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European Heart Journal Advance Access published January 9, 2007
Addition of milk prevents vascular protectiveeffects of tea ´lie von Krosigk1, Peter Martus2, Gert Baumann1, 1 Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charite´—Universita¨tsmedizin Berlin, CCM, Charite´platz 1,D-10117 Berlin, Germany and 2 Institut fu ¨r Biometrie und Klinische Epidemiologie, Charite Received 13 September 2006; revised 28 November 2006; accepted 30 November 2006 Aims Experimental and clinical studies indicate that tea exerts protection against cardiovascular diseases. However, a question of much debate is whether addition of milk modifies the biological activi- ties of tea. We studied the vascular effects of tea, with or without milk, in humans and elucidated the impact of individual milk proteins in cell culture experiments, with isolated rat aortic rings and by HPLC Methods and results A total of 16 healthy female volunteers consumed either 500 mL of freshly brewedblack tea, black tea with 10% skimmed milk, or boiled water as control. Flow-mediated dilation (FMD)was measured by high-resolution vascular ultrasound before and 2 h after consumption. Black teasignificantly improved FMD in humans compared with water, whereas addition of milk completely blunted the effects of tea. To support these findings, similar experiments were performed in isolatedrat aortic rings and endothelial cells. Tea induced vasorelaxation in rat aortic rings and increased theactivity of endothelial nitric oxide synthase by phosphorylation of the enzyme in endothelial cells. Alleffects were completely inhibited by the addition of milk to tea. Of the various kinds of milk proteins,the caseins accounted for these inhibiting effects of milk, probably by formation of complexes with teacatechins.
Conclusion Milk counteracts the favourable health effects of tea on vascular function. This finding indicates the need for particular awareness in the interpretation and design of studies comprising nutri-tional flavonoids.
flow-mediated dilation (FMD) of human blood vessels is ahallmark of a functional endothelium, with impairment of Consumption of tea has been inversely associated with FMD representing an early marker of vascular dysfunction.4,6,7 cardiovascular morbidity and mortality.1 A broad body of We address the question whether milk affects the evidence from experimental and clinical studies indicates beneficial effects of tea on endothelial function. We show that tea exerts antioxidative, anti-inflammatory, and vasodi- here that black tea significantly improves endothelial func- lating effects, thereby rendering protection against cardio- tion in humans, whereas the addition of milk completely vascular diseases.2,3 As the worldwide consumption of tea blunts this amelioration. In our in vitro experiments, we is second only to water, the beneficial effects of tea rep- demonstrate that milk caseins account for these inhibiting resent important public health issues. However, up to now, it is not known whether addition of milk to tea, as widelypracticed in the UK, influences these vasoprotective proper-ties. We therefore investigated the effects of tea with and without milk on endothelial function as a sensitive para- meter of vascular wall homeostasis.4 Many pathophysio-logical Postmenopausal women were recruited by press advertisements.
characterized by attenuated production of protective A total of 229 women responded, and interviews were conductedwith these women. Subjects with chronic diseases and known cardi- vasoactive substances in endothelial cells, resulting in a ovascular risk factors such as high blood cholesterol, diabetes, condition known as endothelial dysfunction.5 Undisturbed arterial hypertension, and obesity as well as with certain food pat-terns (to exclude high regular tea consumption and vegetarian life-style) were excluded. A total of 42 women were invited for clinical * Corresponding author. Tel: þ49 30 450 513153; fax: þ49 30 450 513932.
examination. All measures of clinical parameters were required to & The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: [email protected] be within the normal range to allow study inclusion: lipid profile, For western blots, cells were treated as described earlier, washed blood pressure, body mass index, as well as routine internal para- twice with phosphate buffered saline, and lysed in buffer contain- meters such as thyroidea-stimulating hormone and sexual steroids.
ing, in mmol/L, HEPES 20 (pH 7.9), NaCl 100, Na3VO4 1, sodium A total of 16 healthy women (mean age 59.5 + 5) completed the pyrophosphate 4, EDTA 10, phenylmethylsulfonyl fluoride 1, NaF study. None of the participants had taken medication for at least 10, okadaic acid 0.1, and 1% Triton X-100. Total protein (15 mg per 3 months before entering the study. The participants were asked lane) was subjected to sodium dodecylsulfate polyacrylamide gel to refrain from consuming tea 4 weeks before and during the electrophoresis, and membranes were probed with anti-phospho- study. Study subjects were required to make three clinical visits, eNOS (Ser1179) antibody (1:1000) from Cell Signaling. Bands were at least 3 days apart and at the same time of day (i.e. 8 a.m.
visualized by using BCIP (5-bromo-4-chloro-3-indolyl phosphate, after fasting overnight). Each subject consumed 500 mL of boiled p-toluidine salt) and Nitro Blue Tetrazolium (Sigma).
water, freshly brewed black tea (with 10% water to achieve thesame dilution as tea with milk), or black tea with 10% skimmed Vasorelaxation studies in isolated rat aortic rings milk in a crossover study design. Five grams of tea leaves (Darjeelingblack tea, provided by King’s Teagarden, Berlin, Germany) were Thoracic aortas from male Wistar rats were rapidly excised, cleaned brewed for 3 min with 500 mL of boiled water. All participants of connective tissue, and cut into rings 2–3 mm in length for organ- were required to complete all three visits. FMD and nitro-mediated chamber experiments. The rings were then mounted on platinum dilation (NMD) were measured before and 2 h after consumption of hooks in 10 mL jacketed organ baths containing modified Krebs– the beverages. The participants had a standardized breakfast (one Henseleit solution (composition, in mmol/L, NaCl 144, KCl 5.9, croissant) while they drank the beverage. The study was approved CaCl2 1.6, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and D-glucose 11.1) ´ University Hospital Ethics Committee, and partici- and 1 mmol/L diclofenac. Tension was gradually adjusted to 2 g pants provided their written informed consent.
over 1 h. The solution in the bath was maintained at 378C with agas mixture of 5% CO2 and 95% O2. Following equilibration and sub-maximal precontraction with phenylephrine (0.05 mmol/L), relax- ation to cumulative doses (5–50 mL) of black tea was performed ina volume of 10 mL Krebs–Henseleit solution. Preparation of the Endothelial function was measured by high-resolution vascular ultra- various tea beverages was as described earlier, with the exception sound (Siemens Sonoline Antares), with use of a 13 MHz linear array that 300 mg/mL of individual milk proteins were used. Selected transducer. Endothelium-dependent FMD was assessed by measuring studies were conducted in rings treated with the NOS inhibitor the change in the brachial artery diameter during reactive hyperaemia N-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L) before phenyl- after cuff occlusion of the forearm for 5 min, according to established ephrine exposure. Vasorelaxation is expressed as percentage of guidelines.8 The nature of the stimulus and conditions for the measure- ment of FMD were chosen to obtain a nitric oxide-dependent responseafter a brief period of shear stress in the brachial artery.9,10 Changes in Concentrations of individual tea compounds the arterial diameter were measured every 15 s for up to 2 min. FMDwas defined as the maximum percentage change in diameter compared Tea was prepared as described in study protocols. The concen- with baseline measurement. Endothelium-independent vasodilation trations of individual tea substances in brewed tea, in the super- (NMD) was measured after sublingual application of nitroglycerin natant of tea with or without addition of 10% milk after spray (0.4 mg). The increase in diameter of the brachial artery was centrifugation of the beverage at 13 000 g for 20 min, were deter- measured from 1–6 min, and NMD was defined as the maximum percen- mined as described with slight modifications.12 In brief, tea tage change in diameter compared with baseline measurement.
samples were diluted with 10% acetonitrile containing 500 mg/mL Ultrasound images were digitized online and saved. Analyses of dia- EDTA and ascorbic acid. The samples were analysed by HPLC. The meter changes were conducted offline (Tom Tec Imaging Systems) by HPLC detection system consisted of an Agilent 1100 (Agilent two different investigators blinded to subject treatment.
Technology, San Diego) with a binary pump, thermostatted autosam-pler, column oven, photodiode array detector, and a data system withAgilent 1100 ChemStation software. The column was eluted at 358C Endothelial nitric oxide synthase activity with a binary gradient of 100% solution A (9% acetonitrile, 2% acetic acid, containing 20 mg/mL EDTA) for 10 min, 68% solution Aand 32% of solution B (80% acetonitrile, 2% acetic acid, containing Bovine aortic endothelial cells (BAEC) were maintained in EGM-MV 20 mg/mL EDTA) for 10 min at a flow rate of 1.0 mL/min. The (endothelial cell growth medium), supplemented with 5% fetal eluent was monitored at 278 nm. The signals were verified using UV bovine serum, 10 pg/mL endothelial growth factor, 1 mg/mL hydro- spectra (dioden array detector) and comparisons of the retention cortisone, 12 mg/mL bovine brain extract, and 0.1% gentamicin. For times with reference compounds. Quantification was carried out experiments, cells in 6 cm dishes were incubated for 30 min in using the relative response factor (RRF) concept of ISO 14505:2.
1.5 mL HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]buffer, pH 7.4, containing, in mmol/L, NaCl 145, KCl 5, MgSO4 1, HEPES-Na 10, glucose 10, and CaCl2 1, followed by treatment with100 mL of black tea for 15 min. Tea and tea with milk were prepared A two-step testing procedure was performed for the comparison of as described earlier. Individual milk proteins were added to tea at the three beverages: water, black tea, and tea with milk. In cases of 900 mg/mL. Endothelial nitric oxide synthase (eNOS) activity was overall significance, pairwise tests were performed for the three assessed by formation of L-[3H]citrulline from L-[3H]arginine after possible comparisons between beverages. For three conditions and separation of the amino acids by cation-exchange chromatography, overall significance, no further adjustment for multiple testing as described.11 Briefly, stimulation was initiated by addition of tea was necessary. Since time points and beverages were not balanced, beverages, 10 mmol/L cold L-arginine, and 3 mCi/mL L-[3H]arginine.
all comparisons between beverages were adjusted for a time effect After 15 min, the reaction was terminated with ice-cold stop solution by use of a linear model. The dependent structure of the data was containing 5 mmol/L L-arginine and 4 mmol/L EDTA. Cells were taken into account by use of generalized estimating equations. No denatured with 96% ethanol, and after evaporation, the soluble time effects or time vs. treatment interactions were found. All cellular components were extracted with 20 mmol/L HEPES-Na, pH statistical tests were two-sided (level of significance 0.05). The 5.5. L-[3H]citrulline was separated from L-[3H]arginine by Dowex statistical analysis was performed using SPSS, Release 12.0.1, and chromatography, and L-[3H]citrulline formation was quantified by SAS, Release 9.1. All values are expressed as mean + SD in the table and mean + SEM in the figures.
Measuring of eNOS activity in endothelial cells FMD is evoked by nitric oxide generated by eNOS during ashort period of shear stress.13,14 We therefore measured Baseline characteristics of the study group are summarized generation of nitric oxide in response to tea with or in Table 1. Endothelial function was assessed by measuring without milk in cell culture experiments. Tea increased FMD of the forearm brachial artery in 16 healthy females, eNOS activity to more than 400% in BAEC. Addition of 10% before and 2 h after ingestion of 500 mL water (control), milk to tea completely eliminated the rise in eNOS activity black tea alone, or black tea with 10% milk in a crossover (Figure 2, upper panel). To identify the milk compounds study. Whereas tea significantly increased FMD compared responsible for the inhibiting effects, we performed exper- with control, this effect was completely prevented by inges- iments with single milk proteins. The major proteins in tion of tea with milk (Figure 1A). In contrast, endothelial- milk are a-casein (13 mg/mL), b-casein (9.3 mg/mL), independent vasodilation (NMD) was not affected by k-casein (3.3 mg/mL), a-lactalbumin (1.2 mg/mL), b-lacto- addition of milk to tea (Figure 1B).
globulin (3.2 mg/mL), and serum albumin (0.4 mg/mL).15Individual milk proteins were added to tea at 900 mg/mLto avoid the influence of different amounts of protein perse. This amount corresponds to 9 mg/mL of protein concen- Baseline characteristics of the study population tration in the milk, after 10% of milk is added to tea. Allthree caseins blunted eNOS activity to an extent similar to milk, whereas equal amounts of bovine serum albumin (BSA), a-lactalbumin, and b-lactoglobulin had little effects (Figure 2, upper panel). The activity of eNOS is regulated by phosphorylation of the enzyme.16 Correspondingly, milk and single milk caseins blocked tea-induced eNOS phos- phorylation at Ser1179, whereas the other three milk proteins were without effect (Figure 2, lower panel).
To extend these findings to a functional model, we deter-mined vasodilation in isolated rat aortic rings. Addition oftea cumulatively relaxed precontracted rat aortic rings,which Similarly, addition of 10% milk completely inhibitedtea-induced relaxation (Figure 3A). In corroboration of ourdata in endothelial cells, addition of b-casein to tea Changes in FMD and NMD after consumption of tea with and without milk in humans. (A) A total of 16 volunteers consumed water, blacktea, or black tea with 10% milk, and changes in FMD were measured. FMDsignificantly (P , 0.01 compared with water) increased after consumption Effect of milk and individual milk proteins on tea-induced nitric of black tea. Addition of milk to tea suppressed the vasodilatory effects of oxide production in endothelial cells. Cells were treated for 15 min with tea (P , 0.01 compared with tea alone). (B) Beverages were consumed as the indicated beverages, and eNOS activity was measured (upper panel).
in (A), and NMD was measured after sublingual application of nitroglycerin Western blot showing the phosphorylation of eNOS (lower panel) was per- spray (0.4 mg). FMD and NMD were defined as the maximum percentage formed with an antibody specific to the eNOS Ser1179 phosphorylation site.
change in diameter compared with baseline measurement.
The western blot is representative from three independent experiments.
inhibited relaxation of aortic rings, whereas BSA had little markedly decreased the concentrations of various catechins in tea, whereas the contents, for example, of caffeine,gallic acid, and others were not affected (Table 2).
Tea polyphenols have been shown to interact with pro-teins.17 In an attempt to elucidate the underlying mechan- ism for the diminished biological activities of tea after The most striking finding of our study is that addition of milk addition of milk, we measured the concentrations of to black tea completely prevents the biological activity of tea various tea compounds, including the catechins, in our tea in terms of improvement of endothelial function. Our results preparations. The concentrations of single tea compounds thus provide a possible explanation for the lack of beneficial were determined before, and in the supernatants, after cen- effects of tea on the risk of heart disease in the UK, where trifugation of the tea beverages at 13 000 g for 20 min.
milk is usually added to tea.18 To follow the common practice Contents of individual tea compounds without addition of of drinking tea in the UK, we added 10% milk to our black tea milk did not significantly change upon centrifugation.
preparations. The well-established health benefits of tea, However, addition of 10% milk to tea selectively and as described by numerous studies,1–3 are mainly attributedto various flavonoids, especially catechins.19 Previously,we epigallocatechin-3-gallate (EGCG), induces eNOS activationin endothelial cells and leads to vasorelaxation in rat aorticrings.11 In the present study, we found a particular, selectivedecrease in the concentrations of a number of catechins aftercentrifugation of freshly brewed tea with milk compared withtea without milk, which suggests complex formation between catechins and milk proteins. Concentrations of other teacompounds were not affected by addition of milk. It was pre-viously reported that polyphenols can bind to proteins,20,21and interaction between flavanoids and proteins affects their antioxidant capacity in vitro.22 There is some evidencethat proline-rich proteins such as caseins.23 A very recent studydemonstrated the non-covalent cross-linking of EGCG bycaseins, emphasizing the interaction of tea catechins with Interactions of food-derived flavonoids with milk proteins may impede their physiological effects. An in vitro gastro-intestinal model to simulate the conditions in the humandigestive tract demonstrated that addition of milk to teainhibited its antimutagenic activity.25 Tea possesses strongantioxidant properties, in vitro and in vivo, that areaffected by addition of milk.26,27 In analogy, consumptionof dark chocolate that contains epicatechin, but not milkchocolate or dark chocolate with extra milk, increased theantioxidant capacity of human blood plasma.28 However, itshould be noted that other studies failed to establish aneffect of milk on antioxidant properties of tea.29,30 The Tea-induced nitric oxide-dependent vasorelaxation in rat aortic reasons for these discrepant findings are largely unknown rings after addition of milk and individual milk proteins to tea. (A)Precontracted rat aortic rings were treated with water (control), tea alone, but may be attributed to different physiological/experimen- or tea with 10% milk, and vasorelaxation was determined. L-NAME indicates tal endpoints (e.g. in vitro and in vivo, and assays for NOS-inhibitor. Vasorelaxation is expressed as percent of contraction. (B) measurements of antioxidant activities).
Rat aortic rings were treated with water (control), tea alone, tea with In conclusion, milk may counteract the favourable health b-casein, or tea with BSA, and vasorelaxation was determined as in (A).
effects of tea on vascular function. The finding that the Data are mean + SEM from the indicated numbers of experiments.
Concentrations of tea ingredients (mmol/L) TB, theobromine; TG, theogallin; EC, epicatechin; EGC, epigallocatechin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate.
aTea was centrifuged at 13 000 g for 20 min.
tea-induced improvement of vascular function in humans is activates endothelial nitric oxide synthase by a phosphatidylinositol- completely attenuated after addition of milk may have 3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependentpathway and leads to endothelial-dependent vasorelaxation. J Biol broad implications on the mode of tea preparation and con- sumption. In addition, it indicates that caution is warranted 12. International Organization for Standardization. Determination of sub- in the design of studies involving nutritional flavonoids.
stances characteristic of green and black tea. Part 2. Content ofcatechins in green tea. Method using high-performance liquid chromato-graphy. ISO 14502-2-2005.
13. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, Luscher TF. Nitric oxide is responsible for flow-dependent dilatation of There was no funding or support for this study. We are grateful to human peripheral conduit arteries in vivo. Circulation 1995;91: ¨ft, Angelika Vietzke and Wanda Michaelis for their excellent technical assistance.
14. Meredith IT, Currie KE, Anderson TJ, Roddy MA, Ganz P, Creager MA.
Conflict of interest: none declared.
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