Black Tea Consumption Does Not Protect Low Density Lipoprotein from Oxidative Modi®Cation

Black tea consumption does not protect low density lipoprotein from oxidative modi®cation

GT McAnlis1,2, J McEneny2, J Pearce1 and IS Young2

1Department of Food Science, The Queens University of Belfast, Newforge Lane, Belfast, BT9 5PX, N. Ireland and 2Department of Clinical Biochemistry, The Queens University of Belfast, Royal Victoria Hospital, Belfast, BT12 6BJ, N. Ireland

Objective: To investigate the in vivo and in vitro effects of black tea on the oxidative modi®cation of low density lipoprotein (LDL). Design: The antioxidant activity of the tea was studied in vitro by measuring the resistance of the LDL to oxidative modi®cation in the presence of copper. The effects of tea consumption in vivo were investigated in two settings. Firstly, to assess the acute effects of tea consumption, ®ve fasting healthy subjects ingested 600 mls (50.7 Æ 5.4 mg ¯avonoids) of black tea and peripheral venous blood was collected at 0, 30, 60, 90, 120 and 180 min after consumption. Secondly, to assess the effects of chronic tea consumption, a randomised crossover trial of tea (126.8 Æ 13.5 mg ¯avonoids) and coffee consumption was carried out in ten healthy subjects. Results: Black tea extract increased the resistance of LDL in vitro in a concentration dependent manner. There was no signi®cant change in total plasma antioxidant capacity or susceptibility of the LDL to oxidation over the 3 h period after consumption of black tea. The four-week crossover study in which coffee was used as a control against the black tea showed no signi®cant difference in the total plasma antioxidant capacity or susceptibility of LDL to oxidation between the tea and coffee groups. Serum lipids, including total cholesterol, triglycerides, LDL cholesterol and HDL cholesterol did not change signi®cantly throughout the study. Conclusions: The consumption of moderate quantities of black tea acutely or for one week does not increase plasma total antioxidant capacity or alter the susceptibility of LDL to oxidation. Sponsorship: This work was funded by the Department of Agriculture for Northern Ireland. Descriptors: low density lipoprotein (LDL); antioxidants; tea; ¯avonoids

Introduction found that increased tea consumption may increase susceptibility to heart disease (Hertog et al, 1997). Flavonoids are a group of polyphenolic compounds widely It is currently believed that oxidative modi®cation of distributed throughout the plant kingdom (Kuhnau et al, low density lipoproteins (LDL) by free radicals is a key 1976). They occur naturally in fruit and vegetables and are, early event in the pathogenesis of atherosclerosis therefore, an integral part of the human diet (Hertog et al, (Steinberg et al, 1989). The rapid uptake of oxidatively 1992, 1993a). A number of epidemiological studies show modi®ed LDL via a scavenger receptor leads to the forma- an inverse correlation between dietary ¯avonoid intake and tion of foam cells, and oxidised LDL also has a number of mortality from coronary heart disease (CHD) (Hertog et al, other atherogenic properties (Goldstein et al, 1979). Sev- 1993b, 1995; Knekt et al, 1996; Keli et al, 1996). In the eral dietary ¯avonoids have been shown to inhibit the past, the dietary intake of ¯avonoids in Western Europe oxidative modi®cation of LDL in vitro (de-Whalley et al, was estimated to be in the range of 0.5±1.0 g per day, of 1990) and hence, have the potential to reduce LDL oxida- which about 170 mg consisted of ¯avonol, ¯avonone and tion and atherogenesis in vivo. However, the ef®ciency of ¯avone aglycones (Kuhnau et al, 1976). More recently, the protection of LDL in vivo will depend on several however, it has been suggested that the actual intake is factors, including the absorption of the ¯avonoids and lower than this, around 25 mg of ¯avonol (quercetin, how they interact with lipoproteins. A number of mechan- kaempferol and myricetin) and ¯avone (luteolin and api- isms are likely to contribute to inhibition of LDL oxidation genin) aglycones (Hertog et al, 1993b). by ¯avonoids. Flavonoids may directly scavenge some The main sources of dietary ¯avonoids are tea, onions, radical species, acting as chain-breaking antioxidants (de- apples and red wine (Hertog et al, 1993b). Tea is one of the Whalley et al, 1990). In addition, the ¯avonoids may most frequently consumed beverages in the world, with recycle other chain-breaking antioxidants such as a-toco- around 80% being consumed as black tea and the rest as pherol by donating a hydrogen atom to the tocopheryl green tea (Balentine, 1992). The production of black tea radical (Frankel et al, 1993). Transition metals such as involves a fermentation process to form a complex mix of iron and copper are important pro-oxidants; some ¯avo- catechin esters, thearubigens, thea¯avins and certain ¯avonoids can chelate divalent metal ions, hence preventing free nol glycosides. Recent epidemiological evidence suggests radical formation (de-Whalley et al, 1990). Extensive in that increased black tea consumption may protect against vitro studies have found most of the main dietary ¯avo- stroke (Keli et al, 1996). Conversely the Caerphilly Study noids to be effective in protecting against the oxidative modi®cation of LDL (de-Whalley et al, 1990; Laughton Correspondence: Dr GT McAnlis. Received 11 July 1997; revised 20 September 1997; accepted 2 November et al, 1991; Morel et al, 1993; Rankin et al, 1993; Igarashi 1997 & Ohmuma, 1995; Fuhrman et al, 1995). Speci®cally, the Black tea consumption does not protect LDL GT McAnlis et al 203 ability of tea catechins to protect LDL against oxidative 6 250 mm, 5mm) protected by an Ultracarb ODS 20 C18 modi®cation has been investigated, with (7)-epigallocate- (Phenomenex Ltd. UK) guard column (4.6 6 30 mm, chin (EGC), ()-catechin (C), (7)-epicatechin (EC), (7)- 5 mm). The HPLC system consisted of a TSP spectra sys- epicatechingallate (ECG) and (7)-epigallocatechingallate tem (Thermo Separation Products) P2000 pump, a TSP (EGCG) all having a signi®cantly protective effect (Negre- A1000 autoinjector and a TSP UV1000 detector set at Salvayre et al, 1991). 280 nm. The mobile phase used was acetic acid/metha- Little information is available with regard to the absorption nol/dimethylformamide and water (1:10:35:154) with a or metabolism of ¯avonoids. In vivo studies to determine how ¯ow rate of 1 ml/min. effectively the polyphenols are absorbed from tea suggested a Catechin, epicatechin, epicatechin gallate, epigallocate- peak in plasma polyphenol concentration 60 min after inges- chin and epigallocatechin gallate were all made up to a tion (Takahama, 1985). These ®ndings suggest that signi®- concentration of 500 mg/ml and stored at 4C. Calibration cant polyphenol absorption may occur following tea curves ranging from 10±50 mg/ml were prepared for all the ingestion and point to a possible role of tea polyphenols catechins. Samples were prepared by infusing tea bags for in protecting LDL against oxidative modi®cation and con- 4 min in 300 mls of boiling water. The aqueous tea extract sequently reducing atherogenesis. In view of this, the aim was then ®ltered through a 0.2 mm supor acrodisc syringe of the present study was to investigate the in vivo and in ®lter, 50% methanol was added and the solution was vitro effects of tea on the oxidative modi®cation of LDL. analysed by HPLC. Quercetin, kaempferol and myricetin were quanti®ed using a different technique (Hertog et al, 1992). In short, 15 ml of the aqueous tea extract was Methods hydrolysed with 1.2 M HCl for 2 h at 90C to hydrolyse Chemicals the glycoside. The resulting aglycones were then quanti®ed All chemicals were purchased from Sigma Chemical Co., using the same HPLC system. The mobile phase was unless otherwise stated. All chemicals were > 95% pure ethanol/water (70/30) plus 1% acetic acid, and peaks except myricetin which was > 85%. Catechin, epicatechin, were detected at 375 nm; calibration curves ranging from epicatechin gallate, epigallocatechin and epigallocatechin 1±10 mg/ml were prepared for quercetin, kaempferol and gallate were purchased from Apin Chemicals (Abingdon, myricetin. Inter-assay c.v. was < 10% for all ¯avonoids, Oxon). and intra-assay c.v. < 8%. Total antioxidant capacity assay solutions were purchased from Amersham International Ltd. (England). The LDL Oxidation study was carried out in accordance with the guidelines of Plasma was obtained from heparinised blood samples by the ethics committee of the Queen's University of Belfast. centrifugation at 1200 g for 10 mins at 4C. LDL was isolated from the plasma by rapid ultracentrifugation and Study design oxidised as previously described by McDowell et al (1995). The effects of tea consumption were investigated in two The protein concentration was standardised to 50 mg/l and ways. Firstly, to assess the acute effects of tea consumption, oxidation was catalysed by copper (2 mmol/l) at 37C. ®ve fasting healthy subjects, four males and one female Conjugated diene formation was monitored at aged between 23 and 35 y, ingested 600 mls of black tea l 234 nm. The lag phase prior to the onset of rapid (Marks and Spencer Extra Strong Black tea) prepared as LDL oxidation is seen as a slope with a slow increase in described below, and peripheral venous blood was col- absorbance. The duration of the lag phase is indicative of lected at 0, 30, 60, 90, 120 and 180 min after consumption. the resistance to oxidation of the LDL being examined. Secondly, to assess the effects of chronic tea consumption, Inter-assay c.v. < 5% and intra-assay c.v. < 1%. a randomised crossover trial of tea and coffee consumption was carried out by ten healthy subjects, six males and four In vitro study of the effects of tea on LDL oxidation females aged between 23 and 40 y. On days 1±7 of the Aliquots (10 ml) of black tea (infusion the same as above) study, subjects abstained from drinking tea or coffee. On were freeze-dried. The in vitro antioxidant activity of the day 7 subjects were randomised to drink 5 6 300 mls of tea was then measured by adding increasing concentrations black tea or coffee per day. This was followed by a one of the tea extract to the puri®ed LDL prior to oxidation week `washout' period during which no tea or coffee was (McDowell et al, 1995). A second study was carried out consumed and a ®nal one week period during which with 5 mg/ml freeze dried tea extract being added to the subjects crossed over to the other beverage. Fasting peri- crude LDL and incubated for 2 h at 4C, before puri®cation pheral venous blood samples were taken at the end of each by gel ®ltration and oxidation as described above. one week period. Total antioxidant capacity assay Tea/Coffee preparation The total antioxidant capacity of plasma was assessed using The tea bags (mean 3.3 g) were infused for 4 min in a mug the method described by Whitehead et al (1992). In this of boiling water as suggested by the manufacturer's instruc- method light emission occurs when the chemiluminescent tions (Marks and Spencer Extra Strong Black Tea). Coffee substrate, luminol, is oxidised in the presence of the was made by dissolving one teaspoon (mean 2.1 g) of enhancer, p-iodophenol. The steady emission of light is coffee (Marks and Spencer Decaffeinated Coffee) in one dependent on the production of free radical intermediates mug of boiling water immediately before drinking. Stan- within the reaction mixture. Light emission is, therefore, dard mugs (volume of beverage ingested, 300 mls) were sensitive to interference by chain breaking antioxidants but used throughout the study. will be restored when all such antioxidants have been consumed in the reaction. If the generation of radical HPLC analysis intermediates is constant, then the time period of light The chromatography was carried out using an Ultracarb suppression is directly related to the amount of chain ODS 20 C18 (Phenomenex Ltd. UK) column (4.6 breaking antioxidant present. The assay was calibrated Black tea consumption does not protect LDL GT McAnlis et al 204 against the water-soluble ascorbate and expressed in mmol/l Acute effects of tea consumption ascorbate equivalents. There was no signi®cant change in the susceptibility of the LDL to oxidation over the 3 h period after consumption of black tea (Figure 2). In addition, tea consumption had no Cholesterol and triglycerides signi®cant effect on the total plasma antioxidant capacity Cholesterol and triglycerides were measured using standard (Figure 3). (Kodak Ektachem, Clinical Chemistry Products) enzymatic techniques on a Kodak 750 XRC Ektachem analyser. Chronic effects of tea consumption The four week crossover study in which coffee was used as Results a control against the black tea showed no signi®cant difference in the total plasma antioxidant capacity between HPLC analysis the tea and coffee groups (Figure 4). There was also no The total increased intake of ¯avonoids from the black tea signi®cant difference in the susceptibility of LDL to oxida- was 126.8 Æ 13.5 g per day based on the consumption of tion following coffee or tea consumption (Figure 5). Serum ®ve standard mugs per day (Table 1). lipids, including total cholesterol, triglycerides, LDL cholesterol and HDL cholesterol did not change signi®cantly throughout the study (Figure 6). Effect of tea on LDL oxidation in vitro Black tea extract, when added to the LDL in vitro, increased the resistance of LDL to oxidation in a concen- Discussion tration dependent manner (Figure 1). The lag phase, Epidemiological evidence has shown the intake of ¯avo- increased from 53 min up to 140 min at a concentration noids to be inversely associated with the risk of coronary of 5 mg/ml of the freeze-dried extract. The rate of propaga- heart disease in elderly men. Black tea contributes around tion decreased signi®cantly as the concentration of back tea 60% of the total ¯avonoid intake and recent epidemiologi- extract was increased. Black tea extract, when incubated cal evidence suggests that the consumption of > 4.7 cups of with LDL before puri®cation by gel ®ltration, did not black tea/day may protect against CHD (Keli et al, 1996). protect the LDL from oxidation. The lag phase was 59.4 Æ 4.48 min in the control samples and 63.7 Æ 1.15 min in the samples containing 5 mg/ml freeze-dried tea extract (P > 0.05).

Table 1 Quantities of the main ¯avonoids present in black tea (Marks and Spencer Extra Strong) measured using HPLC. Total increased intake of ¯avonoids from black tea was 126.8 Æ 13.5mg per day. Values expressed as mean Æ s.d. Tea analysis carried out in triplicate.

Tea Flavonoid Concentration (mg/ml)

Epigallocatechin 16.00 Æ 4.21 Catechin 2.08 Æ 0.70 Epicatechin 8.33 Æ 0.98 Epigallocatechin gallate 31.11 Æ 1.73 Epicatechin gallate 17.03 Æ 1.22 Quercetin 4.75 Æ 0.06 Kaempferol 3.51 Æ 0.06 Myricetin 1.71 Æ 0.03 Figure 2 Change in LDL lag time after ingesting 2 mugs (600 mls) of Total 84.52 Æ 8.99 black tea equivalent to 50.7 Æ 5.4 mg total ¯avonoid. Mean of 5 observations Æ s.d.

Figure 1 The effect of increasing concentrations of freeze dried tea Figure 3 Change in total antioxidant capacity of plasma after ingesting extract on the oxidation of LDL in vitro. Experiments carried out in 2 mugs (600 mls) of black tea equivalent to 50.7 Æ 5.4 mg total ¯avonoid. triplicate. Mean of 3 observations Æ s.d. Mean of 5 observations Æ s.d. Black tea consumption does not protect LDL GT McAnlis et al 205 The epidemiological evidence is however not completely clear; a study involving American male health workers (Rimm et al, 1996) found no correlation between ¯avonoid intake and mortality from heart disease and recently the Caerphilly Study (Hertog et al, 1997) found a positive correlation between tea consumption and mortality from heart disease. The object of this study was, therefore, to assess whether the regular consumption of more than 4.7 cups per day of black tea, over a week, caused a rise in plasma antioxidants and/or protected the LDL from oxidation. The commercial tea used throughout all the studies was a rich source of ¯avonoids (Table 1) and during both studies provided a signi®cant increase in estimated average daily intake (average estimated intake 25 mg/d (Hertog et al, 1993b) increased to 126.8 Æ 13.5 mg/d throughout the chronic tea consumption study). The initial in vitro experiment was designed to show whether or not the ¯avonoids from the black tea could protect the LDL from oxidative modi®cation. Several Figure 4 Changes in total antioxidant capacity of plasma after one mechanisms may contribute to the inhibition of LDL weeks consumption of either tea or coffee (5 mugs per day, equivalent oxidation by ¯avonoids, including metal chelation, a to 126.8 Æ 13.5 mg/d total ¯avonoid). Mean of 10 observations Æ s.d. direct chain-breaking antioxidant effect, and recycling of the tocopheryl radical within the LDL particle (Rice- Evans et al, 1996; McAnlis et al, 1997). The addition of increasing concentrations of freeze-dried tea extract had a very highly signi®cant dose dependant effect on the LDL oxidation (Figure 1), demonstrating that the antioxidants present in tea if absorbed in large enough quantities should be able to protect the LDL from oxidation in vivo. The following studies were carried out to see whether or not the tea ¯avonoids are absorbed into the plasma at high enough quantities to cause a rise in total antioxidant capacity and protect the LDL from oxidative modi®cation in vivo. Initially an acute consumption study was carried out to see how quickly the tea ¯avonoids were absorbed into the plasma. After ingestion of 2 mugs (600 mls) of black tea, equivalent to 50.7 Æ 5.4 mg total ¯avonoid, total plasma antioxidant levels (Figure 3) did not change signi®cantly during the 3 h after ingestion, suggesting that tea ¯avonoids made little contribution to plasma antioxidant capacity. Figure 5 Changes in LDL lag times after weeks consumption of either There was also no change in the susceptibility of the tea or coffee (5 mugs per day, equivalent to 126.8 Æ 13.5 mg/d total LDL to oxidative modi®cation over the 3 h after ingestion ¯avonoid). Mean of 10 observations Æ s.d. suggesting the tea ¯avonoids were not present in high enough quantities in the LDL at this time to provide a measurable change in susceptibility to oxidation. This might indicate that ¯avonoids are not absorbed ef®ciently into the plasma after ingestion (Lee et al, 1995). Alterna- tively, the incorporation of ¯avonoids into LDL might require a longer period of time, given that the half life of LDL is 1±2 d. Therefore a chronic tea consumption study was carried out using a quantity of black tea suf®cient to increase total ¯avonoid intake by 126.8 Æ 13.5 mg/d. Coffee was used as a control as it has a negligible ¯avonoid concentration (Hertog et al, 1993a). However, even in this longer term study no signi®cant effect was found on total plasma antioxidant capacity or susceptibility of LDL to oxidation. A further in vitro experiment where tea extract was incubated with LDL which was then passed down a gel ®ltration column showed no signi®cant difference in oxidation parameters. The catechins from the tea were therefore not taken into the LDL particle ef®ciently enough to reduce oxidation. A number of studies (Maramatsu et al, 1986; Figure 6 Change in total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) concentra- Sheriff & Fakhri, 1988) have suggested that increased tions throughout the main study. Mean of 10 observations Æ s.d. ingestion of tea reduced LDL cholesterol concentrations. 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