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Ascorbic acid as a protective agent against peroxidation of LDL
Monday 10 October 1994: Poster Abstracts Lipid peroxidation
50
18-23) have suggested that serum bilirubin (Bu) may be an endogenous antioxidant against LDL oxidation. Our aim here was to examine that hypothesis. Gn agarose gel electrophorcsis, LDL (200-3OO,@ml) which had been oxidized by treatment with 1OrtM Cu*+. had a different mobility from unoxidized LDL. When the experiment was repeated in the presence of >17pM Bu the (normal serum level), the LDL had the same mobility as that of unoxidized LDL. The amount of rnalondialdehyde (a chemical indicator of lipid peroxidation) generated, which was negligible with LDL plus 17pM Bu, increased with diminution of the Bu level. A similar trend was obtained with 2,2’-azobis (2-amidinopropane) hydrochloride as oxidant. These data support the contention that Bu at or near normal serum level inhibits LDL oxidation. Recently, we observed that conjugated and delta bilirubins likewise prevent LDL oxidation. Trolox (a vitamin E analogue) is some 20 times less active an inhibitor of LDL oxidation than Bu under similar conditions. Thus, serum bilirubins can prevent human LDL oxidation, and this may be a hitherto neglected protective role of the bile pigments. 11891 u,
LDL oxidation differences in coronary patients classified according to HDL-cholesterol level Gracia V, Hurtado I, Pinto X, Castiiieiras MJ, Unitat de
Recerca Experimental, Ciutat Sanitdtia Bellvirge (CSVB), Barcelona, Spain
i
Universirciria de
In this study we analyzed the generation of oxidized LDL in coronary patients with different HDL-C levels, to investigate whether protective mechanisms for HDL other than reverse cholesterol transport arc possible. 14 patients with recent coronary disease were divided into 7 with hypoalphalipoproteinemia (HDL-C < 0.9 mmolll, HALP) and 7 without; 22 controls were used. LDL was oxidized with 0.25 mg LDUml in the presence of 40pmol Cu*+ per mg LDL; susceptibility to oxidation (lag phase) was monitored by conjugated diene, fluorescence and TBARS production. The MannWhitney test was used and correlation coefficients calculated. The LDL lag phase was shorter in all patients than in controls (P = O.Ol), and was correlated with HDLC (r = 0.57, P = 0.0004) and the ratio HDL-CTTC (r=O.46, P=O.O06). This correlation persisted within the classified groups (control, r = 0.40, P < 0.05; without HALP, r = 0.51, ns; and with HALP r = 0.91, P c 0.03) and between groups: values (minutes) for lag phase 72 + 21 SD (controls), 57 f 17 (without HALP), 45 f 15 (with HALP). These findings suggest a possible protective role of HDL against atherosclerosis through diminution of LDL oxidation. Ascorbic acid as a protective agent against peroxida11901 tion of LDL m, Hibino T, Iwata S, Yoshikawa M, Sakuma N, Fujinami T, 3rd Dept. of Inr. Med., Nagoya City Univ. Med. Sch.. M&ho-ku. Mizuho-cho, Nagoya, 467 Japan
It has been reported that ascorbic acid (AA) protects LDL against peroxidation. We confirmed this: LDL treated with AA was recognized by the LDL receptor as native LDL and was not taken into macrophages by the scavenger receptor. Native LDL (natLDL) was isolated from normal serum by ultracentrifugation, and incubated with Cu*+ (12.5pmol/l) with or without AA (3Opg/ml) to produce oxidized LDL (oxLDL). Lymphocyte proliferation rates were measured (with blocking endogenous cholesterol synthesis) in the presence of natLDL, oxLDL or LDL treated with AA. Labeled oleate was added to macrophage culture. medium and incubated with the three kinds of LDL, and labeled cholesteryl olcate was estimated as an index of LDL up take by macrophages. The rate of lymphocyte proliferation with OXLDLwas signifi-
cantly lower than that with natLDL, but there was no significant difference between the rates with natLDL and AA-treated LDL. The labeled cholesteryl oleatc in the macrophages after incubation was significantly higher with oxLDL than with natLDL, but not significantly different between natLDL and AA-treated LDL. These results indicate that ascorbic acid protects LDL against peroxidation. Negative correlation of LDL oxidative resistance with peripheral vascular disease Q&y&, O’Neal D, Nuckey T, Lewicki J, Matthews PG, Sikaris K, Best J, Dept. of Pathol., St Vincents Hosp., 41 Victoria Pde, 1191)
Fitzroy 3065, Australia
We wished to investigate the relationship between LDL resistance to oxidation and atherosclerotic peripheral vascular disease (PVD). We selected 74 patients (M45, F29) with a mean age of 57 (SEM 2) years, 38 with Typ II diabetes and 36 without diabetes. LDL was oxidized by Cu + and the lag time before oxidation, seen as an increase in absorption at 234 nm, was used as a measure of LDL resistance to oxidation. For PVD a duplex scan was performed on the leg with the lower ankle brachial index and the arterial system classified as: (1) normal, (2) no stenoses but reduced compliance, (3) stenoses present. We found that for the non-diabetic population there was an inverse association between the lag time and the degree of PVD: PVDl, lag 53(4) min (n = 16, age 39(3) years); PVDZ, lag 41(6) (n = 10, age 55(5)); PVD3, lag 33(7) (n = 10, age 73(3)) (P = 0.02 for lag1 vs. lag3). However for the diabetics there was no significant correlation: PVDl, lag 59(7) (n = IO, age 43(6)); PVDZ, lag 50(6) (n = 14, age 59(3)); PVD3, lag 46(5) (n = 14, age 70(3)) (P = 0.15 for lag1 vs. lag3). The inverse relationship between the length of the lag phase and the degree of PVD in non-diabetic patients is consistent with the hypothesis that the lag time of copper-mediated oxidation is a clinically relevant measure of LDL antioxidant status. In diabetic patients, other factors may influence the relation between coppermediated lag time and PVD. Changes in plasma vitamin E and thiobarbituric acidreactive substances (TBARS) during a single and long-term repeated LDGapheresis Komura T, Yamashita K, Ohba C, Yashiro A, Tasaki WA, H, Takahara K, Nakashima Y, Kuroiwa A, 2nd Dept. of Int. Med.,
1
Univ. of Occupational and Environmental Health, Japan, Iseigaoka, Yahuranishiku, Kirakyushu 807, Japan
l-l
Elimination of vitamin E (Vit E), a major antioxidant in plasma, during LDL-apheresis may raise concern about increased oxidizability of LDL. The purpose of this study was to investigate changes in Vit E and TBARS in plasma during a single and longterm repeated LDL-apheresis. 8 patients with hypercholesterolemia, who had been treated regularly (once per 2 weeks or once per 3 weeks) with LDL-apheresis using dextran sulfate-cellulose (DSC) column, were enrolled in this study. Total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), apolipoprotein B (Apo B), Vit E and TBARS were measured before and after 4 sessions for each patient, at a frequency of approximately once per month. TC, TG, HDLC, LDL-C, Apo B, Vit E and TBARS (averages in 8 patients) were decreased significantly by 47.7, 76.6, 3.1, 56.6, 61.9, 44.1 and 28.6%. respectively, on the contrary, Vit E/APO B and Vit E/LDL-C increased significantly by 55.3% and 36.4%. respectively, by a single LDL-apheresis (P c 0.0001; paired t-test). There was no statistically significant difference in the average pre-treatment level of any parameter between the 1st and the last (4th) LDL-apheresis in the study period (3-4 months). No statistically significant correlation between Vit E or TBARS
Atherosclerosis X, Monrreal, October 1994
50
18-23) have suggested that serum bilirubin (Bu) may be an endogenous antioxidant against LDL oxidation. Our aim here was to examine that hypothesis. Gn agarose gel electrophorcsis, LDL (200-3OO,@ml) which had been oxidized by treatment with 1OrtM Cu*+. had a different mobility from unoxidized LDL. When the experiment was repeated in the presence of >17pM Bu the (normal serum level), the LDL had the same mobility as that of unoxidized LDL. The amount of rnalondialdehyde (a chemical indicator of lipid peroxidation) generated, which was negligible with LDL plus 17pM Bu, increased with diminution of the Bu level. A similar trend was obtained with 2,2’-azobis (2-amidinopropane) hydrochloride as oxidant. These data support the contention that Bu at or near normal serum level inhibits LDL oxidation. Recently, we observed that conjugated and delta bilirubins likewise prevent LDL oxidation. Trolox (a vitamin E analogue) is some 20 times less active an inhibitor of LDL oxidation than Bu under similar conditions. Thus, serum bilirubins can prevent human LDL oxidation, and this may be a hitherto neglected protective role of the bile pigments. 11891 u,
LDL oxidation differences in coronary patients classified according to HDL-cholesterol level Gracia V, Hurtado I, Pinto X, Castiiieiras MJ, Unitat de
Recerca Experimental, Ciutat Sanitdtia Bellvirge (CSVB), Barcelona, Spain
i
Universirciria de
In this study we analyzed the generation of oxidized LDL in coronary patients with different HDL-C levels, to investigate whether protective mechanisms for HDL other than reverse cholesterol transport arc possible. 14 patients with recent coronary disease were divided into 7 with hypoalphalipoproteinemia (HDL-C < 0.9 mmolll, HALP) and 7 without; 22 controls were used. LDL was oxidized with 0.25 mg LDUml in the presence of 40pmol Cu*+ per mg LDL; susceptibility to oxidation (lag phase) was monitored by conjugated diene, fluorescence and TBARS production. The MannWhitney test was used and correlation coefficients calculated. The LDL lag phase was shorter in all patients than in controls (P = O.Ol), and was correlated with HDLC (r = 0.57, P = 0.0004) and the ratio HDL-CTTC (r=O.46, P=O.O06). This correlation persisted within the classified groups (control, r = 0.40, P < 0.05; without HALP, r = 0.51, ns; and with HALP r = 0.91, P c 0.03) and between groups: values (minutes) for lag phase 72 + 21 SD (controls), 57 f 17 (without HALP), 45 f 15 (with HALP). These findings suggest a possible protective role of HDL against atherosclerosis through diminution of LDL oxidation. Ascorbic acid as a protective agent against peroxida11901 tion of LDL m, Hibino T, Iwata S, Yoshikawa M, Sakuma N, Fujinami T, 3rd Dept. of Inr. Med., Nagoya City Univ. Med. Sch.. M&ho-ku. Mizuho-cho, Nagoya, 467 Japan
It has been reported that ascorbic acid (AA) protects LDL against peroxidation. We confirmed this: LDL treated with AA was recognized by the LDL receptor as native LDL and was not taken into macrophages by the scavenger receptor. Native LDL (natLDL) was isolated from normal serum by ultracentrifugation, and incubated with Cu*+ (12.5pmol/l) with or without AA (3Opg/ml) to produce oxidized LDL (oxLDL). Lymphocyte proliferation rates were measured (with blocking endogenous cholesterol synthesis) in the presence of natLDL, oxLDL or LDL treated with AA. Labeled oleate was added to macrophage culture. medium and incubated with the three kinds of LDL, and labeled cholesteryl olcate was estimated as an index of LDL up take by macrophages. The rate of lymphocyte proliferation with OXLDLwas signifi-
cantly lower than that with natLDL, but there was no significant difference between the rates with natLDL and AA-treated LDL. The labeled cholesteryl oleatc in the macrophages after incubation was significantly higher with oxLDL than with natLDL, but not significantly different between natLDL and AA-treated LDL. These results indicate that ascorbic acid protects LDL against peroxidation. Negative correlation of LDL oxidative resistance with peripheral vascular disease Q&y&, O’Neal D, Nuckey T, Lewicki J, Matthews PG, Sikaris K, Best J, Dept. of Pathol., St Vincents Hosp., 41 Victoria Pde, 1191)
Fitzroy 3065, Australia
We wished to investigate the relationship between LDL resistance to oxidation and atherosclerotic peripheral vascular disease (PVD). We selected 74 patients (M45, F29) with a mean age of 57 (SEM 2) years, 38 with Typ II diabetes and 36 without diabetes. LDL was oxidized by Cu + and the lag time before oxidation, seen as an increase in absorption at 234 nm, was used as a measure of LDL resistance to oxidation. For PVD a duplex scan was performed on the leg with the lower ankle brachial index and the arterial system classified as: (1) normal, (2) no stenoses but reduced compliance, (3) stenoses present. We found that for the non-diabetic population there was an inverse association between the lag time and the degree of PVD: PVDl, lag 53(4) min (n = 16, age 39(3) years); PVDZ, lag 41(6) (n = 10, age 55(5)); PVD3, lag 33(7) (n = 10, age 73(3)) (P = 0.02 for lag1 vs. lag3). However for the diabetics there was no significant correlation: PVDl, lag 59(7) (n = IO, age 43(6)); PVDZ, lag 50(6) (n = 14, age 59(3)); PVD3, lag 46(5) (n = 14, age 70(3)) (P = 0.15 for lag1 vs. lag3). The inverse relationship between the length of the lag phase and the degree of PVD in non-diabetic patients is consistent with the hypothesis that the lag time of copper-mediated oxidation is a clinically relevant measure of LDL antioxidant status. In diabetic patients, other factors may influence the relation between coppermediated lag time and PVD. Changes in plasma vitamin E and thiobarbituric acidreactive substances (TBARS) during a single and long-term repeated LDGapheresis Komura T, Yamashita K, Ohba C, Yashiro A, Tasaki WA, H, Takahara K, Nakashima Y, Kuroiwa A, 2nd Dept. of Int. Med.,
1
Univ. of Occupational and Environmental Health, Japan, Iseigaoka, Yahuranishiku, Kirakyushu 807, Japan
l-l
Elimination of vitamin E (Vit E), a major antioxidant in plasma, during LDL-apheresis may raise concern about increased oxidizability of LDL. The purpose of this study was to investigate changes in Vit E and TBARS in plasma during a single and longterm repeated LDL-apheresis. 8 patients with hypercholesterolemia, who had been treated regularly (once per 2 weeks or once per 3 weeks) with LDL-apheresis using dextran sulfate-cellulose (DSC) column, were enrolled in this study. Total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), apolipoprotein B (Apo B), Vit E and TBARS were measured before and after 4 sessions for each patient, at a frequency of approximately once per month. TC, TG, HDLC, LDL-C, Apo B, Vit E and TBARS (averages in 8 patients) were decreased significantly by 47.7, 76.6, 3.1, 56.6, 61.9, 44.1 and 28.6%. respectively, on the contrary, Vit E/APO B and Vit E/LDL-C increased significantly by 55.3% and 36.4%. respectively, by a single LDL-apheresis (P c 0.0001; paired t-test). There was no statistically significant difference in the average pre-treatment level of any parameter between the 1st and the last (4th) LDL-apheresis in the study period (3-4 months). No statistically significant correlation between Vit E or TBARS
Atherosclerosis X, Monrreal, October 1994