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Clinical Chemistry 46: 822-828, 2000;
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(Clinical Chemistry. 2000;46:822-828.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Age-related Increases in Plasma Phosphatidylcholine Hydroperoxide Concentrations in Control Subjects and Patients with Hyperlipidemia

Mikio Kinoshita1,1, Shinichi Oikawa2,a, Kyoko Hayasaka2, Akihiro Sekikawa2, Tazuko Nagashima1, Takayoshi Toyota2 and Teruo Miyazawa1

1 Biodynamic Chemistry Laboratory, Tohoku University Graduate School of Life Science and Agriculture, Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981-8555, Japan.

2 The Third Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai 980-8574, Japan.
a Address correspondence to this author at: The Third Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyoku, Tokyo 113-0022, Japan. Fax 81 3-5685-1793; e-mail shinichi{at}nms.ac.jp


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: The basal lipid peroxide concentration in the plasma of patients with hyperlipidemia may be related to atherosclerosis. Quantitative determination of lipid peroxides in the plasma is an important step in the overall evaluation of the biochemical processes leading to oxidative injury. Unfortunately, the currently available methods for lipid peroxidation lack specificity and sensitivity.

Methods: Hyperlipidemic patients (44 males and 50 females), ages 12–82 years (mean ± SE, 53 ± 2.3 years for males, 58 ± 2.0 years for females, and 56 ± 14 years for total cases), and normolipidemic volunteers (controls, 32 males and 15 females), ages 13–90 years (49 ± 4 years for males, 65 ± 4 years for females, and 55 ± 24 years for total cases), were recruited in the present study. Plasma phosphatidylcholine hydroperoxide (PCOOH) was determined by chemiluminescence-HPLC (CL-HPLC).

Results: Plasma PCOOH concentrations increased with age in both controls and hyperlipidemic patients. However, the mean plasma PCOOH concentration in patients with hyperlipidemia (331 ± 19 nmol/L; n = 94) was significantly (P <0.001) higher than in the controls (160 ± 65 nmol/L; n = 47). Plasma PCOOH concentrations were similar in three hyperlipidemic phenotypes: hypercholesterolemia (IIa), hypertriglyceridemia (IV), and combined hyperlipidemia (IIb). The mean plasma PCOOH in patients with treatment-induced normalized plasma lipids was 202 ± 17 nmol/L. There was no significant correlation between plasma PCOOH concentration and total cholesterol, triglycerides, or phospholipids in hyperlipidemic patients. For all subjects, there was a significantly positive correlation between plasma PCOOH and each lipid (total cholesterol, P = 0.0002; triglycerides, P = 0.0137; and phospholipids, P <0.0001). Analysis of fatty acids composition of plasma phosphatidylcholine showed significantly low concentrations of n-6 fatty acids moieties (linoleic acid and arachidonic acid) in patients compared with controls.

Conclusions: Our results suggest that an increase in plasma PCOOH in patients with hyperlipidemia may be related to the development and progression of atherosclerosis, particularly in the elderly. Measurement of plasma PCOOH is useful for in vivo evaluation of oxidative stress.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Hyperlipidemia is a major risk factor for the development of atherosclerosis (1)(2)(3)(4). In particular, LDL and HDL concentrations are strongly associated with atherosclerosis (5)(6). Recently, other pathophysiological factors of atherosclerosis, apart from cholesterol, have also been recognized (7). The initial pathologic event in atherosclerosis is the formation of foam cells (8). These cells are formed by the conversion of monocytes/macrophages through the scavenger pathway (9)(10)(11)(12)(13). The process of foam cell formation also involves oxidative modification of LDL (7).

Lipid peroxides, which are produced through the action of oxygen radicals or enzymatically through, e.g., lipoxygenase, cause oxidative injury and are related to a pathogenic mechanism involved in the initiation and development of atherosclerosis (9)(14)(15)(16). Quantitative determination of lipid peroxide in the plasma is, therefore, an important step in the overall evaluation of the biochemical processes leading to oxidative injury.

Previous studies looked at peroxidation of whole lipoproteins or total lipids (15)(17)(18), whereas the present study centered on the peroxidation of lipoprotein phospholipids. The phospholipids on the surface of the lipoproteins should be the main site of lipid peroxidation and, therefore, the ideal target for clinical investigation.

In a series of studies, we previously reported a novel method for the quantitative analysis of hydroperoxides in biomembranes by a chemiluminescence-HPLC (CL-HPLC)2 assay (19)(20)(21)(22)(23)(24). We also demonstrated that this method is highly sensitive and specific for lipid hydroperoxides.

In the present study, we used the above method to measure the concentrations of phosphatidylcholine hydroperoxide (PCOOH) in the plasma as a representative phospholipid hydroperoxide. We compared plasma PCOOH concentrations in hyperlipidemic patients with those in controls.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
subjects
Hyperlipidemic patients (44 males and 50 females), ages 12–82 years (mean ± SE, 53 ± 2.3 years for males, 58 ± 2.0 years for females, and 56 ± 14 years for total cases), and normolipidemic volunteers (controls, 32 males and 15 females), ages 13–90 years (49 ± 4 years for males 65 ± 4 years for females, and 55 ± 24 years for total cases), were recruited in the present study. The clinical diagnoses for the hyperlipidemic patients are shown in Table 1 .


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  		Table 1. Clicical diagnosis of hyperlipidemia.

We classified hyperlipidemic phenotypes by plasma total cholesterol (TC) and triglyceride (TG) concentrations (type IIa, TC >2200 mg/L and TGs <1500 mg/L; type IIb, TC >2200 mg/L and TGs >1500 mg/L; type IV, TC <2200 mg/L and TGs >1500 mg/L). Familial hypercholesterolemia was not present in the normalized hyperlipidemic patients and type IV hyperlipidemia. Old myocardial infarctions were observed in all type IIa, IIb, and IV hyperlipidemic patients, and an arteriosclerotic obstruction was present in one type IIa hyperlipidemic patient. Diabetic patients were treated with sulfonyl urea agents or diet alone for diabetic control, and no patients were treated with insulin. The number of smokers were three in type IIa, four in type IV, and none in type IIb hyperlipidemic patients and normalized patients. None of the patients had taken vitamin tablets at least 1 month before the present study. At the time of the study, all patients were on controlled diets and/or hypolipidemic agents in the Lipid Clinic at Tohoku University Hospital.

We used some drugs for treatment. Each drug was effective in decreasing plasma lipids concentrations. When the effect was not sufficient, combination therapy was used, e.g., statin and probucol, or statin and fibrate. The phenotype changed after treatment in some cases. We divided such cases according to the phenotype at the point of experiment after treatment as shown in Table 2 . At entry into the study, the plasma lipid concentrations were within reference values (TC <2200 mg/L and TGs <1500 mg/L) in 13 patients after 3 months of the above therapy. In the remaining patients, plasma lipid concentrations were still high in spite of continued treatment. These patients were divided into three groups according to the phenotype of hyperlipidemia: type IIa hyperlipidemia (n = 34), type IIb hyperlipidemia (n = 24), and type IV hyperlipidemia (n = 23). Plasma lipids concentrations before and after treatment are shown in Table 3 . The study protocol was approved by the Human Ethics Review Committee of Tohoku University, and a signed form was obtained from all subjects.


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  		Table 2. Treatments for hyperlipidemia.


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  		Table 3. Ages and plasma lipids of subjects.

measurement of plasma lipids
Plasma was prepared from venous blood samples obtained from each subject after overnight fasting. Plasma TC and TG concentrations were measured enzymatically with the Cholesterol-E test and the Triglyceride-E test (Wako Pure Chemical Co.), respectively. Phospholipid phosphorus (PL) was determined by the method of Bartlett (25). HDL-cholesterol (HDL-C) was measured with the HDL-cholesterol-E test (Wako).

hydroperoxide assay
Total lipids were extracted from plasma with a mixture of chloroform and methanol containing 0.02 g/L butyl hydroxytoluene as an antioxidant. The samples were submitted to CL-HPLC to determine the concentration of PCOOH. The CL-HPLC system and conditions for measuring plasma PCOOH were identical to those described previously by Miyazawa and co-workers (20)(21). Briefly, the column was a JASCO Finepak SIL NH2–5 [250 x 4.6 mm (i.d.), 5 µm bead size; JASCO]. The column mobile phase was hexane–2-propanol–methanol–water (5:7:2:1, by volume), and the flow rate was maintained at 1.0 mL/min by a JASCO 880PU pump. The column eluate was mixed with the luminescent reagent at a postcolumn mixing joint (Y-type; Kyowa Seimitsu) with the temperature controlled at 40 °C in a JASCO 860 column oven. The luminescent reagent was prepared by dissolving cytochrome C (from horse heart, type IV; Sigma) and luminol (3-aminophytaloyl hydrazine; Wako) in an alkaline borate buffer (pH 10) and was added at a flow rate of 1.1 mL/min by a JASCO 880PU pump. The chemiluminescence was measured by a CLD-100 chemiluminescence detector (Tohoku Electronic Industries). In these HPLC conditions, antioxidants (e.g., {alpha}-tocopherol and ß-carotene) and neutral lipid (e.g., TGs, cholesterol ester, and cholesterol) were eluted rapidly. Therefore, these components did not interfere with this assay.

PCOOH was detected as the predominant lipid hydroperoxide on the chemiluminescent chromatogram. The authentic PCOOH concentration was determined by KI reduction and expressed as pmol of hydroperoxide-O2. The chemiluminescence counts integrated for the chromatographic PCOOH peak in CL-HPLC were linearly proportional to the authentic PCOOH concentrations in a log-log plot. The detection limit was 10 pmol. The authentic PCOOH concentration was significantly proportional to the PL concentration.

fatty acid composition
To determine the fatty acid composition, phosphatidylcholine (PC) was separated from plasma total lipids on thin layer chromatography (Silica gel-60; Merck) with chloroform-methanol-acetic acid-water (25:15:4:2, by volume) as the development solvent. The prepared fatty acid methyl esters were submitted to Shimadzu GC-8A gas-liquid chromatography (26).

statistical analysis
Data are expressed as the mean ± SE. Differences between groups were evaluated using a statistical software package (StatView®, Ver. 4.5). Differences were examined for statistical significance using one-way ANOVA. P <0.05 indicated the presence of a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
After medical treatment, the TG and TC concentrations in the plasma of hyperlipidemic patients decreased. Especially, 13 patients were controlled to normolipidemia (Table 3Up ).

The mean plasma PCOOH concentration in control subjects was 160 ± 65 nmol/L (166 ± 11.1 nmol/L in males and 149 ± 19.3 nmol/L in females; not significant between males and females). The mean concentration in hyperlipidemic patients (331 ± 19 nmol/L) was significantly higher than in controls (P <0.01). There was no difference in plasma PCOOH from patients between males (334 ± 29 nmol/L) and females (330 ± 25 nmol/L). Plasma PCOOH concentrations increased with age (Fig. 1 ). The correlation between age and plasma PCOOH was significant in both control subjects and hyperlipidemic patients. Fig. 2 shows plasma concentrations in different age groups. In each age group, plasma PCOOH concentrations in patients with hyperlipidemia were 2- to 2.5-fold higher than in control subjects. Plasma PCOOH concentration markedly increased in both hyperlipidemic patients and controls >65 years of age.



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  		Figure 1. Correlation between plasma PCOOH and age.

•, control subjects (r = 0.392; P <0.01); {circ}, patients with hyperlipidemia (r = 0.298; P <0.01).



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  		Figure 2. Plasma concentration of PCOOH by age groups.

{square}, control subjects (n = 15 in the group 20–40 years of age, n = 12 in the group 41–64 years of age, and n = 20 in the group >65 years of age); , patients with hyperlipidemia (n = 16 in the group 20–40 years of age, n = 45 in the group 41–64 years of age, and n = 20 in the group >65 years of age). In each age group, the plasma PCOOH concentration was significantly higher in hyperlipidemic patients (P <0.01) than in controls.

We also compared plasma PCOOH concentrations among the four different hyperlipidemic phenotypes (Table 4 ). In patients with normolipidemia who had successfully responded to treatment, plasma PCOOH concentrations were not different from concentrations in controls. PCOOH concentrations in each hyperlipidemic phenotype were significantly higher than the control and normalized groups, but there were no differences in plasma PCOOH concentrations among the hyperlipidemic groups. There was no correlation between plasma PCOOH concentrations and TC, TG, or PL concentrations in hyperlipidemic patients. However, when the analysis was performed for all subjects (n = 141), there was significantly positive correlation between the concentration of PCOOH and each lipid as shown in Table 5 . We calculated the ratio of PCOOH/(TG + TC). The value was significantly (P <0.002) higher in hyperlipidemic patients (mean ± SE, 0.810 ± 0.046) than in controls (0.58 ± 0.038). The ratio for hyperlipidemia patients normalized after treatment was 0.704 ± 0.056, which was between those of the hyperlipidemic patients and the controls. There was no significant difference between the values for normalized patients and hyperlipidemic patients or controls. The results indicated that the increased plasma PCOOH of dyslipidemia was the function of the increased plasma lipid concentration.


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  		Table 4. Plasma PCOOH in hyperlipidemia.


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  		Table 5. Correlation between plasma PCOOH and lipid concentrations.

We also analyzed the fatty acid moieties of plasma PC in each group, as shown in Table 6 . Among the constituent fatty acids, the linoleic acid (18:2, n-6) concentration was significantly lower in each hyperlipidemic group relative to the controls. Similarly, the arachidonic acid (20:4, n-6) concentration tended to be lower in hyperlipidemia. On the other hand, the eicosapentaenoic acid (20:5, n-3) concentration in hyperlipidemia was significantly higher than in controls even when plasma lipid concentrations were normalized. There was no difference for docosahexanoic acid (22:6, n-3) in PC among the five groups.


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  		Table 6. Fatty acid constituents of plasma PC.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that (a) the plasma PCOOH concentration increases with age, (b) plasma PCOOH concentrations in hyperlipidemic patients were significantly higher than in controls, and (c) the linoleic acid and arachidonic acid (n-6 fatty acids) moieties were low, whereas the eicosapentaenoic acid (n-3) moiety of fatty acids was high in hyperlipidemic plasma PC.

Only a few studies have examined the effect of age on lipid peroxide concentrations. Sanderson et al. (27) and Schmuck et al. (18), using thiobarbituric acid and ferrous oxidation-xylenol orange methods, respectively, reported that the total lipid peroxide of LDL was independent of age. One potential cause of the discrepancy between previous studies and our study might be attributable to differences in methodology. Previous studies have looked at peroxidation of whole lipoproteins or total lipids, whereas our study centered on the peroxidation of lipoprotein phospholipids. We previously have shown that PCOOH concentrations in red blood cell membranes were significantly higher in healthy older subjects (56–92 years of age) than in younger subjects (22–27 years of age) (28). The present results indicate that plasma PCOOH increases with age in a manner similar to that in red blood cell membranes, even in normolipidemia. It is interesting that plasma PCOOH concentrations of both hyperlipidemic patients and healthy controls >65 years of age increased as shown in Fig. 2Up . We speculated that the degradation of lipoproteins would be delayed in older patients compared with younger patients, and that food intake in the older subjects might be different from that in the younger subjects. These may contribute to the increase in plasma PCOOH. However, the reason for the dramatic increases observed in subjects >65 years of age is still not known. Further experiments should be performed to clarify this point. The combined effect of aging and hyperlipidemia may have a strong impact on plasma PCOOH concentration.

PC exists on the surface of lipoproteins (9) and is susceptible to peroxidation. Peroxidation can be achieved by both cell-dependent and -independent mechanisms (29). Various cells, e.g., endothelial cells and smooth muscle cells, can induce peroxidation of lipoprotein lipids through both mechanisms, i.e., generation of reactive oxygen species, such as the superoxide anion (30)(31)(32), and the possible action of specific enzymes (33), such as cyclooxygenase and lipoxygenases. Linoleic acid and arachidonic acid are substrates of cyclooxygenase and lipoxygenases, respectively. Our results showed that the composition of plasma fatty acids was different in hyperlipidemic patients with high PCOOH compared with control subjects. In the former group, low concentrations of linoleic acid and arachidonic acid, and high concentrations of eicosapentaenoic acid were noted. These results indicate that oxidation of the n-6 polyunsaturated fatty acids, such as linoleic acid and arachidonic acid, was predominant. On the other hand, the n-3 polyunsaturated fatty acid was not attenuated in plasma PC. Therefore, the possible participation of specific enzymes for PCOOH formation was indicated. To elucidate this mechanism, we hope to conduct structural analysis of PCOOH in future experiments. However, the PCOOH contents in plasma were too low to conduct structural analysis.

Although the formation of PCOOH could occur in the bloodstream and in the arterial wall, it is still controversial where PC peroxidation takes place. Plasma PCOOH concentrations significantly correlated with each lipid. The data suggested that the increased PCOOH concentration would be dependent on lipids mass, including the increased number of lipoprotein particles. The plasma PCOOH concentrations seem to reflect the balance between hydroperoxide formation and its decomposition and clearance. The mechanism by which plasma PCOOH increases in hyperlipidemia is still not known. We hope to clarify the mechanism in future experiments.

The present study showed that plasma PCOOH concentrations in hyperlipidemia did not correlate with those of TC, TGs, and PL. However, there was a significantly positive correlation between plasma concentrations of PCOOH and each lipid in all subjects. The results suggest that lipids mass, not the kinds of lipids, would be correlated to PCOOH concentration. Plasma PCOOH concentrations in patients with hypercholesterolemia (TC >=2200 mg/L) as well as hypertriglyceridemia (TGs >=1500 mg/L) were markedly increased. This suggests that not only LDL but also TG-rich lipoproteins contain abundant PCOOH on the surface and that the plasma PCOOH concentration is dependent on the lipoprotein-lipids mass. Our finding that plasma PCOOH concentrations in patients with treatment-induced normolipidemia were also reduced to control concentrations supports this conclusion. In our drug therapy, antioxidative drugs such as probucol were administered to some patients. However, no patients had received probucol in the treatment-induced normolipidemic group as shown in Table 2Up . Therefore, these results suggested that probucol did not have a significant effect on PCOOH concentrations. On the other hand, it has been reported that HDL is the main carrier of lipid hydroperoxides in plasma (34). Further investigation should be done to determine the PCOOH concentration of each lipoprotein and to clarify the contribution of plasma PCOOH to atherosclerotic diseases in hyperlipidemic patients. The atherogenicity of hypertriglyceridemia (type IV hyperlipidemia) has been discussed (35). In the present study, it was shown that hypertriglyceridemia alone was associated with increased plasma PCOOH concentrations. Thus, the atherogenic effect of hypertriglyceridemia may be mediated by high plasma concentrations of PCOOH. In this regard, formation of PCOOH is a process that involves surface lipid oxidation of lipoproteins. Persistent formation of PCOOH may be associated with oxidation of the core lipids and formation of cholesterol ester hydroperoxide. Thus, increased PCOOH may reflect in vivo oxidative stress or oxidative damage to organs. The effect of the antioxidant system in plasma should also be considered. It has been reported that some kinds of glutathione peroxidase can directly reduce the concentrations of phospholipid hydroperoxide (36). Further studies should be performed to clarify the relationship between plasma PCOOH and antioxidants.

In conclusion, the accumulation of PCOOH in the plasma in hyperlipidemic elderly individuals might be related to the development and progression of atherosclerosis, and measurement of plasma PCOOH is useful for evaluating oxidative stress in vivo.


   Footnotes
 
1 Current address: Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.

2 Nonstandard abbreviations: CL-HPLC, chemiluminescence-HPLC; PCOOH, phosphatidylcholine hydroperoxide; TC, total cholesterol; TG, triglyceride; PL, phospholipid phosphorus; HDL-C, HDL-cholesterol; and PC, phosphatidylcholine.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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J. Lipid Res., November 1, 2005; 46(11): 2514 - 2524.
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J. Adachi, S. Matsushita, N. Yoshioka, R. Funae, T. Fujita, S. Higuchi, and Y. Ueno
Plasma phosphatidylcholine hydroperoxide as a new marker of oxidative stress in alcoholic patients
J. Lipid Res., May 1, 2004; 45(5): 967 - 971.
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J.-H. Oak, K. Nakagawa, and T. Miyazawa
UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples
J. Lipid Res., March 1, 2002; 43(3): 523 - 529.
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