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Biochemical and Biophysical Research Communications 344 (2006) 906–911

BBRC

Cautions in the use of biomarkers of oxidative damage; the vascularand antioxidant effects of dark soy sauce in humans

Chung-Yung J. Lee, Helen B. Isaac, Huansong Wang, Shan Hong Huang, Lee-Hua Long,Andrew M. Jenner, Ronan P. Kelly 1, Barry Halliwell *

National University of Singapore, Yong Loo Lin School of Medicine, Department of Biochemistry, 8 Medical Drive, Singapore 117597, Singapore

Received 29 March 2006Available online 19 April 2006

Abstract

Dark soy sauce (DSS) is a powerful antioxidant in vitro. We investigated whether this effect could occur in vivo and improve vascularfunction. Healthy human subjects were given DSS or placebo meals in a randomized, crossover study. Blood and urine were sampledbefore and 1, 2, 3, and 4 h after the meal for F2-isoprostanes (total, free, and esterified) and 8OHdG measurements. Blood pressure,vascular augmentation index (AIx), and heart rate (HR) were also measured. Plasma total F2-isoprostanes significantly decreased 3 hafter placebo and the decrease was greater for DSS. Plasma free and esterified F2-isoprostanes were also significantly decreased afterDSS. Both placebo and DSS meals increased urinary F2-isoprostanes at 1 h but not thereafter, and lowered urinary 8OHdG levels,DBP and AIx, and increased HR. We conclude that DSS decreases lipid peroxidation in vivo. However, oxidative damage biomarkerschanged after the placebo meal, a phenomenon to consider when designing interventional studies.� 2006 Published by Elsevier Inc.

Keywords: Soy sauce; Isoprostanes; Free-isoprostanes; Total-isoprostanes; Esterified isoprostanes; 8-OHdG; Oxidative stress; AIx; Pulse wave velocity

The use of soy sauce as a condiment is common in theAsian diet and it has been found that much commercial-ly available soy sauce possesses high antioxidant activityin vitro as determined by the ability to scavenge thenitrogen centred ABTS�+ (2,2 0-azino-bis(3-ethylbenzo-thiazoline-6-sulfonate)) radical, an assay that is frequent-ly used to assess the antioxidant activity of beverages,food extracts, and body fluids [1]. Dark soy sauce wasespecially effective [2,3]. High dietary intakes of foodsrich in antioxidants may have significant benefits [re-viewed in 4]. However, while many plant products showantioxidant activity in vitro, few have been proven to actas such in vivo [4,5].

In recent years, many biomarkers for measuring oxida-tive damage to lipids, proteins or DNA in biological sam-

0006-291X/$ - see front matter � 2006 Published by Elsevier Inc.

doi:10.1016/j.bbrc.2006.03.217

* Corresponding author. Fax: +65 6779 1453.E-mail address: bchbh@nus.edu.sg (B. Halliwell).

1 National University Hospital, Department of Medicine, Lower KentRidge Road, Singapore.

ples have been developed [reviewed in 6,7]. In addition, insome (but not all) studies administration of antioxidantshas been reported to improve vascular function [8–10].For example, F2-isoprostanes are a family of eicosanoidsproduced by non-enzymatic free radical oxidation of ara-chidonic acid esterified to phospholipids [11,12]. It is widelyagreed that measurement of F2-isoprostanes in plasma orurine is the best currently available method to assess lipidperoxidation, especially as the method is thought not tobe confounded by diet, in the sense that F2-isoprostanesdo not appear to be absorbed from foods [13–15]. SeveralF2-isoprostanes, including 8-iso-PGF2a, are biologicallyactive and can act as vasoconstrictors [16–19]. Higher levelsof F2-isoprostanes in human body fluids have been relatedto oxidative stress due to smoking, cardiovascular disease,obesity, hypercholesterolemia, diabetes, and liver diseases[12,20–25]. F2-isoprostanes are produced in an esterifiedform within membranes and lipoproteins, and rapidlyhydrolyzed to the free acid form, which is quicklymetabolized [11,12].

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Some reactive species formed in biological systems canoxidatively damage DNA bases and the oxidized basescan take part in mutation and development of cancer [re-viewed in 26,27]. An important product is the guaninederived lesion 8-hydroxyguanine (8OHGua). Its corre-sponding deoxynucleoside 8-hydroxy-2 0-deoxyguanosine(8OHdG) is regarded as an important urinary biomarkerof oxidative damage, which originates both from DNAand the DNA precursor pool [28,29]. Levels of 8OHdGin urine are not thought not to be affected by its presencein the diet [30].

The purpose of this study was to use biomarkers of oxi-dative damage to test whether or not consuming dark soysauce (versus a placebo) would decrease oxidative damageand improve vascular function, i.e., whether or not theantioxidant effects in vitro would be apparent in vivo.

Subjects and methods

Subject and dosing. Twenty-four healthy, non-smoker volunteers wereenrolled into the study (Table 1). All subjects, prior to study commence-ment, signed the informed consent form, which was approved by the localEthics Board (Domain Specific Review Board, Singapore).

This was an observer-blind, randomized, placebo controlled, crossoverstudy. In period 1, the subjects were given either a single dose of 30 mLdark soy sauce (Tiger Brand, Chuen Chong Food Industries, Singapore)mixed with 200 g of plain boiled rice (the DSS group, for ‘‘dark soysauce’’) or 200 g of plain boiled rice with food colorant (placebo meal).After a minimum of 3 days wash out, they were crossed-over to receive theother treatment. The subjects were fasted for at least 6 h prior to the mealand did not consume any foods other than the DSS or placebo meals untilthe end of the study activity for each period. Blood and urine samples werecollected pre-meal and at 1, 2, 3, and 4 h time points after eating. Thesample collections were made approximately the same time of day on eachoccasion for all subjects. Non-invasive vascular measurements—bloodpressure, augmentation index, and heart rates were made at baseline, thenat 1, 2, 3, and 4 h after the meal and just prior to blood sampling.

ABTS assay. The antioxidant activity of dark soy sauce, rice, andcaramel food colorant was measured by the ABTS assay (1). A 2,2 0-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) solution (7 mmol/L) inwater was oxidized by 2.45 mmol/L potassium persulfate in the dark for12 h. The absorbance of the oxidized ABTS solution (ABTSÆ�) wasadjusted to 0.7 ± 0.02 at 734 nm (Beckman UV–VIS Spectrophotometer,DU640B, UK) using phosphate-buffered saline (10 mmol/L, pH 7.4). To

Table 1Basal characteristic of the subjectsa

Value

Age (yr) 22.8 ± 1.6BMI (kg/m2) 21.6 ± 2.2Waist to hip ratio 0.87 ± 0.06% Body fat 23.8 ± 6.5

Fasting glucose (mmol/L) 4.37 ± 0.22Homocysteine (lmol/L) 11.10 ± 4.54Urate (lmol/L) 333 ± 86Total cholesterol (mmol/L) 4.70 ± 0.74LDL-cholesterol (mmol/L) 3.03 ± 0.64HDL-cholesterol (mmol/L) 1.35 ± 0.29Triacylglycerol (mmol/L) 0.71 ± 0.41

Systolic blood pressure (mm Hg) 109 ± 8Diastolic blood pressure (mm Hg) 65 ± 6

a n = 14 male and 10 female.

1 mL ABTS solution, 10 lL of dark soy sauce or rice (extracted in water)or food colorant was added, and after 1 min the absorbance was mea-sured. Using Trolox as the standard, the antioxidant activity of the foodwas expressed as Trolox equivalent antioxidant capacity (TEAC).

Vascular measurements. Blood pressure was measured in the brachialartery using an automated oscillometric device (Omron IntelliSense T8,Omron Healthcare, Illinois, USA).

The technique of pulse wave analysis [31] was used to determine aorticpressure and augmentation index (AIx). Pulse pressure recordings weremade from the radial artery using a Millar tonometer, and data werecollected and analyzed using the SphygmoCor (SphygmoCor 2000 v7.0,PWV Medical, Sydney, Australia), which allowed continuous onlinerecording of the radial artery pressure waveform. An average radialwaveform was calculated and an average aortic pressure waveform derivedat each time point, using a previously validated transfer function. The AIxwas then calculated by dividing the difference in pressure between the firstand second systolic peaks of the aortic pressure waveform by the calcu-lated aortic pulse pressure. This index provides a measure of the contri-bution to afterload of the pulse pressure wave reflected from the peripheryand is an indirect measure of arterial tone [32,33].

Sample preparation. Part of the blood sample was collected in EDTAblood tubes with indomethacin (final concentration 15 lmol/L) forF2-isoprostanes measurement [11,34]. After separation by centrifugation,20 ll of 2 mmol/L butylated hydroxytoluene (BHT) was then added to theplasma and stored at �70 �C for 3 weeks until analysis. Spot mid-streamurine samples were collected and immediately stored at �70 �C for F2-isoprostanes, 8OHdG, and creatinine measurements.

Markers of oxidative stress. Urine and plasma samples for measure-ment of free F2-isoprostanes were prepared as described by Morrow et al.[11] and Lee et al. [34] with some modification. Mixed heavy isotopes,8-iso-PGF2a-d4, and iPF2a-VI-d4 (Cayman Chemicals, Ann Arbor, MI,USA) (0.5 ng each for urine and 0.25 ng each for plasma) in 20 lL ethanolwere added to 1 mL samples and then acidified with 1 mL of 40 mmol/Lformic acid (pH 4.5) for anion exchange solid phase extraction. Foranalysis of total F2-isoprostanes (free + esterified), 0.5 ng of 8-iso-PGF2a-d4 and 0.5 ng iPF2a-VI-d4 in 20 lL ethanol were added to 1 mL plasma,aliquots of which were then hydrolyzed with 1 mL of 1 mol/L potassiumhydroxide prepared in methanol at 37 �C for 30 min and then cooled.Methanol (0.5 mL) was further added. Samples were neutralized with0.2 mL of 5 mol/L HCl, acidified to pH 4.5 with 2.7 mL formic acid(40 mmol/L), and centrifuged at 10,000g, 4 �C for 10 min. The superna-tant was removed for anion exchange solid phase extraction.

Plasma and urine samples were prepared for F2-isoprostanes analysisusing anionic exchange solid phase extraction (SPE) as described by Leeet al. [34], with some modifications. Briefly, MAX-SPE cartridges (Oasis�

Mixed Anion Exchange, Waters Corp., Milford, MA, USA) were washedwith 2 mL methanol and then pre-conditioned with 2 mL of 20 mmol/Lformic acid pH adjusted to 4.5. Acidified plasma or urine containing heavyisotopes was loaded onto the cartridge. The columns were washed with2 mL of 2% (v/v) ammonium hydroxide and then 2 mL metha-nol:20 mmol/L formic acid, pH 4.5 (40:60), 2 mL of 100% hexane, and2 mL hexane:ethyl acetate (70:30) solvents in that order. F2-Isoprostaneswere eluted with 1.6 mL of hexane:ethanol:acetic acid (70:29.4:0.6). Themodified SPE protocol showed CV for 8-iso-PGF2a concentrations in therange 0–2.0 ng/mL to be 3.6% and 5.1% for urine (n = 3) and plasma(n = 3), respectively, and reproducibility to range from 81% to 111% forurine (n = 3) and from 85% to 111% for plasma (n = 3). Recovery of 8-iso-PGF2a of the MAX-SPE extraction from urine and plasma samples wascalculated to be 58 ± 2.3% (n = 3) and 66 ± 2.1% (n = 3), respectively.Although the modified procedure does not show a marked difference inreproducibility and recovery from the previous report [34], the adjustmentof the pH of formic acid and the use of hexane/ethanol/acetic acid in theprocedure produced cleaner F2-isoprostanes chromatograms when ana-lyzed by gas chromatography (GC 6890 Agilent Technologies, USA)interfaced to a mass selective detector in negative chemical ionizationmode (MS-NCI, 5973 Agilent Technologies, USA).

The eluted F2-isoprostanes were dried under nitrogen and derivatized withpentafluorobenzylbromide in the presence of N,N-diisopropylethylamine,

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dried and derivatized again with N, O-bis(trimethylsilyl)trifluoroacetamidewith 1% trimethylchlorosilane (BSTFA + 1%TMCS) in the presence ofacetonitrile. The BSTFA (+1%TMCS) was dried under nitrogen andreconstituted in iso-octane for GC-MS-NCI analysis [34]. Selected ionmonitoring (SIM) was used to monitor the carboxylate ion m/z 569 for 8-iso-PGF2a and at m/z 573 for deuterium labeled (8-iso-PGF2a-d4 andiPF2a-VI-d4) internal standards. F2-isoprostanes concentration was cal-culated by relating the peak area of the F2-isoprostanes with the sum ofthe two different F2-isoprostanes internal standard peaks. Theconcentration of esterified F2-isoprostanes in plasma was calculated bysubtracting the free F2-isoprostanes concentration from the totalF2-isoprostanes concentration. The values are expressed as F2-isopros-tanes ng/mL of plasma, and ng/mg of creatinine for urine.

Oxidative damage to DNA and its precursors was measured bydetermining 8OHdG levels in urine. The samples were prepared andanalyzed according to Lin et al. [35]. Briefly 50 pmol [18O]8OHdG (giftfrom Dr. Miral Dizdaroglu, National Institute of Standards andTechnology, Maryland, USA) was added as internal standard to eachurine sample, which was then acidified with 10% formic acid. Thesample was cleaned by SPE using Oasis� HLB Vac cartridges (WatersCorp., Milford, MA, USA) and dried with a vacuum freeze–dryer. Thedried sample was purged with nitrogen gas and derivatized with 60 lLBSTFA (+1%TMCS) in the presence of 15 lL acetonitrile:ethanethiol(3:1). The derivatized samples were analyzed by gas chromatography(GC 6890, Agilent Technologies, USA) interfaced to a mass selectivedetector (MS 5973, Agilent Technologies, USA) as described in Linet al. [35]. SIM was used in which ions, m/z 383 and 643 for 8OHdG,m/z 385 and 645 for [18O]8OHdG, were monitored for quantification.8OHdG concentrations were achieved by relating the peak area of the8OHdG with [18O]8OHdG internal standard peaks and expressed aslmol/mol of creatinine.

Urinary creatinine. Aliquots of fresh urine samples were sent to theNational Referral Laboratory (NRL, Singapore) for measurement ofcreatinine to standardize urinary F2-isoprostanes and 8OHdG values.

Statistical analysis. Triplicate measurements per subject were made forthe recovery and reproducibility tests (see above) and duplicate mea-surements per subject were made for all time points and study periods. Themeasurements are expressed as means ± SD. One-way ANOVA wasperformed for all measurements and significance at p < 0.05 was furthertested by a two-tailed paired Student’s t test between basal level and timepoints post-intake and between time points of placebo and DSS periods.All statistical methods were performed by StatView version 5.0 (AbacusConcepts Inc., CA, USA).

Results

Compliance

This study aimed to compare the effects of dark soysauce or placebo on parameters of vascular function and

Table 2Vascular measurement of the subjects before and after meal consumption

Time (h) 0 1

AIx (%) Placebo 7.21 ± 11.66 0.42 ± 14.7DSS 8.37 ± 12.77 3.22 ± 11.8

HR (bpm) Placebo 58.78 ± 9.71 61.60 ± 8.30DSS 60.00 ± 9.90 62.29 ± 8.25

Systolic BP (mmHg) Placebo 108.23 ± 8.66 112.21 ± 11.0DSS 109.04 ± 8.75 112.67 ± 11.1

Diastolic BP (mmHg) Placebo 64.92 ± 5.45 64.88 ± 8.19DSS 65.24 ± 6.42 63.29 ± 6.33

Paired, two-tailed Student’s t test indicates ** significance at p < 0.01 versus 0 hNS between placebo and DSS for all time points except for §p < 0.05 DSS ver

oxidative damage in healthy human volunteers to see ifthe high antioxidant activity of dark soy sauce in vitrocould be reflected in vivo. All 24 subjects completed thestudy and there were no significant adverse effects. We con-firmed the high Trolox equivalent antioxidant capacity(TEAC) of the dark soy sauce (122 ± 16 mM, n = 3) bythe ABTS activity. The rice or caramel colorant had no sig-nificant antioxidant activity (data not shown).

Effect of DSS meal on vascular measurements

Augmentation index is an indication of blood flowefficiency in the vascular system. An increase in AIxdenotes vasoconstriction while a decrease denotes areduction in flow resistance (vasodilation). In this study,we found that both meals decreased the AIx substantiallywith a nadir at 1 h for placebo and 2 h for DSS. The heartrate and the systolic blood pressure also increased afterboth meals with significant rises at 1 and 2 h for placeboand 1 h for DSS (Table 2) as compared with baseline.The diastolic blood pressure fell after both placebo andDSS meals, with the latter producing a significantly greaterdrop in DBP at 2 h. There was no significant differencebetween placebo and DSS at any time point for AIx, heartrate or systolic blood pressure.

Effect of DSS on oxidative stress indices

We measured F2-isoprostanes in urine and plasma as abiomarker of lipid peroxidation. Urinary concentrationof F2-isoprostanes was raised at 1 h after the placebo meal(p = 0.01) but then values reverted to baseline. A similartrend (p = 0.09) was observed for the DSS meal (Fig. 1).No significant difference was noticed between the 2 mealsin urinary F2-isoprostanes at any time. Plasma total F2-iso-prostanes, a combined measurement of free and esterifiedforms, were decreased at 3 h after the placebo meal andat 1–4 h after the DSS meal (Fig. 1) when compared withbaseline. When DSS and placebo were compared, therewas a maximal decrease in total F2-isoprostanes 3 hafter consuming DSS at which point the total F2-isopros-tanes concentration was significantly lower than after pla-cebo (0.272 ng/ml vs. 0.310 ng/ml, p = 0.02). Plasma free

2 3 4

3** 1.48 ± 12.99** 3.23 ± 12.08** 2.98 ± 13.84**

3�� �1.31 ± 14.24�� 1.46 ± 13.68�� 3.19 ± 12.37��

** 61.25 ± 8.13** 59.75 ± 7.45 58.88 ± 7.27� 62.13 ± 8.11 61.23 ± 7.70 59.17 ± 6.811** 111.33 ± 10.59** 107.71 ± 10.98 112.42 ± 12.619� 110.21 ± 11.81 109.88 ± 10.93 111.50 ± 12.10

65.75 ± 5.46 64.80 ± 7.73 67.38 ± 8.2062.63 ± 7.04�,§ 64.67 ± 7.14 65.46 ± 6.90

placebo; � p < 0.05 and �� p < 0.01 versus 0 h dark soy sauce meal (DSS).sus placebo.

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Fig. 1. Urinary and plasma F2-isoprostanes concentration after placebo and dark soy sauce meal. Paired, two-tailed Student’s t test indicates **significance at p < 0.01 versus 0 h placebo; � significance at p < 0.05 and �� at p < 0.01 versus 0 h DSS.

C.-Y.J. Lee et al. / Biochemical and Biophysical Research Communications 344 (2006) 906–911 909

F2-isoprostanes were not affected by placebo but consump-tion of DSS decreased levels significantly at 2 h comparedwith baseline (Fig. 1). The DSS meal had greater impactthan placebo on lowering free F2-isoprostanes, with levelsdiffering significantly at 4 h (0.066 ng/ml vs. 0.078 ng/ml,p = 0.04). In order to examine the effect of placebo andDSS on esterified F2-isoprostanes, we subtracted theplasma free F2-isoprostanes concentration from totalF2-isoprostanes. The placebo meal lowered the esterifiedF2-isoprostanes at the 3 h time point (p = 0.02) while theDSS meal tended to reduce esterified F2-isoprostanesthroughout with statistically significant reductions frombaseline at 3 h (p = 0.01) and 4 h (p = 0.04), and a border-line difference (p = 0.07) from placebo values at 3 h(Fig. 1).

The effects of placebo and DSS on oxidative DNA dam-age were determined by measuring 8OHdG in urine. Con-sumption of placebo decreased 8OHdG levels in urine from1 h time point (p = 0.004) and the decrease persistedthroughout the study. A similar trend was noticed afterDSS consumption (p = 0.06) (Table 3). No significant dif-ference was found between placebo and DSS for any timepoint in the study.

Table 3Urinary 8OHdG (lmol/mol creatinine) of the subjects before and after meal c

Time (h) 0 1

Placebo 2.17 ± 1.38 1.43 ± 0.69**

DSS 2.02 ± 1.12 1.62 ± 0.68

Paired, two-tailed Student’s t test indicates * significance at p < 0.05 and ** apoints.

Discussion

Soy sauce is a popular condiment but little is knownabout its health benefits and its contribution as anantioxidant. Our previous studies showed high antioxi-dant activity of dark soy sauce in vitro [2,3] but thereare no published studies of its in vivo effects inhumans.

An important feature of our results is that the placebomeal had effects on parameters of oxidative damage; indeedsuch ‘‘feeding effects’’ have been alluded to earlier in the lit-erature [14,34,36] but not documented to this extent. Forexample Vissers et al. [36] showed positive effects of oliveoil on the sensitivity of LDL to oxidation, but feeding oilwithout antioxidants had the same effect. Thus in subjectson the placebo meal there was a rise in urinary F2-isopros-tanes at 1 h, a fall in plasma total F2-isoprostanes (signifi-cant at 3 h) but no change in free plasma F2-isoprostanes.There was also a drop in urinary 8OHdG. Thus, experi-ments claiming antioxidant effects of foods must be accom-panied by appropriate placebo controls, otherwise amisleading conclusion about in vivo antioxidant effectsmight be drawn. However, DSS had significantly greater

onsumption

2 3 4

1.52 ± 0.67* 1.46 ± 0.67** 1.56 ± 0.59*

1.63 ± 1.18 1.64 ± 0.89 1.47 ± 0.89

t p < 0.01 versus 0 h placebo. NS between placebo and DSS for all time

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effects in decreasing total plasma F2-isoprostanes althoughnot urinary 8OHdG, than placebo.

Before the study reported here, we had performedanother study using a similar protocol (12 subjects), whichproduced a similar result; F2-isoprostanes were measuredin plasma and urine at 2, 6, and 24 h after consumptionof a DSS meal. Compared with the placebo, there was asignificant (p = 0.03) decrease in plasma total F2-isopros-tanes at 2 h, which returned to normal levels at 6 h, andremained normal at 24 h, suggesting that DSS acted rapid-ly in vivo. Therefore in the investigation reported in thepresent paper, we used more subjects and shortened thesampling time intervals to examine how quickly DSS affect-ed F2-isoprostanes levels, which was found to be between 2and 4 h. Levels of urinary F2-isoprostanes were increasedat 1 h after both placebo and DSS meals, but not subse-quently. This may be a general effect of eating, perhapsby promoting hydrolysis of esterified F2-isoprostanes andtheir subsequent excretion, but it could not explain the sig-nificant difference between DSS and placebo in plasmaF2-isoprostanes levels at 3 and 4 h time points. The levelof the free form may depend on phospholipase (PLA2)activity since esterified F2-isoprostanes are released frommembranes and lipoproteins by PLA2, into the circulation,and eventually excreted in the urine [12,15]. Recently, itwas proposed that intracellular platelet-activating factor(PAF) acetylhydrolases could also release esterifiedF2-isoprostanes from phospholipids in vivo [37]. Bothenzymes are associated with LDL, whose levels are affectedby eating. The clearance of free F2-isoprostanes in vivo israpid. It has been shown that when radiolabeled 8-iso-PGF2a

was infused in a male volunteer for 1 h it only took 4.5 h for75% of it to be excreted in urine and the half-life of F2-iso-prostanes was found to be approximately 16 min [38,39].

Thus, the antioxidant effect of DSS in loweringF2-isoprostane levels was greater than placebo and demon-strable in two different human studies. The antioxidanteffects of soy products have often been linked to the isoflav-ones. However, the concentrations of isoflavones are min-imal in soy sauce [40]. In any case, some authors foundthat dietary soy isoflavone given to healthy subjects hadno effect in lowering plasma F2-isoprostanes [41] or urinaryF2-isoprostanes [42], whereas others find [43] decreases inplasma F2-isoprostanes in diets high in soy isoflavone.Regardless, it is difficult to identify the true effect of the iso-flavones on F2-isoprostanes since the study designs [41–43]did not include a placebo meal.

The changes in HR and SBP after the placebo meal rep-resent the well-recognized vasodilatory effect of a meal,with its associated increase in splanchnic blood flow andsympathetic stimulation. DBP decreased further afterDSS than placebo, consistent with the notion that adecrease in oxidative stress could have physiologically rel-evant effects on the vasculature. As isoprostanes can them-selves act as vasoconstrictors [18,19], it is also possible thatthe difference reflects the greater reduction of isoprostanelevels after the DSS meal.

Urinary 8OHdG levels tended to decrease in both place-bo and soy sauce groups in this study but there was no sig-nificant difference between the two groups. 8OHdG isthought to originate from repair of oxidized DNA andthe DNA precursor pool [29,44]. It is thought that 8OHdGis one of the most reliable urinary biomarkers for DNAdamage since 8OHdG cannot be absorbed through thegut [6,30]. However, the data we have obtained suggest thateating can influence either its excretion and/or the creati-nine excretion used to standardize it. Nevertheless, 8OHdGexcretion was not decreased by DSS as compared with pla-cebo. It may be that DSS acts only on lipid peroxidation,or may take longer to protect against DNA oxidation, ifit does so at all.

To summarize, we find that dark soy sauce has arapid (3–4 h) antioxidant effect against lipid peroxida-tion in vivo, and that these effects are accompaniedby vasodilatory haemodynamic changes in vascularfunction consistent with antioxidant effects on the endo-thelium or vascular smooth muscle. The effects weredemonstrable in two separate human studies. The nextstep would be to examine whether long-term feedingof dark soy sauce could prolong the antioxidant activityand ameliorate the adverse consequences of oxidativestress. Several other important issues arise from ourresults. First, one must take care with vascular measure-ments and oxidative stress parameters since feedingalone has an effect. Second, urinary and plasma F2-iso-prostanes levels do not always move in parallel andboth should be determined to allow better interpretationof the data obtained.

Acknowledgment

We are grateful to the Biomedical Research Council(Grant 03/1/21/18/213) for their generous support of thisresearch.

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