Beer increases plasma antioxidant [606979]

Beer increases plasma antioxidant
capacity in humans
Andrea Ghiselli,* Fausta Natella,* Alessia Guidi,* Luigi Montanari,†
Paolo Fantozzi,†and Cristina Scaccini*
* National Institute of Nutrition, Free Radical Research Group, Rome, Italy;†Institute of Agri-
Food Industries, University of Perugia, Perugia, Italy
The positive association of a moderate intake of alcoholic beverages with a low risk for cardiovascular disease,
in addition to ethanol itself, may be linked to their polyphenol content. This article describes the effect of acuteingestion of beer, dealcoholized beer, and ethanol (4.5% v/v) on the total plasma antioxidant status of subjects,and the change in the high performance liquid chromatography profile of some selected phenolic acids (caffeic,sinapic, syringic, and vanillic acids) in 14 healthy humans. Plasma was collected at various times: before (T0),1 hour after (T1), and 2 hours after (T2) drinking. The study is part of a larger research planned to identify boththe impact of brewing on minor components potentially present in beer and their metabolic fate in humans. Beerwas able to induce a significant ( P,0.05) increase in plasma antioxidant capacity at T1 (mean 6SD: T0
1,353 6320mM; T1 1,578 6282mM), returning close to basal values at T2. All phenolic acids measured in
plasmatendedtoincreaseafterbeerintake(20%atT1,40%atT2).Syringicandsinapicacidreachedstatisticalsignificance ( P,0.05 by one-way analysis of variance-Fisher’s test) at T1 and T2, respectively. Plasma
metabolic parameters (glucose, total cholesterol, triglycerides, and uric acid) and plasma antioxidants(a-tocopherolandglutathione)remainedunchanged.Ethanolremovalimpairedtheabsorptionofphenolicacids,
which did not change over the time of the experiment, accounting for the low (and not statistically significant)increase in plasma antioxidant capacity after dealcoholized beer drinking. Ethanol alone did not affect plasmaantioxidant capacity or any of the antioxidant and metabolic parameters measured. (J. Nutr. Biochem. 11:
76–80, 2000) © Elsevier Science Inc. 2000. All rights reserved.
Keywords: polyphenols; antioxidants; human; beer; ethanol; TRAP
Introduction
Several epidemiologic studies have indicated the associa-
tion of moderate ethanol consumption with a reduction inall-cause mortality, particularly with a reduced risk ofcoronaryheartdisease(CHD).
1,2Withregardtotheformby
which ethanol is assumed, Grønbaek et al.1reported that
beer and wine are associated with a reduced mortality fromCHD, whereas spirits lead to an increased risk.
Ethanol is able to increase high density lipoprotein
(HDL)-cholesterol plasma level, to decrease platelet aggre-gation, and to enhance blood fibrinolysis, all events linkedto a low risk of CHD.
3–5However, the protective effects ofsome alcoholic beverages (wine and beer) may result from
(or be implemented by) its nonethanol component, preva-lently consisting of nonvitamin phenolics.
6–8
Once absorbed, phenolic compounds present in beer and
wine seem to be able to contribute to the total antioxidantcapacity of plasma (TRAP)
7,8and possibly of other body
compartments, thus reinforcing the defenses against theoxidative stress. However, there is little scientific informa-tion on their bioavailability, time and site of absorption,influence of ethanol, and metabolic fate in humans.
This study aimed (1) to study the effect of beer drinking
ontheantioxidantcapacityofhumanplasma,(2)toevaluatethe effect of ethanol on the absorption of phenolics, and (3)to study the time course of the absorption of selectedphenolics (caffeic, sinapic, syringic, and vanillic acids).
This article is part of a larger program focusing on the
nutritional properties of beer. A more in-depth analyticaland technological study in close connection with this articleis published elsewhere.
9Address correspondence to Dr. Andrea Ghiselli, Istituto Nazionale della
Nutrizione, Via Ardeatina 543 00178 Rome, Italy.Received March 16, 1999; accepted November 4, 1999.
J. Nutr. Biochem. 11:76–80, 2000© Elsevier Science Inc. 2000. All rights reserved. 0955-2863/00/$–see front matter655 Avenue of the Americas, New York, NY 10010 PII S0955-2863(99)00077-7

Methods and materials
Experimental protocol
Fourteen healthy, fasting nonsmokers (7 males and 7 females;
25–45 years) received in the morning 500 mL of beer. The beersused in this study were 4.5% ethanol lager types produced in Italy,coming from four representative brands and anonymously pro-vided. Each brand was administered to at least three subjects.Subjects were either nondrinkers or were social drinkers ( ,28 and
,14 g ethanol per day, for males and females, respectively), and
they were not taking dietary antioxidant supplements. Because theparameters evaluated in the four beer brands did not differsignificantly, data are presented as a single group. Blood wascollected before (T0) and 1 (T1) and 2 (T2) hours after beerdrinking.
To discriminate the effect of phenolic compounds from that of
ethanol, 500 mL of dealcoholized beer or a 4.5% solution ofethanol in tap water were administered to a smaller group of 7subjects (3 males and 4 females; 25–45 years). Ethanol wasremoved from beer by lyophilization and samples were reconsti-tuted with pure water. No significant changes in the concentrationof phenolic acids were observed after lyophilization (data notshown).
Blood samples were centrifuged and plasma was immediately
analyzed for total antioxidant capacity. Plasma samples for meta-bolic control (glycemia, total cholesterol, triglycerides, and uricacid) and for antioxidants (vitamin E and glutathione) were storedat280°C until the analysis.
Chemicals and reagents
Methanol and acetone [high performance liquid chromatography
(HPLC)grade]wereproducedbyCarloErba(Milan,Italy).Trolox(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) camefrom Aldrich Chemical Co. (Milwaukee, WI USA). 2,2 9-Diazo bis
amidine propane dihydrochloride (AAPH) came from Polyscience(Warrington, PA USA). All other chemicals came from SigmaChemical Co. (St Louis, MO USA).
Blood glycemia, total cholesterol, triglycerides, and urate were
measuredbycommercialkitspurchasedfromSigmaChemicalCo.Vitamin E was analyzed by HPLC as previously described;
10
glutathione was measured in plasma according to Neuschwander-Tetri and Roll.
11
The total antioxidant capacity of beer samples and plasma was
measured as previously described.12In brief, a fluorescent probe
(R-phycoerithrin, R-PE) loses its fluorescence when exposed to aconstant flow of peroxyl radicals, generated by the thermaldecomposition of AAPH. Solutions or body fluids containingantioxidantsareabletoinhibitR-PEoxidationinadose-dependentmanner. Therefore, by standardizing with a known antioxidant(Trolox), it is possible to quantify the antioxidant capacity of thesample. The antioxidant capacity of beer samples is expressed (asconventional for foods) as Trolox equivalents (TE), defined as theantioxidantcapacityof1.0mMTrolox.
13Theantioxidantcapacity
of plasma (TRAP) is expressed as mmoles of peroxyl radicals
trapped b y1Lo f plasma ( mM).
Measure of phenolic compounds in plasma
A reliable HPLC-electrochemical method has been set up for
measuring selected phenolic acids (caffeic, sinapic, syringic, andvanillic). To evaluate the rate of absorption, plasma phenolicpattern was determined at three different times (before and 1 and2 hours after drinking beer, dealcoholized beer, or hydroalcoholicsolution).
The method is able to evaluate plasma concentrations of
phenolic compounds as low as 0.2 nanogram/mL. Samples (500mL of beer or plasma) were acidified with 1.0 N HCl to pH 1.5 6
0.1. Three hundred mg of solid NaCl were added with mixing.Then samples were extracted with four 1.0-mL parts of HPLC-grade diethyl ether on a vortex for 2 minutes. The joined organiclayerswereevaporatedtodrynessandtheresiduewasdissolvedinmobilephase.Mobilephaseconsistedoftwosolutions:SolutionAwas 0.22 M acetic acid and Solution B was methanol. A binarygradient (ranging from 7 to 24% methanol) was applied to areverse phase ODS-2 (150 34.0 mm) analytical column main-
tained at 30°C.
The eluate was monitored with an ESA (Bedford, MA USA)
Coulochem II electrochemical detector equipped with a condition-ing cell (Model 5021) followed by the analytical cell (Model2011). Settings were as follows: the conditioning cell and the firstelectrode of the analytical cell were set at 2100 mV; the second
electrode was the analytical one and was set at 1600 mV. The
output of the detector was registered on a Perkin Elmer Turbo-chrom Chromatography workstation.
Statistical analysis
Data were expressed as mean and standard deviation. Statisticalanalysis was performed by one-way analysis of variance(ANOVA).
Results
As previously reported,14beer contains an appreciable
amountofphenoliccompoundsthatcontributetotheoverallantioxidant capacity of the product. The four brands testedhave an antioxidant capacity of 0.718 60.028 TE (ranging
from 0.634 to 0.800), which is much lower than that of redwines (19.8 TE) and of the same order of magnitude as theantioxidant capacity of white wines (0.950 TE).
7
In our study, the ingestion of 500 mL of beer in bolus
produced a statistically significant increase (approximately17%) in TRAP at T1 ( Table 1), decreasing close to basal
values after 2 hours (T2). Although ethanol removal did notaffect the original phenolic content of the beer, its admin-istration failed to induce a significant increase in plasmaTRAP. The administration of the hydroalcoholic solutiondid not influence plasma TRAP. All treatments did notaffect any of the metabolic parameters measured (totalcholesterol, triglycerides, glycemia, and uricemia) or theantioxidant markers (vitamin E and glutathione) ( Table 2).
The changes in antioxidant capacity did not reflect in
relevant differences in the plasma levels of the selectedphenolic acids after the ingestion of 500 mL of either wholebeer or dealcoholized beer ( Table 3). An unknown peak,
which is present in plasma even in fasting conditions,Table 1 Plasma TRAP values after administration of 500 mL in bolus
of beer, dealcoholized beer, or a 4.5% solution of ethanol in tap water
Whole beer
(mM)Dealcoholized
beer ( mM)Hydroalcoholic
solution ( mM)
Time 0 1,353 6320 1,341 6420 1,440 6261
Time 1 h 1,578 6282* 1,453 6483 1,400 6289
Time 2 h 1,290 6312 1,464 6537 —
*P,0.05 from time 0 by one-way analysis of variance (Fisher’s test).
Values are expressed as mean 6SD.
TRAP–total antioxidant capacity of plasma.Beer increases plasma antioxidant capacity in humans: Ghiselli et al.
J. Nutr. Biochem., 2000, vol. 11, February 77

increases after ingestion of both whole and dealcoholized
beer (approximately 7- and 4-fold, respectively, expressedasarea).Thiscompound,whichisnotpresentinbeer,couldbe a common metabolite of phenolics, still maintaining ahigh antioxidant capacity. In fact, the peak coincides withthe maximum of the antioxidant capacity (T1); ( Figure 1).
The sum of the phenolic acids we measured tended to
increase in the case of the whole beer (24 mg/mL at
baseline, 33 mg/mL at T1, and 42 mg/mL at T2), whereas
that of dealcoholized beer seemed to have no effect (34mg/mL at baseline, 28 mg/mL at T1, and 29 mg/mL at T2);
(Table 3).
Discussion
Large population studies have shown a U-shaped relation
between alcohol and mortality, the mortality rate beinglower in people reporting moderate alcohol intake than ineither nondrinkers or heavier drinkers ( .34 g alcohol/
day).
15,16Moreover, moderate consumption of alcoholic
beverages, in particular wine and beer, is reported to beassociated to a diminished mortality from cardiovasculardiseases.
1,17
Both ethanol (at low doses) and phenolic compounds
play important and different roles in protection againstCHD. Platelet aggregation, for example, which is themechanism underlying myocardial infarction, is reported to
be affected by ethanol,
4but also by some phenolic com-
pounds present in wine and beer.6,18Moreover, ethanol, in
addition to its direct effect on platelet function, HDLmetabolism, and fibrinolysis (all involved in the pathogen-esis of cardiovascular diseases), could play an importantindirect role in the absorption of phenolic compounds.Phenolicsarearomaticcompoundsthatarehardlysolubleinwater, but easy soluble in ethanol. The increased solubilityof these compounds in hydroalcoholic solutions may affectthe rate and the amount of their absorption. This is sup-ported by the results of Miyagi et al,
19who found a
significant in vitro inhibition of human low density lipopro-tein (LDL) oxidation in the presence of both red wine andgrape juice. In vivo ingestion of red wine was able tosignificantly protect LDL, suggesting that flavonoids in redwine can be absorbed more efficiently than flavonoids ingrape juice.
Our results on the plasma antioxidant activity seem to be
in agreement with the data reported by Miyagi et al.
19In
fact, although the intake of whole beer significantly in-creased plasma TRAP, dealcoholized beer showed just aslight tendency to increase the antioxidant capacity, andethanol (as expected) had no effect.
Our data on phenolic absorption suggest that whole beer
is able to transfer its phenolic compounds (at least theTable 2 Plasma values of some metabolic parameters after administration of 500 mL in bolus of beer, dealcoholized beer, or a 4.5% (v/v) solution
of ethanol in tap water
Whole beer Dealcoholized beer Hydroalcoholic solution
Time 0 Time 1 h Time 2 h Time 0 Time 1 h Time 2 h Time 0 Time 1 h Time 2 h
Total cholesterol, mg/dL 192 628 192 630 191 628 162 626 171 627 175 638 213 633 200 638 —
Triglycerides, mg/dL 107 688 119 673 101 660 70 623 73 629 69 621 154 6125 155 6112 —
Glycemia, mg/dL 97 616 93 623 86 689 4 618 78 612 99 63 101 629 102 618 —
Uric acid, mg/dL 4.5 61.8 5.0 61.6 5.2 61.5 4.6 61.1 5.1 61.0 5.2 61.9 4.5 61.4 4.6 62.0 —
Vitamin E, mg/dL 10.7 62.5 10.4 62.5 10.0 62.0 9.3 61.4 10.1 61.5 9.6 61.5 12.3 61.4 10.5 62.0 —
Glutathione, mM 5.9 61.5 6.1 60.8 — 5.6 60.6 5.8 61.1 — 4.9 60.7 5.3 61.6 —
There are no statistically significant differences by one-way analysis of variance (Fisher’s test) among times in the three experimental groups.
Values are expressed as mean 6SD.
Table 3 Plasma levels of phenolic acids after drinking of whole (W) and dealcoholized (D) beer
PhenolicsPlasma T0
(ng/mL)Plasma T1
(ng/mL)Plasma T2
(ng/mL) P
Caffeic acid W 13.7 611.1 16.8 624.6 22.8 627.0 0.7068
D 15.6 614.9 7.7 65.1 6.2 64.7 0.4360
Sinapic acid W 1.5 62.1 1.9 62.5 5.8 65.8* 0.0761
D 0.4 60.8 0.2 60.4 0 0.0961
Syringic acid W 0.9 60.9 5.1 65.9* 1.9 61.9 0.7575
D 7.0 65.5 8.2 66.3 7.9 67.3 0.9507
Vanillic acid W 7.6 66.5 8.8 62.3 11.4 610.6 0.5760
D 10.8 66.6 11.5 66.5 14.7 60.9 0.7548
Unknown peak†W4 9 67 349 665 208 636 0.0003
D7 3 613 296 660 221 625 0.0041
Sum W 23.7 612.1 32.7 625.5 41.9 631.3 0.3466
D 33.8 620.7 27.6 611.5 28.7 61.6 0.8190
*P,0.05 from time 0 by one-way analysis of variance (Fisher’s test).
†The values of the unknown peak are expressed as area *106.
Values are expressed as means 6SD of 8 (W) and 5 (D) subjects.Research Communications
78 J. Nutr. Biochem., 2000, vol. 11, February

molecules detectable in our analytical conditions) to body
fluids more efficiently than dealcoholized beer ( Table 3).
Moreover, although the behavior of the unknown peakfollows that of plasma TRAP, which peaks at T1, plasmaphenolics continue to increase ( Table 1). Although other
compounds that are present in beer and not measured in thepresent study may contribute to the plasma antioxidantcapacity, this peak could represent a key molecule in themodulation of plasma antioxidant capacity. Its presence inplasma even in fasting conditions, and its increase afterphenol’s intake, suggests that the peak can represent eithera long-living metabolite of phenolic compounds or anunidentified molecule endowed with antioxidant activity, assuggested by its electrochemical detectability. The un-known peak also increases to a minor extent in the absenceof ethanol (dealcoholized beer). In this case, it is alsopossible to note a parallel, but not significant, increase inplasma TRAP ( Figure 1).
Gorinstein et al.
20reported a significant beneficial anti-
oxidant effect in rats supplemented for 4 weeks with bothalcohol-containing and alcohol-free beer. The discrepancywith our results in the effect of alcohol-free beer canprobably be explained by the modality of intake: acute, asour case, versus chronic.
Similar results on the variation of plasma antioxidant
capacity after acute administration have been reported bydifferent authors for white wine.
7,21Although the method-
ologies employed were slightly different, the combinedresults indicate a consistent increase of plasma antioxidantcapacity induced by “whole” white wine
21and a lack of
effect by the same amount of “dealcoholized” white wine.7
Inconclusion,ourfindingsindicatedthatbeer,whichhasa moderate antioxidant capacity coupled with a low ethanol
content, is an alcoholic beverage that is able to improveplasma antioxidant capacity without the negative effectsproduced by high doses of ethanol. In fact, although theamountofethanolpresentin500mLofbeer(approximately18 g) did not induce any appreciable change in the markersofmetaboliccontrol(triglycerides,uricacid,andglycemia),it is able to facilitate the transfer of the antioxidant capacityfrom beer to body fluids, probably through the increase ofthe absorption of phenolic compounds.
Acknowledgments
The authors thank Vincenzo Gentili and Maurizio Di Felicefortheirtechnicalassistance.Theresearchwassupportedbygrant of ASSOBIRRA (Italian Brewer’s Association)Rome.
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Figure 1 Relationship between the in-
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