Green tea extract only affects markers of oxidative status postprandiallylasting antioxidant effect of flavonoid-free diet

J F Young1 L O Dragsted2dagger J Haraldsdottir1 B Daneshvar2 M A Kall3 S Loft4 L Nilsson1S E Nielsen2 B Mayer5 L H Skibsted6 T Huynh-Ba7 A Hermetter5 and B Sandstrom1

1Research Department of Human Nutrition Royal Veterinary and Agricultural University Frederiksberg Denmark2Institute of Food Safety and Toxicology Danish Veterinary and Food Administration Soslashborg Denmark

3Institute of Food Chemistry and Nutrition Danish Veterinary and Food Administration Soslashborg Denmark4Institute of Public Health University of Copenhagen Copenhagen Denmark

5Institute of Biochemistry and Food Chemistry Technical University of Graz Graz Austria6Food Chemistry Department of Dairy and Food Science Royal Veterinary and Agricultural University Frederiksberg

Denmark7Nestle Research Centre Nestec Ltd 1000 Lausanne 26 Switzerland

(Received 30 November 2000 ndash Revised 7 September 2001 ndash Accepted 8 December 2001)

Epidemiological studies suggest that foods rich in flavonoids might reduce the risk of cardio-vascular disease and cancer The objective of the present study was to investigate the effectof green tea extract (GTE) used as a food antioxidant on markers of oxidative status after diet-ary depletion of flavonoids and catechins The study was designed as a 2 pound 3 weeks blindedhuman cross-over intervention study (eight smokers eight non-smokers) with GTE correspond-ing to a daily intake of 18middot6 mg catechinsd The GTE was incorporated into meat patties andconsumed with a strictly controlled diet otherwise low in flavonoids GTE interventionincreased plasma antioxidant capacity from 1middot35 to 1middot56 (P0middot02) in postprandially collectedplasma most prominently in smokers The intervention did not significantly affect markersin fasting blood samples including plasma or haemoglobin protein oxidation plasma oxidationlagtime or activities of the erythrocyte superoxide dismutase glutathione peroxidase glutathionereductase and catalase Neither were fasting plasma triacylglycerol cholesterol a-tocopherolretinol b-carotene or ascorbic acid affected by intervention Urinary 8-oxo-deoxyguanosineexcretion was also unaffected Catechins from the extract were excreted into urine with ahalf-life of less than 2 h in accordance with the short-term effects on plasma antioxidantcapacity Since no long-term effects of GTE were observed the study essentially served as afruit and vegetables depletion study The overall effect of the 10-week period without dietaryfruits and vegetables was a decrease in oxidative damage to DNA blood proteins and plasmalipids concomitantly with marked changes in antioxidative defence

Green tea extract intervention Dietary depletion of fruits and vegetables Biomarkers ofoxidative damage Biomarkers of antioxidative defence Protein oxidation

Consumption of fruit and vegetables is widely encouragedbecause of the epidemiological indications of a relationshipbetween increased intake and reduced risk of cancer andcardiovascular diseases A major group of antioxidativecompounds in fruit and vegetables the polyphenols hasbeen suggested to contribute to these beneficial effects

(Hertog et al 1993a 1994 1995) In a Dutch populationtea was found to be the main dietary source of five selectedflavonoids (Hertog et al 1993b) However the beneficialeffects of tea on cancer and cardiovascular diseasesremain controversial (Goldbohm et al 1996 Katan1997 Kohlmeier et al 1997)

The study has been carried out with financial support in part from a Danish Food Technology grant (FOslashTEK2 lsquoAntioxidants from plantsrsquo) and in part from

the Commission of the European Communities Agriculture and Fisheries (FAIR) specific RTD programme CT 95-0158 lsquoNatural Antioxidants from

Foodsrsquo It does not necessarily reflect its views and in no way anticipates the Commissionrsquos future policy in this area

dagger Corresponding author L O Dragsted present address Moslashrkhoslashj Bygade 19 DK-2860 Soslashborg Denmark fax +45 33 95 60 01 email lodfdirdk

Abbreviations AAS 2-amino-adipic semialdehyde EC epicatechin EGC epigallocatechin GTE green tea extract 8-oxo-dG 8-oxo-deoxyguanosine

British Journal of Nutrition (2002) 87 343ndash355 DOI 101079BJN2002523q The Authors 2002

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As much as 26ndash30 of the DM of fresh tea leaves mayconsist of flavonoids (Lin et al 1998) among which thecatechins are the most abundant During the manufactureof black tea (a fermentation process) catechins are pro-gressively oxidised by polyphenol oxidase to form polymer-isation products (Lin et al 1998) The catechins in tea havea potent antioxidative capacity in model systems includingradical scavenging (Yen amp Chen 1995 Gardner et al1998) and provide protection against oxidative changesin plasma (Ishikawa et al 1997 Lotito amp Fraga 1998)and LDL (Ishikawa et al 1997 Zhang et al 1997)Some studies indicate that green tea is a more potent anti-oxidant than black tea in in vitro model systems (Yen ampChen 1995 Gardner et al 1998) probably due to itshigher content of catechins and lack of polymerisation pro-ducts (Lin et al 1998)

If catechins can be incorporated into plasma they may beexpected to affect the antioxidative status Animal studieson pure catechins in rats have shown that these compoundsare absorbed and transported to the tissues (Nakagawa ampMiyazawa 1997) and experiments with human subjectshave shown that catechins are found in plasma (Nakagawaet al 1997) and excreted into the urine (Das 1971) Con-sumption of both green and black tea was found to increaseplasma antioxidant capacity within 30ndash50 min (Serafiniet al 1996) but over a longer time scale (4 weeks) noeffects on antioxidative parameters in blood were observedapart from a slight increase in antioxidative activity inplasma after green tea consumption (van het Hof et al1997)

If tea consumption has an effect on the antioxidativesystem an extract of tea could be expected to exhibit asimilar effect The present study investigated the effectof a green tea extract (GTE) on markers of oxidativestatus in blood and urine in a blinded double cross-overstudy with complete control of all dietary intakes TheGTE was incorporated into meat patties at a concentrationof 1000 mgkg which has been demonstrated to protect

against oxidative changes in dried chicken meat (Nissenet al 2000)

In order to study individuals with different levels of oxi-dative stress the study included both smokers and non-smo-kers

Methods

Subjects

Sixteen men volunteered for the study including eight non-smokers and eight smokers (10ndash15 cigarettesd) Averageage was 23 (range 20ndash31) years and average BMI was22middot7 (range 19middot5ndash25middot7) kgm2 None of the subjects hadany chronic illness Subjects received oral and writteninformation about the study and gave their written con-sent The study was approved by the local ResearchEthics Committee of Copenhagen and Frederiksberg (Jour-nal number KF01-34297)

Study design and diet

The study design is illustrated in Fig 1 It was a double-blind randomised 2 pound 3 week cross-over with 2 weekswash-out before each intervention Subjects were dividedinto groups A and B with four smokers and four non-smo-kers in each group A standardised diet with a low flavo-noid content including 200 g meat patties10 MJ wasserved for the 2 pound 3 week study periods For one of the3-week periods the subjects consumed GTE incorporatedinto meat patties at a concentration of 1000 mgkgDuring the wash-out weeks the subjects excluded flavo-noid-containing foods from their diet Tea wine spiceschocolate cocoa and all products containing fruits berriesand vegetables except potatoes and carrots were excludedfrom their diet A list of allowed dietary items was distrib-uted to the participants which included all meats fishshellfish grain products (including whole grain) such as

Fig 1 Study design (a) Depiction of the overall design over the entire 13-week period including two sequential periods each with an initial2-weeks wash-out followed by a 3-weeks cross-over intervention and a final control period (b) Depiction of the exact sampling times for bloodand urine during each of periods 1 and 2 (+) Intervention with green tea extract (2) sample collection

J F Young et al344

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breads and pasta eggs potatoes carrots freeze driedcoffee mineral water and tap water The freeze driedcoffee was analysed by HPLC and found to be free fromcatechins The diet during intervention consisted of sevenmenus consumed each week from Monday to Sunday(Table 1) The macronutrient and selected micronutrientcontents were calculated using a computer program (Dan-kost) which is based on the Danish Veterinary and FoodAdministration composition database (Moslashller 1989)(Table 2) Each subjectrsquos energy requirement was esti-mated from body weight and degree of physical activityAll meals were prepared at the department in individualportions according to energy requirement One of thedaily main meals was taken at the department while theremaining (cold) meals were provided daily for intake athome Subjects were instructed to return any leftovers tothe department for weighing and subtraction from planned

intake and were asked daily about compliance Freeze-dried coffee powder was provided for preparation ofcoffee Coffee intake was recorded and varied betweensubjects but was held constant over the 6 weeks No foodor drink other than that provided from the departmentwas allowed Fasting blood samples were collectedduring each period on the morning of day 0 1 21 and22 thus reflecting the preceding 24 h ie day 0 and 1samples reflect baseline concentrations before intervention(days 21 and 0 respectively) and days 21 and 22 samplesreflect the last 2 d of each intervention period (days 20 and21 respectively) (see Fig 1(b)) In addition one bloodsample was taken 25 h after the hot meal at midday onday 21 of each period The samples will be referred to inthe text according to the 24 h they are reflecting withineach period A final blood sample was collected afterweek 13 when the subjects had been on their habitualdiet for 3 weeks after the last intervention This samplewas collected to represent the participantrsquos natural back-ground Twenty-four hour urine samples were collectedat baseline ie the 2 d before the intervention (days 21and 0) and during the last 2 d of intervention (days 20and 21) Body weight was determined before and aftereach intervention period

Green tea extract and pork meat patties

Extract of Chinese green tea (Licosa-PThe chinois) wasproduced using the process described in a patent (Aesch-bach amp Rossi 1994) The extract was a hydrophilic mech-anical extract obtained by pressing the tea on a hydrauliclaboratory press using propylene glycol as the carrierThe total polyphenol content of the tea extract was

Table 2 The macronutrient and selected micronutrient content ofthe experimental diet (per 10 MJ)

Content

Nutrient per 10 MJ Energy

Protein (g) 91 16Fat (g) 99 37Carbohydrate (g) 277 47Vitamin E (tocopherol equivalents) 2middot9Vitamin C (mg)dagger 12middot6Vitamin A (retinol equivalents) 3180b-Carotene (mg)dagger 11middot2a-Carotene (mg)dagger 3middot6Lutein (mg)dagger 0middot49

For details of diet and procedures see Table 1 and p 344dagger Analysed by HPLC the remaining nutrients were calculated

Table 1 Composition of the experimental diet (gd) at an energy intake of 10 MJd

Component Menu 1 Menu 2 Menu 3 Menu 4 Menu 5 Menu 6 Menu 7

Breakfast Bread roll with carrots 77 77 77 77 77 77 77Cheese 25 25 25 25 25 25 25Sour milk 170 170 170 170 170 170 170Rye breadcrumbs 30 30 30 30 30 50 50

Lunch Rye bread 100 100 100 100 100 100 100Butter 6 6 6 6 6 6 6Meat patties 50 50 50 50 50 50 50Tuna salad 34 34Potatoes 50 50Ham salad 44Roast beef 30 30Carrot salad 100 100 100 100 100 100 100

Dinner Meat patties 150 150 150 150 150 150 150Mashed potatoes 250 250Pasta 70Potatoes 200 200Rice 70 70Stock 150 150Brown sauce 153 153Cheese sauce 160Chicken sauce 180 180

Snack Bread roll with carrots 77 77 77 77 77 77 77Butter 6 6 6 6 6Sponge cake 100 100 100Carrot cake 100 100 100 100

Milk 250 250 250 250 250 250 250

Green tea and markers of oxidative status 345

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117middot3 mg gallic acid equivalentg extract Catechins con-tent as determined by HPLC are shown in Table 3

GTE was incorporated at 1000 mgkg in pork meat pat-ties which were produced on a pilot scale using pork meat(25 fat) water and salt as ingredients Reference meatpatties containing no added GTE were also producedThe process consisted in tempering of the meat (at228C) mixing all the ingredients grinding (3 mm) pattyforming (approximately 40 g each patty) and frying (beltfryer no use of additional fat or oil) Meat patties werefrozen at 2258C packed under vacuum in plastic pouchesand stored at 2258C until use A microbiological controlwas performed on the finished products

Collection of urine and determination of 8-oxo-deoxyguanosine

Twenty-four hour urine samples were collected in 2middot5 lplastic bottles Fifty ml of 1 moll HCl and 10 ml of 10 ascorbic acid were added to each 2middot5 l bottle to increasestability during collection Urines were weighed densitydetermined pH adjusted to 3ndash4 with 1 moll HCl andstored at 280 8C until analysis Urinary concentrations of8-oxo-deoxyguanosine (8-oxo-dG) a marker of total oxi-datively damaged DNA in the body were determined bycolumn-switching HPLC with electrochemical detectionas described previously (Loft amp Poulsen 1999)

Collection of blood samples and separation of erythrocytesand plasma

Venous blood samples were taken in the morning after atleast 12 h of fasting An additional postprandial sample wasdrawn on day 21 of each intervention 25 h after consump-tion of the cooked meal Blood samples were taken inEDTA-coated tubes after supine resting for 10 min All sub-jects abstained from alcohol for at least 24 h before bloodsampling and the smokers abstained from smoking for atleast 8 h before fasting blood samples and 45 h beforesampling of non-fasting blood samples Subjects wereinstructed to avoid heavy physical activity for 36 h beforeblood sampling Plasma samples for 2-amino-adipic semi-aldehyde (AAS) determination were centrifuged at 1500 gfor 10 min at room temperature Erythrocytes werewashed three times with 3 vol 0middot9 NaCl resuspendedin 1 vol ultrapure (resistance 18 Mohm) water forlysis and then stored at 280 8C until analysis Samples

for a-tocopherol retinol b-carotene antioxidant capacityand vitamin C determination were protected from lightexposure and kept on ice Samples were centrifuged at3000 g for 15 min at 48C and plasma for vitamin C ana-lyses were added to1 volume of 10 meta-phosphoricacid before freezing Plasma was saturated with N2 gasand stored at 280 8C until analysis for antioxidant capacity(maximum 7 d) retinol a-tocopherol and b-carotene(maximum 7 months) cholesterol and triacylglycerol (20months)

Ascorbate plasma protein oxidation and antioxidantenzymes

Plasma and dietary samples were analysed for ascorbate(Kall amp Andersen 1999) and plasma proteins were ana-lysed for oxidised lysine residues (Daneshvar et al 1997)by HPLC as described previously Erythrocyte lysateswere also analysed for protein oxidation products and forthe activities of catalase superoxide dismutase glutathioneperoxidase and glutathione reductase by methods detailedpreviously (Nielsen et al 1999) Inter-series CV was lessthan 10 for all of these analyses

Plasma oxidation lagtime and plasma antioxidant capacity

Oxidation of lipoproteins in unfractionated plasma wascontinuously measured by a fluorescence method asdescribed previously (Hofer et al 1995)

The postprandial samples collected on day 21 of eachperiod were used immediately for determination ofplasma antioxidant activity Fifty ml of plasma (or controls10ml 2 mmoll Trolox +40ml buffer or 50ml buffer) wereadded to 2middot45 ml 1middot33 mmoll methyl linoleate emulsion in5middot0 mmoll oxygen-saturated PBS (pH 7middot4) containing0middot1 Tween 20 and the oxygen consumption was deter-mined as described previously (Joslashrgensen amp Skibsted1993) Antioxidative capacity was estimated by calculatingthe area under oxygen consumption curves and the influ-ence of plasma was expressed as an antioxidative index(I) relative to samples including Trolox

Ioxygen consumption relative to reference including Trolox

frac14 area ethplasmaTHORN=area ethreference including TroloxTHORN

Determination of plasma retinol a-tocopherol b-carotenetriacylglycerol and cholesterol

Plasma retinol a-tocopherol and b-carotene were deter-mined by a contract laboratory (MedindashLab CopenhagenDenmark) using a standard HPLCndashprocedure (Thurnhamet al 1988) Intrandashseries CV was 4 4 and 7 andinterndashseries variation was 5 5 and 10 respectivelyfor retinol a-tocopherol and b-carotene Plasma triacylgly-cerol (kit no 07 36791 Unimate5 Roche) and cholesterol(kit no CHODndashPAP 2016630 Boehringer MannheimMannheim Germany) were determined on a CobasMira thorn (Roche Basel Switzerland) Carotenoids in the

Table 3 Daily intake of some polyphenols from green tea extract(1000 mgkg in the 200 g meat patties served per 10 MJ)

Compound Daily intake (mg10 MJ)

Catechin Not detectedEpicatechin 1middot68Epicatechin gallate 2middot78Epigallocatechin 5middot14Epigallocatechin gallate 9middot04Total phenolicsdagger 23middot5

For details of diet and procedures see Table 1 and pp 344ndash345dagger Total phenolic content was determined by the Folin-Ciocalteu method as

mg gallic acid equivalentg extract Catechins were determined by HPLC

J F Young et al346

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experimental diet were determined as described by Hart ampScott (1995)

Followndashup studies

Two small followndashup studies were performed in order toassess catechin uptake and whether catechins from meatpatties containing GTE were absorbed to the same extentas catechins from GTE given in a waterndashalcohol mixture

In the first study five volunteers consumed an experi-mental diet with a low content of flavonoids as describedin the main study for 4 d On the second day in the morningthey voided a spot urine sample and subsequently ingested2 g GTE in 25 ml waterndashethanol (21) Total urines werecollected 4 24 and 48 h after GTE intake

The second study was a 3 pound 1 d cross-over where threevolunteers consumed a low-catechin diet as described inthe main study for 2 d (day 1ndash2) On days 3ndash5 theywere each day given either 500 mg GTE in 10 ml waterndashethanol (21) 500 mg GTE in meat patties stored at2258C for 12 months (from the main study) or 500 mgGTE mixed into freshly prepared meat patties Urinesamples (24 h) were collected from each volunteer duringdays 2ndash5 The volunteers in these studies were differentfrom those in the main study

The containers for urine collection in both follow-upstudies were prepared as described for the main studyand were additionally filled with Ar Immediately at theend of each collection period the urine samples were satu-rated with Ar and analysed for catechins

HPLC analyses of urine samples green tea extract andinstant coffee for content of epicatechin and

epigallocatechin

The content of epicatechin (EC) and epigallocatechin(EGC) was determined in the GTE in the freeze driedcoffee and in fresh urine samples from the follow-upstudies The assay used for analysis was similar to thosepreviously described for other flavonoids (Nielsen ampDragsted 1998ab) with respect to enzymic hydrolysisand solid phase extraction

Enzymically hydrolysed urine samples GTE or coffeewere suspended in 5 acetonitrile in water before additionof internal standard (10 mg catechinl) and subjected tosolid phase extraction The HPLCndashMS system consistedof a Hewlett Packard (Waldbronn Germany) 1090system with a a MSD 1100 mass spectrometric detectorThe columns used were a Zorbax SB-C8 (4middot6 pound 75 mm3middot5mm) column as column 1 and a Purospher RP-18(4 pound 125 mm 5mm) column with guard cartridge(4 pound 4 mm 5mm) as column 2 (Hewlett Packard)Column temperature was maintained constant at 37 8Cusing a thermostatically controlled column compartmentUv detection was carried out at 280 nm with peakscanning between 190 and 600 nm (2 nm step) The MS-detection was performed by single-ion monitoring usingatmospheric pressure ionisation with electrospray interfacein positive mode The drying gas (N2) was heated to 350 8Cand introduced at a flow-rate of 10middot0 lmin Nebuliser press-ure was set to 30 psig and capillary voltage to 4000 V The

mobile phases used for HPLC were (flow of 0middot4 mlmin)A 1 aqueous formic acid B 100 acetonitrile C100 methanol With the automatic six-port column-switching valve in position 1 the sample was injectedonto column 1 and eluted through the diodearray detectorbypassing column 2 and the MS-detector with a followinglinear gradient of mobile phase B in A (vv) 5 to 15 from 0 to 5 min 15 to 25 from 5 to 14 min Between14middot1ndash15 min 100 B was applied resulting in a short washof column 1 EGC was eluted from column 1 at 7middot5 min Byswitching the column switching valve from position 1 to 2at 7middot2 min and back to position 1 at 7middot8 min this compoundwas eluted onto the second column This procedure wasrepeated between 8middot3ndash8middot8 min and 9middot5ndash10middot0 min for cate-chin (internal standard) and EC respectively From15middot1ndash23 min column 1 was preconditioned with puremobile phase A and after shifting to column 2 at21middot5 min elution of the tea catechins through the MS-detector was initiated using the following gradient ofmobile phase C in A (vv) 24 min 25 C 28ndash37 min32 C 37middot5ndash39 min 100 C 39middot1 min 25 C Afreshly prepared standard solution containing EC EGCand catechin was analysed before and after each run toensure a correct column-switching schedule The EC andEGC detected were corrected for the internal standard(catechin) recovery in each urine sample

Statistics

The statistical analysis of biomarkers in the cross-overstudy was done by paired t tests according to Pocock(1998) Significant period effects were observed forplasma ascorbate 8-oxo-dG in urine and plasma antioxi-dant capacity and corrections were made according toPocock (1998) Plasma oxidation lagtime b-carotene andAAS in erythrocyte lysates were analysed by comparinggroups A and B after the first period using a two-samplet test due to periodndasheffect interactions The effect of smok-ing was analysed by two-sample t tests comparing smo-kers and non-smokers using the baseline samplescollected after run-in The effect of intervention on smo-kers and non-smokers was analysed separately using thesame procedure as outlined above for the whole studyMultivariate ANOVA with a repeated statement for theday on intervention was performed for all biomarkers toadditionally test for time trends during the whole interven-tion trial using the SAS statistical package (SAS 612 SASInstitute Inc Cary NC) A P-value of 0middot05 was con-sidered statistically significant

Results

Subjects baseline comparisons and influence of smokingstatus

All subjects were weight-stable ie body weight changedless than 1 kg during the total experimental period foreach subject Group A and B differed with respect toplasma oxidation lagtime at week 2 There were no otherdifferences between the groups after run-in Smoking didnot influence any of the markers after the 2 week run-in

Green tea and markers of oxidative status 347

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period but in the sample representing habitual diet (13weeks) plasma ascorbate was significantly lower insmokers

Biomarkers of oxidative damage and antioxidant defence

Effects on blood and urine parameters after 3 weeks ofexperimental diet with or without addition of tea extractto a low-flavonoid diet are shown in Tables 4 and 5There was no significant effect of intervention with teaextract on plasma protein oxidation (plasma AAS) orplasma oxidation lagtime analysed as delta change foreach individual from beginning to end of period 1

In erythrocytes no effect of GTE intervention wasobserved on markers of protein oxidation (AAS and g-glu-

tamyl semialdehyde in erythrocytelysates) and theexcretion of 8-oxo-dG in urine was not affected either

There was a significant decrease in plasma AAS and ing-glutamyl semialdehyde in erythrocyte lysates when thesamples representing the participantsrsquo habitual diet (week13) were compared with the samples collected at the endof the periods with restricted diet The downward trendsin plasma AAS (Fig 2(a)) and in AAS in erythrocytelysates during the period with restricted diets were also sig-nificant as determined by repeated measures ANOVAPlasma oxidation lagtimes increased significantly in eachstrictly controlled period and also overall during the 10weeks with a restricted diet as compared with habitualdiet (Table 6 and Fig 2(c)) The urinary excretion of8-oxo-dG decreased significantly during each of the two

Table 5 Comparison of plasma lipid status markers and plasma antioxidant vitamins at the end of3 weeks intake of experimental diet without or with green tea extract (1000 mgkg) in meat patties(200 g10 MJ per d) in sixteen subjects (each represented by the average of blood samples from 2

successive days)

(Mean values with their standard deviations)

Basal diet Tea extract Paired difference

Biomarker Mean SD Mean SD Mean 95 CI

Triacylglycerol (mmoll) 1middot02 0middot23 1middot04 0middot22 0middot019 20middot036 0middot074Cholesterol (mmoll) 4middot25 1middot20 4middot21 1middot03 20middot047 20middot20 0middot11Retinol (mmoll) 1middot73 0middot31 1middot75 0middot25 0middot019 20middot082 0middot121Vitamin C (mgl) 2middot36 0middot88 2middot50 1middot06 0middot14dagger 20middot52 0middot80b-Carotene (mmoll) 1middot43Dagger 0middot58 2middot07Dagger 0middot60 Daggersecta-Tocopherol (mmoll) 17middot6 4middot2 17middot4 4middot2 20middot19 20middot76 0middot38

For details of diet and procedures see Table 1 and p 344dagger There was a significant period effect for this marker and corrected statistical analysis was performed according

to Pocock (1998)Dagger There was significant periodndasheffect interaction for this marker so statistical analysis was performed between

groups in period Isect There was a significant effect of green tea extract intervention (P0middot05)

Table 4 Comparison of oxidative status markers in blood or urine at the end of 3 weeks intake of the experimental diet without andwith green tea extract (1000 mgkg) in meat patties (200 g10 MJ per d) in sixteen subjects (average of blood samples from 2

successive days)

(Mean values with their standard deviations)

Basal diet Tea extract Paired difference

Biomarker Mean SD Mean SD Mean 95 CI

Change in oxidation lagtime (min)dagger 32middot6daggerDagger 26middot0 42middot0daggerDagger 30middot0 daggerDagger8-Oxo-deoxyguanosine (pmol24 h per kg body wt) 257 79 261 81 4middot3sect 231middot4 39middot9Plasma antioxidant capacity (Iref

k) 1middot35 0middot25 1middot56 0middot34 0middot21sect 0middot021 0middot41AASpl (nmolmg protein) 18middot8 3middot8 19middot5 3middot7 0middot75 21middot78 3middot28AAShb (nmolmg protein) 36middot2Dagger 4middot9 36middot4Dagger 4middot5 DaggerGGShb (nmolmg protein) 21middot9 4middot0 21middot5 3middot7 20middot39 22middot25 1middot46Gpx (Ug Hb) 37middot9 9middot8 37middot4 10middot8 20middot48 25middot58 4middot61Glutathione reductase (Ug Hb) 7middot05 1middot2 7middot06 1middot2 0middot01 20middot13 0middot16Superoxide dismutase (Ug Hb) 1009 149 1035 139 25middot9 23middot7 55middot5Catalase (Ug Hb) 12middot0 2middot1 11middot4 1middot2 20middot65 21middot54 0middot24

AASpl Plasma 2-amino-adipic semialdehyde AAShb 2-amino-adipic semialdehyde in erythrocyte lysates GGShb g-glutamyl semialdehyde in erythro-cyte lysates Gpx glutathione peroxidase Hb haemoglobin

For details of diet and procedures see Table 1 and pp 344ndash345dagger Due to a significant difference between the groups at baseline the change in plasma oxidation lagtime from start to end of period I is shownDagger There was significant periodndasheffect interaction for this marker so statistical analysis was performed between groups in period I onlysect There was a significant period effect for this marker and statistical analysis was performed according to Pocock (1998)k Iref antioxidative capacity relative to reference including Trolox (see p 346)There was a significant effect of green tea extract intervention (P0middot02)

J F Young et al348

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Table 6 Overall changes in biomarkers of oxidative status and in blood lipids from start to end of periods with controlled diets

(Mean values with their standard deviations)

Restricted diet period I Restricted diet period I Habitual diet

Start (week 2) End (week 5) Start (week 7) End (week 10) End (week 13)

Biomarker Mean SD Mean SD Mean SD Mean SD Mean SD

Glutathione peroxidase (Ug Hb) 40middot3 9middot0 38middot7 13middot8 40middot8 14middot8 41middot9 9middot4 35middot6Dagger 12middot1Glutathione reductase (Ug Hb) 7middot20 1middot11 7middot05 1middot12 7middot01 1middot08 7middot06 1middot21 7middot50DaggerDagger 0middot83Superoxide dismutase (Ug Hb) 1035 177 1011 158 1019 154 1034 128 1098Dagger 105Catalase (Ug Hb) 11middot8 1middot8 11middot7 2middot3 12middot1 2middot6 11middot4 1middot4 9middot92DaggerDagger 1middot16AASpl (nmolmg protein) 21middot6 3middot4 20middot0 4middot2 18middot6 4middot4 18middot3 3middot0 29middot0DaggerDaggerDagger 8middot3AAShb (nmolmg protein) 37middot4 5middot1 35middot9 4middot9 36middot1 5middot3 36middot6 4middot5 38middot9 4middot9GGShb (nmolmg protein) 23middot3 3middot7 22middot0 3middot6 20middot8 3middot3 21middot5 4middot0 23middot4Dagger 3middot2Plasma lagtime for lipid marker (min) 373 46 457daggerdaggerdagger 56 430 59 468daggerdaggerdagger 65 203DaggerDaggerDagger 31Plasma antioxidant capacity (Iref) ND 1middot35 0middot21 ND 1middot57sect 0middot22 ND8-Oxo-dG (pmol24 h per kg body wt) 337 131 277daggerdaggerdagger 86 262 90 235 69 NDPlasma cholesterol (mmoll) 4middot13 0middot98 4middot24 1middot04 4middot39 1middot08 4middot22 1middot20 4middot15 1middot03Plasma triacylglycerol (mmoll) 1middot14 0middot24 1middot03 0middot25 1middot31 1middot41 1middot03daggerdagger 0middot19 1middot30DaggerDaggerDagger 0middot40Retinol (mmoll) 1middot88 0middot36 1middot75 0middot26 2middot06 0middot29 1middot73daggerdaggerdagger 0middot30 2middot01DaggerDaggerDagger 0middot32Vitamin C (mmoll) 43middot5 18middot4 16middot7daggerdaggerdagger 5middot2 14middot1 6middot5 10middot0 3middot46 78middot4DaggerDaggerDagger 25middot6b-Carotene (mmoll) 0middot18 0middot10 1middot75daggerdaggerdagger 0middot56 0middot55 0middot26 1middot76daggerdaggerdagger 0middot61 0middot45DaggerDaggerDagger 0middot17a-Tocopherol (mmoll) 18middot5 4middot1 17middot7 4middot1 19middot6 4middot5 17middot4daggerdaggerdagger 4middot3 19middot5DaggerDaggerDagger 5middot2

Hb haemoglobin AASpL plasma 2-amino-adipic semialdehyde AAShb 2-amino-adipic semialdehyde in erythrocyte lysates GGShb g-glutamyl semialdehyde in erythrocyte lysates Iref antioxidative capacity relativeto reference including Trolox in postprandial plasma samples 8-oxo-dG 8-oxo-deoxyguanosine ND not determined

For details of diet and procedures see Table 1 and p 344Mean values were significantly different from those at the start of the period by paired t test daggerdagger P0middot01 daggerdaggerdagger P0middot001Mean values were significantly different from those at the end of the restricted diet period by paired t test (week 10) Dagger P0middot05 DaggerDagger P0middot01 DaggerDaggerDagger P0middot001Mean value was significantly different from that obtained in week 5 sect P0middot05

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intervention periods and also exhibited a significant over-all decrease of 29 (95 CI 14 44) over time during the8 weeks on restricted diet (Fig 2(b)) At baseline (week 2)there were no significant differences between smokers andnon-smokers for any of the biomarkers of oxidative stressalthough the urinary excretion of 8-oxo-dG was 17 (95 CI 24 48) higher in smokers than in non-smokersThere were no significant differences between smokersand non-smokers with respect to the changes observedover time for the oxidative damage markers (Figs 2 and 3)

Enzymes in antioxidant defence

No changes were observed in activities of the antioxidativeenzymes superoxide dismutase glutathione peroxidaseglutathione reductase and catal in erythrocytes after inter-vention and none of these markers were affected by smok-ing status at baseline

All the enzyme activities were fairly stable during theperiods with restricted diets but changed markedly as thevolunteers returned to their habitual diets Superoxide dis-mutase and glutathione reductase increased when therestricted diet was substituted for the habitual diet as deter-mined by paired comparisons Glutathione peroxidase andcatalase decreased (Table 6)

Total antioxidative capacity in postprandially collectedplasma

There was a significant period effect for plasma antioxida-tive capacity The capacity was higher in the secondperiod Plasma antioxidative capacity increased signifi-cantly overall as determined by paired t test and theincrease remained after correction for the period effect(Table 4) The effect was most pronounced in group Bwhich had GTE in the second period The effect was notsignificant overall in the non-smokers but was significantlyincreased among the smokers after intervention with teaextract

Plasma cholesterol and triacylglycerol

Cholesterol and triacylglycerol in plasma were not affectedby tea extract intervention (Table 5) Tea extract did notaffect these markers in either smokers or non-smokers

Plasma cholesterol and triacylglycerol did not differbetween the groups A and B or between smokers andnon-smokers at baseline (week 2) Triacylglycerols inplasma decreased significantly in the last interventionperiod and also increased when the volunteers returnedto their habitual diets

Vitamin C in diet and plasma

The calculated content of vitamin C in the diet is listed inTable 2 Plasma ascorbate was significantly lower in smo-kers than in non-smokers on their habitual diets The actualascorbic acid content of the intervention diet was below thedetection limit ie 1 mg100 g diet but the dehydro-ascorbic acid content was 12middot6 mg10 MJ per d Therewas no effect of the GTE intervention on plasma ascorbateneither on total plasma vitamin C (Table 5) nor when smo-kers and non-smokers were analysed separately

Compared with the reference blood sample reflectinghabitual diet (week 13) the plasma vitamin C concen-tration decreased steeply within the first 2 weeks of thestudy by 36 resulting in a total decrease by 85 atthe end of the 10th week (Table 6) similar for smokersand non-smokers (Fig 3(a))

Fig 2 Biomarkers of oxidative stress (mean values and standarddeviations) (a) plasma protein oxidation (2-amino-adipic semi-aldehyde AAS) (b) urinary excretion of 8-oxo-deoxyguanosine(8-oxo-dG) (c) plasma total lipoprotein lagtime in sixteen subjectsthroughout the study period with restricted diets expressed as per-centage of the value obtained 3 weeks after its completion (reflect-ing habitual diet depicted as day 0) Values for smokers (--B--)non-smokers (--V--) and both (mdashOmdash) are shown

J F Young et al350

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Plasma a-tocopherol retinol and b-carotene

Plasma a-tocopherol and retinol were not affected by inter-vention with tea extract in either smokers or non-smokers(Table 5)

There was a small decrease by 16 and 11 respect-ively from start to end of the second intervention period inplasma retinol and a-tocopherol (Table 6) Similardecreases were observed in smokers and non-smokersbetween habitual and intervention diet (Fig 3)

The basal intervention diet had a relatively high content ofb-carotene 11middot2 mg10 MJ per d which meant that meandaily intake was 16middot2 mgd similar in group A and B(range 13middot4ndash20middot2 mgd) and that plasma b-caroteneincreased significantly during both intervention periodsThere was a significant decrease in b-carotene after the lastintervention when compared with the level after 3 weekswith habitual diet The b-carotene plasma levels did notreach baseline levels within the wash-out period giving riseto carry-over from the first to the second interventionperiod (Fig 3) When tested at the end of the first interventionperiod tea extract had an increasing effect upon the plasmab-carotene level (Table 5) Group A had a uniformly largerincrease in b-carotene than group B in both periods indicat-ing that this was not due to the tea extract The response wasnot influenced by smoking status

Catechins in urine

The analysis for catechins was very sensitive with a detec-tion limit of 0middot5 ngml urine and linearity in the range of

0middot5ndash50 ngml (r 2 0middot998) The standards were stable inthe HPLC vials for up to 1 week at room temperatureand at 2208C for 3 months

In the first follow-up study we observed that 80 of theexcreted EC and EGC were detected in urine within 4 hwhereas 20 were excreted between 4 and 24 h At 48 hno catechins were detectable (see Fig 4) This points toan excretion half-life of about 16 h for both substancesThere was good concordance between double determi-nations for each individual (CV was 6middot1 for EC and5middot3 for EGC) but relatively large inter-individual vari-ations in total excretion (CV was 27 for EC and 19 for EGC) A mean of 3middot2 of EC and 1middot3 of EGCwas excreted in the first follow-up study

In the second follow-up study EC and EGC wereobserved in 24-h urine samples from each of the three vol-unteers on the days where GTE was ingested whereas onlya trace was found in urine from day 2 where the volunteershad abstained from catechin-containing foods for 24 h (seeTable 7) After correcting for the recovery of catechin ineach sample 3middot7 of the EC and 2middot8 of the EGCfrom the ingested GTE were found in the urine EC andEGC were excreted in urine after ingestion of the sameGTE-containing meat patties which were used in themain study indicating that EC and EGC are absorbablefrom enriched meat patties

Discussion

It is still controversial whether green tea can protect againstcancer and heart disease (Goldbohm et al 1996 Katan

Fig 3 Plasma concentrations of antioxidant vitamins (mean values and standard deviations) (a) vitaminC (b) a-tocopherol (c) retinol (d)b-carotene in sixteen subjects throughout the study period with restricted diets expressed as percentage of the value obtained 3 weeks afterits completion (reflecting habitual diet depicted as day 0) Values for smokers (--B--) non-smokers (ndash ndashVndash ndash) and both (mdashOmdash) are shown

Green tea and markers of oxidative status 351

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1997 Kohlmeier et al 1997) but the high content of anti-oxidants in green tea catechins in particular has beensuggested to cause such effects through a protective mech-anism against radical-mediated damage (Imai amp Nakachi1995 Benzie et al 1999 Klaunig et al 1999) Althoughgreen or black tea tea extracts or isolated catechins haveshown potent protective effects against oxidative stress invarious model systems in vitro and in animal experiments(Loft amp Poulsen 1999) recent studies with human subjectshave recorded limited effects in accordance with our pre-sent results Van het Hof et al (1997) did not observeany effects on resistance to LDL oxidation plasma lipidoxidation or antioxidative enzyme activities in volunteers

after intake of tea for 2 weeks Similarly black tea poly-phenols increased the resistance of human plasma to lipidperoxidation in vitro but not ex vivo after ingestion ofblack tea (Cherubini et al 1999)

The present study included a group of smokers with apotentially higher oxidative stress In a cross-sectionalstudy Imai amp Nakachi (1995) observed a decrease inlipid peroxides in fasting blood samples from heavy smo-kers who took ten cups of green tead or more but Princenet al (1998) did not observe any effect on LDL-oxidationcholesterol levels or plasma triacylglycerols in a random-ised controlled study on smokers receving six cups of blackor green tea a day for 4 weeks In the present study we didnot observe differences between smokers and non-smokersin fasting blood samples using any of the selected markersof oxidative status or lipid status either before or afterintervention with catechin-rich tea extract after what corre-sponds to a more ordinary intake of about 1ndash2 cups oftead which is within the estimated median intake of20ndash50 mg daily of catechins in Denmark (Dragsted et al1997) As expected the smokers in the present study hadlower ascorbate levels in plasma samples representinghabitual diet In the postprandial samples we observed asignificant effect of GTE intervention on plasma anti-oxidant capacity in smokers only when smokers andnon-smokers were analysed separately indicating thatGTE has a more prominent effect in oxidatively stressedindividuals Thus our observations in smokers is in fairagreement with the results of others although the dose ofcatechins in the present study may not have been suffi-ciently large for an effect on lipid oxidation

The unexpectedly high bioavailability of b-carotenecaused significant periodndasheffect interactions and whenanalysed at the end of the first intervention period teaextract seemed to increase plasma b-carotene A sparingeffect of GTE catechins on plasma b-carotene can there-fore not be excluded to be present also in vivo Howeverthere is a large inter-individual variation in response tob-carotene and extreme subjects are sometimes character-ised as lsquonon-respondersrsquo and lsquohigh-respondersrsquo respect-ively (Bowen et al 1993 Parker 1996) In the presentstudy the larger response in group A observed in bothintervention periods seemed to be due to a largernumber of lsquohigh-respondersrsquo in this group and we thereforeconclude that GTE probably had no true sparing effect onb-carotene in the present study This is in line with theobservation that plasma b-carotene did not have a time-course similar to any markers of free-radical mediateddamage in the present study

Results of our follow-up studies indicate that as much as2middot7 of the dose of EC and 2middot0 of EGC may have beenabsorbed from the meat patties fortified with catechins butthat excretion is very fast with urinary excretion half-livesof less than 2 h The protein-rich matrix did not seem togreatly influence catechin uptake in accordance with resultsconcerning other polyphenols (Hollman et al 1997) Naka-gawa et al (1997) have shown that up to 2 epigallocate-chin gallate and 13 EGC are incorporated into plasma90 min after ingestion of GTE in capsules in fair agreementwith our results Our results on antioxidant vitamins inplasma also confirm the results of others who did not

Fig 4 Urinary excretion of (a) epicatechin and (b) epigallocatechinin five volunteers ( A) at various intervals up to 48 h afteringestion of 2 g green tea extract containing 16middot3 mg epicatechinand 51middot4 mg epigallocatechin Each sample was analysed in dupli-cate and the total amounts excreted in each interval are shown asbars

Table 7 Excretion of epigallocatechin (EGC) and epicatechin (EC)after intake of green tea extract in three different regimens

EGCdagger ECdagger

Diet Mean SD Mean SD

Enriched meat pattiesDagger 2middot02 1middot00 2middot73 1middot26Meat patties with added extractsect 2middot12 0middot06 2middot62 1middot75Extract only 2middot68 0middot83 3middot20 0middot57

For details of procedures see p 344dagger Expressed as percentage of ingested amountDagger Enriched during production and stored for 12 months at 2258Csect Green tea extract added right before ingestion

J F Young et al352

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see any changes in tocopherols b-carotene ascorbic acidor uric acid after consumption of teas or tea extracts at5ndash20 times higher doses than ours but with less controlover dietary intakes (Nakagawa et al 1997 van het Hofet al 1997)

We observed that the antioxidant capacity increased asmeasured 2 h after a tea extract-containing meal Thiseffect on the antioxidative capacity in plasma confirmsthe results of several others (van het Hof et al 1997Pietta et al 1998 Benzie et al 1999 Sung et al 2000)who also observed an increased total antioxidant activityin plasma shortly after consumption of a single or multipledoses of green or black tea The presence of only a short-term effect may readily be explained by the short half-lifeof catechins from GTE as seen in Fig 4

The lack of long-term effects from the GTE interventionallows for a post-hoc analysis of the longitudinal effects ofa flavonoid-free diet on the biomarkers of oxidativedamage and defence in the present study We have pre-viously observed a significant increase in plasma AASafter high intakes of juices rich in ascorbate and plant phe-nols (Young et al 1999) and Castenmiller et al (1999)observed a significant correlation between plasma ascor-bate and plasma AAS among sixty-four volunteers in aspinach intervention study (Castenmiller et al 1999) Wehave also observed a decrease in plasma ascorbate and inplasma protein oxidation in a recent study over a periodof 2 weeks with a fruit- and vegetable-free diet similar tothe one used in the present study (Young et al 2000) Itis possible that dietary changes induced a change in proteinturnover or degradation However a similar decrease inprotein oxidation in 8-oxo-dG excretion and in theincreased resistance of plasma lipoproteins to oxidationin the present study points to a more general relief of oxi-dative stress after depletion of flavonoid- and ascorbate-rich fruits and vegetables from the diet contrary tocommon beliefs

The decrease in the urinary excretion of 8-oxo-dG mightalso have resulted from a decrease in the efficiency ofDNA-repair Other studies on DNA damage in relation tofruit and vegetable intakes show an equivocal picture(Velthuis-te Wierik et al 1995 Hertog et al 1997Djuric et al 1998) whereas no significant effect of vitaminC vitamin E or b-carotene on the excretion of 8-oxo-dGhas been observed in placebo-controlled interventionstudies and in meta-analyses (van Poppel et al 1995Prieme et al 1997 Poulsen et al 1998 Loft amp Poulsen2000) Studies on plasma lipid oxidation plasma oxidationlagtimes and plasma malondialdehyde in relation to dietgenerally point to an increase in oxidative damage withdiets low in fruits vegetables or vitamin C in disagree-ment with our findings (Fuller et al 1996 Omaye et al1996 Anderson et al 1997 Miller et al 1998 Maskarinecet al 1999) Complex pro- and antioxidant effects couldtogether with large differences in study material and proto-cols possibly explain much of the apparent extensive vari-ation and discrepancies regarding the effects ofantioxidants fruits and vegetables on DNA lipid and pro-tein oxidation

In conclusion GTE incorporated into meat patties indoses realistic for addition to processed foods only leads

to a short-term change in plasma antioxidant capacityand has no long-term effects on oxidation parameterswithin the blood or urine compartments in smokers or innon-smokers The study can therefore be seen as a 10weeks controlled study with dietary depletion of all foodantioxidants derived from fruits and vegetables exceptfor carrots and potatoes During the depletion we observeda decrease in oxidative damage to proteins DNA andlipids concomitantly with a major reduction in plasmaascorbate and minor changes in other vitamins and in anti-oxidant enzyme activities We speculate that these seem-ingly positive effects on oxidative status are partly due todepletion of some pro-oxidant compounds co-existingwith vitamin C in fruits and vegetables and this underlinesthe general lack of solid knowledge of the mechanisms bywhich a diet rich in fruits and vegetables cause a decreasein the risk of chronic diseases

Acknowledgements

The authors wish to thank Anni Schou Vibeke Kegel JoanFrandsen Hanne Fenger Eriksen Susanne M Hansen Bir-gitte Alstrup Joslashrgensen and Ella Jessen for excellentassistance and Nestle RampD Bjuv (Sweden) for providingthe meat products used in this study

References

Aeschbach R amp Rossi P (1994) Alkylene glycol extraction of anti-oxidants from vegetable matter Nestec Ltd (US patentUS5795609)

Anderson D Phillips BJ Yu TW Edwards AJ Ayesh R amp But-terworth KR (1997) The effects of vitamin C supplementationon biomarkers of oxygen radical generated damage in humanvolunteers with low or high cholesterol levels Environmentaland Molecular Mutagenesis 30 161ndash174

Benzie IFF Szeto YT Strain JJ amp Tomlinson B (1999) Consump-tion of green tea causes rapid increase in plasma antioxidantpower in humans Nutrition and Cancer 34 83ndash87

Bowen PE Garg V Stacewicz-Sapuntzakis M Yelton L ampSchreiner RS (1993) Variability of serum carotenoids inresponse to controlled diets containing six servings of fruitsand vegetables per day Annals of the New York Academy ofSciences 691 241ndash243

Castenmiller JJM Lauridsen ST Dragsted LO van het Hof KHLinssen JPH amp West CE (1999) Beta-carotene does not changemarkers of enzymatic and non-enzymatic antioxidant activityin human blood Journal of Nutrition 129 2162ndash2169

Cherubini A Beal MF amp Frei B (1999) Black tea increases theresistance of human plasma to lipid peroxidation in vitro butnot ex vivo Free Radical Biology and Medicine 27 381ndash387

Daneshvar B Frandsen H Autrup H amp Dragsted LO (1997)Gamma-glutamyl semialdehyde and 2-amino-adipic semialde-hyde biomarkers of oxidative damage to proteins Biomarkers2 117ndash123

Das NP (1971) Studies of flavonoid metabolism Absorption andmetabolism of (+)-catechin in man Biochemical Pharmacology20 3435ndash3445

Djuric Z Depper JB Uhley V Smith D Lababidi S Martino S ampHeilbrun LK (1998) Oxidative DNA damage levels in bloodfrom women at high risk for breast cancer are associatedwith dietary intakes of meats vegetables and fruits Journalof the American Dietetics Association 98 524ndash528

Dragsted LO Strube M amp Leth T (1997) Dietary levels of plant

Green tea and markers of oxidative status 353

httpswwwcambridgeorgcoreterms httpsdoiorg101079BJN2002523Downloaded from httpswwwcambridgeorgcore University of Basel Library on 10 Jul 2017 at 151712 subject to the Cambridge Core terms of use available at

phenols and other non-nutritive components could they pre-vent cancer European Journal of Cancer Prevention 6522ndash528

Fuller CJ Grundy SM Norkus EP amp Jialal I (1996) Effect ofascorbate supplementation on low density lipoprotein oxidationin smokers Atherosclerosis 119 139ndash150

Gardner PT McPhail DB amp Duthie GC (1998) Electron spinresonance spectroscopic assessment of the antioxidant potentialof teas in aqueous and organic media Journal of the Science ofFood and Agriculture 76 257ndash262

Goldbohm RA Hertog MGL Brants HAM van Poppel G amp vanden Brandt PA (1996) Consumption of black tea and cancerrisk A prospective cohort study Journal of the NationalCancer Institute 88 93ndash100

Hart DJ amp Scott KJ (1995) Development and evaluation of anHPLC method for the analysis of carotenoids in foods andthe measurement of the carotenoid content of vegetables andfruits commonly consumed in the UK Food Chemistry 54101ndash111

Hertog MGL de Vries A Ocke MC Schouten A Bueno-de-Mes-quita HB amp Verhagen H (1997) Oxidative DNA damage inhumans comparison between high and low habitual fruit andvegetable consumption Biomarkers 2 259ndash262

Hertog MGL Feskens EJM Hollman PCH Katan MB amp Kromh-out D (1993a) Dietary antioxidant flavonoids and risk of corn-ary heart disease the Zutphen elderly study Lancet 3421007ndash1011

Hertog MGL Feskens EJM Hollman PCH Katan MB amp Kromh-out D (1994) Dietary flavonoids and cancer risk in the Zutphenelderly study Nutrition and Cancer 22 175ndash184

Hertog MGL Hollman PCH Katan MB amp Kromhout D (1993b)Estimation of daily intake of potentially anticarcinogenic flavo-noids and their determinats in adults in The Netherlands Nutri-tion and Cancer 20 21ndash29

Hertog MGL Kromhout D Aravanis C Blackburn H Buzina RFidanza F Giampaoli S Jansen A Menotti A Nedeljkovic SPekkarinen M Simic BS Toshima H Feskens EJM HollmanPCH amp Katan MB (1995) Flavonoid intake and long-termrisk of coronary heart disease and cancer in The SevenCountries Study Archives of Internal Medicine 155 381ndash386

Hofer G Lichtenberg D amp Hermetter A (1995) A new fluor-escence method for the continuous determination of surfacelipid oxidation in lipoproteins and plasma Free RadicalResearch 23 317ndash327

Hollman PC Tijburg LB amp Yang CS (1997) Bioavailability offlavonoids from tea Critical Reviews of Food Science andNutrition 37 719ndash738

Imai K amp Nakachi K (1995) Cross sectional study of effects ofdrinking green tea on cardiovascular and liver diseases BritishMedical Journal 310 693ndash696

Ishikawa T Suzukawa M Ito T Yoshida H Ayaori M NishiwakiM Yonemura A Hara Y amp Nakamura H (1997) Effect of teaflavonoid supplementation on the susceptibility of low-densitylipoprotein to oxidative modification American Journal ofClinical Nutrition 66 261ndash266

Joslashrgensen K amp Skibsted L (1993) Carotenoid scavenging of rad-icals Effects of carotenoid structure and oxygen partial press-ure on antioxidative activity Zeitschrift fur LebensmittelUntersuchung und Forschung 196 423ndash429

Kall M amp Andersen C (1999) Improved method for simultaneousdetermination of ascorbic acid and dehydroascorbic acid isoas-corbic acid and dehydroisoascorbic acid in food and biologicalsamples Journal of Chromatography B Biomedical Sciencesand Applications 730 101ndash111

Katan MB (1997) Flavonoids and heart disease American Jour-nal of Clinical Nutrition 65 1542ndash1543

Klaunig JE Xu Y Han C Kamendulis LM Chen J Heiser C

Gordon MS amp Mohler ER III (1999) The effect of tea con-sumption on oxidative stress in smokers and nonsmokers Pro-ceedings of the Society Experimental Biology and Medicine220 249ndash254

Kohlmeier L Weterings KG Steck S amp Kok FJ (1997) Tea andcancer prevention an evaluation of the epidemiologic litera-ture Nutrition and Cancer 27 1ndash13

Lin J-K Lin C-L Liang Y-C Lin-Shiau S-Y amp Juan I-M (1998)Survey of catechins gallic acid and methylxanthines in greenoolong pu-erh and black teas Journal of Agricultural FoodChemistry 46 3635ndash3642

Loft S amp Poulsen HE (1999) Markers of oxidative damage toDNA antioxidants and molecular damage Methods in Enzy-mology 300 166ndash184

Loft S amp Poulsen HE (2000) Antioxidant intervention studiesrelated to DNA damage DNA repair and gene expressionFree Radical Research 33 S67ndashS83

Lotito SB amp Fraga CG (1998) (+)-Catechin prevents humanplasma oxidation Free Radical Biology and Medicine 24435ndash441

Maskarinec G Chan CL Meng L Franke AA amp Cooney RV(1999) Exploring the feasibility and effects of a high-fruitand -vegetable diet in healthy women Cancer EpidemiologyBiomarkers and Prevention 8 919ndash924

Miller ER III Appel LJ amp Risby TH (1998) Effect of dietary pat-terns on measures of lipid peroxidation results from a random-ized clinical trial Circulation 98 2390ndash2395

Moslashller A (1989) Food Composition tables Copenhagen NationalFood Agency

Nakagawa K amp Miyazawa T (1997) Absorption and distributionof tea catechin (-)- epigallocatechin-3-gallate in the rat Jour-nal of Nutritional Science and Vitaminology 43 679ndash684

Nakagawa K Okuda S amp Miyazawa T (1997) Dose-dependentincorporation of tea catechins (-)- epigallocatechin-3-gallateand (-)-epigallocatechin into human plasma Bioscience Bio-technology Biochemistry 61 1981ndash1985

Nielsen SE amp Dragsted LO (1998a) Column-switching high-per-formance liquid chromatographic assay for the determination ofquercetin in human urine with ultraviolet absorbance detectionJournal of Chromatography 707B 81ndash89

Nielsen SE amp Dragsted LO (1998b) Column-switching high-per-formance liquid chromatographic assay for determination ofapigenin and acacetin in human urine with ultraviolet absor-bance detection Journal of Chromatography 713B 379ndash386

Nielsen SE Young JF Daneshvar B Lauridsen ST Knuthsen PSandstrom B amp Dragsted LO (1999) Effect of parsley (Petro-selinum crispum ) intake on urinary apigenin excretion bloodantioxidant enzymes and biomarkers for oxidative stress inhuman subjects [see comments] British Journal of Nutrition81 447ndash455

Nissen LR Mansson L Bertelsen G Huynh-Ba T amp Skibsted LH(2000) Protection of dehydrated chicken meat by natural anti-oxidants as evaluated by electron spin resonance spectrometryJournal of Agricultural and Food Chemistry 48 5548ndash5556

Omaye ST Burri BJ Swendseid ME Henning SM Briggs LABowen HT amp Ota RB (1996) Blood antioxidants changes inyoung women following beta-carotene depletion and repletionJournal of the American College of Nutrition 15 469ndash474

Parker RS (1996) Absorption metabolism and transport of caro-tenoids FASEB Journal 10 542ndash551

Pietta P Simonetti P Gardana C Brusamolino A Morazzoni P ampBombardelli E (1998) Relationship between rate and extent ofcatechin absorption and plasma antioxidant status Biochemis-try and Molecular Biology International 46 895ndash903

Pocock SJ (1998) Clinical Trials A Practical Approach Chiches-ter UK Wiley amp Sons

Poulsen HE Prieme H amp Loft S (1998) Role of oxidative DNA

J F Young et al354

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damage in cancer initiation and promotion European Journalof Cancer Prevention 7 9ndash16

Prieme H Loft S Nyyssonen K Salonen JT amp Poulsen HE(1997) No effect of supplementation with vitamin E ascorbicacid or coenzyme Q10 on oxidative DNA damage estimatedby 8-oxo-78-dihydro-20-deoxyguanosine excretion in smokersAmerican Journal of Clinical Nutrition 65 503ndash507

Princen HM van Duyvenvoorde W Buytenhek R Blonk C Tij-burg LB Langius JA Meinders AE amp Pijl H (1998) No effectof consumption of green and black tea on plasma lipid and anti-oxidant levels and on LDL oxidation in smokers Arteriosclero-sis Thrombosis and Vascular Biology 18 833ndash841

Serafini M Ghiselli A amp Ferro LA (1996) In vivo antioxidanteffect of green and black tea in man European Journal ofClinical Nutrition 50 28ndash32

Sung H Nah J Chun S Park H Yang SE amp Min WK (2000) Invivo antioxidant effect of green tea (In Process Citation) Euro-pean Journal of Clinical Nutrition 54 527ndash529

Thurnham DI Smith E amp Flora PS (1988) Concurrent liquid-chromatographic assay of retinol alpha-tocopherol beta-caro-tene alpha-carotene lycopene and beta-cryptoxanthin inplasma with tocopherol acetate as internal standard ClinicalChemistry 34 377ndash381

van het Hof KH de Boer HSM Wiseman SA Lien N WeststrateJA amp Tijburg LBM (1997) Consumption of green or black tea

does not increase resistance of low-density lipoprotein to oxi-dation in humans American Journal of Clinical Nutrition 661125ndash1132

van Poppel G Poulsen H Loft S amp Verhagen H (1995) No influ-ence of beta-carotene on oxidative DNA damage in male smo-kers Journal of the National Cancer Institute 87 310ndash311

Velthuis-te Wierik EJ van Leeuwen RE Hendriks HF VerhagenH Loft S Poulsen HE amp van den BH (1995) Short-term mod-erate energy restriction does not affect indicators of oxidativestress and genotoxicity in humans Journal of Nutrition 1252631ndash2639

Yen G amp Chen H (1995) Antioxidant activity of various teaextracts in relation to their antimutagenicity Journal of Agri-cultural and Food Chemistry 43 27ndash32

Young JF Dragsted LO Daneshvar B Lauridsen ST Hansen Mamp Sandstrom B (2000) The effect of grape skin extract on oxi-dative status British Journal of Nutrition 84 505ndash513

Young JF Nielsen SE Haraldsdottir J Daneshvar B LauridsenST Knuthsen P Crozier A Sandstrom B amp Dragsted LO(1999) Effect of fruit juice intake on urinary quercetinexcretion and biomarkers of antioxidative status AmericanJournal of Clinical Nutrition 69 87ndash94

Zhang A Chan PT Luk YS Ho WKK amp Chen ZY (1997) Inhibi-tory effect of jasmine green tea epicatechin isomers on LDL-oxidation Nutritional Biochemistry 8 334ndash340

Green tea and markers of oxidative status 355

httpswwwcambridgeorgcoreterms httpsdoiorg101079BJN2002523Downloaded from httpswwwcambridgeorgcore University of Basel Library on 10 Jul 2017 at 151712 subject to the Cambridge Core terms of use available at