Elementos conceptuales

Oxidative modification of low-density lipoprotein (LDL) has been proposed to enhance its atherogenicity (11). It is quoted that antioxidants, such as vitamin E, provide resistance to this process (12) and that habitual intake of them will lower the incidence of coronary artery disease.

Dietary flavonoid intake has also been reported to be in- versely associated with mortality from coronary artery disease (13). The low incidence of coronary artery disease in the French, who consume a high-fat diet (French paradox), has been attributed, in part, to regular consumption of red wine. In spite of the high smoking rate and susceptibility of oxida- tive modification of LDL in smokers, the mortality rates from coronary artery disease are much lower in China and in Japan than in the West (Far East paradox) where people drink a lot of tea. Tea polyphenols and red wine polyphenols are repre- sentative plant polyphenols that are consumed on a regular daily basis. Potent antioxidative properties of tea polyphenols have been described elsewhere. Ishikawa et al. (14) examined in vitro and in vivo the inhibitory effects of tea polyphenols on oxidative stress on LDL as follows.

A. In Vitro Study

Plasma was treated with tea polyphenols in order to adsorb them to LDL particles and the oxidizability of LDL isolated thereafter was measured. Blood collected from a normolip- idemic healthy male volunteer was centrifuged to obtain se- rum. Tea catechins and theaflavins were mixed with serum in different concentrations (25µM/L to 400µM/L) and incubated for 3 hours at 37°C. Thereafter, LDLs were isolated from the serum by ultracentrifugation method and dialyzed for 12

hours at 4°C with degassed PBS (1L⫻ 4 times) to obtain tea polyphenol-adsorbed LDL. Control LDL was treated without tea polyphenols. The oxidizability of LDL thus obtained was estimated by measuring three indexes (conjugated dienes, lipid peroxides and thiobarbituric acid reactive substances [TBARS]). By initiating the diene formation by Cu⫹⫹, the lag time before the oxidative propagation was measured as shown in Fig. 6. Galloyl catechins and theaflavins (EGCg, ECg, TF2 and TF3) increased lag time markedly and significantly before the onset of oxidative propagation in a dose dependent man- ner. Particularly, EGCg and TF3 prolonged lag time almost three times more than that of the control by 200µM/L treat-

ment, while vitamin E prolonged it only twice by 400 µM/L

treatment (data not shown). Suppression of TBARS and lipid peroxides formation was also observed in the LDL samples with catechins(Fig. 7)and with theaflavins(Fig. 8).As shown in the figures, TBARS and lipid peroxide formation were sig-

FIG. 6 Effect of in vitro addition of catechins (0-400µM/L) to plasma on susceptibility of low-density lipoprotein to copper-induced oxidation.

FIG. 7 Effect of in vitro addition of catechins to plasma on susceptibility of low-density lipoprotein to TBARS and macrophage-mediated oxidation. n⫽ 4. One representative experiment of three is shown; the other two exper- iments yielded similar results. TBARS, thiobarbituric acid-reactive sub- stance; MDA, malondialdehyde. TBARS and lipid peroxide differ signifi- cantly between groups, p⬍ 0.01 (ANOVA).

Significantly different from control: *p⬍ 0.05, **p ⬍ 0.0001.

nificantly reduced particularly in those LDL samples treated by 400µM/L tea polyphenols.

B. In Vivo Study

Normolipidemic healthy male volunteers (n ⫽ 22) aged 22 ⫾

1 years old, who abstained from tea for more than 4 weeks, were divided into two groups: tea and water. The tea group consumed five cups of black tea/day (11 g/750 mL/day) for four

FIG. 8 Effect of in vitro addition of theaflavins to plasma on susceptibility of low-density lipoprotein to TBARS and macrophage-mediated oxidation. n⫽ 4. One representative experiment of three is shown; the other two exper- iments yielded similar results. TBARS, thiobarbituric acid-reactive sub- stance; MDA, malondialdehyde. TBARS and lipid peroxide differ signifi- cantly between groups, p⬍ 0.01 (ANOVA).

Significantly different from control: *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.0001.

weeks and the water group consumed the same amount of wa- ter. Blood samples were collected at the start and at the end of the study. After tea consumption for four weeks, significant prolongation of lag time before LDL oxidation was noted from 54 to 62 min, whereas no significant change was observed in the water group(Fig. 9).Other parameters showed no signifi- cant changes such as plasma total cholesterol, HDL choles- terol, triglyceride, apolipoprotein B, and plasma vitamin E be- tween baseline and the end of the study in both groups.

FIG. 9 Changes in oxidative susceptibility of low-denisty lipoprotein by black tea consumption in human volunteers. Significantly different from lag time at week 0, p⬍ 0.01.

In a separate experiment, when 220 mg of EGCg was in- gested in capsule form by a volunteer 1, 2, 3 h after ingestion, EGCg was present in the plasma at concentrations of 250, 230, and 180ηg/mL respectively. The concentration was highest 1 or 2 h after ingestion and had decreased slightly by 3 h after ingestion.

C. Discussion

In the in vitro experiment, oxidation of LDL that was isolated from plasma was significantly inhibited by preincubation with tea polyphenols in a dose-dependent manner. In the case of

EGCg,ⱖ200 µM/Lⱌ 100 ηg/mL was necessary for a signifi-

cant suppressive effect on LDL oxidizability.

Since only a part of EGCg seems to have been adsorbed to LDL and the rest was washed away, the effective amount of EGCg must be very trace in this experimental system. In the in vivo study, the mean lag time before LDL oxidation was significantly prolonged from 54 to 62 min after the subjects

had consumed tea for four weeks, whereas no significant change was observed in the control group. The concentration of catechins in plasma was in the range of hundreds ofηg/mL a few hours after the ingestion, suggesting that a very similar situation will occur as it did in the in vitro experiment. Re- peated exposure of LDL particles to tea polyphenols may en- rich the LDL particles sufficiently to make them less suscepti- ble to oxidative stress. Ascertaining the concentrations of tea polyphenols incorporated into LDL would contribute enor- mously to clarifying the mechanism by which tea affects LDL oxidizability. In conclusion, tea polyphenols may have favor- able effects in ameliorating atherosclerosis by decreasing the susceptibility of LDL to oxidative modification.

V. SUPPRESSION OF THE ACCUMULATION