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1 From the Servicio de Bioquímica-Investigación (PC, AD, FC, HO, DG-C, and MAL) and the Servicio de Nefrología (RE, JLT, MFL, and JO), Hospital Ramón y Cajal, Madrid; the Instituto de Fermentaciones Industriales, CSIC, Madrid (AD); and the Universidad de Alcalá, Alcalá de Henares (JO, MAL), Spain
2 Supported by grants from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (VIN00-004 and VIN03-027) and the Fundación para la Investigación del Vino y la Nutrición and Fundación Lair, Spain, and by a fellowship from the Fundación Carolina, Spain (to AD). 3 Reprints not available. Address correspondence to MA Lasunción, Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Ctra de Colmenar, km 9, E-28034 Madrid, Spain. E-mail: miguel.a.lasuncion{at}hrc.es.
ABSTRACT
Background: Patients treated with hemodialysis frequently experience cardiovascular complications attributed, among other causes, to dyslipidemia, increased oxidative stress, and inflammation.
Objective: The aim of the study was to study the effects of dietary supplementation with concentrated red grape juice (RGJ), a source of polyphenols, on lipoprotein profile, antioxidant capacity, LDL oxidation, and inflammatory biomarkers.
Design: Twenty-six patients receiving hemodialysis and 15 healthy subjects were instructed to drink 100 mL RGJ/d for 14 d. Blood was drawn at baseline, twice during RGJ supplementation, and twice during the 6-mo follow-up period. As a control, 12 other randomly recruited hemodialysis patients not receiving RGJ were studied. Lipids, apolipoproteins, oxidized LDL, and antioxidant vitamins were measured in plasma. The bioavailability of RGJ polyphenols was assessed in healthy subjects.
Results: The maximum plasma concentration of quercetin was achieved 3 h after RGJ ingestion, which indicates that supplement-derived polyphenols are rapidly absorbed. In both healthy subjects and hemodialysis patients, RGJ consumption increased the antioxidant capacity of plasma without affecting concentrations of uric acid or ascorbic acid; reduced the concentration of oxidized LDL; and increased the concentration of cholesterol-standardized -tocopherol. RGJ supplementation also caused a significant decrease in LDL-cholesterol and apolipoprotein B-100 concentrations, while increasing the concentrations of HDL cholesterol and apolipoprotein A-I. In a further study in hemodialysis patients, RGJ supplementation for 3 wk significantly reduced plasma monocyte chemoattractant protein 1, an inflammatory biomarker associated with cardiovascular disease risk.
Conclusion: Dietary supplementation with concentrated RGJ improves the lipoprotein profile, reduces plasma concentrations of inflammatory biomarkers and oxidized LDL, and may favor a reduction in cardiovascular disease risk.
Key Words: Antioxidants • cholesterol • oxidized LDL • inflammation • hemodialysis • cardiovascular disease
INTRODUCTION
Patients with end-stage renal disease frequently have advanced atherosclerosis, and cardiovascular complications are the principal cause of death in this population. Although hypercholesterolemia is infrequent in these patients, most of them exhibit slight hypertriacylglycerolemia and reduced HDL-cholesterol and apolipoprotein (apo) A-I concentrations (1, 2). Oxidative stress is another important cardiovascular risk factor in chronic renal insufficiency (3-5), and plasma concentrations of oxidized LDL are a predictor of atherosclerosis in both hemodialysis patients (6, 7) and the general population (8-11). Moreover, LDL from hemodialysis patients is highly susceptible to oxidation in vitro (12). Interestingly, vitamin E supplementation has been shown to reduce cardiovascular disease events in hemodialysis patients (13).
In addition to vitamins E and C, the most common and active antioxidant compounds that occur naturally in foods are flavonoids. The so-called French paradox attributes the relatively low cardiovascular mortality rate observed in Mediterranean countries to the regular consumption of red wine, a product that is rich in flavonoids (14). However, it remains to be determined whether it is the flavonoid content, the alcohol, or both that account for the protective effects of red wine. The bioavailability of dietary polyphenols, including those from grape derivatives, has been shown previously by many authors (15-19). After being ingested, polyphenols are detected in plasma both in their soluble form and physically associated with lipoproteins (20-22). Oral administration of grape flavonoids has been shown to confer antioxidant protection (23, 24), inhibit platelet function (25, 26), reduce thrombus formation (27) and the concentration of inflammatory biomarkers (28), and inhibit the activation of nuclear transcription factor B (29). At the vascular wall level, grape polyphenols exert vasorelaxant effects (30), inhibit the adhesion of monocytes to the endothelium (31, 32), and improve endothelial function (23). Finally, red wine polyphenols have been shown to reduce the development of atherosclerosis in animal models (33-35).
Hemodialysis patients are subjected to severe dietary restrictions to reduce potassium intake, which may limit the intake of other micronutrients. In the present work, we studied the effects of a dietary supplement of concentrated red grape juice (RGJ), a source of polyphenols that is devoid of ethanol, in hemodialysis patients and healthy subjects.
SUBJECTS AND METHODS
Subjects and study design
Thirty-eight clinically stable patients who had been receiving maintenance hemodialysis for >3 mo were recruited from patients attending the morning shift in our dialysis unit at Hospital Ramón y Cajal, Madrid. The etiology of the patients’ renal disease was as follows: 10 patients with glomerulonephritis, 10 with interstitial chronic nephropathy, 7 with vascular nephropathy, 1 with polycystic kidney disease, 1 with light-chain disease, 1 with amyloidosis, 1 with lupus nephropathy, and 7 with disease of unknown causes. Patients with diabetes mellitus were excluded because of the high carbohydrate content of RGJ. Patients with unstable angina pectoris, recent myocardial infarction, or congestive heart failure were also excluded. All patients were dialyzed 3 times per week for 3.5 to 4.5 h per session with membranes of intermediate (22 patients) or high (16 patients) biocompatibility and had been treated for an average of 5.3 y. All patients were receiving erythropoietin. To compensate for losses of low molecular weight molecules as a result of the dialysis procedure, the patients also received a multivitamin supplement (Becozyme C Forte; Roche Diagnostics, Sant Cugat del Vallés, Spain) with vitamin C (200 mg/d) as the only antioxidant. Nine subjects were treated with ß-blockers. These treatments were not altered during the course of the study. A diet consisting of 15% protein, 50% carbohydrates, 35% lipids (35% saturated fatty acids, 52% monounsaturated fatty acids, and 13% polyunsaturated fatty acids), 320 mg cholesterol, 25 g fiber, and 1.2 L of water was recommended in all subjects. Special care was taken to limit the intake of potassium (100 mEq/d), and patients were instructed to avoid foods with a high potassium content, such as green-leaf vegetables and dried fruit, and to generally restrict their consumption of fruit and vegetables. Patients declared that no changes were made to their diet during the course of the study.
The patients were randomly distributed in 2 groups: RGJ and control. The RGJ group consisted of 26 patients, 13 men and 13 women, aged 62.0 ± 3.4 y (
In an additional study, used to evaluate the effects of RGJ consumption on inflammatory biomarkers, 10 hemodialysis patients (4 men and 6 women) were recruited and instructed to consume the RGJ supplement (50 mL, twice daily) for 3 consecutive weeks. Blood samples were taken at baseline and every 7 d during the RGJ consumption period.
Finally, to study the bioavailability of polyphenols present in RGJ, 6 healthy volunteers were given 100 mL of RGJ in a single dose at 1900 along with a meal consisting of 2 cheese and ham sandwiches. Six other volunteers not taking the RGJ supplement were studied as controls. The plasma concentration of quercetin was determined in blood samples withdrawn immediately before ingestion and 1.5, 3, and 14 h later. No food was taken during the sampling period.
The study complied with the principles of the Declaration of Helsinki. The study protocols were approved by the Ethics Committee on Clinical Research of the Hospital Ramón y Cajal, and informed consent was obtained from all participants.
Concentrated red grape juice
The RGJ concentrate was purchased from Dream Fruits (Quero, Toledo, Spain). It was prepared from natural RGJ (from the bobal grape varietal) by dehydration at low pressure and heating (–0.8 bar, 65 °C). As certified by the provider, the specifications of the RGJ concentrate were as follows: 68 degrees Brix, equivalent to 84 g of sugars/100 mL, with approximately equal amounts of glucose and fructose. The polyphenol composition was assessed at the Instituto de Fermentaciones Industriales, CSIC, Madrid (by Begoña Bartolomé and M Carmen Gómez-Cordovés) and was as follows: total polyphenols, 0.64 g/100 mL (using gallic acid as calibrator); quercetin-3 rutinoside, 4.13 mg/100 mL; quercetin-3 glucoside, 0.256 mg/100 mL; quercetin aglycon, traces; myricetin aglycon, 3.13 mg/100 mL; catechin aglycon, 0.018 mg/100 mL; procyanidin B2, 0.348 mg/100 mL; anthocyanidines, 12.4 mg/100 mL (using malvidin-3-glucoside as calibrator). The potassium content was 6.8 mEq per daily dose (100 mL), 6% of the estimated daily intake of potassium in hemodialysis patients. The same batch of RGJ was used in all the studies. The RGJ was bottled in 750-mL bottles and stored in a cold room at 4 °C until it was given to the participants (one bottle per week).
Biochemical measurements
Routine biochemical analyses were performed by using automated procedures (RA-1000 Autoanalyzer; Technicon Ltd, Dublin, Ireland). Concentrations of cholesterol and triacylglycerols were measured in EDTA-treated plasma by using enzymatic methods (Menarini Diagnostici, Firenze, Italy). HDL-cholesterol concentrations were measured after precipitation of apo B–containing lipoproteins with phosphotungstic acid and magnesium (Roche Diagnostics, Madrid, Spain). LDL-cholesterol concentration were calculated according to the Friedewald formula. Concentrations of apo A-I and apo B were assessed by immunonephelometry (Dade Behring, Frankfurt, Germany).
Total antioxidant capacity (TEAC) was determined as the capacity to reduce the ABTS (2,2- azino-di-[3-ehtylbenzthiazoline sulphonate) oxidation induced by hydrogen peroxide in the presence of ferryl myoglobin and is expressed as Trolox equivalents (36). The concentration of oxidized LDL was measured by sandwich enzyme-linked immunosorbent assay by using the monoclonal antibody mAb-4E6 as the capture antibody and peroxidase-conjugated anti-apolipoprotein B for detection (Mercodia AB, Uppsala, Sweden).
Concentrations of soluble vascular cell adhesion molecule 1 (VCAM-1), soluble intercellular adhesion molecule 1 (ICAM-1), and monocyte chemoattractant protein 1 (MCP-1) were measured in serum with commercially available enzyme-linked immunosorbent assay kits (from Diaclone SAS, Besançon, France, for VCAM-1 and ICAM-1, and PeproTech EC Ltd, London, United Kingdom, for MCP-1). Concentrations of high-sensitivity C-reactive protein and complement C3 protein were measured by immunonephelometry (Dade Behring, Frankfurt, Germany).
The lipid-soluble antioxidants -tocopherol, -tocopherol, lycopene, -carotene, and ß-carotene were measured by gradient HPLC with photodiode-array detection (Beckman Coulter Inc, Fullerton, CA) after extraction with ethanol:methanol with the use of retinol palmitate and tocopherol acetate as internal standards (37).
For vitamin C determinations, blood was collected in EDTA-coated tubes and was immediately centrifuged at 1200 x g at 4 °C for 10 min to obtain plasma. An equal volume of 10% (wt:vol) metaphosphoric acid was added to a 0.2-mL aliquot of plasma, the mixture was centrifuged, and the supernatant fluid was stored at –80 °C before use. L-(+)-Ascorbic acid concentrations were measured by using an HPLC-fluorimetric method described previously by Speek et al (38). Briefly, L-(+)-ascorbic acid was oxidized to dehydro- l-(+)-ascorbic acid with cholesterol oxidase and condensed with o-phenylenediamine to its quinoxaline derivative, and the product was separated by HPLC and detected (HP 1046A programmable fluorescence detector; Agilent, Wilmington, DE).
Total plasma quercetin was processed by using the method proposed by Erlund et al (39) with certain modifications. Briefly, thawed EDTA-treated plasma (1 mL in duplicate) was hydrolyzed for 17 h at 37 °C by incubation with 110 µL of 0.78 mol sodium acetate buffer/L (pH 4.8), 100 µL of 0.1 mol ascorbic acid/L and 4000 U of ß-glucuronidase/200 U of sulfatase (ß-glucuronidase from Helix pomatia type HP-2; Sigma, St Louis, MO). Then, 100 µL of 5 mol formic acid/L was added and quercetin was extracted 3 times with ethyl acetate (2 + 1 + 1 mL), the phases being separated each time by centrifugation (3500 x g, 5 min, 4 °C). The supernatant portions were combined and evaporated to dryness under nitrogen. The residue was dissolved in 300 µL of a methanol:water mixture (1:1, by vol), filtered (0.45 µm), and injected (200 µL) into a reversed-phase Nova-pack C18 column (30 cm x 3.9 mm internal diameter; Waters, Milford, MA). Detection was performed by scanning from 220 to 380 nm, and quercetin quantitation was carried out by area measurements at 366 nm (996 photodiode-array, Waters).
Statistical analysis
Data are presented as means ± SEMs. Comparisons of plasma variables at baseline between hemodialysis patients and healthy subjects were made by using Student’s t test. In hemodialysis patients, the interaction between time (different periods) and treatment (RGJ supplementation or control) was analyzed by two-factor repeated-measures ANOVA. In this case, only 3 study periods could be considered: baseline, RGJ II, and follow-up II in the patients receiving RGJ and the corresponding visits in the patients not receiving RGJ (control group). To evaluate the effect of RGJ supplementation and follow-up over time, variables were compared by one-way repeated-measures ANOVA, and multiple post-hoc comparisons were performed by using Tukey’s test. In the study of RGJ polyphenol assimilation, changes in the plasma concentration of quercetin over time were analyzed by two-factor repeated-measures ANOVA and post-hoc multiple comparisons were made by using Tukey’s test. Pearson correlation coefficients were calculated for linear regression analyses. The effect of RGJ supplementation on the relations between plasma concentrations of oxidized LDL and LDL-cholesterol in hemodialysis patients compared with healthy subjects was evaluated by multiple regression. The variables considered were oxidized LDL as the dependent variable, and LDL-cholesterol concentration, group (hemodialysis patients and healthy subjects), and study periods (baseline, RGJ I, RGJ II, follow-up I, and follow-up II) as the independent variables. Study periods were introduced as dummy variables. The 3-factor (LDL-cholesterol x group x study period) and 2-factor (LDL-cholesterol x group, LDL-cholesterol x study period, and group x study period) interactions were calculated. Significance was set at P < 0.05. Calculations were performed by using SIGMASTAT STATISTICAL ANALYSIS, system V. 1.00 (Jandel Corporation, San Rafael, CA), and STATGRAPHICS PLUS v5.0 (Statistical Graphics Corporation, Rockville, MD).
RESULTS
To assess the assimilation of RGJ polyphenols, 6 healthy volunteers were given 100 mL RGJ in a single dose along with an evening meal low in polyphenolic compounds, and plasma quercetin was measured at different times after ingestion. Another 6 subjects who did not receive the RGJ concentrate were studied as a control group. At baseline, the plasma concentration of quercetin varied markedly among the volunteers, ranging from undetectable to as high as 136 µg/L. For the sake of clarity, the results are given as % of basal (Figure 1). Ingestion of RGJ led to a transient increase in the plasma concentration of total quercetin; this increase was statistically significant at 3 h after ingestion and represented an average increase of 25%. In contrast, in the control group, plasma concentrations of quercetin declined progressively over the study period.
FIGURE 1.. Mean (±SEM) plasma concentrations of quercetin in healthy subjects after receiving 100 mL of concentrated red grape juice () or placebo (). n = 6 per group. Values are expressed as % of basal (time 0). Statistical analyses were performed with crude values. Time-dependent changes were evaluated by repeated-measures ANOVA: red grape juice group, P < 0.05; placebo group, P < 0.01. Interaction between time and treatment: P = 0.0029. Tukey’s test was applied for post hoc statistical comparisons versus time 0: *, P < 0.05.
The effects of RGJ supplementation were studied in both healthy subjects and patients affected by renal disease. Of the 39 hemodialysis patients initially recruited, one refused to take the RGJ supplement because of its "unpleasant" sweet taste and was dropped from the study. In 3 cases, we could not obtain a blood sample corresponding to the follow-up II period (6 mo after cessation of RGJ supplementation) because of kidney transplantation (2 patients) or death (1 patient). All other participants successfully completed the study. Overall, the supplement was well tolerated and no side effects, including diarrhea, were reported. Both hemodialysis patients and healthy subjects declared that they did not change their food intake habits and neither group experienced a change in body mass during the study: hemodialysis patients, 67.9 ± 6.6 and 67.9 ± 6.7 kg at baseline and follow-up II, respectively; healthy subjects, 64.3 ± 3.6 and 64.6 ± 3.7 kg, respectively.
Plasma concentrations of glucose, uric acid, albumin, and total proteins did not change significantly in response to RGJ supplementation in either healthy subjects or patients receiving hemodialysis (Table 1). Plasma concentrations of lipids and apolipoproteins are shown in Table 2. The interaction between RGJ supplementation and time (study periods) could be studied only in the hemodialysis patients, considering 3 study periods: baseline, RGJ II, and follow-up II in the patients receiving the RGJ concentrate and the corresponding periods in the patients not receiving the RGJ concentrate (control group). There were no significant RGJ supplementation x time interactions for the concentrations of triacylglycerols or total cholesterol. However, there were significant interactions for LDL cholesterol, HDL cholesterol, the ratio of LDL to HDL cholesterol, apo A-I, and apo B. RGJ supplementation produced a significant reduction in the concentrations of both LDL cholesterol and apo B and an increase in the concentrations of HDL cholesterol and apo A-I. As a result, a marked decrease in the LDL-HDL ratio was observed in response to RGJ consumption. In general, the changes were larger in the second week than in the first week of intervention and, with the exception of apo A-I, persisted for 4 wk after supplement withdrawal (follow-up I). Six months later (follow-up II), all lipid and lipoprotein concentrations had returned to baseline values (Table 2).
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TABLE 1. General biochemical variables in plasma in healthy subjects and in hemodialysis (HD) patients receiving or not receiving (control) a concentrated red grape juice (RGJ) supplement1
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TABLE 2. Plasma lipids and apolipoproteins in healthy subjects and in hemodialysis (HD) patients receiving or not receiving (control) a concentrated red grape juice (RGJ) supplement1
In healthy subjects, the changes associated with RGJ consumption and follow-up were similar to those observed in the hemodialysis patients, with a decrease in the concentrations of total cholesterol, LDL cholesterol, and apo B, and an increase in concentrations of HDL cholesterol and apo A-I (Table 2). These changes returned to basal values at the follow-up II period. No changes in any of these lipoprotein-related variable were observed in the hemodialysis patients not receiving RGJ (control) who were analyzed in parallel (Table 2).
Oxidation-related variables are shown in Table 3. At baseline, both TEAC and plasma concentrations of oxidized LDL were significantly higher in patients undergoing hemodialysis than in healthy subjects. There was a significant RGJ consumption x time interaction for both variable in the hemodialysis patients. In both hemodialysis patients and healthy subjects, RGJ consumption led to a significant increase in TEAC within the first week of intervention, and TEAC remained elevated in the second week before returning to baseline values during the follow-up period (Table 3). In contrast, oral supplementation with RGJ caused a decrease in the plasma concentration of oxidized LDL of 35% in both groups. No changes were observed in TEAC or oxidized LDL concentrations over the study period in the hemodialysis patients not receiving RGJ (Table 3).
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TABLE 3. Total antioxidant capacity (TEAC) and concentrations of oxidized LDL in plasma from healthy subjects and from hemodialysis (HD) patients receiving or not receiving (control) a concentrated red grape juice (RGJ) supplement1
At baseline, plasma concentrations of oxidized LDL were directly correlated with those of LDL cholesterol in both the hemodialysis patients and the healthy subjects (Figure 2). The effect of RGJ consumption was studied by multiple regression analysis, and the interaction terms were calculated. The 3-factor interaction between LDL cholesterol, group, and study period was not significant. However, the LDL cholesterol x group interaction was significant (P < 0.05), which indicated a distinct relation between oxidized LDL and LDL cholesterol in the hemodialysis patients compared with the healthy subjects. Moreover, the study period had a significant effect in the whole model (P < 0.001) and also in the hemodialysis patients (P < 0.001) and the healthy subjects (P < 0.05) when considered separately. Consistent with this finding, a tendency for the slope of the regression lines to decline as a result of RGJ supplementation and then to return progressively to baseline values during the follow-up period was clearly observed when the different periods were studied separately by simple regression (Figure 2). In contrast, the absolute decrease in the concentration of oxidized LDL caused by RGJ supplementation did not correlate with that of LDL cholesterol in any group (hemodialysis patients, r = –0.0636, P > 0.05; healthy subjects, r = 0.0894, P > 0.05). This suggests that, in addition to the reduction of LDL cholesterol concentration, other factors may contribute to the reduction of oxidized LDL caused by RGJ supplementation.
FIGURE 2.. Correlation between oxidized LDL and LDL-cholesterol concentrations in hemodialysis patients and healthy subjects receiving a dietary red grape juice (RGJ) supplement. Study periods were as follows: baseline; RGJ I, 7 d of RGJ supplementation (• and solid line); RGJ II, 14 d of RGJ supplementation ( and dashed line); follow-up I, 4 wk after withdrawal of RGJ supplementation (• and solid line); follow-up II, 6 mo after withdrawal of RGJ supplementation ( and dashed line). Multiple regression analysis was performed to evaluate the effect of LDL cholesterol on the plasma concentration of oxidized LDL and to calculate the 3-factor and 2-factor interactions between LDL cholesterol, group (hemodialysis patients and healthy subjects), and study periods (baseline, RGJ I, RGJ II, follow-up I, and follow-up II). After backward selection, the variables significantly contributing to the model were LDL cholesterol (P < 0.001), study period (introduced as a dummy variable; P < 0.001), and the LDL cholesterol x group interaction (P < 0.05). Regression line equations for hemodialysis patients are as follows: baseline, y = 5.834x + 1.962 (r = 0.6595, P < 0.001); RGJ I, y = 3.016x + 3.457 (r = 0. 5321, P < 0.01); RGJ II, y = 3.421x + 1.317 (r = 0. 5352, P < 0.01); follow-up I, y = 4.581x + 0.857 (r = 0. 7266, P < 0.001); and follow-up II, y = 7.168x – 3.175 (r = 0. 6746, P < 0.001). Regression line equations for healthy subjects are as follows: baseline, y = 3.193x + 5.090 (r = 0.4491, P < 0.001); RGJ I, y = 2.637x + 4.049 (r = 0. 4250, P < 0.01); RGJ II, y = 2.304x + 2.553 (r = 0. 4690, P < 0.01); follow-up I, y = 3.522x + 1.180 (r = 0. 4442, P < 0.001); and follow-up II, y = 3.325x + 3.703 (r = 0. 4494, P < 0.001).
Concentrations of vitamin C, vitamin E, and carotenoids were measured as the antioxidants that could contribute to the effects on TEAC and oxidized LDL (Table 4). At baseline, the mean concentration of ascorbic acid in plasma was significantly higher in the hemodialysis patients than in the healthy subjects. This finding is consistent with the regular ingestion of a polyvitamin supplement in the patients treated with hemodialysis. There were no significant RGJ consumption x study period interactions for these variables in the hemodialysis patients. In fact, no significant changes in the concentrations of vitamin C or lipid-soluble antioxidants were detected during the treatment period in any group (Table 4).
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TABLE 4. Antioxidant vitamins in plasma from healthy subjects and from hemodialysis (HD) patients receiving or not receiving (control) a concentrated red grape juice (RGJ) supplement1
Fat-soluble antioxidants circulate in blood bound to lipoproteins, and their concentrations are highly dependent on the concentrations of lipids (40). Thus, to better compare the load of these antioxidants between individuals with different plasma lipid concentrations, the data were standardized by dividing the antioxidant concentration by that of cholesterol (Table 5). At baseline, there was no significant difference in the standardized concentrations of -tocopherol and -tocopherol between the hemodialysis patients and the healthy subjects. In contrast, standardized concentrations of carotenoids were lower in the hemodialysis patients than in the healthy subjects. In the hemodialysis patients, the interaction between RGJ supplementation and study period was not significant for any of the lipophilic antioxidants studied (Table 5). When the interaction term was removed, the standardized concentration of -tocopherol significantly increased in response to RGJ supplementation, both in hemodialysis patients and in healthy subjects, whereas other lipid-soluble antioxidants did not change significantly (Table 5).
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TABLE 5. Standardized concentrations of lipophilic antioxidants in plasma from healthy subjects and from hemodialysis (HD) patients receiving or not receiving (control) a concentrated red grape juice (RGJ) supplement1
In a further study in 10 hemodialysis patients, RGJ consumption was prolonged for 3 wk. As in the previous study, no significant changes were observed for general plasma variable (glucose, 5.32 ± 0.60 and 5.31 ± 0.60 mmol/L; uric acid, 0.40 ± 0.03 and 0.43 ± 0.02 mmol/L; total proteins, 65.9 ± 1.68 and 63.9 ± 1.29 g/L; albumin, 40.7 ± 0.79 and 39.9 ± 0.75 g/L, at baseline and after the third week of RGJ consumption, respectively). No effect was observed on triacylglycerol concentration (1.99 ± 0.30 and 1.73 ± 0.16 mmol/L). The concentrations of total cholesterol (4.19 ± 0.34 and 3.66 ± 0.29 mmol/L; P < 0.01), LDL cholesterol (2.62 ± 0.31 and 2.12 ± 0.25 mmol/L; P < 0.01), and apo B (0.764 ± 0.061 and 0.657 ± 0.056 g//L; P < 0.01) decreased significantly in response to RGJ consumption. The concentration of HDL cholesterol increased significantly (0.665 ± 0.06 and 0.796 ± 0.077 mmol/L; P < 0.05), whereas the change in apo A-I was not significant (1.09 ± 0.07 and 1.12 ± 0.09 g/L). Finally, RGJ consumption caused a marked reduction in the plasma concentration of oxidized LDL, from 17.7 ± 1.95 mU/L at baseline to 6.5 ± 1.13 mU/L during the third week of intervention (P < 0.001). All these changes were similar to those observed in the first series of hemodialysis patients described above.
Finally, the effects of RGJ consumption on inflammatory markers in serum were studied. As shown in Table 6, concentrations of VCAM-1, ICAM-1, complement C3 protein, and C-reactive protein did not change significantly in response to RGJ supplementation during the 3-wk study period. In contrast, the concentration of MCP-1 decreased progressively, reaching a value roughly one-half that observed at baseline in the third week of intervention (Table 6).
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TABLE 6. Biomarkers of inflammation in plasma from hemodialysis patients receiving a concentrated red grape juice (RGJ) supplement for 3 consecutive weeks1
DISCUSSION
Previous studies reported the assimilation of grape polyphenols (15-19) and their ability to confer antioxidant protection in vivo (23, 24, 26, 41, 42). In the present work, in addition to confirming that quercetin, a major polyphenol in RGJ, is absorbed, we show for the first time that RGJ supplementation improves the lipoprotein profile by decreasing plasma concentrations of LDL and increasing those of HDL and greatly reduces the plasma concentrations of oxidized LDL and MCP-1, an inflammatory biomarker. This is particularly important in hemodialysis patients, who are at high risk of developing cardiovascular disease, probably caused by excessive oxidative stress (3-5) and systemic inflammation (43, 44).
Circulating oxidized LDL is a sensitive marker of coronary artery disease, both in the general population (8, 10, 11) and in hemodialysis patients (7). Importantly, the oxidized LDL concentration is directly correlated with the severity of the underlying acute coronary syndrome (9). We found that plasma concentrations of oxidized LDL were significantly higher in the hemodialysis patients than in the healthy subjects, and that dietary supplementation with RGJ markedly reduced this variable in both groups.
The mechanism by which RGJ consumption reduces oxidized LDL is unclear. Flavonoids have been shown to spare vitamin E in vitro (45). We found that RGJ consumption was associated with an increase in the concentration of cholesterol-standardized -tocopherol, but not total -tocopherol. Other authors found either no change (24) or an increase in the plasma concentration (26) of total -tocopherol after consumption of grape-derived products. Interestingly, when the data from all the hemodialysis patients were considered together, an inverse correlation was apparent between standardized -tocopherol and oxidized LDL concentrations in plasma (r = –0.2273, P < 0.01). Therefore, RGJ polyphenols may protect LDL from oxidation either directly or indirectly by sparing vitamin E. This possibility, however, must be considered with caution, because recent studies have shown that long-term administration of vitamin E fails to alter the concentration of oxidized LDL (46). On the other hand, shortening of the residence time of LDL within the vascular compartment, as indicated by the decrease in the number of circulating LDL particles, may also contribute to the reduction of oxidized LDL caused by RGJ consumption (47).
Despite having a higher concentration of oxidized LDL, hemodialysis patients exhibited a higher antioxidant capacity in plasma than did healthy subjects. This agrees with previously reported results (3, 48, 49) and has been attributed to differences in uric acid concentration (48). In the hemodialysis patients studied here, who were taking vitamin C supplements, the increased concentration of ascorbic acid could also contribute to these differences in antioxidant capacity. In any case, the increased presence of soluble antioxidants in the plasma of the hemodialysis patients did not appear to offer effective protection against lipid peroxidation.
Dietary RGJ supplementation produced no detectable changes in plasma concentrations of urate or ascorbate in any of the groups studied (Tables 1 and 4). This is consistent with the results of other studies using purple grape juice (24, 26). Lotite and Frei (50) recently reported that the rapid increase in plasma antioxidant capacity after the ingestion of 1 kg of apples was mainly due to urate produced as a consequence of the apples’ high fructose content (>64 g/kg). These effects of fructose were very rapid: maximum values were reached 1 h after ingestion and disappeared 3 h later (50). With RGJ, we did not detect any significant change in uric acid concentration after acute administration in healthy subjects: concentrations were 0.25 ± 0.02, 0.24 ± 0.02, 0.24 ± 0.02, and 0.22 ± 0.02 mmol/L, at 0, 1.5, 3, and 14 h, respectively. Therefore, other antioxidants, probably polyphenols, are likely to be responsible for the observed effects of RGJ consumption on antioxidant capacity.
Evidence is accumulating that inflammation plays a role in atherosclerosis (51, 52). Moderate consumption of red wine has been shown to inhibit the expression of MCP-1 and other adhesion molecules in monocytes and to reduce the ability of these cells to adhere to the endothelium (32, 53). Moreover, consumption of red wine, but not gin, reduces the serum concentration of C-reactive protein and several soluble adhesion molecules (28). In our study, we did not observe significant variation in the concentration of VCAM-1, ICAM-1, or C-reactive protein, but the concentration of MCP-1 decreased to one-half the baseline value at the third week of intervention with concentrated RGJ. Because serum concentrations of MCP-1 are usually elevated in hemodialysis patients (54, 55) and have been recognized to be an important factor in the progression of atherosclerosis (56), this effect of RGJ must be considered as beneficial in the reduction of cardiovascular risk in these patients.
One of the most striking results of the present study is the improvement of the lipoprotein profile in response to RGJ supplementation, with parallel reductions in the concentrations of both LDL cholesterol and apo B, and increases in HDL cholesterol and apo A-I. These effects were observed in both hemodialysis patients and in healthy subjects. Some other authors, however, have not observed this effect in response to ingestion of unconcentrated grape juice in healthy subjects (24). Differences in the composition and concentration of polyphenols in the grape juice may account for this discrepancy: 644 mg/d in the present study compared with 432 mg/d in the other study (24).
Because both hemodialysis patients and healthy subjects declared that they had not changed their food intake habits during the study period, the observed effects on plasma lipids can be directly attributed to the ingestion of RGJ. Consistent with this observation, ingestion of polyphenol-rich extracts from either green tea (57) or pine bark (58) have been shown to reduce plasma LDL-cholesterol concentrations in humans. A similar effect was found for red wine polyphenols in hamsters (59). Very recently, administration of procyanidins from grape seeds was shown to lead to a reduction in the plasma concentrations of LDL cholesterol and apo B and to a slight increase in that of HDL cholesterol in rats (60). The active compounds and the mechanism or mechanisms underlying this hypolipidemic effect, however, are unclear. Red wine polyphenols (61) and green tea catechins (62) increase LDL-receptor expression in a human hepatocyte cell line in culture. In addition, pure epigallocatechin gallate interferes with micellar solubilization of cholesterol in the digestive tract of rats, thereby reducing cholesterol absorption (63). In women, both red wine and dealcoholized red wine attenuate postprandial chylomicron concentrations, possibly by delaying intestinal absorption of fat (64). In this respect, saponins, which can interfere with dietary fat assimilation, were recently reported to be present in grapes and wines (65). Therefore, both the inhibition of intestinal absorption of cholesterol and the accelerated clearance of plasma LDL may account for the observed hypolipidemic action of RGJ.
Although the antioxidant capacity of plasma returned to baseline values immediately after RGJ withdrawal, other variable took a longer time to return to normal values. This was particularly true for the concentrations of LDL cholesterol and oxidized LDL, which remained significantly reduced 4 wk after cessation of RGJ ingestion (follow-up I period). When high doses of quercetin glycosides are orally administered, the elimination of quercetin from plasma has been shown to be slow, taking longer than 24 h, which suggests that quercetin may accumulate throughout the day with repeated intake (66). This may also be the case for dietary supplementation with RGJ; in this case, the flavonoids it contains or their metabolites would be accumulated in the body and their effects would last for an extended period of time.
In conclusion, dietary supplementation with concentrated RGJ exerts hypolipidemic, antioxidant, and antiinflammatory actions in both healthy subjects and patients with end-stage renal disease. This effect may be considered to be beneficial for the prevention of cardiovascular disease.
ACKNOWLEDGMENTS
We thank Miguel Martín for excellent technical assistance, Víctor Abraira and Alfonso Muriel for their help with the statistical analyses, and Iain Patten and Olive Waldron for editorial advice. We are also indebted to Begoña Bartolomé and María del Carmen Gómez-Cordovés from the Instituto de Fermentaciones Industriales, CSIC, Madrid, for analysis of the composition of the red grape juice concentrate.
MAL and JLT designed the study. PC performed the determinations of TEAC, oxidized LDL, vitamins, and antiinflammatory markers. RE participated in sample collection. FC and DG-C performed the lipid and apolipoprotein determinations. AD performed the quercetin determinations. HO contributed to TEAC determinations. JLT, MFL, and JO were responsible for patient care. MAL supervised the study and prepared the manuscript, which was reviewed by all authors. None of the authors had a financial or personal conflict of interests.
REFERENCES