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Correlation between susceptibility of LDL subfractions to in vitro oxidation and in vivo oxidized LDL
Clinical Biochemistry, Vol. 33, No. 1, 71–73, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter
PII S0009-9120(99)00065-X
Correlation Between Susceptibility of LDL Subfractions to In Vitro Oxidation and In Vivo Oxidized LDL WANG JUN-JUN, LIU XIAO-ZHUAN, ZHUANG YI-YI, and LI LU-YAN Biochemistry Department, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China Introduction pidemiologic and clinical studies have indicated that elevated levels of plasma cholesterol, associated with circulating low density lipoprotein (LDL), are correlated with an increased risk of coronary artery diseases (CAD) (1). LDL particles are heterogeneous in size, density, and composition. Small, dense LDL in subjects with the atherogenic pattern B are more susceptible to oxidative modification and have been established as a risk factor of atherosclerosis (2). In addition, oxidation of LDL is believed to be an important step in the process of pathogenesis (1). Several lines of evidence support the existence of in vivo oxidized LDL (Ox-LDL). Ox-LDL has been demonstrated in atherosclerotic lesion (3). Increased level of autoantibodies against Ox-LDL and Ox-LDL appear to be associated with CAD (4,5). It was also reported that oxidatively modified plasma LDL is found largely among the dense LDL fractions (6,7). That indicates oxidatively modified plasma LDL tends to associate with dense LDL. However, no studies about the association between susceptibility of LDL oxidation and in vivo Ox-LDL were reported. In the present study we analyzed oxidative susceptibility of total, dense, and light LDL to Cu2⫹mediated oxidation from CAD patients. The relationship between the oxidative susceptibility of LDL and level of plasma Ox-LDL was also explored.
E
which were confirmed by eletrocardiography and laboratory tests. The patients were studied 6 months after their alleviation from acute attacks. The 20 male control subjects (55.4 ⫾ 4.1 years) without dylipidemia, hypertension, diabetes mellitus, or any clinically evident sign of atherosclerosis were selected from routine health examination, physical, eletrocardiography, and laboratory tests. The blood was sampled at least 12 h after fasting and collected into EDTA-containing tubes (1 mg/ mL), and plasma was seperated immediately and stored in ⫺70° C until analysis. ISOLATION
OF
LDL
LDL (d ⫽ 1.019 to 1.063 g/mL) was isolated from plasma by preparative ultracentrifugation in the presence of EDTA (8). Then light (d ⬍ 1.040 g/mL) and dense LDL (d ⬎ 1.040 g/mL) were separated from the total LDL by ultracentrifugation again. All LDL were dialyzed against PBS containing 1 mg/mL EDTA and 0.1 mg/mL NaN3 (pH7.6). They were filtered and sterilized through a 0.45-m cellulose filter and stored at 4° C in the dark under nitrogen gas. The EDTA-containing LDL samples were all extensively dialyzed against PBS-free EDTA in the dark for 48 h at 4° C, then the EDTA-free LDL were used for oxidation study immediately.
Materials and methods
OXIDATION
SUBJECTS
The oxidation experiments were performed essentially as described by Esterbauer et al. (9). Briefly, the EDTA-free LDL or LDL subfractions were diluted to 0.05 g/L. Oxidation was initiated by addition of freshly prepared 10-mol/L CuSO4 solution (final concentration). The kinetics of the oxidation of LDL was determined by monitoring the changes in 234-nm absorbance at 37° C on a Shimadzu UV-300 spectrophotometer every 5 min for 4 h. The amount of conjugated diene was used ε234 ⫽ 29,500 L/mol per centimeter as molar extinction coefficient.
AND BLOOD COLLECTION
Twenty-two male patients (56.8 ⫾ 3.7 years) in this study from Jinling Hospital were myocardial infarction survivors showing typical symptoms, Correspondence: Wang Jun-Jun, Biochemistry Department, Jinling Hospital, 305#, East Zhong Shan Road, Nanjing, 210002, P. R. China. Manuscript received April 27, 1999; revised July 30, 1999; accepted August 12, 1999. CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
OF
LDL
71
JUN-JUN
ET AL.
TABLE 1 Plasma Lipids, Oxidized LDL, and Apolipoproteins Concentrations in Patients and Controls Lipoprotein
CAD (n ⫽ 22)
Control (n ⫽ 20)
Total cholesterol (mmol/L) Total triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Ox-LDL (g/L)
5.27 ⫾ 1.03 1.93 ⫾ 0.91** 1.27 ⫾ 0.24* 3.11 ⫾ 0.87 539.1 ⫾ 155.6**
4.96 ⫾ 0.89 1.16 ⫾ 0.41 1.49 ⫾ 0.31 3.04 ⫾ 0.75 318.0 ⫾ 159.6
Compared with control: *p ⬍ 0.05; **p ⬍ 0.01.
SANDWICH ELISA
FOR
OX-LDL
Monoclonal antibodies against Cu2⫹ oxidized LDL presented by Professor Kong (10) was diluted to 5-g/mL IgG with 0.05-mol/mL (pH 9.5) sodium carbonate/bicarbonate buffer, then 100 L volume of the solution for each well was used to coat microtiter plates (Corning Co., Ltd., Corning, NY) that were kept at 4° C overnight and quenched with 10% (v/v) BSA in washing solution afterwards. The 100-L volume of samples and serial standard diluted in PBS containing 5% BSA and 0.5 mL/L Tween 20 were added to the wells and incubated for 1 h at 37° C. After washing, 100-L volume of diluted HRPlabeled sheep antihuman apolipoprotein B antibody was added to each well and incubated for 1 h at 37° C. The wells were washed and color was developed by adding 100 L of substrate (o-phenylenediamine dihydrochloride and hydrogen peroxide). After 15 min at room temperature, the enzyme reaction was stopped by adding 100 L of 2 mol/L H2SO4, and the absorbence of each well was read at 495 nm. Copperoxidized LDL was prepared as described (10) and used as Ox-LDL standard. The sensitivity of this assay was between 100 and 1600 g/L. Intra- and interassay coefficients of variation were 4.9% and 10.2%, respectively. When copper-oxidized LDL was added to human plasma at a final concentration of 200 and 1200 g/L, respectively, recoveries were 90% and 96%, respectively. LIPOPROTEIN
ANALYSIS
Total cholesterol, HDL cholesterol, and triglycerides were measured enzymatically with reagents
from Boehringer Mannheim (Mannheim, Germany), adapted to 7150 Automatic Analyzer (Hitachi, Japan). LDL cholesterol was calculated according to Friedewald et al. (11) Protein concentration of the LDL was determined according to modified Lowry’s method. STATISTICAL
ANALYSIS
Results were given as mean ⫾ standard deviation. Student t-test, Spearman’s rank correlation coefficient analysis, and multivariate regression analysis were applied in the processing of data obtained. Results Plasma total triglycerides and HDL cholesterol concentrations in CAD patients were significantly different from that of the controls. No significant difference was found in the levels of total cholesterol and LDL cholesterol in the patients, whereas the Ox-LDL concentration was significantly elevated in comparison with the controls (Table 1). Susceptibility of total LDL and its subfractions to in vitro oxidation are presented in Table 2. A significant difference in lag time and total amount of conjugated dienes in the test for total LDL oxidizabilty between CAD patients and controls were found, whereas the difference in oxidation rate did not reach statistical significance. It was also observed that lag time of dense LDL from the CAD patients was significantly shorter than that of light LDL, and total amount of conjugated dienes was significantly increased, while no difference was found in the oxidation rate of two LDL subfractions.
TABLE 2 Characteristics of Absorbance Curves During Oxidation of Total LDL and Its Subfractions From the CHD Patients and Controls CAD (n ⫽ 22) Group Lag time (min) Rate of oxidation (nmol CD/mg LDL 䡠 min⫺1) Amount of dienes (nmol CD/mg LDL)
Dense LDL
Light LDL
Total LDL
Control (n ⫽ 220) (Total LDL)
66.7 ⫾ 20.1** 1.65 ⫾ 0.75
85.5 ⫾ 21.0 1.42 ⫾ 0.75
79.5 ⫾ 20.4* 1.49 ⫾ 0.62
92.9 ⫾ 22.2 1.16 ⫾ 0.53
179.2 ⫾ 73.5**
114.2 ⫾ 34.1
139.4 ⫾ 51.9**
82.9 ⫾ 35.3
Dense LDL compared with Light LDL, total LDL group compared with control: *p ⬍ 0.05; **p ⬍ 0.01. 72
CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
SUSCEPTIBILITY OF LDL SUBFRACTIONS TO IN VITRO OXIDATION
This demonstrates that dense LDL is more susceptible to oxidative modification. To reveal the possible association between susceptibility of LDL oxidation and level of in vivo Ox-LDL, we analyzed the relation between characteristics of LDL from the CAD patients. A significantly inverse correlation was found between the lag time of total LDL oxidation and plasma Ox-LDL level (r ⫽ ⫺0.44, p ⬍ 0.05). We further found that the Ox-LDL level was negatively correlated with the lag time of dense LDL (r ⫽ ⫺0.48, p ⬍ 0.05), but not with that of light LDL. The correlation between the lag time of dense LDL and plasma Ox-LDL level remained significant (p ⬍ 0.05) after adjustment for the lag time of total and light LDL by multivariate regression analysis. Neither the amount of conjugated dienes nor the oxidation rate in dense, light, or total LDL was found correlated with plasma Ox-LDL. It seems that dense LDL was correlated to in vivo Ox-LDL so we analyzed the correlation between plasma Ox-LDL and LDL protein level. Neither light nor total LDL protein level was correlated with OxLDL level, although dense LDL protein level tended to be correlated with Ox-LDL level (r ⫽ 0.42, p ⫽ 0.053). Discussion This is the first study that reveals significant association between in vivo Ox-LDL and susceptibility of dense LDL oxidization in the patients with CAD. Our data show not only plasma Ox-LDL level is correlated with the lag time of dense LDL to in vitro oxidation, but also tends to correlate with plasma concentration of dense LDL protein. The studied CAD patients had significantly high levels of triglycerides. Hypertriglyceridemia can promote formation of small, pattern B LDL with a high atherogenic potential, as mediated by the cholesteryl ester transfer protein and lipase. Our previous data also showed that the distributions of LDL subfractions in the CAD patients tended to be small, dense LDL. Oxidation of LDL may play a causal role in atherosclerosis. We found that enhanced susceptibility to in vitro oxidation of the dense LDL subfractions in the patients. Elevated levels of this pattern of dense LDL fraction prone to oxidization may attribute to the higher oxidizability of total LDL in CHD patients than that in control. Meanwhile, plasma levels of Ox-LDL increased significantly in the CAD patients. Sevanian et al. (6) found oxidatively modified plasma LDL largely among the dense LDL fractions. Kotani et al. (7) detected malondialdehyde-modified LDL (MDA-LDL) in the sera of healthy individuals using ELISA. Furthermore, their assays for lipoprotein subfractions revealed that MDA-LDL was mainly distributed in the LDL fraction, and MDA-LDL/apoB ratio showed a peak at dense LDL fractions. These seem in vivo Ox-LDL association with dense LDL.
CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
Maggi et al. (4) compared in vitro and in vivo LDL oxidation markers in the patients with severe atherosclerosis. However, no correlation was found between antibody titer calculated as anti-MDA-LDL/antiMDA-HSA ratio and the lag time of LDL oxidation. On the other hand, a highly significant negative correlation was detected between the antibody titer and vitamin E level in LDL. Interestingly, the present study revealed the plasma Ox-LDL level was significant negatively correlated with the lag time of dense LDL but not with that of light LDL. We also found dense LDL protein tends to be correlated with in vivo Ox-LDL level. Our study confirms the CAD patients have elevated plasma levels of dense LDL, and the dense LDL were more susceptible to oxidative modification and associated with degree of plasma Ox-LDL. References 1. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implication for cholesterol deposition in atherosclerosis. Ann Rev Biochem 1983; 52: 223– 61. 2. Chait A, Brazg RL, Tribble DK, Krauss RM. Susceptibility of small, dense, low density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype, pattern B. Am J Med 1993; 94: 350 – 6. 3. Yla¨-Herttuala S, Palinski W, Rosenfeld M, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lession of rabbit and man. J Clin Invest 1989; 84: 1086 –95. 4. Maggi E, Chiesa R, Melissano G, et al. LDL oxidation in patients with severe carotid atherosclerosis—a study of in vitro and in vivo oxidation markers. Arterioscler Thromb 1994; 14: 1892–9. 5. Holveot P, Stassen JM, Van Cleemput J, et al. Oxidized low density lipoproteins in patients with transplant-associated coronary artery disease. Arterioscler Thromb Vasc Biol 1998; 18: 100 –7. 6. Sevanian A, Hwang J, Hodis H, et al. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subfractions. Arterioscler Thromb Vasc Biol 1996; 16: 784 –93. 7. Kotani K, Kondo A, Manabe M, et al. Determination of malondialdehyde-modified LDL (MDA-LDL) and its potential usefulness. Rinsho Byori 1997; 45: 47–54. 8. Ma˜rz W, Gro W. Analysis of plasma lipoproteins by ultracentrifugation in a new fixed angle rotor: evaluation of a phosphotungstic acid/Mgcl precipitation and a quantitative lipoprotein electrophoresis assay. Clin Chim Acta 1986; 160: 1–18. 9. Esterbauer H, Striegl G, Puhl H, et al. Continuous monitoring of in vitro oxidation of human low-density lipoproteins. Free Radic Res Commun 1989; 6: 67–75. 10. Wang HL, Chen SC, Kong XT, et al. Quantitation of plasma oxidatively modified low density lipoprotein by sandwich enzyme linked immunosorbent assay. Clin Chem Acta 1993; 218: 97–103. 11. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 449 –502.
73
PII S0009-9120(99)00065-X
Correlation Between Susceptibility of LDL Subfractions to In Vitro Oxidation and In Vivo Oxidized LDL WANG JUN-JUN, LIU XIAO-ZHUAN, ZHUANG YI-YI, and LI LU-YAN Biochemistry Department, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China Introduction pidemiologic and clinical studies have indicated that elevated levels of plasma cholesterol, associated with circulating low density lipoprotein (LDL), are correlated with an increased risk of coronary artery diseases (CAD) (1). LDL particles are heterogeneous in size, density, and composition. Small, dense LDL in subjects with the atherogenic pattern B are more susceptible to oxidative modification and have been established as a risk factor of atherosclerosis (2). In addition, oxidation of LDL is believed to be an important step in the process of pathogenesis (1). Several lines of evidence support the existence of in vivo oxidized LDL (Ox-LDL). Ox-LDL has been demonstrated in atherosclerotic lesion (3). Increased level of autoantibodies against Ox-LDL and Ox-LDL appear to be associated with CAD (4,5). It was also reported that oxidatively modified plasma LDL is found largely among the dense LDL fractions (6,7). That indicates oxidatively modified plasma LDL tends to associate with dense LDL. However, no studies about the association between susceptibility of LDL oxidation and in vivo Ox-LDL were reported. In the present study we analyzed oxidative susceptibility of total, dense, and light LDL to Cu2⫹mediated oxidation from CAD patients. The relationship between the oxidative susceptibility of LDL and level of plasma Ox-LDL was also explored.
E
which were confirmed by eletrocardiography and laboratory tests. The patients were studied 6 months after their alleviation from acute attacks. The 20 male control subjects (55.4 ⫾ 4.1 years) without dylipidemia, hypertension, diabetes mellitus, or any clinically evident sign of atherosclerosis were selected from routine health examination, physical, eletrocardiography, and laboratory tests. The blood was sampled at least 12 h after fasting and collected into EDTA-containing tubes (1 mg/ mL), and plasma was seperated immediately and stored in ⫺70° C until analysis. ISOLATION
OF
LDL
LDL (d ⫽ 1.019 to 1.063 g/mL) was isolated from plasma by preparative ultracentrifugation in the presence of EDTA (8). Then light (d ⬍ 1.040 g/mL) and dense LDL (d ⬎ 1.040 g/mL) were separated from the total LDL by ultracentrifugation again. All LDL were dialyzed against PBS containing 1 mg/mL EDTA and 0.1 mg/mL NaN3 (pH7.6). They were filtered and sterilized through a 0.45-m cellulose filter and stored at 4° C in the dark under nitrogen gas. The EDTA-containing LDL samples were all extensively dialyzed against PBS-free EDTA in the dark for 48 h at 4° C, then the EDTA-free LDL were used for oxidation study immediately.
Materials and methods
OXIDATION
SUBJECTS
The oxidation experiments were performed essentially as described by Esterbauer et al. (9). Briefly, the EDTA-free LDL or LDL subfractions were diluted to 0.05 g/L. Oxidation was initiated by addition of freshly prepared 10-mol/L CuSO4 solution (final concentration). The kinetics of the oxidation of LDL was determined by monitoring the changes in 234-nm absorbance at 37° C on a Shimadzu UV-300 spectrophotometer every 5 min for 4 h. The amount of conjugated diene was used ε234 ⫽ 29,500 L/mol per centimeter as molar extinction coefficient.
AND BLOOD COLLECTION
Twenty-two male patients (56.8 ⫾ 3.7 years) in this study from Jinling Hospital were myocardial infarction survivors showing typical symptoms, Correspondence: Wang Jun-Jun, Biochemistry Department, Jinling Hospital, 305#, East Zhong Shan Road, Nanjing, 210002, P. R. China. Manuscript received April 27, 1999; revised July 30, 1999; accepted August 12, 1999. CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
OF
LDL
71
JUN-JUN
ET AL.
TABLE 1 Plasma Lipids, Oxidized LDL, and Apolipoproteins Concentrations in Patients and Controls Lipoprotein
CAD (n ⫽ 22)
Control (n ⫽ 20)
Total cholesterol (mmol/L) Total triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Ox-LDL (g/L)
5.27 ⫾ 1.03 1.93 ⫾ 0.91** 1.27 ⫾ 0.24* 3.11 ⫾ 0.87 539.1 ⫾ 155.6**
4.96 ⫾ 0.89 1.16 ⫾ 0.41 1.49 ⫾ 0.31 3.04 ⫾ 0.75 318.0 ⫾ 159.6
Compared with control: *p ⬍ 0.05; **p ⬍ 0.01.
SANDWICH ELISA
FOR
OX-LDL
Monoclonal antibodies against Cu2⫹ oxidized LDL presented by Professor Kong (10) was diluted to 5-g/mL IgG with 0.05-mol/mL (pH 9.5) sodium carbonate/bicarbonate buffer, then 100 L volume of the solution for each well was used to coat microtiter plates (Corning Co., Ltd., Corning, NY) that were kept at 4° C overnight and quenched with 10% (v/v) BSA in washing solution afterwards. The 100-L volume of samples and serial standard diluted in PBS containing 5% BSA and 0.5 mL/L Tween 20 were added to the wells and incubated for 1 h at 37° C. After washing, 100-L volume of diluted HRPlabeled sheep antihuman apolipoprotein B antibody was added to each well and incubated for 1 h at 37° C. The wells were washed and color was developed by adding 100 L of substrate (o-phenylenediamine dihydrochloride and hydrogen peroxide). After 15 min at room temperature, the enzyme reaction was stopped by adding 100 L of 2 mol/L H2SO4, and the absorbence of each well was read at 495 nm. Copperoxidized LDL was prepared as described (10) and used as Ox-LDL standard. The sensitivity of this assay was between 100 and 1600 g/L. Intra- and interassay coefficients of variation were 4.9% and 10.2%, respectively. When copper-oxidized LDL was added to human plasma at a final concentration of 200 and 1200 g/L, respectively, recoveries were 90% and 96%, respectively. LIPOPROTEIN
ANALYSIS
Total cholesterol, HDL cholesterol, and triglycerides were measured enzymatically with reagents
from Boehringer Mannheim (Mannheim, Germany), adapted to 7150 Automatic Analyzer (Hitachi, Japan). LDL cholesterol was calculated according to Friedewald et al. (11) Protein concentration of the LDL was determined according to modified Lowry’s method. STATISTICAL
ANALYSIS
Results were given as mean ⫾ standard deviation. Student t-test, Spearman’s rank correlation coefficient analysis, and multivariate regression analysis were applied in the processing of data obtained. Results Plasma total triglycerides and HDL cholesterol concentrations in CAD patients were significantly different from that of the controls. No significant difference was found in the levels of total cholesterol and LDL cholesterol in the patients, whereas the Ox-LDL concentration was significantly elevated in comparison with the controls (Table 1). Susceptibility of total LDL and its subfractions to in vitro oxidation are presented in Table 2. A significant difference in lag time and total amount of conjugated dienes in the test for total LDL oxidizabilty between CAD patients and controls were found, whereas the difference in oxidation rate did not reach statistical significance. It was also observed that lag time of dense LDL from the CAD patients was significantly shorter than that of light LDL, and total amount of conjugated dienes was significantly increased, while no difference was found in the oxidation rate of two LDL subfractions.
TABLE 2 Characteristics of Absorbance Curves During Oxidation of Total LDL and Its Subfractions From the CHD Patients and Controls CAD (n ⫽ 22) Group Lag time (min) Rate of oxidation (nmol CD/mg LDL 䡠 min⫺1) Amount of dienes (nmol CD/mg LDL)
Dense LDL
Light LDL
Total LDL
Control (n ⫽ 220) (Total LDL)
66.7 ⫾ 20.1** 1.65 ⫾ 0.75
85.5 ⫾ 21.0 1.42 ⫾ 0.75
79.5 ⫾ 20.4* 1.49 ⫾ 0.62
92.9 ⫾ 22.2 1.16 ⫾ 0.53
179.2 ⫾ 73.5**
114.2 ⫾ 34.1
139.4 ⫾ 51.9**
82.9 ⫾ 35.3
Dense LDL compared with Light LDL, total LDL group compared with control: *p ⬍ 0.05; **p ⬍ 0.01. 72
CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
SUSCEPTIBILITY OF LDL SUBFRACTIONS TO IN VITRO OXIDATION
This demonstrates that dense LDL is more susceptible to oxidative modification. To reveal the possible association between susceptibility of LDL oxidation and level of in vivo Ox-LDL, we analyzed the relation between characteristics of LDL from the CAD patients. A significantly inverse correlation was found between the lag time of total LDL oxidation and plasma Ox-LDL level (r ⫽ ⫺0.44, p ⬍ 0.05). We further found that the Ox-LDL level was negatively correlated with the lag time of dense LDL (r ⫽ ⫺0.48, p ⬍ 0.05), but not with that of light LDL. The correlation between the lag time of dense LDL and plasma Ox-LDL level remained significant (p ⬍ 0.05) after adjustment for the lag time of total and light LDL by multivariate regression analysis. Neither the amount of conjugated dienes nor the oxidation rate in dense, light, or total LDL was found correlated with plasma Ox-LDL. It seems that dense LDL was correlated to in vivo Ox-LDL so we analyzed the correlation between plasma Ox-LDL and LDL protein level. Neither light nor total LDL protein level was correlated with OxLDL level, although dense LDL protein level tended to be correlated with Ox-LDL level (r ⫽ 0.42, p ⫽ 0.053). Discussion This is the first study that reveals significant association between in vivo Ox-LDL and susceptibility of dense LDL oxidization in the patients with CAD. Our data show not only plasma Ox-LDL level is correlated with the lag time of dense LDL to in vitro oxidation, but also tends to correlate with plasma concentration of dense LDL protein. The studied CAD patients had significantly high levels of triglycerides. Hypertriglyceridemia can promote formation of small, pattern B LDL with a high atherogenic potential, as mediated by the cholesteryl ester transfer protein and lipase. Our previous data also showed that the distributions of LDL subfractions in the CAD patients tended to be small, dense LDL. Oxidation of LDL may play a causal role in atherosclerosis. We found that enhanced susceptibility to in vitro oxidation of the dense LDL subfractions in the patients. Elevated levels of this pattern of dense LDL fraction prone to oxidization may attribute to the higher oxidizability of total LDL in CHD patients than that in control. Meanwhile, plasma levels of Ox-LDL increased significantly in the CAD patients. Sevanian et al. (6) found oxidatively modified plasma LDL largely among the dense LDL fractions. Kotani et al. (7) detected malondialdehyde-modified LDL (MDA-LDL) in the sera of healthy individuals using ELISA. Furthermore, their assays for lipoprotein subfractions revealed that MDA-LDL was mainly distributed in the LDL fraction, and MDA-LDL/apoB ratio showed a peak at dense LDL fractions. These seem in vivo Ox-LDL association with dense LDL.
CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000
Maggi et al. (4) compared in vitro and in vivo LDL oxidation markers in the patients with severe atherosclerosis. However, no correlation was found between antibody titer calculated as anti-MDA-LDL/antiMDA-HSA ratio and the lag time of LDL oxidation. On the other hand, a highly significant negative correlation was detected between the antibody titer and vitamin E level in LDL. Interestingly, the present study revealed the plasma Ox-LDL level was significant negatively correlated with the lag time of dense LDL but not with that of light LDL. We also found dense LDL protein tends to be correlated with in vivo Ox-LDL level. Our study confirms the CAD patients have elevated plasma levels of dense LDL, and the dense LDL were more susceptible to oxidative modification and associated with degree of plasma Ox-LDL. References 1. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implication for cholesterol deposition in atherosclerosis. Ann Rev Biochem 1983; 52: 223– 61. 2. Chait A, Brazg RL, Tribble DK, Krauss RM. Susceptibility of small, dense, low density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype, pattern B. Am J Med 1993; 94: 350 – 6. 3. Yla¨-Herttuala S, Palinski W, Rosenfeld M, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lession of rabbit and man. J Clin Invest 1989; 84: 1086 –95. 4. Maggi E, Chiesa R, Melissano G, et al. LDL oxidation in patients with severe carotid atherosclerosis—a study of in vitro and in vivo oxidation markers. Arterioscler Thromb 1994; 14: 1892–9. 5. Holveot P, Stassen JM, Van Cleemput J, et al. Oxidized low density lipoproteins in patients with transplant-associated coronary artery disease. Arterioscler Thromb Vasc Biol 1998; 18: 100 –7. 6. Sevanian A, Hwang J, Hodis H, et al. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subfractions. Arterioscler Thromb Vasc Biol 1996; 16: 784 –93. 7. Kotani K, Kondo A, Manabe M, et al. Determination of malondialdehyde-modified LDL (MDA-LDL) and its potential usefulness. Rinsho Byori 1997; 45: 47–54. 8. Ma˜rz W, Gro W. Analysis of plasma lipoproteins by ultracentrifugation in a new fixed angle rotor: evaluation of a phosphotungstic acid/Mgcl precipitation and a quantitative lipoprotein electrophoresis assay. Clin Chim Acta 1986; 160: 1–18. 9. Esterbauer H, Striegl G, Puhl H, et al. Continuous monitoring of in vitro oxidation of human low-density lipoproteins. Free Radic Res Commun 1989; 6: 67–75. 10. Wang HL, Chen SC, Kong XT, et al. Quantitation of plasma oxidatively modified low density lipoprotein by sandwich enzyme linked immunosorbent assay. Clin Chem Acta 1993; 218: 97–103. 11. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 449 –502.
73