blood plasma. J. Lipid Res. 1998. 39: 1133–1140.

Supplementary key words: phosphatidylcholine hydroxide, selenium-deficient, selenium-supplemented, plasma glutathione peroxidase, high density lipoprotein, peroxidase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS and DISCUSSION REFERENCES

The mechanisms of protection against oxygen radicals have attracted extensive attention since oxygen radicals have been suggested to be a causative factor in aging and are implicated in degenerative diseases such as heart attack, diabetes, cancer, and others (1). Despite the presence of various antioxidants and antioxidant enzymes, human and rat plasmas contain nanomolar levels of cholesteryl ester hydroperoxide (CE-OOH) (2) (3) (4) and this is regarded as one of the direct pieces of evidence of oxygen radicals-mediated injury in vivo as lipid hydroperoxides are the primary oxidation products of lipids. However, we could not detect phosphatidylcholine hydroperoxide (PC-OOH) in human and rat plasmas (3) (5) (6) although in vitro oxidation of lipoprotein gives both PC-OOH and CE-OOH (4) (7) (8) (9). The instability of PC-OOH in plasma (7) (9) is likely to be the reason. We (10) and others (11) (12) have shown that plasma glutathione peroxidase (GSHPx) can reduce PC-OOH to its hydroxide (PC-OH) in the presence of glutathione. Moreover, we have shown that PC-OOH in high density lipoprotein (HDL) can be converted to CE-OOH by the action of lecithin:cholesterol acyltransferase (LCAT) (13).

Human and rat plasma GSHPx have been characterized as a tetrameric protein of identical subunits of 22.5 kDa molecular mass containing selenium (14) (15) (16) (17). Therefore, the selenium content in diet can manipulate not only the enzymatic activity (18) but also protein levels of plasma GSHPx in blood plasma. GSHPx requires two molecules of glutathione for the reduction of one molecule of hydroperoxide (19). This implies that in vitro human plasma can reduce only about 2.5 µM hydroperoxides because the level of glutathione usually available in human plasma is 5 µM (20). However, we have noticed that human plasma has a capacity to reduce 20 µM PC-OOH (13) or more. To understand why human plasma can reduce such large amounts of PC-OOH, we confirmed that human plasma can reduce to a large degree the concentration of 36 µM PC-OOH. We also assessed the possibility that albumin may work as a reducing substrate for plasma GSHPx. We compared the rates of the reduction of PC-OOH in sera from rats with selenium-deficient and selenium-supplemented diets. Finally, we isolated the proteins that can reduce PC-OOH. Here we report the inability of albumin to act as a reducing substrate for plasma GSHPx, a lack of difference in the reduction of 50 µM PC-OOH between sera from selenium-deficient and selenium-supplemented rats, and the isolation of two protein fractions that can reduce PC-OOH. We have identified one of the proteins by amino acid sequence as apolipoprotein A-I (apoA-I).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS and DISCUSSION REFERENCES

Reagents
Soybean phosphatidylcholine (PC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), rat serum albumin, tert -butyl hydroperoxide (BOOH), cumene hydroperoxide, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma (Tokyo). 2,2'-Azobis(2,4-dimethylvarelonitrile) (AMVN) and choline chloride were obtained from Wako Pure Chemical (Osaka). HiLoad Q Sepharose 26/10 HP, Phenyl Sepharose 6FF (low sub), HiTrap Heparin, Protein A Sepharose CL-4B, and PD-10 columns were from Pharmacia (Tokyo). Ultrafiltration membrane CENTRIFLO CF-25 was purchased from Amicon (Tokyo). Solvents and other reagents were of the highest grade commercially available. Mouse monoclonal antibody raised against human apoA-I was a kind gift from Japan Immunoresearch Laboratory (Takasaki).

PC-OOH was prepared by the aerobic oxidation of soybean PC (50 µmol) with AMVN (5 µmol) in 100 ml hexane at 37°C for 2 h. After exchanging solvent from hexane to methanol, PC-OOH was purified on a semipreparative octadecylsilyl column (25 cm x 10 mm i.d., Japan Spectroscopic, Tokyo) using methanol containing 0.02% triethylamine as the mobile phase at a flow rate of 4 ml/min. PLPC-OOH was prepared as described previously (13).

Plasma GSHPx was separated from rat serum as described previously (21). Fractions that decomposed cumene hydroperoxide were concentrated on a CF25 membrane and stored at -80°C. 2-Mercaptoethanol was present during all the purification steps to preserve the enzyme activity but was removed by gel filtration prior to activity determination because this thiol works as a reducing substrate of the enzyme (10). Enzyme activity was measured by the oxidation of NADPH in the presence of 0.23 mM cumene hydroperoxide, 0.25 mM GSH, 0.12 mM NADPH, and 1 unit/ml glutathione reductase at 37°C under aerobic conditions (21). One unit of the enzyme activity is defined as 1 µmol NADPH oxidized/min at 37°C.

Animals
Male Wistar rats were fed a selenium-deficient diet (less than 0.05 ppm) obtained from Oriental Yeast (Tokyo) or a conventional diet containing 1 ppm of sodium selenite for 12 weeks after weaning. The activity of plasma GSHPx in sera from these animals was measured as described above.

Incubation of PLPC-OOH in human plasma or rat serum
5 µl of 1.2 mM PLPC-OOH in methanol was mixed with 95 µl of human heparinized plasma obtained from a 26-year-old healthy male donor and incubated at 37°C under aerobic conditions. Incubation of PLPC-OOH in sera obtained from rats fed selenium-deficient or selenium-supplemented diets was carried out similarly. Aliquots (5 µl) of human plasma or rat serum were withdrawn during the course of incubation and stored at -80°C until analysis. Analyses of PLPC-OOH and PLPC-OH were carried out by a reported HPLC method (22) with slight modification. Briefly, aliquots (5 µl) of human plasma or rat serum were mixed with 4 volumes of methanol. After centrifugation at 12,000 g for 3 min, the supernatant (5 µl) was injected onto a CAPCELL PAK ODS column (5 µm, 4.6 x 250 mm; Shiseido, Tokyo) with UV detection at 234 nm. The mobile phase used was acetonitrile–methanol–water 100:99:1 (v/v/v) containing 10 mM choline chloride and the flow rate was 2 ml/min.

Albumin as a reducing substrate of plasma GSHPx
Sulfhydryl groups on albumin (albumin-SH) were measured by a reported method (23). Thirty µM BOOH was incubated in 50 mM Tris buffer (pH 7.6) containing 100 µM EDTA in the presence of 1.0 unit/ml rat plasma GSHPx and in the absence or presence of either 100 µM rat serum albumin-SH or glutathione at 37°C under aerobic conditions. The concentration of BOOH was determined by a hydroperoxide-specific chemiluminescence method (24) after separation on an octadecylsilyl column (CAPCELL PAK ODS; 5 µm, 4.6 x 250 mm; Shiseido, Tokyo) using acetonitrile–water 1/3 (v/v) as an eluant at a flow rate of 1 ml/min. A 70% aqueous solution of BOOH was used as a standard.

PC-OOH-reducing activity in high and low molecular weight materials in human plasma
High and low molecular weight materials were separated from human heparinized plasma by a PD-10 column using 10 mM phosphate-buffered saline containing 1 mM EDTA (PBS, pH 7.4) as the eluant. Five µl of a solution of 1.0 mM PLPC-OOH in methanol was mixed with 95 µl of either high molecular or low molecular weight fraction after diluting them with an equal volume of PBS. PLPC-OOH was also mixed with the combined solution of equal volume of high molecular weight fraction and low molecular weight fraction. The decay of PLPC-OOH and the formation of PLPC-OH were monitored as described above.

Purification of PC-OOH-reducing proteins from human blood plasma
All the purification steps were carried out at 4°C or on ice. The PC-OOH-reducing activity of protein fractions was assessed as follows. Five µl of a 100 µM solution of soybean PC-OOH in methanol was added to 100 µl of protein fractions and incubated at 37°C for 1 h under aerobic conditions. Then, 400 µl of methanol was added to the reaction solution to precipitate salts and proteins. After centrifugation at 12,000 g for 3 min, aliquots (100 µl) of the supernatant were injected onto HPLC equipped with a hydroperoxide-specific, chemiluminescence detection system (24). PC-OOH was separated on a silica gel column (LC-Si, 5 µm, 25 cm x 4.6 mm i.d., Supelco Japan, Tokyo) using methanol/40 mM monobasic sodium phosphate (9:1, v/v) as an eluant at a flow rate of 1 ml/min.

Ammonium sulfate precipitation. Heparinized human plasma (20 ml) obtained from a 26-year-old healthy male donor was mixed with the same volume of ice-cold buffer A (20 mM sodium phosphate (pH 8.0) containing 1 mM EDTA, 0.2 mM PMSF, and 0.1 M NaCl). Solid ammonium sulfate was then added slowly to the above solution to give a final concentration of 50% saturation. This protein solution was stirred on ice for 30 min, then kept on ice for a further 30 min. After centrifugation at 15,000 g for 10 min at 4°C, the supernatant was decanted, the precipitate was collected and resuspended in buffer A (20 ml), and the ammonium sulfate precipitation and the centrifugation were repeated.

Q Sepharose column chromatography. The precipitate was desalted by a PD-10 column gel filtration using buffer A and subsequently loaded onto a HiLoad Q Sepharose 26/10 HP column (50 ml) equilibrated with buffer A. After washing thoroughly with buffer A (100 ml), proteins were fractionated by elution with a linear gradient of 0.1–0.4 M NaCl in buffer B (20 mM sodium phosphate (pH 8.0) containing 1 mM EDTA and 0.2 mM PMSF) using an FPLC system (Pharmacia) over a period of 50 min at a flow rate of 5 ml/min. Then, 1.1 M NaCl in buffer B was used as an eluant. Ten-ml fractions of the eluant were collected. The PC-OOH-reducing activity eluted around 0.2–0.3 M NaCl as a broad peak.

Phenyl Sepharose column chromatography. Fractions with PC-OOH-reducing activity were combined and mixed with solid ammonium sulfated to give a final concentration of 40% saturation. This turbid protein suspension was then applied onto a Phenyl Sepharose 6FF (low sub) column (1.6 x 5 cm) equilibrated with buffer B containing 40% ammonium sulfate. Proteins were eluted by a stepwise gradient with 40, 20, and 0% ammonium sulfate in buffer B at a flow rate of 1 ml/min. The PC-OOH-reducing proteins, which remained bound under these conditions, were eluted using 50% propylene glycol in buffer B, and exchanged into buffer A using a PD-10 column.

HiTrap Heparin chromatography. The resulting fraction with PC-OOH-reducing activity was purified further using a HiTrap Heparin column (1 ml). After loading and washing extensively with buffer A, proteins were eluted with a linear 20 min gradient of 0.1–2.1 M NaCl in buffer B at a flow rate of 1 ml/min using an FPLC system. One-ml fractions of the eluant were collected. One fraction with PC-OOH-reducing activity (fraction A) eluted without retention and was concentrated (640 µg protein/ml) using a CF-25 membrane. The other PC-OOH-reducing fraction (fraction B) was eluted with 0.5–1.0 M NaCl in buffer B.

Amino acid sequence determination
The purity of proteins in fraction A was assessed by a 10% SDS-polyacrylamide gel electrophoresis (PAGE) and the protein bands were transblotted onto polyvinylidene difluoride membrane (Bio-Rad). The blotted proteins were detected by staining with Coomassie Brilliant Blue R-250. The major band was excised and subjected to automated gas–liquid phase sequencer (model PPSQ-10, Shimadzu, Kyoto).

Immunoprecipitation
Five hundred µl of mouse anti-human apoA-I antibody (2 mg/ml), 200 µl (wet gel) of Protein A Sepharose CL-4B (Pharmacia), and 500 µl of 1.5 M glycine buffer (pH 8.9) containing 3 M NaCl were incubated for 1 h at room temperature to bind the antibody to the gel. After centrifugation at 12,000 g for 3 min, the gel was washed with PBS; and centrifugation and washing were repeated 3 times. Then, the antibody-fixed Protein A Sepharose gel (about 60 µl) was mixed with 60 µl of fraction A (26 µg protein/ml) in PBS, left to stand for 10 min at room temperature, and centrifuged at 12,000 g for 3 min. Fifty µl of the supernatant or PBS was mixed with 2.5 µl of a 100 µM PLPC-OOH in methanol and incubated for 1 h at 37°C under aerobic conditions. Then, 200 µl of methanol was added, the mixture was centrifuged at 12,000 g for 3 min, and 100 µl of the supernatant was injected onto an ODS column to measure the concentrations of PLPC-OOH and PLPC-OH. HPLC conditions were as described above.

	RESULTS and DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS and DISCUSSION REFERENCES

Lipid hydroperoxides are the primary oxidation product of lipids and are detoxified by reduction because they can initiate further oxidation of lipids, proteins, and other biological components in the presence of metal ions. In the course of our study on the fate of PC-OOH in human plasma, we demonstrated that plasma GSHPx can reduce PC-OOH in the presence of glutathione. The capacity for this reduction should be about 2.5 µM as plasma of healthy humans contains 5 µM glutathione (20) and two mole GSH are needed to reduce one mole PC-OOH. However, we found that human plasma can reduce more than the 20 µM level of PC-OOH (13). To confirm this observation, we incubated 58 µM PLPC-OOH in human plasma for 2 h under aerobic conditions at 37°C. Figure 1 shows the resulting decrease in PLPC-OOH and the formation of PLPC-OH, as measured by an HPLC method.

View larger version (15K): [in this window] [in a new window]   Figure 1. Reduction of PLPC-OOH to PLPC-OH by human plasma isolated from a 26-year-old healthy male donor at 37°C under aerobic conditions. Two HPLC chromatograms obtained from 0 (right after the addition of 58 µM PLPC-OOH to the plasma) and 2 h incubation were compared. The HPLC conditions were described in Materials and Methods.

Figure 2 shows the time course of this reaction and that the total concentration of PLPC-OOH and PLPC-OH was almost constant, indicating that the two-electron reduction is the predominant reaction because one-electron reduction of PC-OOH gives alkoxyl radical (PC-O.) and this gives aldehydes in high yields by ß-scission reaction (1). After 2 h incubation, the concentration of PLPC-OH was 36 µM. This observation may be explained if albumin, in addition to GSH, works as a reducing substrate of plasma GSHPx as albumin is present at about 600 µM in human plasma and has one free sulfhydryl group at position 34 from the N-terminal. However, we failed to demonstrate that rat serum albumin accelerated the reduction of BOOH in the presence of rat plasma GSHPx ( Figure 3). Human albumin freshly isolated from plasma using a Blue Sepharose column (25) also failed to function as a reducing substrate (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2. Time course changes in the levels of PLPC-OOH (), PLPC-OH (), and PLPC-OOH + PLPC-OH () during the incubation of 58 µM PLPC-OOH in human plasma at 37°C under aerobic conditions. Plasma was isolated from a 26-year-old healthy male donor. Values are the mean of two independent and reproducible analyses.

View larger version (16K): [in this window] [in a new window]   Figure 3. The decay of tert-butyl hydroperoxide (BOOH) by rat plasma glutathione peroxidase (1.0 unit/ml) in the absence or the presence of 100 µM glutathione (GSH) or rat serum albumin at 37°C under aerobic conditions. Values are the mean of two independent and reproducible analyses.

To assess the involvement of plasma GSHPx in the reduction of large amounts of PC-OOH, we further compared the PC-OOH-reducing activity of sera from rats fed selenium-deficient or selenium-supplemented diets. The activity of the selenium-dependent plasma GSHPx in the sera was 0.07 and 9.92 units/ml, respectively, as measured by the decrease of NADPH in the presence of glutathione reductase, cumene hydroperoxide, and glutathione (21), indicating that rats fed a selenium-deficient diet were indeed deficient in selenium. Despite such a big difference in plasma GSHPx activity, there was a reduction in concentration of 50 µM PLPC-OOH by sera from both groups that proceeded at the same rate as shown in Figure 4. After 2 h of incubation the concentration of PLPC-OH was about 40 µM (Figure 4). The above data strongly suggest the presence in rat serum of the hydroperoxide-reducing activity other than plasma GSHPx.

View larger version (22K): [in this window] [in a new window]   Figure 4. Time course changes in the levels of PLPC-OOH (), PLPC-OH (), and PLPC-OOH + PLPC-OH () during the aerobic incubation at 37°C of 50 µM PLPC-OOH in sera from rats fed a selenium-deficient or selenium-supplemented diet for 12 weeks; their plasma GSHPx activities were 0.07 and 9.92 units/ml, respectively. Values are the mean of two independent and reproducible analyses.

We next separated human plasma into high and low molecular weight fractions by gel filtration using a PD-10 column and measured the PC-OOH-reducing activity in both fractions. We observed the activity only in the high molecular weight fraction ( Figure 5). Moreover, the addition of the low molecular weight fraction to the high molecular weight fraction instead of PBS did not accelerate the reduction of PC-OOH (Figure 5), indicating that the PC-OOH-reducing activity does not require low molecular weight cofactors.

View larger version (20K): [in this window] [in a new window]   Figure 5. Formation of PLPC-OH during the incubation of 50 µM PLPC-OOH in equal volume of the high and/or low molecular weight fractions from human plasma and 10 mM phosphate-buffered saline containing 1 mM EDTA (pH 7.4) at 37°C under aerobic conditions. Values are the mean of two independent and reproducible analyses.

Purification of PC-OOH-reducing proteins
We therefore tried to isolate the PC-OOH-reducing proteins from human plasma by a combination of ammonium sulfate precipitation, and chromatography in HiLoad Q Sepharose, Phenyl Sepharose and HiTrap Heparin columns ( Table 1). Ammonium sulfate (50%) precipitation increased the PC-OOH-reducing activity (pmol/h per mg) 3-fold. Figure 6 shows the elution profiles of proteins, the PC-OOH-reducing activity, and plasma GSHPx by anion exchange chromatography using a HiLoad Q Sepharose column. The PC-OOH-reducing activity eluted as a broad peak while plasma GSHPx eluted as a single, sharp peak with maximal activities in fractions 28 and 31, respectively, indicating that the PC-OOH-reducing activity was different from plasma GSHPx.

View larger version (19K): [in this window] [in a new window]   Figure 6. Elution profiles of proteins (–––; absorbance at 280 nm), plasma GSHPx (), and PC-OOH-reducing proteins () from HiLoad Q Sepharose 26/10 HP column eluted with 20 mM sodium phosphate (pH 8.0) containing 1 mM EDTA and 0.2 mM PMSF at a flow rate of 5 ml/min. Concentration of NaCl was indicated (- - - - -).

Fractions 24 to 28 eluting from the Q Sepharose column were combined and applied to a Phenyl Sepharose column. Ammonium sulfate was added to the fractions to a final concentration of 40% as the PC-OOH-reducing activity did not bind to the hydrophobic Phenyl Sepharose gel in the absence of 40% of ammonium sulfate (data not shown). The PC-OOH-reducing activity was eluted as a single peak by a 50% propylene glycol in buffer B after a stepwise decrease of ammonium sulfate from 40 to 0 % in buffer B. This purification step decreased the amount of protein recovered from 73 to 3.2 mg (4.4%) while the specific activity increased from 828 to 5588 pmol/h per mg (Table 1).

The PC-OOH-reducing activity was separated into two fractions (fractions A and B) by a HiTrap Heparin column; the protein(s) in fraction A were not retained by the column whereas the protein(s) in fraction B eluted with 0.5–1.0 M NaCl. Fractions A and B both yielded 6% of the initial PC-OOH-reducing activity with an increase in specific activity of 55- and 143-fold, respectively (Table 1). However, these numbers should be judged with caution as the specific PC-OOH-reducing activity of fraction A was dependent on the protein concentration; i.e., the activity increased with increasing protein concentration of fraction A but the relationships were not linear (data not shown).

Fractions A and B were applied to SDS-PAGE to assess the purity of the proteins. It was found that fraction A contained a 28 kDa protein as the major protein with several minor proteins as shown in Figure 7. Fraction B yielded a single band (data not shown).

View larger version (45K): [in this window] [in a new window]   Figure 7. 10% SDS-polyacrylamide gel electrophoresis (PAGE) of fraction A and molecular markers.

To demonstrate the PC-OOH-reducing activity of fraction A, fraction A (64 µg protein/ml) was incubated with PLPC-OOH (7 µM) in PBS at 37°C under aerobic conditions. Figure 8 shows the stoichiometric reduction of PLPC-OOH to PLPC-OH. As 0.8 µM PLPC-OOH was reduced in 1 h, the specific activity can be calculated as 0.8 x 1000000/64 = 12500 pmol/h per mg. This number is much higher than 1962 pmol/h per mg shown in Table 1 where the protein concentration of fraction A was 640 µg/ml. However, this is not surprising because of the reason described above.

View larger version (18K): [in this window] [in a new window]   Figure 8. Time course changes in the levels of PLPC-OOH (), PLPC-OH (), and PLPC-OOH + PLPC-OH () during the aerobic incubation of 7 µM PLPC-OOH in 10 mM phosphate-buffered saline (pH 7.4) containing fraction A (64 µg protein/ml) and 1 mM EDTA at 37°C. Values are the mean of two independent and reproducible analyses.

The N-terminal amino acid sequence of the 28 kDa protein in fraction A was found to be XXPPQSPXDRVKD LATVY (X denotes an unidentified amino acid), matching that of apoA-I (26). The amino acid sequence of minor proteins was not determined because of their insufficient amounts. Characterization of the protein in fraction B is ongoing.

Figure 9A shows that PLPC-OOH (5 µM) was stable in PBS for 1 h. Although the addition of fraction A resulted in the formation of 0.38 µM PLPC-OH (Figure 9B), the treatment of fraction A with anti-human apoA-I monoclonal antibody prior to the addition reduced the formation of PLPC-OH almost completely (Figure 9C), indicating that the PC-OOH-reducing activity of fraction A is due to apoA-I.

View larger version (18K): [in this window] [in a new window]   Figure 9. HPLC chromatograms after 1 h aerobic incubation of 5 µM PLPC-OOH in (A) 10 mM phosphate- buffered saline containing 1 mM EDTA (PBS, pH 7.4), (B) PBS containing fraction A (13 µg protein/ml), and (C) PBS containing fraction A pretreated with anti-human apoA-I monoclonal antibody. Same results were obtained in triplicate experiments. Details were described in Materials and Methods. 1, PLPC-OOH; 2, PLPC-OH.

Mechanism of the reduction
Apo A-I is the major protein of high density lipoprotein (HDL) and accelerates the transport of peripheral free cholesterol (reverse cholesterol transport) by activating LCAT which catalyzes the esterification of free cholesterol to cholesteryl esters (27). ApoA-I is a 28 kDa protein of a single peptide with 243 amino acids and contains three methionine residues at the 86-, 112-, and 148-positions from the N-terminal (26). The oxidation of apoA-I by hydrogen peroxide converts methionine at the 112- and 148-positions to methionine sulfoxide but not methionine at the 86-position (28). Methionine residues at the 112- and 148-positions locate in the amphipathic region of -helix of apoA-I molecule (26) where hydrogen peroxide is accessible. Accessibility of hydroperoxide should be very important for the reduction of hydroperoxide by methionine. In fact, albumin did not reduce BOOH (Figure 3) and PC-OOH (data not shown) although it contains six methionine residues (29).

Recently, Garner et al. (30) (31) observed the reduction of CE-OOH to CE-OH by apoA-I with a concomitant formation of methionine sulfoxide. It is reasonable to assume that the reduction of PC-OOH to PC-OH by apo A-I proceeds by the same reaction mechanism. It has been observed that HDL can reduce CE-OOH (32) and PC-OOH (13) to their corresponding alcohols.

Role of apo A-I-dependent reduction of hydroperoxides
Under physiological conditions the levels of lipid hydroperoxides in human plasma are likely to be submicromolar as the detected levels of CE-OOH are 3 nM (2). Under these conditions, plasma GSHPx is likely to play the most important role in reducing lipid hydroperoxides except CE-OOH (10) as plasma contains micromolar GSH and plasma GSHPx reduces hydroperoxides very rapidly (20). However, the reduction of lipid hydroperoxides by apoA-I may play a significant role under pathophysiological conditions such as inflammation and plasma GSHPx deficiency (33). Concentration of apoA-I in human plasma is about 1.27 g/l (45 µM) (34). This implies that human plasma has a capacity of reducing at least 90 µM hydroperoxides if two methionine residues in apoA-I are active (28). If we could detect methionine sulfoxide-containing apoA-I in biological samples, it would be good evidence of apoA-I-dependent reduction of hydroperoxides; further investigation is being considered. The presence of methionine sulfoxide reductase in humans (35) (36) may also suggest a role of apoA-I-dependent reduction of hydroperoxides.

In summary, we have demonstrated that human plasma and rat serum contain PC-OOH-reducing activities. We have succeeded in isolating two purified fractions of PC-OOH-reducing activities and apoA-I has been identified in one of them as a major PC-OOH-reducing protein.

	ACKNOWLEDGMENTS

The authors are grateful to Dr. Jun Kondo for conducting the amino acid sequence analyses and Dr. Roland Stocker for careful reading of the manuscript and helpful comments.

Manuscript received July 28, 1997; and in revised form October 27, 1997; and in revised form February 3, 1998.

Abbreviations: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; GSHPx, glutathione peroxidase; PC-OOH, phosphatidylcholine hydroperoxide; PC-OH, phosphatidylcholine hydroxide; PLPC, 1-palmitoy1-2-linoleoylphosphatidylcholine; PLPC-OOH, 1-palmitoyl-2-linoleoylphosphatidylcholine hydroperoxide; PLPC-OH, 1-palmitoyl-2-linoleoylphosphatidylcholine hydroxide

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS and DISCUSSION REFERENCES

1. Halliwell, B., and J. M. C. Gutteridge. 1989. Free Radicals in Biology and Medicine. 2nd ed. Clarendon, Oxford.

2. Yamamoto, Y., Niki, E. 1989. Presence of cholesteryl ester hydroperoxide in human blood plasma. Biochem. Biophys. Res. Commun. 165:988-993[Medline].

3. Yamamoto, Y., Wakabayashi, K., Niki, E., Nagao, M. 1992. Comparison of plasma levels of lipid hydroperoxides and antioxidants in hyperlipidemic Nagase analbuminemic rats, Sprague-Dawley rats and humans. Biochem. Biophys. Res. Commun. 189:518-523[Medline].

4. Bowry, V. W., Stanley, K. K., Stocker, R. 1992. High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors. Proc. Natl. Acad. Sci. USA. 89:10316-10320[Abstract/Free Full Text].

5. Yamamoto, Y., Ames, B. N. 1987. Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay. Free Radical Biol. Med. 3:359-361[Medline].

6. Frei, B., Yamamoto, Y., Niclas, D., Ames, B. N. 1988. Evaluation of an isoluminol chemiluminescence assay for the detection of hydroperoxides in human blood plasma. Anal. Biochem. 175:120-130[Medline].

7. Frei, B., Stocker, R., Ames, B. N. 1988. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc. Natl. Acad. Sci. USA. 85:9748-9752[Abstract/Free Full Text].

8. Stocker, R., Bowry, V. W., Frei, B. 1991. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does a-tocopherol. Proc. Natl. Acad. Sci. USA. 88:1646-1650[Abstract/Free Full Text].

9. Yamamoto, Y., M. Kawamura, K. Tatsuno, S. Yamashita, E. Niki, and C. Naito. (1991) Formation of lipid hydroperoxides in the cupric ion-induced oxidation of plasma and low density lipoprotein. In Oxidative Damage and Repair. Chemical, Biological and Medical Aspects. K. J. A. Davis, editor. Pergamon Press, Oxford. 287–291.

10. Yamamoto, Y., Nagata, Y., Niki, E., Watanabe, K., Yoshimura, S. 1993. Plasma glutathione peroxidase reduces phosphatidylcholine hydroperoxide. Biochem. Biophys. Res. Commun. 193:133-138[Medline].

11. Yamamoto, Y., Takahashi, K. 1993. Glutathione peroxidase isolated from plasma reduces phosphatidylcholine hydroperoxides. Arch. Biochem. Biophys. 305:541-545[Medline].

12. Esworthy, R. S., Chu, F-F., Geiger, P., Girotti, A. W., Doroshow, J. H. 1993. Reactivity of plasma glutathione peroxidase with hydroperoxide substrates and glutathione. Arch. Biochem. Biophys. 307:29-34[Medline].

13. Nagata, Y., Yamamoto, Y., Niki, E. 1996. Reaction of phosphatidylcholine hydroperoxide in human plasma: the role of peroxidase and lecithin:cholesterol acyltransferase. Arch. Biochem. Biophys. 329:24-30[Medline].

14. Maddipati, K. R., Marnett, L. J. 1987. Characterization of the major hydroperoxide-reducing activity of human plasma. J. Biol. Chem. 262:17398-17403[Abstract/Free Full Text].

15. Takahashi, K., Avissar, N., Whitin, J., Cohen, H. 1987. Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme. Arch. Biochem. Biophys. 256:677-686[Medline].

16. Broderick, D. J., Deagen, J. T., Whanger, P. D. 1987. Properties of glutathione peroxidase isolated from human plasma. J. Inorg. Biochem. 30:299-308[Medline].

17. Yoshimura, S., Watanabe, K., Suemizu, H., Onozawa, T., Mizoguchi, J., Tsuda, K., Hatta, H., Moriuchi, T. 1991. Tissue specific expression of the plasma glutathione peroxidase gene in rat kidney. J. Biochem. 109:918-923[Abstract/Free Full Text].

18. Hill, K. E., Burk, R. F., Lane, J. M. 1986. Effect of selenium depletion and repletion on plasma glutathione and glutathione-dependent enzymes in the rat. J. Nutr. 117:99-104.

19. Flohe, L. 1982. Glutathione peroxidase brought into focus. In Free Radicals in Biology. W. A. Pryor, editor. Academic Press, New York. 223–254.

20. Mansoor, M. A., Svardal, A. M., Ureland, P. M. 1992. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal. Biochem. 200:218-229[Medline].

21. Yoshimura, S., Komatsu, N., Watanabe, K. 1980. Purification and immunohistochemical localization of rat liver glutathione peroxidase. Biochim. Biophys. Acta. 621:130-137[Medline].

22. Bao, Y., Chambers, S. J., Williamson, G. 1995. Direct separation of hydroperoxy- and hydroxy-phosphatidylcholine derivatives: application to the assay of phospholipid hydroperoxide glutathione peroxidase. Anal. Biochem. 224:395-399[Medline].

23. Ellman, G. L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70-77[Medline].

24. Yamamoto, Y., Brodsky, M. H., Baker, J. C., Ames, B. N. 1987. Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography. Anal. Biochem. 160:7-13[Medline].

25. Travis, J., Bowen, J., Tewksbury, D., Johnson, D., Pannell, R. 1976. Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem. J. 157:301-306[Medline].

26. Karathanasis, S. K., Zannis, V. I., Breslow, J. L. 1983. Isolation and characterization of the human apolipoprotein A-I gene. Proc. Natl. Acad. Sci. USA. 80:6147-6151[Abstract/Free Full Text].

27. Pieters, M. N., Schouten, D., Van Berkel, T. J. C. 1994. In vitro and in vivo evidence for the role of HDL in reverse cholesterol transport. Biochim. Biophys. Acta. 1225:125-134[Medline].

28. von Eckardstein, A., Water, M., Holz, H., Benninghoven, A., Assmann, G. 1991. Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III. J. Lipid Res. 32:1465-1476[Abstract].

29. Carter, D. C., Ho, J. X. 1994. Structure of serum albumin. Adv. Protein Chem. 45:153-203[Medline].

30. Garner, B., Witting, P. K., Waldeck, A. R., Christison, J. K., Raftery, M., Stocker, R. 1998. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apoproteins A-I and A-II is an early event that accompanies lipid peroxidation and can be enhanced by -tocopherol. J. Biol. Chem. 273:6080-6087[Abstract/Free Full Text].

31. Garner, B., Waldeck, A. R., Witting, P. K., Rye, K., Stocker, R. 1998. Oxidation of high density lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins A-I and A-II. J. Biol. Chem. 273:6088-6095[Abstract/Free Full Text].

32. Sattler, W., Christison, J., Stocker, R. 1995. Cholesteryl ester hydroperoxide reducing activity associated with isolated high- and low-density lipoproteins. Free Radical Biol. Med. 18:421-429[Medline].

33. Yoshimura, S., Shimizu, H., Nomoto, Y., Sakai, H., Kawamura, N., Moriuchi, T. 1996. Plasma glutathione peroxidase deficiency caused by renal dysfunction. Nephron. 73:207-211[Medline].

34. Stampfer, M. J., Sacks, F. M., Salvini, S., Willett, W. C., Hennekens, C. H. 1991. A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. N. Engl. J. Med. 325:373-381[Abstract].

35. Brot, N., Weissbach, H. 1983. Biochemistry and physiological role of methionine sulfoxide residues in proteins. Arch. Biochem. Biophys. 223:271-281[Medline].

36. Vogt, W. 1995. Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radical Biol. Med. 18:93-105[Medline].