A Quercetin Supplemented Diet Does Not Prevent Cardiovascular by nikeborome


									        The Journal of Nutrition
        Nutrition and Disease

A Quercetin Supplemented Diet Does Not
Prevent Cardiovascular Complications
in Spontaneously Hypertensive Rats1
Justin Carlstrom,2,5 J. David Symons,2,5* Tzu Ching Wu,2 Richard S. Bruno,4 Sheldon E. Litwin,3
and Thunder Jalili2,6*
  College of Health, 3Division of Cardiology, University of Utah, Salt Lake City, UT 84112 and 4Department of Nutritional
Sciences, University of Connecticut, Storrs, CT 06269

Diets high in quercetin may decrease the risk of developing cardiovascular disease. We tested whether quercetin delays or
reduces the severity of hypertension, vascular dysfunction, or cardiac hypertrophy in the spontaneously hypertensive rat
(SHR). Normotensive, 5-wk–old SHR consumed standard (n ¼ 18) or quercetin-supplemented diet (1.5 g quercetin/kg diet,

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n ¼ 22, SHR-Q) for 5 or 11 wk. Wistar Kyoto rats (WKY, n ¼ 19), fed a standard diet, served as controls. At 16 wk, plasma
quercetin, measured by HPLC, was 2.09 6 0.33 mmol/L in SHR-Q and below assay detection limits in SHR and WKY rats.
At 10 and 16 wk of age, arterial blood pressure and heart weight:body weight were not different between SHR and SHR-Q.
At 16 wk, cardiac function (echocardiography), vascular morphology (hematoxylin and eosin staining of aortae), and
resistance and conductance vessel reactivity (wire myography) was unchanged in SHR vs. SHR-Q. Thus, a quercetin-
supplemented diet does not delay the onset or lessen the severity of cardiovascular complications that develop in SHR.
These findings contrast with previous reports of cardiovascular protection when quercetin was delivered via oral gavage.
To determine whether the efficacy of quercetin depends on its method of delivery, 15-wk–old SHR were given quercetin
(10 mg/kg) once daily via oral gavage for 4 consecutive days. Arterial blood pressure (mm Hg) was lower in gavaged SHR
(148 6 5) than in SHR-Q (162 6 2, P , 0.02) and SHR (168 6 3, P , 0.001). These data suggest that mode of delivery is a
critical determinant in whether quercetin provides cardiovascular benefits. J. Nutr. 137: 628–633, 2007.

                                                                                   and cardiac hypertrophy in the adult spontaneously hyperten-
Hypertension is a strong risk factor for the development of heart                  sive rat (SHR)7 (11). In the second, quercetin treatment delayed
failure, myocardial infarction, kidney failure, stroke, and death                  the onset and attenuated the severity of hypertension produced
(1). Although effective pharmacological strategies for the treat-                  by 6 wk of nitric oxide synthase inhibition using NW-nitro-L-
ment of hypertension exist, there is a great deal of interest in                   arginine methyl ester (L-NAME) (10). Given that quercetin
using dietary agents (e.g., phytochemicals) to prevent or reduce                   lowered indices of oxidative stress in both studies, the antiox-
hypertension. Epidemiological studies indicate that quercetin                      idant potential of quercetin was hypothesized to have contrib-
consumption is inversely related to the risk for cardiovascular                    uted to the protective effects.
disease (2–8). In addition, laboratory investigations using sev-                       SHR are normotensive at ;6 wk of age and develop hyper-
eral experimental animal models suggest that quercetin is bene-                    tension, cardiac hypertrophy, vascular dysfunction, and in-
ficial in this regard. For example, we showed that hypertension                     creased oxidative stress by ;12 wk (12,13) and progress to heart
was normalized and cardiac hypertrophy was attenuated in rats                      failure by 18–24 mo (14). In this study we sought to determine
that consumed quercetin-supplemented vs. a standard diet fol-                      whether a quercetin-enriched diet delays the onset of hyperten-
lowing abdominal aortic banding to produce pressure overload                       sion and cardiac growth in SHR. Furthermore, we tested
(9). Two additional studies reported protective effects of quer-                   whether a quercetin-enriched diet attenuates the severity of hy-
cetin when administered once daily via oral gavage (10,11). In                     pertension, cardiac growth, vascular remodeling, vascular dys-
the first, quercetin treatment regressed established hypertension                   function, and oxidant stress that develops in the adult SHR.

  Supported by the University of Utah Research Council (T.J.), and AHA, National
Affiliate, Scientist Development Grant (0130099N) (J.D.S.).
5                                                                                  7
  Authors contributed equally to this project.                                       Abbreviations used: AAC, abdominal aortic constriction; ACh, acetylcholine;
  Conflict of interest disclosure: Patent pending on the use of quercetin as an     L-NAME, NW-nitro-L-arginine methyl ester; L-NMMA, NG monomethyl-L-arginine;
antihypertensive agent to T.J.                                                     MDA, malondialdehyde; NE, norepinephrine; SHR, spontaneously hypertensive
* To whom correspondence should be addressed. E-mail: thunder.jalili@utah.         rat; SHR-Q, spontaneously hypertensive rat fed 0.15 g quercetin/kg diet; SNP,
edu or 00295675.acs.unc@hsc.utah.edu.                                              sodium nitroprusside; WKY, Wistar Kyoto rat.

628                                                                                                  0022-3166/07 $8.00 ª 2007 American Society for Nutrition.
                                        Manuscript received 1 September 2006. Initial review completed 26 October 2006. Revision accepted 29 November 2006.
Materials and Methods                                                        for 5 min. Then, a portion of the supernatant was removed, dried under
                                                                             nitrogen, reconstituted with mobile phase A, and injected onto the HPLC.
Animals and diets. All protocols were approved by the University of              The injected sample (50 mL) was separated (MD-150 column, ESA;
Utah Institutional Animal Care and Use Committee. Four-week–old              150 mm 3 3.0 mm i.d., 3 mm) and detected using a binary gradient
male SHR and Wistar-Kyoto rats (WKY) were obtained from Harlan               (0.6 mL/min) with Coularry detector settings of 100, 200, 300, and
and acclimated for 1 wk. SHR were given free access to standard rodent       400 mV. The following gradient profile was used: 0–35 min, linear
diet (SHR) or diet enriched with purified quercetin aglycone (SHR-Q;          gradient from 10–80% B; 35–37 min, 80–10% B; and 37–50 min, 10%
1.5 g quercetin/kg diet) for 5 (SHR, n ¼ 6; SHR-Q, n ¼ 6) or 11 wk           B to allow for sufficient system equilibration prior to the next sample
(SHR, n ¼ 12; SHR-Q, n ¼ 16). WKY rats were allowed free access to           injection. The identification of quercetin (;17.5 min) was achieved
standard rodent diet for 5 (n ¼ 6) or 11 (n ¼ 13) wk. Therefore, all rats    by comparing their retention times and electrochemical responses to
were studied at 10 or 16 wk of age. Diets were prepared based on             purified standards (Sigma).
AIN-93 recommendations (Research Diets) (15). The quercetin dose
was chosen because we previously demonstrated that it: 1) produces           Gavage protocol. To determine whether the method used to administer
detectable plasma and liver levels, 2) does not affect blood pressure or     quercetin affects the cardiovascular outcomes, 15-wk–old male WKY
myocardial function in normotensive rats, but 3) attenuates hypertension     rats (n ¼ 3) and SHR (n ¼ 3) were given quercetin aglycone (10 mg/kg)
and cardiac hypertrophy in rats subjected to 14 d of pressure overload       suspended in 1% methylcellulose (Sigma) once daily via oral gavage for
evoked by abdominal aortic constriction (9).                                 4 consecutive days as described (11). Twenty-four hours after the last
                                                                             gavage (i.e., d 5), blood pressure was measured and arterial samples were
Myocardial function. Echocardiography was performed at 16 wk using           taken for analysis of plasma quercetin concentrations.
methods we have described (9,16).
                                                                             Statistical analyses. A 1-way ANOVA was used to detect differences
Arterial blood pressure and tissue sampling. Rats were evaluated at          among groups at 10 and 16 wk of age, using SPSS, version 10. If a
10 and 16 wk of age. A fluid-filled catheter was placed into the caudal        significant P-value was obtained, post-hoc (least significant differences)
artery of rats anesthetized with 2–5% isoflurane, and, 60 min after           tests were performed to identify individual group differences. In case of
regaining consciousness, arterial blood pressure and heart rate were         unequal variances, Tamhane’s T2 test was performed. Significance was

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measured over ;20 cardiac cycles (Biopac Systems) (9,17–19). Next,           accepted at P , 0.05. Dose response curves generated from vascular
rats were anesthetized, the chest opened, and were killed by removing the    function experiments were compared using 2-way (dose 3 experimental
heart. Sections of liver, segments of thoracic aorta, and mesenteric         group) repeated measures ANOVA. Planned comparisons were made at
arteries, which were removed for analysis as previously detailed (9).        each drug dose to determine whether differences existed among groups.
                                                                             Results are presented as means 6 SEM. Vasorelaxation to ACh and SNP
Measurement of vascular reactivity. Segments of thoracic aortae,             is expressed as the percentage of relaxation from precontraction tension.
mesenteric arteries, and coronary arteries from 16-wk–old rats were          Vasocontractile responses to NE, L-NMMA, KCl, and endothelin-1 are
mounted on wire-type myographs as described previously (9,18). For           presented as mg of developed tension or as the percentage of increase
thoracic aortae and mesenteric arteries, concentration-response curves to    from precontraction tension (L-NMMA only).
acetylcholine (ACh, 10 nmol–1 mmol/L) and sodium nitroprusside (SNP,
10 nmol–10 mmol/L) were performed after vessels were precontracted
with 1 mmol/L norepinephrine (NE) to estimate endothelium-dependent          Results
and independent vasorelaxation, respectively. Dose-response curves to
NE (100 nmol–10 mmol/L) and potassium chloride (KCl, 20–100 mmol/L)          Plasma quercetin levels, blood pressure, cardiac hyper-
were completed to assess receptor-mediated and nonreceptor-mediated          trophy, and aortic morphology. Plasma quercetin concentra-
vasoconstriction, respectively. In aortae, basal nitric oxide synthase       tions (unconjugated, b-glucuronidated, and sulfated forms of
activity was estimated by adding NG monomethyl-L-arginine (L-NMMA,           quercetin) were below assay detection limits in SHR and WKY
10 mmol/L) to vessels precontracted using 1 mmol/L NE. After the             rats fed control diets, but markedly elevated in SHR-Q (Table 1).
maximal response to L-NMMA was recorded, the degree of nitric oxide          To determine whether quercetin delayed the onset or lessened
synthase inhibition was quantified by adding 1 mmol/L ACh and noting          the severity of hypertension, we compared arterial blood pres-
the resultant vasorelaxation. In coronary arteries, vasorelaxation to ACh    sure among groups at 10 and 16 wk. Systolic and diastolic blood
and SNP was evaluated on arteries precontracted with endothelin-1
                                                                             pressures (Table 1), and mean arterial pressure were increased to
(;3 3 100 nmol/L). All protocols were separated by ;30 min and stan-
dard time, volume, and vehicle controls were performed (9,18).
                                                                             a similar degree in SHR and SHR-Q compared with WKY rats at
                                                                             both time points. Heart rate was higher in SHR and SHR-Q than
Vascular morphology. Four mm thick thoracic aortae sections from             WKY rats at 16 wk (Table 1).
16-wk–old rats were prepared as previously detailed (9,18). Images were          To assess whether quercetin delayed the onset or lessened the
taken with a microscope (Nikon E6000) equipped with a digital camera         severity of cardiac hypertrophy, heart weight was normalized to
(Q imaging, Micro Publisher 5.0 RTV) using 23 objective.                     body weight at both time points. Although heart weight:body
                                                                             weight was not different among groups at 10 wk, it was similarly
Estimates of hepatic oxidant load. Lipid oxidation was determined in
segments of liver by fluorescence detection of malondialdehyde (MDA)
                                                                             increased in SHR and SHR-Q compared with WKY rats at 16
equivalents (nmol/mg protein) as previously described (18,20). Protein       wk (Table 1).
oxidation was estimated by quantifying protein carbonyls (nmol/mg                Vascular remodeling resulting from chronic hypertension was
protein) via spectrophotometric quantification of the dinitrophenylhy-        assessed by examining thoracic aortae of 16-wk–old rats. Med-
drazine adduct (18,20). For all assays, protein concentrations were deter-   ial thickness, lumen diameter, and mean cross-sectional area
mined using bovine serum albumin as the standard (21).                       were increased to the same extent in SHR and SHR-Q compared
                                                                             with WKY rats (Table 2, Fig. 1). Collectively, these data dem-
Plasma quercetin analysis. Plasma quercetin (unconjugated,
                                                                             onstrate that although plasma quercetin was elevated in SHR-Q,
b-glucuronidated, and sulfated forms) were extracted and measured by
                                                                             it did not delay the onset or lessen the severity of arterial hyper-
HPLC-Coularry as described (22), with modifications. Briefly, plasma
(200 mL) was mixed with 375 U of b-glucuronidase and 10 U sulfatase in       tension, cardiac hypertrophy, or aortic remodeling.
0.4 mol/L NaH2PO4 (pH 5.0), 20 mL of 2% ascorbic acid (w:v) and
2.5 mmol/L DTPA in 0.4 mol/L NaH2PO4 (pH 5.0). Samples were in-              In vivo myocardial function. Echocardiography was per-
cubated (37°C, 45 min) with gentle agitation and extracted with aceto-       formed prior to the terminal experiments at 16 wk to determine
nitrile by vigorous mixing, followed by centrifugation (14,000 3 g, 4°C),    the pattern of cardiac hypertrophy and to assess whether
                                                                                                          Quercetin and hypertension in SHR       629
TABLE 1         Morphological characteristics of rats fed control or quercetin supplemented diets beginning at 5 wk of age1

                                            WKY                   SHR                  SHR-Q                   WKY                      SHR                SHR-Q

n                                            6                       6                      6                     13                  12                     16
Age, wk                                      9                      11                     10                     16                  16                     16
Heart wt, g                             0.97 6   0.03a         1.18 6 0.2b          1.03   6 0.02a         1.04   6 0.2a         1.26 6 0.03b           1.20 6 0.02b
Body wt, g                              245 6    5a            297 6 4b             263    6 4a            306    6 10           333 6 6                325 6 5
Heart wt:body wt, mg:g                  3.96 6   0.11          3.94 6 0.10          4.03   6 0.10          3.40   6 0.08a        3.79 6 0.09b           3.72 6 0.10b
Heart rate, beats/min                   398 6    10            410 6 8              436    6 15            411    6 13a          473 6 11b              451 6 9b
Caudal blood pressure, mm Hg
   Systolic                              127 6 3a              162 6 3b              155 6 3b              140    6 2a           182    6 5b             184 6 2b
   Diastolic                             111 6 3a              148 6 3b              142 6 3b              117    6 2a           158    6 3b             151 6 2b
Arterial pressure, mm Hg                 115 6 1a              152 6 2b              147 6 4b              125    6 2a           168    6 3b             162 6 2b
Liver MDA, pmol/mg                                                                                         116    6 8a           181    6 48a,b          228 6 17b
Liver protein carbonyls, nmol/mg                                                                           2.47   6 0.23         3.04   6 0.14          2.68 6 0.22
Plasma total quercetin, mmol/L                                                                                    ND                    ND              2.09 6 0.33
 Values are means 6 SEM. Means in a row with superscripts without a common letter differ, P , 0.05. MDA, malondialdehyde equivalents. Total plasma quercetin composed of
unconjugated, b-glucuronidated and sulfated forms extracted from plasma. ND, not dectectable.

myocardial function is improved in SHR-Q vs. SHR. The hearts                          degree of nitric oxide synthase enzyme inhibition was similar
of SHR and SHR-Q had a greater mean wall thickness (P ,                               among groups. Aortae were precontracted to the same degree
0.01), and interventricular septum systolic dimension (P , 0.03)                      among groups before adding ACh, SNP, or L-NMMA. Maximal

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than WKY rats and tended to have a smaller left ventricular                           responses to nonreceptor mediated vasocontraction (i.e., KCl)
diastolic dimension (P ¼ 0.09) (Fig. 2, Table 3). The posterior                       and receptor-mediated vasocontraction (i.e., NE) did not differ
wall dimension and fractional shortening did not differ among                         among groups (data not shown).
the groups (Table 3). Taken together, these data show that
concentric cardiac hypertrophy and myocardial function are                            Mesenteric arteries. Maximal relaxation produced by ACh
similar in 16-wk–old SHR and SHR-Q.                                                   was less in mesenteric arteries from SHR and SHR-Q compared
                                                                                      with WKY rats (Fig. 3B, upper panel). Maximal endothelium-
Vascular reactivity. To determine whether quercetin could                             independent responses to 1 mmol/L SNP did not differ among
prevent or lessen vascular dysfunction, ex vivo examinations of                       WKY rats, SHR, and SHR-Q. Mesenteric arteries were more re-
aortic (conductance sized vessel), mesenteric (resistance sized                       sponsive in SHR and SHR-Q than in WKY rats at 2 of 7 doses
vessel), and coronary (resistance sized vessel) arterial reactivity                   administered [i.e., 1 mmol/L and 10 mmol/L (Fig. 3B, lower
were performed.                                                                       panel)]. Mesenteric arteries were precontracted to the same degree
                                                                                      among groups before adding ACh or SNP. Nonreceptor-mediated
Aortae. Maximal ACh-evoked vasorelaxation in aortae was                               vasocontraction (i.e., KCl) and receptor-mediated vasocontraction
reduced similarly in SHR and SHR-Q compared with WKY rats,                            (i.e., NE) did not differ among the groups (data not shown).
indicating that quercetin did not prevent or lessen endothelium-
dependent dysfunction (Fig. 3A, upper panel). Maximal                                 Coronary arteries. Vasorelaxation evoked by ACh was blunted
endothelium-independent responses to SNP (1 mmol/L) did not                           in SHR and SHR-Q compared with WKY rats at concentrations
differ among groups, indicating that vascular smooth muscle                           between 100 nmol/L and 10 mmol/L (Fig. 3C, upper panel).
function was intact (Fig. 3A, lower panel). Aortae from SHR                           Endothelium-independent vasorelaxation in response to SNP
were more responsive to SNP than SHR-Q and WKY rats at 100                            was similar among WKY, SHR, and SHR-Q (Fig. 3C, lower
nmol/L. Nitric oxide synthase inhibition of precontracted seg-                        panel). Coronary arteries were precontracted to the same degree
ments of aortae produced a similar degree of tension develop-                         among groups before adding ACh or SNP. Taken together,
ment among groups, indicating basal activity of this enzyme                           dysfunction observed in resistance and conductance-sized vessels
was not affected by quercetin treatment. ACh-evoked relaxation                        was not improved by quercetin treatment.
in the presence of L-NMMA was performed to verify that the

TABLE 2         Morphological parameters of aortae from
                16-wk–old rats fed control or quercetin
                supplemented diets beginning at 5 wk of age1,2

                       WKY                  SHR                   SHR-Q
                             a                      b
MT, mm               80 6 1                93 6 1                93   6 2b
ED, mm            1436 6 13a            1710 6 7b             1651    6 47b
LD, mm            1131 6 15a            1388 6 5b             1338    6 34b
MCSA, mm2       628,860 6 13,402a     782,305 6 10,299b     741,253   6 59,848b
M:L                7.06 6 0.14           6.73 6 0.10           6.72   6 0.21
  Values are means 6 SEM. Means in a row with superscripts without a common           Figure 1 Hematoxylin and eosin stained aortic cross sections from 16-wk–old
letter differ, P , 0.05, n ¼ 8.                                                       Wistar-Kyoto rats (WKY), spontaneously hypertensive rats that consumed
  MT, medial thickness; ED, external diameter; LD, lumen diameter; MCSA, mean         standard rodent diet (SHR), and spontaneously hypertensive rats that consumed
cross sectional area; M:L, media:lumen ratio.                                         quercetin-supplemented (0.15%) diet (SHR-Q), n ¼ 8.

630    Carlstrom et al.
                                                                                            16 wk. These results are in contrast to 2 previous studies where
                                                                                            protective effects of quercetin supplementation were reported
                                                                                            (10,11). In those investigations, quercetin was delivered once
                                                                                            daily via oral gavage. To determine whether the efficacy of
                                                                                            quercetin was dependent upon the method of delivery, 15-wk–
                                                                                            old male SHR and WKY were given quercetin via oral gavage for
                                                                                            4 consecutive days. Mean arterial blood pressure (mm Hg) was
                                                                                            greater in quercetin gavaged SHR (148 6 5) than in gavaged
                                                                                            WKY rats (120 6 6) but was still significantly lower than both
                                                                                            SHR (168 6 3) and SHR-Q (162 6 2). Plasma total quercetin
                                                                                            (mmol/L) was similar in SHR and WKY gavaged with quercetin
                                                                                            (SHR ¼ 0.61 6 0.01, WKY ¼ 0.80 6 0.10). Heart weight (1.26 6
                                                                                            0.01 mg) and heart:body weight (3.70 6 0.04, mg/g) in the
                                                                                            quercetin-gavaged SHR was similar to SHR and SHR-Q (Table
                                                                                            1). These data are in agreement with a previous report (11)
                                                                                            demonstrating that oral gavage of quercetin is efficacious in
                                                                                            reducing blood pressure in SHR.

                                                                                            We hypothesized that if spontaneously hypertensive rats were
                                                                                            fed quercetin-enriched diets prior to the onset of hypertension,
                                                                                            i.e., at 5 wk, then the expected increases in arterial pressure and

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                                                                                            cardiac growth might be delayed, and/or the extent of these and
                                                                                            related cardiovascular complications would be less severe.
                                                                                            Rationale for our hypotheses was based on results from several
                                                                                            investigations suggesting that quercetin: 1) attenuated the de-
                                                                                            velopment of hypertension and cardiac hypertrophy in response
                                                                                            to 14 d of pressure overload hypertrophy evoked by abdominal
                                                                                            aortic constriction (9) or 6 wk of nitric oxide synthase inhibition
                                                                                            (10); and 2) regressed established hypertension and cardiac
Figure 2 M-mode echocardiograph from 16-wk–old Wistar-Kyoto rats (WKY),                     hypertrophy in 19-wk–old SHR (11). Contrary to our hypoth-
spontaneously hypertensive rats that consume standard rodent diet (SHR), and spon-
taneously hypertensive rats that consume quercetin-supplemented (0.15%) diet (SHR-
                                                                                            eses, arterial blood pressure and cardiac growth were similar
Q), n ¼ 8. IVS, intraventricular septum; LV, left ventricular cavity; PW, posterior wall.   between SHR-Q and SHR compared with WKY rats at 10 wk.
                                                                                            Furthermore, the severity of hypertension, cardiac hypertrophy,
Oxidative stress. Previous studies have reported a reduction in                             vascular remodeling, vascular dysfunction, and oxidant stress
oxidative stress in quercetin-treated rats (10,11,23), therefore,                           that developed in 16-wk–old SHR vs. WKY rats was not at-
we assessed whether increases in oxidant stress usually present in                          tenuated in SHR-Q rats. Therefore, cardioprotection was not
the SHR were attenuated in SHR-Q compared with SHR. Liver                                   provided in 10 or 16-wk–old SHR that consumed quercetin
protein carbonyls and MDA were measured in segments of liver                                enriched diet. In contrast to our results, a recent study by
from 16-wk–old rats. Compared with WKY rats, MDA was                                        Sanchez et al. (24) demonstrated that 5-wk–old SHR treated by
elevated in SHR-Q and tended to be higher in SHR (P ¼ 0.06;                                 oral gavage with quercetin for 13 wk had lower blood pressure,
Table 1). Protein carbonyl levels tended to be greater in SHR                               heart rate, and improved endothelium dependent arterial relax-
than in SHR-Q and WKY rats (P ¼ 0.06) (Table 1).                                            ation compared with untreated SHR.
                                                                                                Possible reasons for the lack of efficacy of quercertin in the
Gavage protocol. We observed no differences in blood pressure                               current study compared with others (10,11,24) could be related
or cardiac hypertrophy in SHR-Q compared with SHR at 10 or                                  to: 1) the magnitude and severity of arterial hypertension and
                                                                                            cardiac hypertrophy, 2) the particular form of quercetin admin-
TABLE 3          Echocardiographic assessment of cardiac structure                          istered, and/or 3) the method of quercetin delivery. First, re-
                 and function from 16-wk–old rats fed control                               garding the magnitude of hypertension, this flavonoid was
                 or quercetin supplemented diets beginning                                  shown to attenuate systolic blood pressures of greater (i.e.,
                 at 5 wk of age1,2                                                          ;200 mm Hg) (9,11) and lesser (;170 mm Hg) (10) severity
                                                                                            than experienced by 16-wk–old SHR (i.e., ;180 mm Hg) in the
                         WKY                       SHR                     SHR-Q            present study. Based on these data, we believe the degree of
FS, %                  56 6 4                   49 6 4                    46 6 2            hypertension in our rats was neither too severe nor insufficient
LVDd, mm              7.2 6 0.2                6.4 6 0.2                 6.1 6 0.5          for a beneficial effect of quercetin to be realized.
IVSd, mm              2.1 6 0.1a               2.6 6 0.1b               2.5. 6 0.1b             Second, efficacy of quercetin could depend on the particular
PWd, mm               1.9 6 0.1                2.4 6 0.1                 2.3 6 0.2          form utilized (e.g., quercetin aglycone, rutin, or quercetin-3-
MWT                  0.20 6 0.01a             0.25 6 0.01b              0.24 6 0.01b        glucoside). We enriched the rat diet with quercetin aglycone in a
                                                                                            previous study (9) and in the present investigation. Because we
  Values are means 6 SEM. Means in a row with superscripts without a common                 (9) and others (10,11,24,25) demonstrated cardiovascular pro-
letter differ, P , 0.05, n ¼ 8.
  FS, fractional shortening; LVDd, left ventricular diastolic dimension; IVSd, interven-
                                                                                            tection using quercetin aglycone, this choice of isoform is not
triclular septum diastolic thickness; PWd, posterior wall diastolic thickness; MWT,         likely responsible for the lack of effect observed in the present
mean wall thickness.                                                                        study.
                                                                                                                      Quercetin and hypertension in SHR    631
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Figure 3 Endothelium-dependent (top panels) and independent (bottom panels) vasorelaxation of aortae (A), mesenteric (B), and coronary (C) arteries from 16-wk–
old Wistar-Kyoto rats (WKY, n ¼ 13), spontaneously hypertensive rats that consume standard rodent diet (SHR, n ¼ 12), and spontaneously hypertensive rats that
consume quercetin-supplemented (0.15%) diet (SHR-Q, n ¼ 16). Results are means 6 SEM. *Different from SHR and SHR-Q, P , 0.05.

    Finally, the method of quercetin delivery may be an impor-                   24 h after quercetin supplementation is terminated (26,27). Even
tant consideration. In contrast to our method of incorporating                   though plasma quercetin concentrations obtained in this study
quercetin directly into the diet and allowing ad libitum con-                    were low in SHR 24 h after gavage, previous literature indicates
sumption, others administered quercetin once daily via oral                      that peak quercetin levels achieved after gavage are likely much
gavage (10 mg/kg) (10,11,23,24). Although plasma and tissue                      higher than those achieved in diet-supplemented SHR-Q. In this
quercetin concentrations were not reported in those studies, the                 regard, da Silva et al. (28) reported plasma quercetin concen-
protective effects were clearly evident. Specifically, when 14-wk–                trations of 9.6 mmol/L 6 h after 10 mg/kg quercetin was de-
old SHR were treated for 1 wk with quercetin via oral gavage,                    livered via oral gavage to the rats. Unfortunately, the lack of a
systolic blood pressure was reduced (;218 mm Hg) compared                        comprehensive evaluation regarding the time-related decay of
with SHR treated with vehicle (;230 mm Hg)(11). Another                          plasma quercetin concentrations after the last gavage is a lim-
study from the same laboratory showed that compared with                         itation of our study. However, the findings of da Silva et al. (28)
vehicle administration, the onset of L-NAME-induced hyper-                       as well as the cardiovascular protection observed in SHR after
tension was delayed, and the severity of hypertension and car-                   5 wk of quercetin gavage by Duarte et al. (11), strongly suggest
diac growth were attenuated when quercetin was administered                      that the route of delivery is a critical determinant in producing
to rats via oral gavage. The protection afforded by quercetin                    efficacious concentrations of this flavonoid that may result in
when delivered in this manner, compared with the absence of                      cardiovascular protection. As such, the route or method of
similar effects in the present study, prompted us to test whether                compound delivery should be an important consideration when
the route of quercetin delivery (i.e., once daily oral gavage vs. ad             studies are designed to examine the biological effect(s) of phyto-
libitum consumption of quercetin supplemented diet) is a critical                chemicals.
determinant of biological effect. Strikingly, we observed that                       In a previous study, we reported that quercetin-supplemented
mean arterial pressure in 16-wk–old gavaged SHR was signif-                      diets prevent increases in blood pressure and cardiac hypertro-
icantly lower than 16-wk–old SHR and SHR-Q. The blood                            phy in rats with abdominal aortic constriction (AAC) (9). AAC
pressure reduction we observed is comparable to that reported                    is a surgical procedure that places a physical constriction, via
by Duarte et al. (11) when 14-wk–old SHR were gavaged for 7 d                    ligature or hemoclip, on the abdominal aorta. This creates pres-
with quercetin. In that study, however, more robust decreases in                 sure overload in the heart and in the vasculature located prox-
blood pressure were evident after 5 wk of quercetin treatment.                   imal to the mechanical hindrance to blood flow, but not the rest
    Given these findings concerning blood pressure, we expected                   of the body (9). It is not clear why a dietary approach identical to
quercetin-gavaged SHR to exhibit higher plasma quercetin con-                    that described in the present study produced cardiovascular
centrations than SHR-Q. Surprisingly, plasma quercetin levels                    protection in the AAC rat but not the SHR. However, this dis-
were lower in gavaged SHR (0.61 mmol/L) than in SHR-Q                            crepancy may be due to the local (AAC rat) vs. systemic (SHR)
(2.09 mmol/L). One potential explanation is that SHR-Q con-                      nature of hypertension in these models, the duration of
sumed the quercetin-supplemented diet up to the time they were                   hypertension (2 wk in AAC vs. 5–10 wk in SHR), or the genetic
killed, whereas gavaged SHR were 24 h removed from the last                      background of the rats (AAC Sprague Dawley background vs.
dose, as done in previous studies (11). This time gap may have                   SHR Wistar Kyoto background).
resulted in a gradual decrease in quercetin levels due to the on-                    In summary, we show that arterial hypertension, cardiac
going metabolism in the liver. Indeed, previous studies reported                 hypertrophy, vascular dysfunction, vascular remodeling, and
that plasma total quercetin concentrations return to baseline                    indices of oxidant stress are similar in SHR regardless of whether
632   Carlstrom et al.
they consumed quercetin-supplemented or standard diet. These                    13. Zanchi A, Brunner HR, Hayoz D. Age-related changes of the mechan-
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                                                                                    J Hypertens. 1997;15:1415–22.
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                                                                                14. Boluyt MO, Bing OH, Lakatta EG. The ageing spontaneously hyper-
protection was observed in models of pharmacologically                              tensive rat as a model of the transition from stable compensated
induced hypertension (10) and SHR (11). We think that the                           hypertrophy to heart failure. Eur Heart J. 1995;16: Suppl N:19–30.
mode of delivery, i.e., a dietary approach of enriching standard                15. Reeves PG. Components of the AIN-93 diets as improvements in the
rodent diet with quercetin aglycone compared with administer-                       AIN-76A diet. J Nutr. 1997;127:838S–41S.
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building peak plasma concentrations that would result in                            PS. Serial echocardiographic-Doppler assessment of left ventricular
                                                                                    geometry and function in rats with pressure-overload hypertrophy.
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